Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Cerebral metabolism in anoxia and the effects of some neurotropic drugs Shankar, Raj 1971

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1971_A1 S33_5.pdf [ 12.66MB ]
Metadata
JSON: 831-1.0101871.json
JSON-LD: 831-1.0101871-ld.json
RDF/XML (Pretty): 831-1.0101871-rdf.xml
RDF/JSON: 831-1.0101871-rdf.json
Turtle: 831-1.0101871-turtle.txt
N-Triples: 831-1.0101871-rdf-ntriples.txt
Original Record: 831-1.0101871-source.json
Full Text
831-1.0101871-fulltext.txt
Citation
831-1.0101871.ris

Full Text

CEREBRAL METABOLISM IN ANOXIA AND THE EFFECTS OF SOME NEUROTROPIC DRUGS by RAJ SHANKAR B . S c , U n i v e r s i t y of Gorakhpur, 1964 M.Sc., U n i v e r s i t y of Lucknow, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Biochemistry We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1971 In present ing t h i s thes i s in p a r t i a l f u l f i lmen t of the requirements fo r an advanced degree at the Un iver s i t y of B r i t i s h Columbia, I agree that I fu r ther agree that permission for extens ive copying of th i s thes i s fo r s cho la r l y purposes may be granted by the Head of my Department or by h i s representat ives . It is understood that copying or pub l i c a t i on of th i s thes i s f o r f i n anc i a l gain sha l l not be allowed without my wr i t ten permiss ion. the L ib ra ry sha l l make i t f r e e l y ava i l ab le fo r reference and study. Department of The Un ivers i ty of B r i t i s h Columbia Vancouver 8, Canada Date %tvA < ^ W W V^l | \ ABSTRACT The e f f e c t s o f t e t r o d o t o x i n (TTX) a n d o t h e r n e u r o t r o p i c d r u g s on a n a e r o b i c g l y c o l y s i s , and on t r a n s p o r t p r o c e s s e s , o f i n c u b a t e d c e r e b r a l c o r t e x s l i c e s h a v e b e e n s t u d i e d i n an e f f o r t t o u n d e r s t a n d more f u l l y c e r e b r a l m e t a b o l i c p r o c e s s e s d u r i n g a n o x i a , and t h e mode o f a c t i o n o f c e r t a i n n e u r o t r o p i c d r u g s . The g e n e r a l a p p r o a c h h a s b e e n t o s t u d y t h e a c t i o n o f v a r i o u s d r u g s on t h e r a t e s o f a n a e r o b i c g l y c o l y s i s o f i n c u b a t e d b r a i n s l i c e s u n d e r a v a r i e t y + + o f c o n d i t i o n s . The c a t i o n (Na , K ) c o n t e n t s were a l s o s t u d i e d u n d e r t h e s e c o n d i t i o n s and t h e c h a n g e s i n t h e s e c o n t e n t s were r e l a t e d t o c o n c o m i t a n t c h a n g e s i n c e r e b r a l m e t a b o l i s m . E x p e r i m e n t s were a l s o c a r r i e d o u t on t h e c e r e b r a l t r a n s p o r t o f amino a c i d s and g l u c o s e u n d e r a v a r i e t y o f i n c u b a t i o n c o n d i t i o n s . M e a s u r e m e n t o f t h e r a t e s o f a n a e r o b i c g l y c o l y s i s i n t h e p r e s e n c e o f TTX showed t h a t t h e d r u g , a t low c o n c e n t r a t i o n s s u c h as 2 yM, e n h a n c e s t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s two t o t h r e e f o l d , t h e e f f e c t s b e i n g g r e a t e r i n t h e a b s e n c e o f C a + + . S u c h an e f f e c t o f TTX i s f a r g r e a t e r t h a n t h a t o b t a i n e d on t h e a e r o b i c m e t a b o l i s m o f t h e c e r e -b r a l c o r t e x s l i c e s . The a n a e r o b i c g l y c o l y s i s o f k i d n e y m e d u l l a s l i c e s , 2-day o l d r a t b r a i n s l i c e s o r o f a c e t o n e powder e x t r a c t s f r o m b r a i n a r e n o t a f f e c t e d by t h e d r u g , i n d i c a t i n g t h a t t h e e f f e c t s o f TTX on t h e a n a e r o b i c g l y c o l y s i s a r e s p e c i f i c f o r m a t u r e c e r e b r a l t i s s u e , and r e q u i r e s i n t e g r i t y o f t h e b r a i n c e l l s f o r i t s a c t i o n . TTX h a s l i t t l e o r no e f f e c t i n i n c r e a s i n g r a t e o f a n a e r o b i c g l y c o l y s i s when i t i s a d d e d 10 m i n u t e s , o r l a t e r , a f t e r t h e o n s e t o f a n o x i a , o r when h i g h K +, p r o t o v e r a t r i n e , L - g l u t a m a t e o r NH^ + a r e p r e s e n t i n t h e i n c u b a t i o n medium. Under t h e s e c o n d i t i o n s , t h e r e i s e i t h e r an i n f l u x o f N a + i n t o , O r «n4 l o s s o f K + f r o m , t h e i n c u b a t e d c e r e b r a l t i s s u e . I n t h e p r e s e n c e o f TTX u n d e r a n o x i c c o n d i t i o n s , a much s l o w e r d e c l i n e i n t h e K /Na r a t i o o f t h e c e r e -b r a l c o r t e x s l i c e s i s o b s e r v e d . T h e s e a n d o t h e r e x -p e r i m e n t s l e a d t o t h e c o n c l u s i o n t h a t t h e e f f e c t s o f TTX on a n a e r o b i c g l y c o l y s i s a r e due t o i t s a c t i o n a t t h e b r a i n c e l l membrane r e s u l t i n g i n t h e p r e v e n t i o n o f t h e c h a n g e s i n b r a i n c e l l p e r m e a b i l i t i e s t o N a + and K + b r o u g h t a b o u t by t h e o n s e t o f a n o x i a . I n t h e p r e s e n c e o f TTX, t h e i n i t i a l h i g h r a t e o f g l y c o l y s i s t e n d s t o be m a i n t a i n e d due t o o n l y a s l o w d e c l i n e i n t h e c e l l u l a r K + / N a + r a t i o . The e f f e c t s o f K + and N a + on t h e a n a e r o b i c g l y c o l y s i s a r e t h o u g h t t o be m e d i a t e d i v . l a r g e l y by changes i n the pyruvate k i n a s e a c t i v i t y , which i s enhanced by K + and d i m i n i s h e d by N a + . TTX appears to a f f e c t the a e r o b i c and ana e r o b i c metabolism o f b r a i n i n v i t r o i n the same way as i t a f f e c t s the g e n e r a t i o n a c t i o n p o t e n t i a l s i . e . by + + d i m i n i s h i n g the i n f l u x of Na and e f f l u x o f K . These r e s u l t s l e a d t o the c o n c l u s i o n t h a t a c t i o n p o t e n t i a l s are generated i n the i n c u b a t e d c e r e b r a l t i s s u e a t the onset o f anoxia . These are b l o c k e d by TTX which mani-f e s t s i t s e f f e c t by a h i g h e r r a t e o f a n a e r o b i c g l y c o l y s i s . The e f f e c t o f TTX on the N a + and K + contents may be g r e a t e r i n the neurons than i n g l i a l c e l l s because the former are the s i t e of a c t i o n o f TTX. Consequently, the changes i n the ne u r o n a l K +/Na + r a t i o brought about by TTX are pro b a b l y much g r e a t e r than those o f the K +/ Na + r a t i o found i n the t i s s u e as a whole. In a d d i t i o n t o i t s e f f e c t s on the N a + and K + f l u x e s , TTX a l s o p r e v e n t s the e f f l u x o f amino a c i d s from the i n c u b a t e d c e r e b r a l c o r t e x s l i c e s t h a t o c c u r s a t the onset o f anoxia . T h i s e f f e c t o f TTX i s i n -dependent of the a c t i v i t y o f the t r a n s p o r t p r o c e s s e s n o r m a l l y o p e r a t i n g on the amino a c i d uptake i n t o the b r a i n . V. L o c a l a n e s t h e t i c s , ouabain, amytal and r e s e r p i n e a l s o i n c r e a s e the r a t e of anaerobic g l y c o l y s i s of cere-b r a l c o r t e x s l i c e s . L o c a l a n e s t h e t i c s a c t i n a manner s i m i l a r to TTX, although much higher c o n c e n t r a t i o n s are r e q u i r e d . The e f f e c t s of ouabain i n a C a + + - f r e e medium are much greater than i n a C a + + - c o n t a i n i n g medium. I t i s suggested t h a t the in c r e a s e i n the r a t e of anaerobic g l y c o l y s i s due t o ouabain i s p o s s i b l y mediated by an i n -crease i n c e l l ATP c o n c e n t r a t i o n under anoxia, as a r e s u l t of i n h i b i t i o n of Na + , K +-ATPase. The a c t i o n s of amytal and r e s e r p i n e on the anaerobic g l y c o l y s i s of c e r e b r a l c o r t e x s l i c e s are p o s s i b l y mediated by mem-brane c a t i o n changes, but f u r t h e r work i s necessary to support t h i s c o n c l u s i o n . TABLE OF CONTENTS v i Page TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS v i LIST OF FIGURES x i i LIST OF TABLES x v i LIST OF SCHEMES xx ABBREVIATIONS.. x x i ACKNOWLEDGEMENTS. x x i i Chapter 1 INTRODUCTION 1 1.1 E f f e c t o f Anoxia on B r a i n Metabolism 1 1.2 Glucose Metabolism i n B r a i n 6 1.3 E f f e c t s of L o c a l A n e s t h e t i c s 19 1.4 E f f e c t s o f T e t r o d o t o x i n on the Nervous System 26 1.5 E f f e c t s o f o t h e r N e u r o t r o p i c Drugs on the Nervous System 31 1.6 B i o c h e m i s t r y o f the Developing B r a i n 43 1.7 T r a n s p o r t o f Amino A c i d s and Sugars a c r o s s the B r a i n C e l l Membrane 45 1.8 O b j e c t i v e s 48 2 MATERIALS AND METHODS 49 2.1 E x p e r i m e n t a l Animals 49 2.2 Chemicals . 49 2.3 P r e p a r a t i o n o f B r a i n S l i c e s 50 2.4 I n c u b a t i o n Procedure 51 2.5 I n c u b a t i o n Medium 52 2.6 P r e p a r a t i o n o f Kidney Medulla 54 2.7 P r e p a r a t i o n o f Caudate Nucleus 54 2.8 P r e p a r a t i o n o f Acetone Powder E x t r a c t s 54 2.9 P r e p a r a t i o n o f Synaptosomes 55 2.10 P r e p a r a t i o n o f Microsomes and Assay o f Na +, K +-ATPase 55 2.11 Measurement of G l y c o l y s i s 57 2.12 Measurement o f Amino A c i d E f f l u x and Uptake.. 59 2.13 Measurement of NAD+ and NADI-1 60 2.14 Measurement o f CAMP P r o d u c t i o n 62 2.15 Measurement of ATP L e v e l s 63 v i i 22 2.16 Measurement of Na I n f l u x 63 2.17 Measurement of Na + and K + 64 2.18 Miscellaneous Methods 64 2.19 R e p r o d u c i b i l i t y of the Results 66 3 RATE LIMITING FACTORS IN ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES AND ACETONE POWDER EXTRACTS 67 3.1 E f f e c t s of Calcium on the Anaerobic G l y c o l y s i s of C e r e b r a l Cortex S l i c e s 67 3.2 E f f e c t s of E x t e r n a l l y Added NAD+ and ATP on the Anaerobic G l y c o l y s i s of Ce r e b r a l Cortex S l i c e s . . 72 3.3 Movement of NAD + Across the C e l l Membrane.. 74 3.4 E f f e c t s of NAD+ on Aerobic G l y c o l y s i s 77 3.5 E f f e c t s of C i t r a t e and AMP on the Anaerobic G l y c o l y s i s of C e r e b r a l Cortex S l i c e s 78 3.6 E f f e c t s of C y c l i c AMP and D i b u t y r y l c y c l i c AMP on the Anaerobic G l y c o l y s i s c f the Ce r e b r a l Cortex S l i c e s 81 3.7 E f f e c t s of v a r y i n g C a t i o n Concentrations on the Anaerobic G l y c o l y s i s of Ce r e b r a l Cortex S l i c e s 83 3.8 E f f e c t s of L-Glutamate and of Ammonium Ions on the Anaerobic G l y c o l y s i s of Ce r e b r a l Cortex S l i c e s 85 3.9 E f f e c t of NAD+ and ATP, i n a Sodium-Free Medium, on the Anaerobic G l y c o l y s i s of Ce r e b r a l Cortex S l i c e s 87 3.10 Rate L i m i t i n g F a ctors of G l y c o l y s i s i n the Acetone Powder E x t r a c t s 89 3.11 Summary of Chapter 3 91 4 EFFECTS OF TETRODOTOXIN ON CEREBRAL METABOLISM AND TRANSPORT IN ANOXIA 94 4.1 E f f e c t s of Tet r o d o t o x i n on the Anaerobic G l y c o l y s i s of Guinea P i g and Rat Cer e b r a l 94 Cortex S l i c e s 4.2 E f f e c t s of Calcium on Tet r o d o t o x i n Stimulated Anaerobic G l y c o l y s i s 9 5 4.3 E f f e c t of Te t r o d o t o x i n on MAD+ Movements Across the C e l l Membrane 9 8 4.4 E f f e c t s of Tet r o d o t o x i n on the Amino A c i d E f f l u x from the C e r e b r a l Cortex S l i c e s . . . 100 4.5 E f f e c t of Tet r o d o t o x i n on the Uptake of Amino Acids under Anaerobic C o n d i t i o n s . . . 102 4.6 E f f e c t of Tet r o d o t o x i n at D i f f e r e n t Glucose Concentrations on the Anaerobic G l y c o l y s i s of C e r e b r a l Cortex S l i c e s 105 4.7 Glucose Transport i n C e r e b r a l Cortex S l i c e s 105 4.8 E f f e c t s of Te t r o d o t o x i n , i n the Presence of Some Amino A c i d s , on the Anaerobic G l y c o l y s i s of C e r e b r a l Cortex S l i c e s 107 4.9 E f f e c t s of C i t r a t e , AMP and NH^+ on the Tetr o d o t o x i n S t i m u l a t i o n of G l y c o l y s i s of Rat C e r e b r a l Cortex S l i c e s 109 v i i i 4.10 E f f e c t s of Phospholipases on the Tet r o -d o t o x i n S t i m u l a t e d G l y c o l y s i s of the r a t C e r e b r a l Cortex S l i c e s 112 4.11 E f f e c t s of Tet r o d o t o x i n on Anaerobic G l y c o l y s i s of Kidney Medulla S l i c e s and Acetone Powder E x t r a c t s 114 4.12 E f f e c t s of Tet r o d o t o x i n on the Anaerobic G l y c o l y s i s of Developing B r a i n Cortex S l i c e s 117 4.13 E f f e c t of Tet r o d o t o x i n i n Presence of Glutamate, Aspartate and NH4+ on the Anaerobic G l y c o l y s i s of Developing Cortex S l i c e s 117 4.14 E f f e c t s of Tet r o d o t o x i n on the Aerobic G l y c o l y s i s of A d u l t Rat C e r e b r a l Cortex S l i c e s 120 4.15 Summary of Chapter 4 124 5 FURTHER STUDIES ON THE MECHANISM OF ACTION OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES 127 5.1 E f f e c t s of P r e - i n c u b a t i o n i n oxygen on the Tet r o d o t o x i n S t i m u l a t e d G l y c o l y s i s of Ce r e b r a l Cortex S l i c e s 127 5.2 E f f e c t s of Tet r o d o t o x i n a f t e r Various Periods of Anaerobiosis on the Anaerobic G l y c o l y s i s of C e r e b r a l Cortex S l i c e s 128 5.3 E f f e c t s of Te t r o d o t o x i n i n the Presence of Pyruvate on the Anaerobic G l y c o l y s i s of Ce r e b r a l Cortex S l i c e s 130 5.4 E f f e c t s of Glucose A d d i t i o n under Various C o n d i t i o n s on the S t i m u l a t i o n of Anaerobic G l y c o l y s i s by Tet r o d o t o x i n 134 5.5 E f f e c t of Aerobic Incubation w i t h D i n i t r o -phenol on the Te t r o d o t o x i n S t i m u l a t i o n of Anaerobic G l y c o l y s i s , and on the ATP l e v e l of the Rat C e r e b r a l Cortex S l i c e s . . 136 5.6 E f f e c t s of T e t r o d o t o x i n and Ouabain on the ATP Content of Guinea P i g C e r e b r a l Cortex S l i c e s 139 5.7 E f f e c t s of R a i s i n g ATP L e v e l by Aerobic Incubation w i t h Adenosine on the Tet r o -d o t o x i n Stimulated G l y c o l y s i s of the Rat Ce r e b r a l Cortex S l i c e s 139 5.8 E f f e c t s of Tetro d o t o x i n on cAMP Production i n C e r e b r a l Cortex S l i c e s 142 5.9 E f f e c t of P r o t o v e r a t r i n e on the Tetrodo-t o x i n Stimulated G l y c o l y s i s 143 i x 22 5.10 E f f e c t s of Te t r o d o t o x i n on the Na Transport i n the Rat C e r e b r a l Cortex S l i c e s 145 5.11 E f f e c t s of T e t r o d o t o x i n , a t Various C a t i o n Concentrations of the Medium, on the Anaero-b i c G l y c o l y s i s of C e r e b r a l Cortex S l i c e s 150 5.12 E f f e c t s of Tetro d o t o x i n on the Na+ and K + L e v e l s of Incubated C e r e b r a l Cortex S l i c e s under Anoxia 155 5.13 E f f e c t s of Tetro d o t o x i n on the Anaerobic G l y c o l y s i s and Na^2 Transport i n the Caudate Nucleus of Rat 157 5.14 E f f e c t s of Te t r o d o t o x i n on the Anaerobic G l y c o l y s i s of Synaptosomal P r e p a r a t i o n s of Rat B r a i n 157 5.15 E f f e c t s of P r e - i n c u b a t i o n i n Oxygen on the Na + and K + Contents of C e r e b r a l Cortex S l i c e s under Anoxia i n the Presence of Tetr o d o t o x i n 159 5.16 E f f e c t s of T e t r o d o t o x i n on the Na + and K + l e v e l s of I n f a n t Rat and Guinea P i g C e r e b r a l Cortex S l i c e s 161 5.17 E f f e c t s of Te t r o d o t o x i n on the Na + and K + Levels of Kidney Medulla S l i c e s 16 5 5.18 E f f e c t s of Te t r o d o t o x i n i n the Presence of C h e l a t i n g Agents on the Anaerobic G l y c o l y s i s of C e r e b r a l Cortex S l i c e s 165 5.19 E f f e c t s of Aerobic P r e - i n c u b a t i o n w i t h Ethanol on the Tet r o d o t o x i n S t i m u l a t i o n of Anaerobic G l y c o l y s i s of C e r e b r a l Cortex S l i c e s 167 5.20 E f f e c t s of Te t r o d o t o x i n on the Na+ and K + Content of the C e r e b r a l Cortex S l i c e s i n the Presence of Glutamate, A s p a r t a t e , Homo-c y s t e i a t e and NH.+ 170 5.21 E f f e c t s of Te t r o d o t o x i n on the Pyruvate and Phosphoenol-pyruvate Contents of C e r e b r a l Cortex s l i c e s under Anoxia 170 5.22 Summary of Chapter 5 174 6 EFFECTS OF OUABAIN AND LOCAL ANESTHETICS ON THE CEREBRAL METABOLISM AND TRANSPORT UNDER ANOXIA 177 6.1 E f f e c t s of ouabain on the Anaerobic G l y c o l y s i s of C e r e b r a l Cortex S l i c e s 178 6.2 E f f e c t s of Ouabain on the Anaerobic G l y c o l y s i s of I n f a n t Rat B r a i n Cortex S l i c e s 180 6.3 E f f e c t s of Ouabain and Tetro d o t o x i n on the Anaerobic G l y c o l y s i s of Rat C e r e b r a l Cortex S l i c e s a t D i f f e r e n t Concentrations of Na + i n the Incubation Medium 183 6.4 E f f e c t s of Ouabain on Anaerobic G l y c o l y s i s of Acetone Powder of B r a i n 183 6.5 E f f e c t s o f Ouabain i n the Presence o f L-Glutamate, C i t r a t e , AMP and N H 4 + on the Anaerobic G l y c o l y s i s o f C e r e b r a l Cortex S l i c e s 6.6 E f f e c t s o f Ouabain on the A e r o b i c G l y c o l y s i s o f C e r e b r a l Cortex S l i c e s 6.7 E f f e c t s of Ouabain on the ATP Contents o f C e r e b r a l C o r t e x S l i c e s 6.8 E f f e c t s of A d d i t i o n o f Ouabain a f t e r V a r i o u s P e r i o d s o f A n a e r o b i o s i s on the Anaerobic G l y c o l y s i s o f Rat C e r e b r a l Cortex S l i c e s . . 6.9 E f f e c t s o f A d d i t i o n o f Ouabain and T e t r o d o -t o x i n t o g e t h e r on the Anaerobic G l y c o l y s i s of C e r e b r a l Cortex S l i c e s 6.10 E f f e c t s o f Ouabain on the N a + and K + Contents o f Rat C e r e b r a l Cortex S l i c e s Under Anoxia 6.11 E f f e c t s o f Ouabain, C a + + and NAD + on the Microsomal Na +, K +-ATPase 6.12 E f f e c t s o f Ouabain on Amino A c i d E f f l u x e s from the C e r e b r a l Cortex S l i c e s under Anoxia 6.13 E f f e c t s o f P r o c a i n e and L i d o c a i n e on the Anaerobic G l y c o l y s i s o f Rat C e r e b r a l C o r t e x S l i c e s 6.14 E f f e c t s o f L i d o c a i n e on the Ana e r o b i c G l y c o l y s i s of I n f a n t Rat and Guinea P i g C e r e b r a l Cortex S l i c e s 6.15 E f f e c t s of L i d o c a i n e on the N a + and K + Contents o f C e r e b r a l Cortex S l i c e s 6.16 Summary o f Chapter 6 EFFECTS OF VARIOUS NEUROTROPIC DRUGS ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES 7.1 A c t i o n o f P y r r o l e and P y r i d i n e on the Anaerobic G l y c o l y s i s o f C e r e b r a l Cortex S l i c e s 7.2 E f f e c t s o f P y r r o l e on the Na+ and K + Contents of Guinea P i g C e r e b r a l Cortex S l i c e s under Anoxia 7.3 E f f e c t s o f Amytal and P e n t o t h a l on the Anaerobic G l y c o l y s i s of C e r e b r a l Cortex S l i c e s 7.4 E f f e c t s o f Chlorpromazine on the Anaerobic G l y c o l y s i s o f C e r e b r a l Cortex S l i c e s 7.5 E f f e c t s o f Amphetamine and Nialamide on the Anaerobic G l y c o l y s i s o f Rat C e r e b r a l Cortex S l i c e s x i 7.6 E f f e c t of Reserpine on the Anaerobic G l y c o l y s i s of C e r e b r a l Cortex S l i c e s 219 7.7 Summary of Chapter 7 222 8 DISCUSSION...... 224 8.1 E f f e c t of Calcium Ions on C e r e b r a l Anaerobic G l y c o l y s i s 224 8.2 E f f e c t s of Exogenous Nuc l e o t i d e s on the Anaerobic G l y c o l y s i s 226 8.3 E f f e c t s of Cations on the Anaerobic G l y -c o l y s i s 228 8.4 Anaerobic G l y c o l y s i s of Acetone Powder E x t r a c t s of B r a i n 229 8.5 E f f e c t s of T e t r o d o t o x i n on Anaerobic G l y c o l y s i s of B r a i n 230 8.6 E f f e c t s of Ouabain on C e r e b r a l Metabolism and Transport 246 8.7 E f f e c t s of L o c a l A n e s t h e t i c s on the Anaerobic G l y c o l y s i s 250 8.8 E f f e c t s of Other Neurotropic Drugs on the Anaerobic G l y c o l y s i s 251 8.9 General Conclusions 253 BIBLIOGRAPHY 258 x i i LIST OF FIGURES FIGURE Page 1 E f f e c t o f c a l c i u m on the an a e r o b i c g l y c o l y s i s of guinea p i g c e r e b r a l c o r t e x s l i c e s 6 8 2 E f f e c t o f d i f f e r e n t c o n c e n t r a t i o n s o f c a l c i u m on the anaerobic g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s 69 3 E f f e c t o f exogenous NAD + on the NAD + and NADH c o n c e n t r a t i o n s o f r a t c e r e b r a l c o r -t e x s l i c e s under anoxia 75 4 E f f e c t o f exogenous NAD + on the NAD + and NADH c o n c e n t r a t i o n s o f a e r o b i c a l l y i n -cubated c e r e b r a l c o r t e x s l i c e s from r a t . . 76 5 E f f e c t o f NAD + i n the presence o f v a r y i n g c o n c e n t r a t i o n s o f 2 , 4 - d i n i t r o p h e n o l on the a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s 79 6 E f f e c t s o f c i t r a t e and AMP on the an-a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s 80 7 E f f e c t s o f c y c l i c AMP and d i b u t y r y l c y c l i c AMP on the anaerobic g l y c o l y s i s o f guinea p i g c e r e b r a l c o r t e x s l i c e s 82 8 E f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f K +, L i + and Na + on the anae r o b i c g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s 84 9 E f f e c t s o f glutamate and N H 4 + on the ana e r o b i c g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s 8 6 10 Anaerobic g l y c o l y s i s by acetone powder e x t r a c t s o f r a t b r a i n 90 11 E f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f r a t e l i m i t i n g f a c t o r s on the anaerobic g l y -c o l y s i s o f b r a i n acetone powder e x t r a c t s 92 x i i i 12 E f f e c t s of t e t r o d o t o x i n on the anaerobic g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s . . 96 13 E f f e c t s of d i f f e r e n t concentrations of t e t r o d o t o x i n on the anaerobic g l y c o l y s i s of c e r e b r a l c o r t e x s l i c e s 97 14 E f f e c t s of d i f f e r e n t concentrations of calcium on the anaerobic g l y c o l y s i s of c e r e b r a l c o r t e x s l i c e s i n the presence of t e t r o d o t o x i n 99 15 E f f e c t of v a r y i n g glucose concentrations on the anaerobic g l y c o l y s i s of r a t cere-b r a l c o r t e x s l i c e s i n the presence and absence of t e t r o d o t o x i n 106 16 E f f e c t s of t e t r o d o t o x i n on the anaerobic g l y c o l y s i s of r a t kidney medulla s l i c e s . . . 115 17 E f f e c t s of t e t r o d o t o x i n and ouabain on the anaerobic g l y c o l y s i s of acetone powder e x t r a c t s from r a t b r a i n 116 18 E f f e c t s of d i f f e r e n t concentrations of t e t r o d o t o x i n on the anaerobic g l y c o l y s i s of newly born guinea p i g c e r e b r a l c o r t e x s l i c e s 119 19 E f f e c t s of t e t r o d o t o x i n and ouabain on the a e r o b i c g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s 121 20 E f f e c t of a e r o b i c p r e - i n c u b a t i o n on the t e t r o d o t o x i n s t i m u l a t i o n of anaerobic g l y c o l y s i s of r a t c e r e b r a l cortex s l i c e s . . 129 21 E f f e c t of a d d i t i o n of t e t r o d o t o x i n a f t e r v a r y i n g time periods of anoxia on the anaerobic g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s 131 22 E f f e c t s of t e t r o d o t o x i n , i n the presence of p r o t o v e r a t r i n e , on the anaerobic g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s . . 146 x i v 23 E f f e c t s of a d d i t i o n of h i g h c o n c e n t r a t i o n s o f c a t i o n s on the t e t r o d o t o x i n s t i m u l a t e d anaerobic g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s 154 24 E f f e c t s of t e t r o d o t o x i n on the sodium and potassium c o n c e n t r a t i o n s o f guinea p i g c e r e b r a l c o r t e x s l i c e s under anoxia 156 25 E f f e c t s of t e t r o d o t o x i n , c a l c i u m and ouabain on the a n a e r o b i c g l y c o l y s i s of r a t synaptosomes 160 26 E f f e c t s of t e t r o d o t o x i n and c a l c i u m on the sodium and potassium c o n c e n t r a t i o n s of r a t c e r e b r a l c o r t e x s l i c e s 162 27 E f f e c t s of t e t r o d o t o x i n on the sodium and potassium c o n c e n t r a t i o n s of newly born guinea p i g c e r e b r a l c o r t e x s l i c e s 163 28 E f f e c t s of t e t r o d o t o x i n on the sodium and potassium c o n c e n t r a t i o n s o f two day o l d r a t c e r e b r a l c o r t e x s l i c e s 164 29 E f f e c t s of t e t r o d o t o x i n on the sodium and potassium c o n c e n t r a t i o n s of r a t kidney medulla s l i c e s 166 30 E f f e c t s o f d i f f e r e n t c o n c e n t r a t i o n s of ouabain on the a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s 179 31 E f f e c t of v a r y i n g c a l c i u m c o n c e n t r a t i o n i n the presence of ouabain on the anaerobic g l y c o l y s i s of c e r e b r a l c o r t e x s l i c e s 181 32 E f f e c t s of ouabain and t e t r o d o t o x i n on the a n a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r -tex s l i c e s a t v a r y i n g c o n c e n t r a t i o n s o f sodium 184 33 E f f e c t o f a d d i t i o n of ouabain a f t e r v a r y i n g time p e r i o d s o f anoxia on the a n a e r o b i c g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s . . . 189 34 E f f e c t s of ouabain and t e t r o d o t o x i n t o -gether on the a n a e r o b i c g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s 190 X V 35 E f f e c t s of ouabain and t e t r o d o t o x i n and ouabain together on the sodium and potassium concentrations of r a t c e r e b r a l c o r t e x s l i c e s 192 36 E f f e c t of Li d o c a i n e on the anaerobic g l y c o l y s i s of c e r e b r a l c o r t e x s l i c e s 199 37 E f f e c t s of p y r r o l e and p y r i d i n e on the anaerobic g l y c o l y s i s of c e r e b r a l c o r t e x s l i c e s 206 38 E f f e c t of p y r r o l e on the sodium and potassium concentrations of guinea p i g c e r e b r a l c o r t e x s l i c e s under anoxia 210 39 E f f e c t of amytal on the anaerobic g l y -c o l y s i s of c e r e b r a l c o r t e x s l i c e s 212 40 E f f e c t of chlorpromazine on the anaerobic g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s . 217 41 E f f e c t s of amphetamine and nialamide on the anaerobic g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s 218 42 E f f e c t s of r e s e r p i n e on the anaerobic g l y c o l y s i s of c e r e b r a l c o r t e x s l i c e s 220 x v i LIST OF TABLES TABLE Page 1 E f f e c t o f c a l c i u m on the an a e r o b i c g l y c o l y s i s of i n f a n t r a t and guinea p i g c e r e b r a l c o r t e x s l i c e s 71 2 E f f e c t o f exogenous n u c l e o t i d e s on the an a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r -tex s l i c e s 73 3 E f f e c t s of c a l c i u m and NAD + on the an-a e r o b i c g l y c o l y s i s o f guinea p i g c e r e -b r a l c o r t e x s l i c e s i n a sodium f r e e medium 88 4 E f f e c t s of t e t r o d o t o x i n on the amino a c i d c o n t e n t o f r a t c e r e b r a l c o r t e x s l i c e s under anoxia 103 5 E f f e c t s of t e t r o d o t o x i n on the uptake of amino a c i d s under anaerobic con-d i t i o n s 104 6 E f f e c t s o f t e t r o d o t o x i n on the gl u c o s e t r a n s p o r t i n r a t c e r e b r a l c o r t e x s l i c e s under anoxia 10 8 7 E f f e c t s o f t e t r o d o t o x i n , i n the presence of some amino a c i d s , on the anae r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . . . . 110 8 E f f e c t s o f c i t r a t e , AMP and N H 4 + i n the presence o f t e t r o d o t o x i n on the anaero-b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s I l l 9 E f f e c t s o f t e t r o d o t o x i n i n the presence of p h o s p h o l i p a s e s on the anaerobic g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s 113 10 E f f e c t of t e t r o d o t o x i n on the anae r o b i c g l y c o l y s i s o f i n f a n t r a t c e r e b r a l c o r -tex s l i c e s 118 11 E f f e c t s o f t e t r o d o t o x i n i n the presence o f some amino a c i d s o r N H 4 + on the an-a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s from i n f a n t animals 122 x v i i 11A E f f e c t s o f t e t r o d o t o x i n on the a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s i n the presence of cyanide 123 12 E f f e c t s o f t e t r o d o t o x i n i n the presence of pyruvate on the ana e r o b i c g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s 133 13 E f f e c t s o f a d d i t i o n of glu c o s e under v a r i o u s c o n d i t i o n s on the t e t r o d o t o x i n s t i m u l a t i o n o f anaerobic g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s 135 14 E f f e c t s o f i n c u b a t i o n w i t h 2 , 4 - d i n i t r o -phenol on the t e t r o d o t o x i n s t i m u l a t i o n of a n a e r o b i c g l y c o l y s i s and the ATP con-t e n t s o f r a t c e r e b r a l c o r t e x s l i c e s 138 15 E f f e c t s o f t e t r o d o t o x i n and ouabain on the ATP content o f guinea p i g c e r e b r a l c o r t e x s l i c e s under anoxia 140 16 E f f e c t o f a e r o b i c p r e - i n c u b a t i o n i n ad-enosine on the t e t r o d o t o x i n s t i m u l a t i o n of a n a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s 141 17 E f f e c t s o f t e t r o d o t o x i n on the cAMP f o r -mation i n the c e r e b r a l c o r t e x s l i c e s 144 22 18 E f f e c t o f some n e u r o t r o p i c drugs on Na i n f l u x i n r a t c e r e b r a l c o r t e x s l i c e s 148 19 E f f e c t s o f t e t r o d o t o x i n on the anaerobic g l y c o l y s i s and Na22 t r a n s p o r t i n r a t c e r e b r a l c o r t e x s l i c e s i n a c h l o r i d e -f r e e medium 149 20 E f f e c t o f presence o f d i f f e r e n t concen-t r a t i o n o f K + on the t e t r o d o t o x i n stimu-l a t i o n o f an a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s 1^1 21 E f f e c t s o f t e t r o d o t o x i n a t v a r y i n g c a t i o n c o n c e n t r a t i o n o f the medium on the an-a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s -*-52 x v i i i 22 E f f e c t s o f t e t r o d o t o x i n on the anaerobic g l y c o l y s i s and Na22 t r a n s p o r t i n caudate nucleus o f r a t 158 23 E f f e c t s of EDTA and EGTA on the t e t r o d o -t o x i n s t i m u l a t i o n o f a n a e r o b i c g l y c o l y s i s of c e r e b r a l c o r t e x s l i c e s 16 8 24 E f f e c t o f e t h a n o l on the t e t r o d o t o x i n s t i m u l a t i o n o f ana e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s 169 25 E f f e c t s o f t e t r o d o t o x i n on the sodium and potassium c o n c e n t r a t i o n s o f r a t c e r e b r a l c o r t e x s l i c e s i n the presence of some amino a c i d s and NH^* 171 26 E f f e c t s o f t e t r o d o t o x i n on the pyruvate and phosphoenol pyruvate content o f r a t c e r e b r a l c o r t e x s l i c e s 173 27 E f f e c t o f ouabain on the anaerobic g l y c o l y -s i s o f d e v e l o p i n g r a t c e r e b r a l c o r t e x s l i c e s . 182 28 E f f e c t s of ouabain i n the presence of g l u t a -mate, c i t r a t e , N H 4 + and AMP on the ana e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s 186 ++ + 29 E f f e c t s o f ouabain, Ca and NAD on the microsomal Na +, K +-ATPase 19 4 30 E f f e c t s o f ouabain and ouabain + t e t r o d o -t o x i n t o g e t h e r on the amino a c i d e f f l u x from r a t c e r e b r a l c o r t e x s l i c e s under anoxia 196 31 E f f e c t s o f p r o c a i n e and l i d o c a i n e on the an a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s ± J O 32 E f f e c t s of l i d o c a i n e on the sodium and potassium contents o f c e r e b r a l c o r t e x s l i c e s . 2 ° 1 33 E f f e c t o f p y r r o l e on the anaerobic g l y c o l y s i s of guinea p i g c e r e b r a l c o r t e x s l i c e s i n the presence of some amino a c i d s 2^8 34 E f f e c t s o f some n e u r o t r o p i c drugs on the sodium and potassium c o n t e n t s o f r a t c e r e b r a l c o r t e x s l i c e s ^14 x i x 35 E f f e c t s of some ne u r o t r o p i c drugs on the sodium and potassium contents of newly born guinea p i g c e r e b r a l c o r t e x s l i c e s . . . . 216 36 E f f e c t s of some amines on the anaerobic g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s . . 221 X X P a g e LIST OF SCHEMES 1 The G l y c o l y t i c Pathway 9 2 P r e p a r a t i o n of Synaptosomes by Sucrose Gradient C e n t r i f u g a t i o n 56 xx i ABBREVIATIONS AMP ADP ATP cAMP, c y c l i c AMP D i b u t y r y l c y c l i c AMP DNP EDTA EGTA a-KG NAD + NADH Adenosine monophosphate Adenosine diphosphate Adenosine t r i p h o s p h a t e 3 ' , 5 ' - c y c l i c adenosine monophosphate 6 2 * N , 0 - D i b u t y r y l adenosine-s' -5' - c y c l i c phosphate 2,4-Dinitrophenol E t h y l e n e d i a m i n e t e t r a a c e t i c a c i d E t h a n e d i o x y b i s ( e t h y l a m i n e ) t e t r a a c e t a t e c t - K e t o g l u t a r a t e N i c o t i n a m i d e adenine d i n u c l e o t i d e N i c o t i n a m i d e adenine d i -n u c l e o t i d e , reduced form x x i i ACKNOWLE DGEMENTS I am most g r a t e f u l to my s u p e r v i s o r , P r o f e s s o r J.H. Quastel, F.R.S., f o r h i s c a r e f u l guidance and encouragement throughout the course of t h i s work. I would l i k e to express my s i n c e r e thanks to Pr o f e s s o r s V.J. O'Donnell and S.C. Sung f o r c r i t i c a l reading of the manuscript. I am indebted t o my f r i e n d , Dr. C.B.R. Sa s t r y f o r h i s u s e f u l advice d u r i n g the p r e p a r a t i o n of the manuscript. Thanks are due to Dr. D.S. Grewaal and Mr. A.M. Benjamin f o r reading p a r t s of the t h e s i s . Mr. Benjamin a l s o helped me i n the amino a c i d analyses. Thanks are extended to the Department of S o i l Science, the U n i v e r s i t y of B r i t i s h Columbia, f o r per-m i t t i n g me t o use t h e i r Atomic Absorption Spectro-photometer . I wish a l s o to express my thanks to Miss Joanne A l l a n f o r help i n t y p i n g the manuscript. I wish to thank the Medical Research C o u n c i l of Canada f o r f i n a n c i a l a s s i s t a n c e through grants made to Pr o f e s s o r Quastel. Thanks are a l s o due to the Uni-v e r s i t y of B r i t i s h Columbia f o r f i n a n c i a l a s s i s t a n c e i n the form of a U.B.C. Graduate F e l l o w s h i p (1968-1971). CHAPTER 1 INTRODUCTION 1.1 EFFECTS OF ANOXIA ON BRAIN METABOLISM The m a j o r a i m o f i n v e s t i g a t i o n s on t h e b r a i n m e t a b o l i s m i s t o g a i n i n f o r m a t i o n c o n c e r n i n g b i o c h e m i c a l mechanisms o p e r a t i n g i n t h e l i v i n g b r a i n . A l l t h e i n f o r m a t i o n d e s i r e d c a n n o t be o b t a i n e d by d o i n g i n v i v o e x p e r i m e n t s by t e c h n i q u e s p r e s e n t l y a v a i l a b l e and a number o f t e c h n i q u e s h a v e b e e n d e v e l o p e d t o s t u d y t h e b r a i n in_ v i t r o . Removal o f t h e b r a i n f r o m t h e a n i m a l may r e s u l t i n a v a r i e t y o f m e t a b o l i c and s t r u c t -u r a l c h a n g e s . L a c k o f o x y g e n * i s one m a j o r f a c t o r i n f l u e n c i n g t h e s e c h a n g e s . T h i s i s s p e c i a l l y i m p o r t a n t d u r i n g t h e f i r s t few m i n u t e s a f t e r t h e d e a t h o f t h e a n i m a l d u r i n g t h e p r e p a r a t i o n o f t h e c e r e b r a l t i s s u e ( s l i c e s ) f o r i n v i t r o s t u d i e s , e v e n when o x y g e n i s made a v a i l a b l e t o t h e t i s s u e d u r i n g s u b s e q u e n t i n c u b a t i o n p r o c e d u r e . ( i ) E f f e c t s o f H y p o x i a on B r a i n M e t a b o l i s m i n v i v o Many i n v e s t i g a t i o n s have b e e n c a r r i e d o u t on t h e e f f e c t s o f o x y g e n d e p r i v a t i o n on t h e b r a i n i n v i v o . When o x y g e n s u p p l y t o t h e b r a i n i s r e d u c e d o r c u t o f f , c e r e b r a l f u n c t i o n may s t i l l be m a i n t a i n e d f o r a s h o r t t i m e t h r o u g h u t i l i z a t i o n o f e n e r g y r e s e r v e s t h a t h a v e , so f a r as i s known a t p r e s e n t , f o u r m a j o r c o m p o n e n t s - p h o s p h o c r e a t i n e , ATP, g l y c o g e n and g l u c o s e . Changes i n t h e s e s u b s t a n c e s d u r i n g b r i e f p e r i o d s o f h y p o x i a h a ve b e e n * A n o x i a - o x y g e n l a c k ; h y p o x i a - o x y g e n d e f i c i e n c y ; i s c h e m i a -l o c a l a n e m i a due t o m e c h a n i c a l o b s t r u c t i o n ( m a i n l y a r t e r i a l n a r r o w i n g ) t o t h e b l o o d s u p p l y . - 2 -s t u d i e d as m e a s u r e s o f m e t a b o l i c r a t e s u n d e r t h e s e c o n d i t i o n s . As e a r l y as i n 1944, G u r d j i a n , S t o n e and Webster"*" r e p o r t e d t h a t i n t h e d o g , c e r e b r a l l a c t i c a c i d l e v e l r i s e s a n d p h o s p h o c r e a t i n e breakdown o c c u r s when t h e o x y g e n s a t u r a t i o n o f 2 t h e c e r e b r a l v e n o u s b l o o d i s r e d u c e d . I n a l a t e r s t u d y , t h e s e w o r k e r s showed t h a t c e r e b r a l ATP l e v e l s r e m a i n u n c h a n g e d u n t i l t h e p h o s p h o c r e a t i n e i s a l m o s t c o m p l e t e l y decomposed. 3 J i l e k , F i s c h e r , K r u l i c h and T r o j a n s t u d i e d t h e f u n c t i o n a l , b i o c h e m i c a l a n d s t r u c t u r a l r e a c t i o n o f t h e b r a i n t o h y p o x i a and a n o x i a d u r i n g d e v e l o p m e n t and showed t h a t t h e newborn r a t i s more r e s i s t a n t t h a n t h e a d u l t t o a n o x i a . T h i s r e s i s t a n c e d e c r e a s e s r a p i d l y a t a b o u t t h e 2 0 t h day o f l i f e . I n y o u n g e r r a t s (4-12 d a y s ) t h e r e i s a l m o s t c o m p l e t e e x h a u s t i o n o f g l y c o g e n and a t w o - t o t h r e e - f o l d r i s e i n l a c t i c a c i d i n h y p o x i a . I n o l d e r r a t s t h e d e c r e a s e i n g l y c o g e n and t h e i n c r e a s e i n l a c t i c a c i d a r e much l e s s . T h e i r r e s u l t s l e d them t o t h e c o n c l u s i o n t h a t immature n e r v o u s t i s s u e i s a b l e t o a d j u s t t o h y p o x i c c o n d i t i o n s by i n c r e a s i n g t h e i n t e n s i t y o f a n a e r o b i c g l y c o l y s i s , w h e r e a s t h e t i s s u e s o f a d u l t a n i m a l s do n o t p o s s e s s 4 t h i s a b i l i t y . T h o r n , S c h o l l , P f l e i d e r e r and M u e l d e n e r s t u d i e d t h e m e t a b o l i c p r o c e s s e s o f t h e b r a i n u n d e r a n o x i a and i s c h e m i a , a n d o b s e r v e d s i m i l a r c h a n g e s t a k i n g p l a c e u n d e r b o t h t h e s e c o n d i t i o n s . F o r e x a m p l e , i n o r g a n i c p h o s p h a t e ( P i ) and l a c t i c a c i d i n c r e a s e d , t h e c o n t e n t o f c r e a t i n e p h o s p h a t e d e c r e a s e d r a p i d l y w h i l e ATP c o n t e n t d e c r e a s e d g r a d u a l l y . L o l l e y a n d Samson^ e x t e n d e d t h e s e s t u d i e s f u r t h e r t o i n c l u d e o t h e r n u c l e o -t i d e s . T h e y c o n f i r m e d t h e g e n e r a l o b s e r v a t i o n t h a t t h e r e i s a s h i f t d u r i n g a n o x i a o f t r i p h o s p h a t e n u c l e o t i d e s t o monophos-- 3 -p h a t e s and a v e r y r a p i d breakdown o f p h o s p h o c r e a t i n e . The p a t t e r n o f c h a n g e s i n GTP and UTP c o n t e n t was more c o m p l e x t h a n t h a t o f ATP. NAD l e v e l s r e m a i n e d u n a f f e c t e d u n t i l l a t e a n o x i a when t h e y d e c r e a s e t o some e x t e n t . S t u d i e s c a r r i e d o u t by L o w r y , Passonreau, H a s s e l b e r g e r 6 7 and S c h u l z , and S t e w a r t , P a s s o n n e a u and Lowry , w i t h t h e w h o l e b r a i n and w i t h t h e r a b b i t s c i a t i c n e r v e showed t h a t d u r i n g i s c h e m i a c h a n g e s i n t h e h i g h e n e r g y compounds and e n e r g y r e s e r v e s a r e m easure o f t h e m e t a b o l i c r a t e s . G a t e f i e l d , L o wry, g S c h u l z and Passonneau , u s e d t h e method o f b l o c k i n g b l o o d s u p p l y t o s e v e r a l d i f f e r e n t r e g i o n s o f mouse b r a i n , and t h r e e h i s t o l o g i c a l l y d e f i n e d l a y e r s o f mouse c e r e b e l l u m t o i n v e s t i g a t e c h a n g e s i n t h e h i g h e n e r g y compounds. D u r i n g i s c h e m i a t h e r e was d e p l e t i o n o f g l y c o g e n , p h o s p h o c r e a t i n e and ATP f r o m n e a r l y a l l r e g i o n s a n d t h e r e was an a c c u m u l a t i o n o f l a c t a t e . The m e t a b o l i c r a t e s a s s e s s e d f r o m c h a n g e s i n m e t a b o l i t e s i n d i c a t e d t h a t t h e y were a b o u t t h e same i n c e r e b r a l c o r t e x , c e r e b e l l a r c o r t e x and m e d u l l a b u t much more d e p r e s s e d i n t h e Amnion's h o r n . ( i i ) E f f e c t s o f A n o x i a on t h e M e t a b o l i s m o f t h e C e r e b r a l  C o r t e x S l i c e s Many s t u d i e s h a v e been c a r r i e d o u t w i t h c e r e b r a l c o r t e x s l i c e s and some have b e e n c o n c e r n e d e x c l u s i v e l y w i t h t h e e f f e c t s 9 o f a n o x i a on c e r e b r a l m o r p h o l o g y and c h e m i s t r y . Cohen d e v e l o p -e d a method whereby t h e m o r p h o l o g y o f t h e c e r e b r a l c o r t e x s l i c e s m e t a b o l i z i n g i n v i t r o m i g h t be c o r r e l a t e d w i t h b i o c h e m i c a l f i n d i n g s . U s i n g t h i s method C o h e n 1 ^ made d e t a i l e d s t u d y o f t h e c h e m i c a l b e h a v i o u r o f c e r e b r a l c o r t e x s l i c e s i n a n o x i a and c o n f i r m e d t h e o b s e r v a t i o n s o f a number o f w o r k e r s t h a t ATP l e v e l - 4 -d e c l i n e s s l o w l y d u r i n g a n o x i a . T h i s was i n c o n t r a s t t o t h e r e s u l t s o f Albaum, N o e l l and Chinn"*""*" who c l a i m e d t h a t t h e c o n c e n t r a t i o n o f p h o s p h o c r e a t i n e d e c r e a s e s more s l o w l y t h a n ATP. As ATP i s t h e i m m e d i a t e s o u r c e o f e n e r g y t o meet met a -b o l i c r e q u i r e m e n t s i n t h e b r a i n w h i l e p h o s p h o c r e a t i n e s e r v e s as a r e s e r v o i r , t h e t e n d e n c y s h o u l d be f o r ATP t o r e m a i n e l e v a t e d a t t h e e x p e n s e o f p h o s p h o c r e a t i n e . C o hen"^ a l s o o b s e r v e d t h a t a n o x i a r e s u l t s i n w i d e s p r e a d n e u r o n a l damage; however, s c a t t e r e d i s l a n d s o f r e l a t i v e l y i n t a c t n e u r o n s s t i l l p e r s i s t a f t e r 40 m i n u t e s . D e s p i t e m o r p h o l o g i c a l a l t e r a t i o n s i n d i c a t i n g N i s s l s u b s t a n c e d i m i n u t i o n , t h e amount o f RNA d o e s n o t d e c r e a s e d u r i n g a two h o u r p e r i o d o f a n o x i a . O t h e r meta-b o l i c e f f e c t s o f a n o x i a , s u c h a s a d e c r e a s e i n t h e i n c o r p o r -a t i o n o f r a d i o a c t i v e p h o s p h a t e i n t o l i p i d s and n u c l e i c a c i d s , were c o n s i d e r e d t o be t h e r e s u l t o f d i m i n i s h e d ATP s y n t h e s i s . The g e n e r a l c o n c l u s i o n was t h a t h y p o x i a damages c e r e b r a l mechanisms f o r e n e r g y p r o d u c t i o n . 12 Swanson s t u d i e d t h e e f f e c t s o f o x y g e n d e p r i v a t i o n on t h e e l e c t r i c a l l y s t i m u l a t e d g u i n e a p i g c e r e b r a l c o r t e x s l i c e s a nd he s u g g e s t e d t h a t h y p o x i a damages t h e e n e r g y u t i l i z i n g s y s t e m s i n t h e c e r e b r a l c o r t e x s l i c e s . He a t t e m p t e d t o e x p l a i n 13 t h e r e s u l t s o f M c l l w a i n t h a t d e c r e a s e d a n a e r o b i c g l y c o l y s i s p e r s i s t s a f t e r c e s s a t i o n o f e l e c t r i c a l s t i m u l a t i o n a n d t h e t i s s u e l o s e s i t s a b i l i t y t o r e s p o n d a e r o b i c a l l y , b o t h i n 12 r e s p i r a t i o n and g l y c o l y s i s , t o e l e c t r i c a l s t i m u l a t i o n . Swanson s p e c u l a t e d t h a t , w i t h h y p o x i a , a l t e r a t i o n s i n t h e membrane s t r u c t u r e o c c u r s so t h a t t h e c e l l s c a n no l o n g e r m a i n t a i n c a t i o n g r a d i e n t s a c r o s s t h e i r membranes o r t h a t t h e c e l l membranes - 5 -become i n e x c i t a b l e by i n t e r f e r e n c e w i t h the t r a n s i e n t s t r u c t u r a l changes t h a t occur upon passage o f a nerve impulse. He s t u d i e d the f u n c t i o n a l d e f i c i t i nduced by hypoxia w i t h the h e l p of e l e c t r i c a l s t i m u l a t i o n technique and concluded t h a t d u r i n g the p e r i o d of hypoxia, p r o d u c t i o n o f h i g h energy phosphates, through n o n - o x i d a t i v e pathways, i s i n s u f f i c i e n t t o m a i n t a i n the energy u t i l i z a t i o n p r o c e s s e s such as those i n v o l v e d i n a c t i v e c a t i o n t r a n s p o r t . Lack of energy i n the b r a i n c e l l r e s u l t s i n i n c r e a s e d N a + i n f l u x and K + e f f l u x . T h i s e f f e c t i s made more apparent i f e l e c t r i c a l s t i m u l a t i o n accompanies the hypoxia. However, 12 experiments c a r r i e d out w i t h reoxygenated s l i c e s , a f t e r o n l y s h o r t p e r i o d of hypoxia (15 m i n u t e s ) , suggest t h a t there i s some p r e s e r v a t i o n of the mechanisms o f both a c t i v e c a t i o n t r a n s p o r t and energy p r o d u c t i o n . Experiments w i t h p r o l o n g e d p e r i o d s of hypoxia (60 minutes) suggest t h a t an i r r e v e r s i b l e damage t o the p h o s p h o r y l a t i n g mechanisms takes p l a c e . J e n n i n g s , Kaltenbach 14 and Sommers have found t h a t m i t o c h o n d r i a , i s o l a t e d from i s c h e m i c canine myocardium, show a 60 p e r c e n t decrease i n o x i d a t i v e and p h o s p h o r y l a t i n g c a p a c i t y a f t e r 15 minutes of i s c h e m i a . Other changes have a l s o been observed i n the c e r e b r a l c o r t e x s l i c e s a f t e r p e r i o d s of a n a e r o b i o s i s . Thus M c l l w a i n , 15 Thomas and B e l l found t h a t the l e v e l of cozymase i n the guinea p i g c e r e b r a l c o r t e x s l i c e s decreases d u r i n g a n a e r o b i o s i s w h i l e i t remains c o n s t a n t d u r i n g a e r o b i c p e r i o d a f t e r an i n i t i a l drop. 16 Thomas concluded t h a t c r e a t i n e phosphate i s l o s t from the i n c u b a t e d c e r e b r a l c o r t e x s l i c e s d u r i n g a n a e r o b i o s i s and t h a t uptake o f K +, glutamate, a s c o r b a t e and c r e a t i n e i s d i m i n i s h e d . - 6 -13 7 \ Q u a s t e l and h i s coworkers have shown t h a t accumulation i n the b r a i n c e l l , a g a i n s t a c o n c e n t r a t i o n g r a d i e n t , of t h i a m i n e , a s c o r b i c a c i d and amino a c i d s i s completely suppressed under a n a e r o b i c c o n d i t i o n s . 1.2 GLUCOSE METABOLISM IN BRAIN As noted e a r l i e r , l a c t a t e f o r m a t i o n from g l u c o s e and endogenous glycogen i n c r e a s e s s e v e r a l f o l d d u r i n g a n o x i a . T h i s p r o c e s s , the a n a e r o b i c g l y c o l y s i s , and the e f f e c t s t h e r e o n of v a r i o u s e n v i r o n m e n t a l c o n d i t i o n s i s the major t o p i c of the p r e s e n t study. I t i s proposed, t h e r e f o r e , to d i s c u s s i n the f i r s t p l a c e , mechanisms o f g l y c o l y s i s and i t s r e g u l a t i o n . One of the major consequences of g l u c o s e metabolism, i n the mammalian t i s s u e , i s the s y n t h e s i s of ATP. ATP can be o b t a i n e d from g l u c o s e through w e l l known g l y c o l y t i c sequence e i t h e r by f o r m a t i o n o f a c e t y l CoA and o x i d a t i o n of t h i s substance by o p e r a t i o n of the c i t r i c a c i d c y c l e , o r , i n the absence of oxygen, through the f o r m a t i o n of l a c t i c a c i d . Whereas the former p r o c e s s produces 3 8 moles of ATP, the l a t t e r produces 2 moles of ATP per mole of g l u c o s e consumed. Most animal t i s s u e s are capable o f c a r r y i n g out the p r o c e s s o f g l y c o l y s i s , though t o v a r y i n g e x t e n t s . In b r a i n , the end p r o d u c t of g l y c o l y s i s under a n a e r o b i c c o n d i t i o n s i s l a c t i c a c i d . The p r o c e s s and c o n d i t i o n s a f f e c t i n g carbohydrate metabolism i n b r a i n have r e c e n t l y been 17-20 reviewed . In t h i s s e c t i o n we w i l l . b e mostly concerned w i t h the n o n - o x i d a t i v e metabolism of g l u c o s e i n b r a i n . B r a i n i s c h a r a c t e r i z e d by the presence of h i g h concent-r a t i o n o f g l y c o l y t i c enzymes and i t has been e s t i m a t e d t h a t more than 90 p e r c e n t of the t o t a l g l u c o s e consumed i n b r a i n - 7 -21 proceeds through the g l y c o l y t i c pathway . The r e v e r s e f o r m a t i o n 17 of g l u c o s e from pyruvate i s n e g l i g i b l e i n b r a i n (see B a l a z s ). The enzymic makeup of b r a i n f o r g l y c o l y t i c enzymes 17 d i f f e r s q u a n t i t a t i v e l y from t h a t of o t h e r t i s s u e s . B a l a z s has compared the r e l e v a n t d a t a on the enzyme a c t i v i t i e s i n b r a i n w i t h those p r e s e n t i n l i v e r . Thus the a v a i l a b l e data show t h a t hexo-k i n a s e a c t i v i t y i s about twenty times h i g h e r i n b r a i n than i n 22 l i v e r . A c t i v i t i e s of a l l o t h e r k i n a s e s (phosphofructokinase, phosphoglycerate k i n a s e and pyruvate kinase) are a l s o h i g h e r i n b r a i n than i n l i v e r . These enzymic steps are c o n t r o l p o i n t s i n g l y c o l y s i s and have been d i s c u s s e d l a t e r . I t has been mentioned t h a t the s y n t h e s i s of g l u c o s e from pyruvate i s n e g l i g i b l e i n b r a i n as compared to t h a t i n the l i v e r or kidney. Formation of g l u c o s e from pyruvate r e q u i r e s r e v e r s a l of c e r t a i n r e a c t i o n s of 23 24 the g l y c o l y t i c pathway ' . The a c t i v i t i e s o f enzymes 17 c a t a l y z i n g these r e a c t i o n s seem t o be v e r y low i n b r a i n so are those of enzymes needed f o r the s y n t h e s i s of glycogen, and of 17 pentose phosphate c y c l e . A number of i n v e s t i g a t i o n s have l e d t o the c o n c l u s i o n t h a t pentose phosphate pathway p l a y s o n l y a minor r o l e i n the c e n t r a l nervous system (CNS) of a d u l t mammals. I t s h o u l d be p o i n t e d out t h a t f o r an u n d e r s t a n d i n g of g l y c o l y t i c p r o c e s s i n the b r a i n , the l o c a l i z a t i o n of v a r i o u s enzymes i n v o l v e d i n such a heterogeneous organ i s most important. 25 As p o i n t e d out by Lowry and Passonneau , t r u e l o c a l concent-r a t i o n s of i n t e r m e d i a t e s i n v a r i o u s p a r t s of the b r a i n undoubt-e d l y v a r y g r e a t l y and are q u i t e d i f f e r e n t from average concent-r a t i o n s . An u n d e r s t a n d i n g , t h e r e f o r e , of the f i n e r d e t a i l s of g l y c o l y t i c changes i n the b r a i n must w a i t h i s t o c h e m i c a l s t u d i e s - 8 -o f i n d i v i d u a l c e l l s and p a r t o f c e l l s . ( i ) P r o p e r t i e s o f Some Enzymes i n v o l v e d i n G l y c o l y s i s  o f t h e B r a i n (a) H e x o k i n a s e ; B r a i n h e x o k i n a s e (HK) r e q u i r e s ATP a n d M g + + f o r i t s a c t i v i t y and i t p h o s p h o r y l a t e s s e v e r a l s u g a r s u b s t r a t e s i n c l u d i n g g l u c o s e , f r u c t o s e and mannose as w e l l a s D - g l u c o s a m i n e 2 6 and 2 - d e o x y g l u c o s e . I t i s i n h i b i t e d by g l u c o s e 6 -phosphate 27-29 (G-6-P) and ADP . G-6-P a c t s as a n o n - c o m p e t i t i v e i n h i b i t o r and i s t h o u g h t t o p l a y a r e g u l a t o r y r o l e . G-6-P and ADP i n h i b i t HK a c t i v i t y by c o m p e t i n g f o r ATP s i t e . G-6-P i n h i b i t i o n may be p a r t i a l l y r e l i e v e d by p h o s p h a t e i o n s . Km f o r g l u c o s e i s i n t h e 18 r a n g e o f 0.04-0.1 mM . However, enzymes w i t h h i g h e r Km h a v e 32 33 b e e n r e p o r t e d f r o m s h e e p b r a i n and human b r a i n . Thompson 34 a n d B a c h e l a r d were a b l e t o s e p a r a t e b o t h m i t o c h o n d r i a l HK and c y t o p l a s m i c HK i n t o two f r a c t i o n s b u t c o u l d n o t d e t e c t any m a j o r d i f f e r e n c e b e t w e e n t h e a c t i v i t i e s o f t h e m i t o c h o n d r i a l a n d c y t o -p l a s m i c enzymes. (b) P h o s p h o f r u c t o k i n a s e ; P h o s p h o f r u c t o k i n a s e (PFK) f r o m b r a i n , as w e l l as f r o m a v a r i e t y o f t i s s u e s , h a s b e en e x t e n s i v e l y s t u d i e d . T h i s enzyme c a t a l y z e s t h e t r a n s f e r o f t h e t e r m i n a l g r o u p o f ATP t o f r u c t o s e 6 - p hosphate (F-6-P) i n t h e p r e s e n c e o f ++ Mg . PFK a c t s s p e c i f i c a l l y on F-6-P and does n o t c a t a l y z e any o t h e r t r a n s f e r r e a c t i o n . I t shows k i n e t i c p r o p e r t i e s w h i c h may a c c o u n t f o r i t s r a t e l i m i t i n g e f f e c t s on t h e g l y c o l y s i s i n b r a i n . W h i l s t ATP and c i t r a t e a r e s t r o n g i n h i b i t o r s o f PFK, + t h e enzyme i s s t i m u l a t e d by F-6-P, ADP, AMP, NH^, p h o s p h a t e i o n s 35 * 17 as w e l l as c y c l i c AMP ( s e e L owry and P a s s o n n e a u and B a l a z s ) . 35 Lowry and Passonneau have p r o p o s e d a m odel f o r b r a i n PFK t h a t - 9 Scheme 1 : THE GLYCOLYTIC PATHWAY A c t i v a t o r s I n h i b i t o r s GLUCOSE + GLUC0SE-6-PH0SPHATE 1 i FRUCTOSE-6-PHOSPHATE cAMP, AMP, ADP, P i + . ATP, C i t r a t e FRUCTOSE-l-6-DIPHOSPHATE GLYCERALDEHYDE-3- ^ ^ DIHYDROXYACETONE PHOSPHATE * PHOSPHATE NAD^" ^ * I o d o a c e t a t e 1,3-DIPHOSPHOGLYCERIC ACID J f 3-PH0SPH0GLYCERIC ACID 2 - 3 - D i p h o s p h o g l y c e r a t e J ^ 2-PHOSPHOGLYCERIC ACID J F7 P P i , C a + + PHOSPHOENOL PYRUVIC ACID K + , F r u c t o s e - 1 - 6 - I t C a + + , N a + d i p h o s p h a t e PYRUVIC ACID NAD + + Oxamate LACTIC ACID - 10 -may account f o r a number of k i n e t i c p r o p e r t i e s o f the enzyme. They suggest t h a t the enzyme possess a t l e a s t seven, and p o s s i b l y as many as twelve, separate s i t e s f o r combination w i t h i n h i b i t o r s or a c t i v a t o r s . T h e i r arrangement on the enzyme are such t h a t b i n d i n g o f one i n h i b i t o r may l e a d to g r e a t e r a f f i n i t y of ot h e r i n h i b i t o r s ; the a c t i v a t o r s may a l s o a c t s y n e r g i s t i c a l l y : these p r o p e r t i e s o f the enzyme are thought t o p l a y an important r o l e i n the r e g u l a t i o n of g l y c o l y s i s (see l a t e r i n t h i s s e c t i o n ) . (c) Pyruvate Kinase; Pyruvate k i n a s e (PK), which i s one o f the most important of the g l y c o l y t i c enzymes, c a t a l y z e s the convers-i o n o f phosphoenol pyruvate (PEP) to pyruvate. I t s a c t i v i t y i n the b r a i n , as compared t o o t h e r t i s s u e s , i s v e r y h i g h . One of the i n t e r e s t i n g p r o p e r t i e s of t h i s enzyme i s t h a t i t s a c t i v i t y + + ++ 36 i s s t i m u l a t e d by K , NH^ and Rb (see A x e l r o d ) and i s + 37-39 i n h i b i t e d . b y Na (d) L a c t i c Dehydrogenase: L a c t i c dehydrogenase (LDH) i s p r e s e n t 17 i n the b r a i n a t a v e r y h i g h a c t i v i t y : there i s v e r y l i t t l e p y r u v a t e accumulation i n the i s o l a t e d i n c u b a t e d b r a i n c o r t e x or i n v i v o even d u r i n g c o n v u l s i o n s , which shows the g r e a t c a p a c i t y 40 of t h i s enzyme . LDH from b r a i n i s a tetramer and i s p r e s e n t 17 i n s e v e r a l d i f f e r e n t m o l e c u l a r forms (isozymes) . In a d u l t mammalian b r a i n , type I (heart type, H) i s the dominant form w h i l e type V (muscle type, M) and h y b r i d s of d i f f e r e n t s u b u n i t s (H and M type) are p r e s e n t t o a l e s s e r e x t e n t . Excess pyruvate 17 i n h i b i t s H type a t lower c o n c e n t r a t i o n s than M type . There i s a p r o g r e s s i v e change i n the LDH isozyme p a t t e r n d u r i n g b r a i n m a t u r a t i o n which c o r r e l a t e s w i t h the r e s i s t a n c e o f the animal to . 41 anoxia - 11 -( i i ) L o c a l i z a t i o n o f G l y c o l y t i c Enzymes i n t h e B r a i n The g l y c o l y t i c enzymes, w i t h t h e e x c e p t i o n o f HK, a r e a l m o s t e n t i r e l y p r e s e n t i n t h e s u p e r n a t a n t f r a c t i o n s o b t a i n e d by h i g h s p e e d c e n t r i f u g a t i o n o f b r a i n homogenates (see Q u a s t e l 19 ) . S e v e r a l g r o u p s o f w o r k e r s h ave s t u d i e d t h e d i s t r i b u t i o n o f g l y c o l y t i c enzymes i n t h e s u b - c e l l u l a r p r e p a r a t i o n s o f b r a i n 42-45 C e r e b r a l HK o c c u r s p a r t l y i n t h e c y t o p l a s m and p a r t l y b o u n d t o m i t o c h o n d r i a " ^ ' ^ . However, s u f f i c i e n t c y t o p l a s m i c 34 HK i s a v a i l a b l e t o a c c o u n t f o r n o r m a l r a t e s o f g l y c o l y s i s P a r t i c u l a t e HK a c t i v i t y i s e x c l u s i v e l y m i t o c h o n d r i a l a n d v a r i e s 43-44 46 f r o m 30 t o o v e r 75 p e r c e n t o f t h e t o t a l a c t i v i t y ' E a r l i e r w o r k e r s h a v e shown t h a t some g l y c o l y t i c a c t i v i t y o f r a t b r a i n homogenates i s a s s o c i a t e d w i t h t h e m i t o -43 c h o n d r i a l f r a c t i o n . S u c h i n i t i a l o b s e r v a t i o n s a r e r e l a t e d t o t h e p r e s e n c e o f n e r v e e n d i n g p a r t i c l e s , w h i c h c o n t a i n s g l y c o l y t i c 17 49 enzymes, i n t h i s f r a c t i o n . Abood, B r u n n g r a b e r and T a y l o r h a v e shown t h a t , w i t h h y p e r t o n i c s u c r o s e , i t i s p o s s i b l e t o o b t a i n a f r a c t i o n r i c h i n m i t o c h o n d r i a w h i c h i s a l m o s t d e v o i d o f 45 g l y c o l y t i c a c t i v i t y . T a n a k a and Abood e s t a b l i s h e d t h a t t h e p r e s e n c e o f g l y c o l y s i s i n t h e m i t o c h o n d r i a l f r a c t i o n , p r e p a r e d c o n v e n t i o n a l l y f r o m r a t b r a i n , may be due l a r g e l y t o c o n t a m i n a t i o n o f t h i s f r a c t i o n w i t h o t h e r c e l l c o m p o n e n t s . S t u d i e s have b e e n done on t h e d i s t r i b u t i o n o f g l y c o l y t i c enzymes i n d i f f e r e n t a r e a s o f b r a i n and i n t h e v a r i o u s l a y e r s o f c e r e b r a l and c e r e b e l l a r c o r t e x , and Ammons' h o r n . T h e s e i n v e s t -i g a t i o n s show t h a t t h e c o n t e n t s o f g l y c o l y t i c enzymes v a r y i n d i f f e r e n t p a r t s o f t h e b r a i n . ( i i i ) C o n t r o l s o f C a r b o h y d r a t e M e t a b o l i s m i n t h e B r a i n The r a t e of g l u c o s e u t i l i z a t i o n i n c e r e b r a l t i s s u e i n 50 v i t r o i s much l e s s than t h a t i n v i v o . Even i n v i t r o , under an o x i a , the r a t e of glu c o s e u t i l i z a t i o n i s much l e s s than the maximum p o s s i b l e by the enzymes i n v o l v e d . From t h i s i t i s e v i d e n t t h a t the v a r i o u s r a t e s must be s u b j e c t to c o n t r o l by i n d i v i d u a l r e g u l a t o r y mechanisms. Most of the r e g u l a t o r y p r o c e s s e s i n c e r e b r a l g l y c o l y s i s have been i n v e s t i g a t e d w i t h the use o f b r a i n c o r t e x s l i c e s i n v i t r o . G l y c o l y t i c r a t e s i n . t h e c e r e b r a l t i s s u e s are c o n t r o l l e d by two d i f f e r e n t mechanisms: (1) r e g u l a t i o n o f the c o n c e n t r a t i o n s of m e t a b o l i c i n t e r m e d i a t e s through c o n t r o l o f enzyme a c t i v i t i e s and (2) r e g u l a t i o n o f the i o n i c c o n t e n t s . (a) P a s t e u r E f f e c t : The r a t e of oxygen consumption i n b r a i n s l i c e s can account f o r the o x i d a t i o n o f approximately 0.4 mmole l a c t a t e Kg min S i n c e , under a n o x i a , approximately 1 mmole l a c t a t e Kg "*"min ^ i s formed, i t would be expected t h a t a e r o b i c -1 -1 l a c t a t e p r o d u c t i o n would be l e s s than 1 mmole Kg min . However, the a c t u a l r a t e i s o n l y 10-20 p e r c e n t of t h i s v a l u e . The phenom-ena of i n h i b i t i o n of g l y c o l y s i s by r e s p i r a t i o n i s u s u a l l y c a l l e d the P a s t e u r e f f e c t . The p r o c e s s o f r e s p i r a t i o n alone i s not, however, respons-i b l e f o r the Pa s t e u r e f f e c t . I t i s p o s s i b l e t o i n c r e a s e the a e r o b i c r a t e o f l a c t a t e p r o d u c t i o n w h i l e s t i l l m a i n t a i n i n g the r e s p i r a t o r y r a t e a t a c o n s t a n t v a l u e . Under these c o n d i t i o n s t h e r e i s found to be a decrease i n the ATP c o n t e n t o f the t i s s u e w h i l e the c o n t e n t s o f ADP, AMP and P i i n c r e a s e . -These changes i n the c o n c e n t r a t i o n s are c o n s i d e r e d t o b r i n g about an i n c r e a s e d r a t e o f g l y c o l y s i s by s t i m u l a t i o n o f some of the g l y c o l y t i c enzymes (see l a t e r ) . R a c k e r h a s , i n f a c t , c o n c l u d e d t h a t i n h i b i t i o n o f PFK by ATP t o g e t h e r w i t h t h e r a t e l i m i t i n g c o n c e n t r a t i o n s o f ADP and P i may a c c o u n t f o r t h e P a s t e u r e f f e c t . 6 25 (b) R e g u l a t i o n o f G l y c o l y t i c Enzymes: Lowry and h i s c o w o r k e r s ' h a v e s t u d i e d t h e e f f e c t s o f i s c h e m i a on t h e amount o f t h e known s u b s t r a t e s and c o f a c t o r s o f t h e g l y c o l y t i c p a t h way i n b r a i n i n v i v o . C o m p a r i s o n o f t h e s t e a d y s t a t e l e v e l s o f i n t e r m e d i a t e s f o u n d u n d e r a e r o b i c c o n d i t i o n s w i t h t h o s e f o u n d u n d e r a n o x i a l e d t o t h e c o n c l u s i o n t h a t t h e c h a n g e s r e s u l t e d l a r g e l y f r o m a c c e l -e r a t i o n o f t h e PFK s t e p . T h i s was a t t r i b u t e d t o t h e i n c r e a s e i n ADP, AMP and P i t h a t a r e c a p a b l e o f o v e r c o m i n g t h e ATP i n h i b i t i o n o f PFK. K i n e t i c e v i d e n c e a l s o p r o v i d e d s u p p o r t i n g e v i d e n c e f o r t h e c o n t r o l l i n g r o l e s o f HK and PFK. 5 1 5 2 R o l l e s t o n and Newsholme ' s t u d i e d t h e c o n t r o l o f g l y c o l y s i s i n g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . T h e y c o r r e l a t e d t h e c h a n g e s i n c o n c e n t r a t i o n s o f s u b s t r a t e s f o r enzymes c a t a l y z -i n g " n o n - e q u i l i b r i u m " r e a c t i o n s w i t h t h e c h a n g e s i n r a t e s o f g l y c o l y s i s c a u s e d by a l t e r a t i o n o f t h e c o n d i t i o n s o f i n c u b a t i o n . T h e y c o n c l u d e d t h a t HK, PFK, PK a n d p o s s i b l y g l y c e r a l d e h y d e 3 - p h o s p h a t e d e h y d r o g e n a s e (G -3-PDH) a r e s u b j e c t t o m e t a b o l i c c o n t r o l i n t h e c e r e b r a l c o r t e x s l i c e s . T h e y f u r t h e r s u g g e s t e d t h a t HK a n d PFK t o g e t h e r f o r m a r e g u l a t o r y s y s t e m . I t h a s b e e n known f o r a l o n g t i m e t h a t ATP i n h i b i t s PFK a n d t h a t t h i s 35 5 3 - 5 6 i n h i b i t i o n i s r e l i e v e d b y AMP ' . B e c a u s e o f t h e p r e s e n c e o f a d e n y l a t e k i n a s e , a s m a l l c h a n g e i n t h e ATP c o n c e n t r a t i o n may c a u s e l a r g e i n c r e a s e i n t h e c o n c e n t r a t i o n o f AMP. R o l l e s t o n and 51 52 Newsholme ' p o i n t e d o u t t h a t any c o n d i t i o n d e c r e a s i n g t h e c o n c e n t r a t i o n o f ATP w i l l t e n d t o i n c r e a s e t h e a c t i v i t y o f PFK - 14 -w h i l e t h e c h a n g e s i n AMP c o n c e n t r a t i o n w i l l a m p l i f y t h e e f f e c t o f ATP. The r e s u l t a n t i n c r e a s e d a c t i v i t y o f PFK w i l l t e n d t o d e c r e a s e t h e c o n c e n t r a t i o n o f F-6-P w h i c h i s i t s e l f an i n h i b i t o r o f HK. T h u s , a c c o r d i n g t o t h e s e w o r k e r s , t h e a c t i v i t y o f HK and PFK a r e l i n k e d t o g e t h e r i n a r e g u l a t o r y s y s t e m . The c o n t r o l l i n g r o l e o f PFK has been e s t a b l i s h e d i n a 35 w i d e v a r i e t y o f t i s s u e s . I t h a s b e e n a l r e a d y m e n t i o n e d t h a t a w i d e v a r i e t y o f compounds a f f e c t PFK a c t i v i t y . I n h i b i t i o n o f PFK b y c i t r a t e may r e s u l t i n a m o d i f y i n g a f f e c t o f t h e c i t r i c a c i d c y c l e on t h e r a t e o f g l y c o l y s i s " ^ . The NH^ f o r m e d d u r i n g a n o x i a , o r d u r i n g e l e c t r i c a l a c t i v i t y , may be e x p e c t e d t o . . 35 r e s u l t i n i n c r e a s e d PFK a c t i v i t y The r a t e o f g l y c o l y s i s may a l s o be a f f e c t e d by t h e c o n c e n t r a t i o n o f p h o s p h a t e i n t h e medium"*"^ ' ' ^ ® . L a c t a t e f o r m a t i o n i n c e r e b r a l e x t r a c t s i s d e p e n d e n t upon t h e p r e s e n c e o f a d d e d p h o s p h a t e and i s p r o p o r t i o n a l t o t h e p h o s p h a t e c o n c e n t -5 0 r a t i o n up t o 1 5 - 2 0 mM . F u r t h e r , p h o s p h a t e i s a s u b s t r a t e f o r G-3-PDH and i t i s an a c t i v a t o r o f PFK as w e l l a s HK 3 (^' 3^. I t h a s b e e n known f o r a l o n g t i m e t h a t b r a i n HK i s 27 29 2 8 s e n s i t i v e t o i n h i b i t i o n by ADP ' and b y G-6-P . I n h i b i t i o n 2 8 o f HK b y G-6-P i s n o n - c o m p e t i t i v e and c a n be r e l i e v e d by p h o s p h a t e ' a ' . G-6-P, by i t s e l f , i s u n l i k e l y t o s u p p r e s s t h e e n z y m a t i c a c t i v i t y i n t h e b r a i n c e l l t o t h e e x t e n t i t i s r e q u i r e d b e c a u s e t h e r a t e o f g l y c o l y s i s i s o n l y 1 -3% o f t h e m a x i m a l r a t e p o s s i b l e , a s c a l c u l a t e d f r o m t h e t o t a l HK a c t i v i t y , o r a b o u t 5 - 6 % o f t h e m a x i m a l r a t e p o s s i b l e f r o m t h e amount o f HK 18 f o u n d i n t h e c y t o p l a s m . G-6-P, a t t h e c o n c e n t r a t i o n s p r e s e n t i n v i v o , p r o d u c e a b o u t 5 0 - 7 0 % i n h i b i t i o n o f HK u n d e r o p t i m a l - 15 -28 30 31a. c o n d i t i o n s ' ' . From the ex p e r i m e n t a l data on the degrees of i n h i b i t i o n of HK by G-6-P and ADP, i t i s q u i t e c l e a r t h a t the t o t a l i n h i b i t i o n o f HK by these substances i s not s u f f i c i e n t t o account f o r the observed c e r e b r a l g l y c o l y t i c r a t e s . The l o c a l i z a t i o n of the enzymes and i n h i b i t o r s i n the b r a i n c e l l may be j u s t as important as t h e i r q u a n t i t i e s f o r an e x p l a n a t i o n o f the r e g u l a t i o n o f c e r e b r a l g l y c o l y s i s . I t has a l r e a d y been p o i n t e d out t h a t the a c t i v i t y o f PK i s g r e a t l y a f f e c t e d by the presence o f c a t i o n s , n o t a b l y K + and Na +: K + b e i n g s t i m u l a t o r y and N a + b e i n g i n h i b i t o r y . C a + + i s a l s o known t o i n h i b i t the a c t i v i t y of PK, both i n the b r a i n and other mammalian t i s s u e s ^ 8 / 5 9 ^ B y g r a v e ^ observed t h a t the i n h i b i t i o n o f g l y c o l y s i s by added C a + + i n e x t r a c t s o f E h r l i c h a s c i t e s c e l l s i s l a r g e l y accounted f o r by the i n h i b i t i o n of PK a c t i v i t y due t o c o m p e t i t i o n w i t h M g + + and K +. C o n s i d e r i n g the e f f e c t s o f c a t i o n s 59 on PK a c t i v i t y i n c e r e b r a l t i s s u e s , Takagaki concluded t h a t + i n the presence o f K c o n c e n t r a t i o n normally found i n the cerebral t i s s u e , PK i s f u l l y a c t i v e and a s m a l l change i n the c o n c e n t r a t i o n of K + i n t h i s range, may not change the enzyme a c t i v i t y . Howsver, ++ 61 the c o n c e n t r a t i o n o f Ca (1.3mM) , which i n h i b i t s PK i n v i t r o , i s s i m i l a r to t h a t found i n the i n t a c t t i s s u e and, t h e r e f o r e , s m a l l changes i n C a + + c o n c e n t r a t i o n may b r i n g about l a r g e changes 59 i n the enzyme a c t i v i t y . Takagaki has f u r t h e r concluded t h a t C a + + i n the a e r o b i c a l l y i n c u b a t e d c e r e b r a l t i s s u e may, i n p a r t , be r e s p o n s i b l e f o r the presence o f PK i n a h i g h l y i n h i b i t e d state, i n a d d i t i o n to the c e l l u l a r o r g a n i z a t i o n o f the s l i c e s , (c) E f f e c t s of C a t i o n Contents and of E l e c t r i c a l S t i m u l a t i o n on G l y c o l y s i s i n C e r e b r a l Cortex S l i c e s : - 16 -19 Q u a s t e l has reviewed the e f f e c t s o f v a r i o u s i o n s on g l y c o l y s i s i n b r a i n and has p o i n t e d out t h a t the e f f e c t s of c a t i o n s must be i n t e r p r e t e d i n the l i g h t o f two f o l d a c t i o n -i o n t r a n s p o r t changes a t the c e l l membrane ca u s i n g changed i o n c o n c e n t r a t i o n i n the c e l l , and d i r e c t e f f e c t s on the g l y c o l y t i c p r o c e s s i t s e l f . I t has been known f o r a long time t h a t carbohydrate metabolism o f i s o l a t e d b r a i n t i s s u e i n v i t r o i s dependent on the i o n i c environment. As e a r l y as i n 1935, A s h f o r d and Dixon 6 2 + found t h a t the presence of lOOmM K i n c r e a s e both the r a t e of oxygen consumption and of a e r o b i c g l y c o l y s i s of r a b b i t b r a i n c o r t e x s l i c e s , r e s p i r i n g i n a Ringer medium a t 37°. Dickens 6 3 and G r e v i l l e f u r t h e r s t u d i e d the e f f e c t o f n e u t r a l s a l t s on r e s p i r a t i o n and a e r o b i c g l y c o l y s i s of r a t b r a i n c o r t e x s l i c e s . 6 3 They confirmed the o b s e r v a t i o n s of A s h f o r d and Dixon and i n a d d i t i o n found t h a t o m i t t i n g C a + + from the medium i n c r e a s e s the r a t e o f r e s p i r a t i o n . The e f f e c t o f C a + + i n lo w e r i n g the r e s p i r a t i o n c o u l d be overcome by u s i n g h i g h c o n c e n t r a t i o n s o f K + (up to lOOmM). With N a + as the o n l y c a t i o n , the r a t e o f a e r o b i c g l y c o l y s i s i s i n c r e a s e d but t h a t o f ana e r o b i c g l y c o l y s i s i s suppressed. Dickens and G r e v i l l e were unable t o e x p l a i n c l e a r l y the e f f e c t of n e u t r a l s a l t s on a e r o b i c g l y c o l y s i s . 6 4 With c e l l - f r e e b r a i n p r e p a r a t i o n s , Racker and Krimsky observed t h a t N a + i s a s t r o n g i n h i b i t o r of r e s p i r a t i o n and 37 65 g l y c o l y s i s (see U t t e r )• Takagaki and Tsukada s t u d i e d the e f f e c t s o f lOOmM K + on c e r e b r a l c o r t e x s l i c e s m e t a b o l i z i n g i n a N a + - f r e e medium. They observed t h a t i n response t o lOOmM K +, ther e i s no i n c r e a s e i n the r a t e o f oxygen uptake, l a c t i c a c i d f o r m a t i o n or gl u c o s e u t i l i z a t i o n , although i n the u s u a l medium - iv -such an i n c r e a s e i n K + i n c r e a s e d the metabolism c o n s i d e r a b l y . When N a + i s om i t t e d from the medium, the oxygen uptake i s not changed but the a e r o b i c l a c t i c a c i d f o r m a t i o n and glu c o s e u t i l i z a t i o n i n c r e a s e s i g n i f i c a n t l y ; the omis s i o n o f K + causes an i n c r e a s e i n the glu c o s e u t i l i z a t i o n and the l a c t i c a c i d f o r m a t i o n , w h i l e the r e s p i r a t i o n i s not changed. Ana e r o b i c g l y c o l y s i s i n b r a i n s l i c e s i s i n c r e a s e d i f N a + i s r e p l a c e d by c h o l i n e or K + but i s s l i g h t l y d epressed i n a L i + medium^. In c o n t r a s t to a e r o b i c g l y c o l y s i s , a n a e r o b i c g l y c o l y s i s i s depressed by the a d d i t i o n of h i g h K + to a Ringer , . 6 2 medium The e f f e c t o f K +, under a e r o b i c c o n d i t i o n s , on the metabolism of c e r e b r a l c o r t e x s l i c e s may be p a r t l y due to the f a c t t h a t K + s t i m u l a t e s the i n t e r a c t i o n of PEP and ADP 6^ and p a r t l y due t o i t s s t i m u l a t i o n of the a c t i v i t y o f membrane ATPase, c a u s i n g an i n c r e a s e i n the c o n c e n t r a t i o n o f ADP and 19 + phosphate . The e f f e c t of h i g h K may a l s o p a r t l y be the r e s u l t o f a d i m i n i s h e d c e l l l e v e l o f ATP. B r a i n s l i c e s meta-b o l i z i n g g l u c o s e o r pyruvate i n a normal balanced media m a i n t a i n h i g h c o n c e n t r a t i o n o f pho s p h o c r e a t i n e . In the presence of h i g h *4~ 6 8 K , the con t e n t o f phosphocreatine i s d i m i n i s h e d . F i n d l a y , 69 + Magee and R o s s i t e r found t h a t h i g h K i n h i b i t s the m c o r p o r a -32 t i o n of P i i n the v a r i o u s phosphate f r a c t i o n of the b r a i n c o r t e x s l i c e s . 70 In 1937, Q u a s t e l and Wheatley observed t h a t the r a t e of a n a e r o b i c g l y c o l y s i s of b r a i n c o r t e x s l i c e s i s markedly s t i m u l a t e d by the a d d i t i o n of C a + + , and t o a l e s s e r e x t e n t by ++ + + Mg , t o a medium i n which Na and K are the o n l y c a t i o n s . 71 ++ Adams and Q u a s t e l made a d e t a i l e d study of the e f f e c t s of Ca on the anaerobic g l y c o l y s i s of guinea p i g c e r e b r a l c o r t e x and r . T . m, 71 confirmed the o b s e r v a t i o n s of Q u a s t e l of tumour s l i c e s . They and Wheatley and f u r t h e r observed t h a t the i n h i b i t o r y e f f e c t on the r a t e of b r a i n g l y c o l y s i s e s t a b l i s h e d i n the absence of Ca from the medium, i n which the b r a i n s l i c e s are suspended, i s l a r g e l y overcome by d e c r e a s i n g the pH t o 7.0. The same e f f e c t i s o b t a i n e d by s u b j e c t i n g the b r a i n s l i c e s t o oxygenation a f t e r a p e r i o d of a n e r o b i c treatment. T h i s was thought to be due t o pyruvate accumulation d u r i n g the the p e r i o d of oxygenation 71 - pyruvate b e i n g a w e l l known a c c e l e r a t o r of c e r e b r a l 19 g l y c o l y s i s . Q u a s t e l suggested t h a t i t i s q u i t e p o s s i b l e t h a t the d i m i n i s h e d r a t e of c e r e b r a l a n a e r o b i c g l y c o l y s i s r e s u l t i n g ++ + from the absence of Ca i s due to an i n f l o w of Na i n t o the b r a i n c e l l s w i t h c o r r e s p o n d i n g s u p p r e s s i o n of g l y c o l y t i c r a t e . A number of o r g a n i c bases such as p y r r o l e , p y r i d i n e and a n i l i n e a c c e l e r a t e a n a e r o b i c b r a i n g l y c o l y s i s i n a C a + + - f r e e medium"^ "*". T h e i r e f f i c i e n c y i n r e p l a c i n g C a + + f o r b r a i n a n a e r o b i c g l y c o l y s i s can be, i n g e n e r a l , c o r r e l a t e d w i t h t h e i r d i s s o c i a t i o n 4 - 4 - 7 1 c o n s t a n t s S t u d y i n g a e r o b i c g l y c o l y s i s i n the c e r e b r a l c o r t e x 59 ++ s l i c e s , Takagaki observed t h a t Ca i n h i b i t s the a c t i v i t i e s of many g l y c o l y t i c enzymes i n c l u d i n g f o u r c o n t r o l l i n g enzymes, namely HK, PFK, G-3-PDH and PK. I t has a l r e a d y been mentioned t h a t C a + + has i n h i b i t o r y e f f e c t on the g l y c o l y s i s of E h r l i c h a s c i t e s tumour c e l l e x t r a c t s ^ w h i l e they have no e f f e c t on the g l y c o l y s i s of i n t a c t tumour c e l l s ^ ' ^ . A p p l i c a t i o n of e l e c t r i c a l impulses to the nerve or - 19 -c e r e b r a l t i s s u e l e a d s t o w e l l known movements o f c a t i o n s a c r o s s t h e c e l l membrane. E v e n a few m i n u t e s e x c i t a t i o n l e a d s t o 2-3 f o l d i n c r e a s e i n t h e N a + c o n t e n t . T h i s r e s u l t s i n + + g r e a t e r a c t i v i t y o f Na , K A T P a s e and h e n c e g r e a t e r u t i l i z -a t i o n o f ATP. T h e r e i s a f a l l i n t h e c e l l l e v e l o f ATP and i n c r e a s e i n ADP. As a r e s u l t , d u r i n g e l e c t r i c a l s t i m u l a t i o n o f t h e t i s s u e s l i c e s , r e s p i r a t i o n a s w e l l a s a e r o b i c g l y c o l y -s i s i s i n c r e a s e d . On t h e o t h e r h a n d a n a e r o b i c g l y c o l y s i s i s 50 s u p p r e s s e d by e l e c t r i c a l s t i m u l a t i o n . 1.3 EFFECTS OF LOCAL ANESTHETICS Much o f t h i s t h e s i s i s c o n c e r n e d w i t h t h e e f f e c t s o f t e t r o d o t o x i n (TTX) on b r a i n m e t a b o l i s m . TTX h a s p o t e n t l o c a l 12 8 a n e s t h e t i c e f f e c t s . I n t h i s s e c t i o n t h e mode o f a c t i o n a n d t h e e f f e c t s o f l o c a l a n e s t h e t i c s on t h e n e r v o u s s y s t e m w i l l be d i s c u s s e d . As t h e p r i m a r y a c t i o n o f t h e l o c a l a n e s t h e t i c i s t h e b l o c k a d e o f n e r v e i m p u l s e c o n d u c t i o n , a s h o r t a c c o u n t o f t h e t r a n s m i s s i o n o f t h e n e r v e i m p u l s e w i l l f o l l o w . E f f e c t s o f TTX w i l l be d i s c u s s e d i n a s e p a r a t e s e c t i o n . ( i ) T r a n s m i s s i o n o f t h e N e r v e I m p u l s e : E l e c t r o c h e m i c a l a s p e c t s o f e x c i t a t i o n h a s b e e n e x t e n s i v e -72-74 l y r e v i e w e d . I n e x c i t a b l e a s w e l l a s i n o t h e r c e l l s , t h e r e i s a s t e a d y d i f f e r e n c e o f p o t e n t i a l b e t w e e n t h e i n n e r a n d o u t e r p a r t o f t h e c e l l ( t h e r e s t i n g p o t e n t i a l ) . T h i s i s i n c o n t r a s t t o t h e t r a n s i t o r y c h a n g e s i n t h i s p o t e n t i a l d u r i n g p r o p a g a t i o n o f a n e r v e i m p u l s e ( t h e a c t i o n p o t e n t i a l ) . The p o t e n t i a l d i f f e r e n c e s e t up a c r o s s t h e n e u r o n a l membrane i s t h e r e s u l t o f t h e d i f f e r e n c e i n t h e c o n c e n t r a t i o n o f N a + a n d K + a c r o s s t h e membrane. F o r t h e c a t m o t o n e u r o n s , t h e c o n c e n t r a t i o n o f K + - 20 -i n s i d e the neurons i s 27 times h i g h e r than t h a t o u t s i d e w h i l e the c o n c e n t r a t i o n s o f N a + i s 10 times h i g h e r o u t s i d e than 75 t h a t i n s i d e The r e s t i n g membrane i s a t l e a s t 50 times more permeable to K + than t o Na +. During the g e n e r a t i o n of an a c t i o n p o t e n t i a l , + + the nerve becomes more permeable t o Na . As a r e s u l t Na f l u s h e s i n s i d e the c e l l and almost a t the same magnitude K + moves 76 + o u t s i d e the c e l l . Hodgkin has c a l c u l a t e d t h a t Na and K movements a s s o c i a t e d w i t h the e l e c t r i c a l a c t i v i t y o f the unmyel--12 2 i n a t e d nerve f i b e r s are i n the range of 3.5-4.5 x 10 M per cm . The changes i n the membrane p e r m e a b i l i t y d u r i n g the g e n e r a t i o n of an a c t i o n p o t e n t i a l have been v i s u a l i z e d as r e s u l t i n g from 77 a change i n the pore s i z e of c e r t a i n channels . Using TTX as a t o o l , i t has been c a l c u l a t e d t h a t t h e r e are about 13, or + 2 78 7 9 fewer, Na channels per u of the membrane s u r f a c e ' . I t i s + + 75 s t i l l u n c e r t a i n whether Na and K t r a v e r s e the same channels . + + 75 Na and K channels can be d i f f e r e n t i a l l y b l o c k e d ( i i ) C a lcium ions and E x c i t a t i o n : I t has been known f o r a long time t h a t nerves f i r e s pontaneously when the c o n c e n t r a t i o n of C a + + i s reduced and t h i s has l e d to the s u g g e s t i o n t h a t the i n c r e a s e i n the N a + perm-++ e a b i l i t y o c c u r s because d e p o l a r i z a t i o n removes Ca from s i t e s 80 81 + or c a r r i e r s i n the membrane ' . One can v i s u a l i z e t h a t Na cr o s s the membrane through s p e c i a l channels which are b l o c k e d i n the presence o f Ca On the o t h e r hand, i f a nerve i s bathed i n a s o l u t i o n c o n t a i n i n g h i g h e r than normal C a + + c o n c e n t r a t i o n , the f i r i n g 75 t h r e s h o l d i s r a i s e d and the nerve may even become i n e x c i t a b l e . - 21 -I t h a s been p r o p o s e d t h a t C a + + may be t h o u g h t o f as b l o c k i n g t ransmembrane c h a n n e l s t o N a + b y l o o s e b i n d i n g t o t h e p o l a r h e a d s o f t h e e x t e r n a l p h o s p h o l i p i d s l a y e r ; d i s l o c a t i o n o f C a + + f r o m t h i s bond w o u l d t h e n p e r m i t r o t a t i o n a l movement o f t h e + 82 83 p o l a r h e a d and f r e e p a s s a g e o f Na ' ( i i i ) The Sodium Pump; As has been d i s c u s s e d e a r l i e r i n t h i s c h a p t e r , t h e g e n e r a t i o n o f an i m p u l s e r e s u l t s i n t h e i n f l u x o f N a + and e f f l u x o f K + f r o m t h e n e r v e c e l l . A f t e r t h e c o n d u c t i o n o f t h e n e r v e i m p u l s e , t h e r e s t i n g p o t e n t i a l i s r e - e s t a b l i s h e d by move-+ + ment o f Na t o o u t s i d e t h e n e r v e c e l l , a nd o f K t o i n s i d e o f t h e c e l l . T h i s i s a c h i e v e d by t h e s o c a l l e d "Sodium Pump" w h i c h u t i l i z e s ATP t o e j e c t N a + and r e t u r n K + . I t i s b e l i e v e d + + t h a t t h e s o d i u m pump i s o p e r a t e d by Na , K - a c t i v a t e d A T P a s e 84 w h i c h i s p r e s e n t i n t h e c e l l membrane . T h i s w i l l be d i s c u s s e d l a t e r i n d e t a i l . ( i v ) S i t e a n d Mode o f A c t i o n o f L o c a l A n e s t h e t i c s : T h e r e i s v e r y l i t t l e d o u b t t h a t s i t e o f a c t i o n o f l o c a l 8 5 a n e s t h e t i c s i s a t t h e n e r v e membrane . Thus when an i s o l a t e d n e r v e membrane i s b a t h e d w i t h a l o c a l a n e s t h e t i c s o l u t i o n , t h e a c t i o n p o t e n t i a l becomes s m a l l e r a n d s m a l l e r and e v e n t u a l l y 75 d i s a p p e a r s . I t r e a p p e a r s o n l y when t h e d r u g i s washed o u t F u r t h e r , t h e e x t e r n a l s u p p o r t i n g s t r u c t u r e o f t h e f i b e r , i t s s h e a t / a n d Schwann c e l l , i s n o t r e q u i r e d f o r t h e b l o c k i n g e f f e c t . The l o c a l a n e s t h e t i c s may i n f a c t b l o c k t h e i m p u l s e c o n d u c t i o n 8 6 q u i c k e r i n u n s t r i p p e d n e r v e s . I n c o n t r a s t t o n o n - m y e l i n a t e d f i b e r s , t h e m y e l i n a t e d f i b e r s p e r m i t a c c e s s t o l o c a l a n e s t h e t i c s o n l y a t t h e nodes o f R a n v i e r ; t h i s e x p o s u r e i s s t i l l s u f f i c i e n t - 22 -87 88 to b l o c k impulse con d u c t i o n ' I t i s g e n e r a l l y b e l i e v e d t h a t the l o c a l a n e s t h e t i c s b l o c k the co n d u c t i o n by i n t e r f e r i n g w i t h the process fundamental to the g e n e r a t i o n o f an a c t i o n p o t e n t i a l , namely the l a r g e • t r a n s i e n t r i s e i n the p e r m e a b i l i t y of the membranes t o Na +, 89 which a r i s e s on d e p o l a r i z a t i o n of the membrane . I t has been w e l l e s t a b l i s h e d t h a t the e f f e c t i s the r e s u l t o f r e d u c t i o n i n the c a r r y i n g c a p a c i t y o f the system t o N a + d u r i n g an a c t i o n 90 96 p o t e n t i a l ' . L o c a l a n e s t h e t i c s a l s o reduce the i n c r e a s e i n + + K conductance but the e f f e c t on K conductance i s much l e s s + 90 91 97 98 than t h a t on the Na conductance ' ' ' I n c r e a s i n g a n e s t h e t i c c o n c e n t r a t i o n has an i n c r e a s i n g e f f e c t on the K + 99 + conductance . Thus, 0.5mM l i d o c a i n e has no e f f e c t on the K conductance but 3.5mM l i d o c a i n e lowers i t t o 75% of i t s o r i g i n a l v a l u e ; l i d o c a i n e i s 100 times more e f f e c t i v e on N a + channels than on K + channels. The e f f e c t s o f l o c a l a n e s t h e t i c s on the N a + and K + conductance i n e x c i t a b l e c e l l s d i f f e r from those i n i n e x c i t a b l e c e l l s such as human r e d bloo d c e l l s ( R B C ) 1 0 0 . High c o n c e n t r a -t i o n of the l o c a l a n e s t h e t i c causes hemolysis and i t has been suggested t h a t l o c a l a n e s t h e t i c base makes the r e d c e l l membrane more p e r v i o u s . In a d d i t i o n to nerve f i b e r s , l o c a l + + a n e s t h e t i c s b l o c k the movements of Na and K i n the membrane of muscle (both i n the r e s t i n g s t a t e and d u r i n g g e n e r a t i o n o f the 89 a c t i o n p o t e n t i a l ) , f r o g s k i n and othe r t i s s u e s A number of n a t u r a l l y o c c u r r i n g substances such as a c e t y l c h o l i n e , ATP, C a + + , and thiamine have been thought t o p l a y key r o l e s i n the con d u c t i o n of the nerve impulse and the r e have - 23 -been a number of r e p o r t s on i n t e r a c t i o n o f l o c a l a n e s t h e t i c s 89 wxth the above agents. However, as R i t c h i e and Greengard have p o i n t e d o u t, any t h e o r y on the mode of a c t i o n o f l o c a l a n e s t h e t i c s , based on an antagonism between l o c a l a n e s t h e t i c s and a n a t u r a l l y o c c u r r i n g substance, i s weakened by the ve r y d i v e r s i t y o f the s t r u c t u r e s capable of a n t a g o n i z i n g l o c a l a n e s t h e t i c a c t i v i t y . In many r e s p e c t s l o c a l a n e s t h e t i c s behave l i k e C a + + i n s t a b i l i z i n g the e x c i t a b l e membranes. They i n c r e a s e the time constants f o r the r i s e and f a l l of N a + c o n d u c t a n c e 1 ^ 2 . In a d d i t i o n t o i n c r e a s i n g the e x c i t a t i o n t h r e s h o l d , i f s u f f i c -i e n t q u a n t i t y i s p r e s e n t , the l o c a l a n e s t h e t i c w i l l b l o c k the conductance completely w i t h no change i n the r e s t i n g p o t e n t i a l 72,73,91,103^ I t ^ a s keen proposed t h a t the l o c a l a n e s t h e t i c and C a + + both a c t on the same system which i s r e s p o n s i b l e f o r c a r r y i n g the N a + through the nerve membrane 1^. In f r o g desheathed m y e l i n a t e d nerves, d e p o l a r i z a t i o n i n the absence of C a + + can be prevented by l o c a l a n e s t h e t i c s 1 ^ ' 1 ( ^ . B l a u s t e i n 92 ++ and Goldman have shown t h a t , i n l o b s t e r g i a n t axons, Ca and l o c a l a n e s t h e t i c s compete f o r the same s i t e on the 107 membrane. On the o t h e r hand F e i s t e i n has proposed t h a t l o c a l a n e s t h e t i c s may a c t p r i m a r i l y by i n h i b i t i n g r e l e a s e of C a + + from the s i t e s t o which i t i s bound i n the membrane. In t h i s way, l o c a l a n e s t h e t i c s may pr e v e n t the secondary changes + + i n the Na and K p e r m e a b i l i t y , and thus prevent p r o p a g a t i o n of the wave of e x c i t a t i o n along the c e l l membrane. There i s s t i l l a c o n t r o v e r s y as t o whether the u n d i s -s o c i a t e d m o l e c u l a r form of the drug or i t s c a t i o n i c form i s r e s p o n s i b l e f o r the nerve b l o c k i n g a c t i o n . De Jong concluded t h a t i n a l l s t u d i e s i n which the nerve sheath was l e f t i n t a c t , l o c a l a n e s t h e t i c s were found to be most e f f e c t i v e i n a l k a l i n e s o l u t i o n where the base predominates, whereas i n most s t u d i e s i n which the nerve sheath was removed, a n e s t h e t i c s were more e f f e c t i v e i n n e u t r a l o r s l i g h t l y a c i d s o l u t i o n where the c a t i o n predominates. (v) B i o c h e m i c a l E f f e c t s of L o c a l A n e s t h e t i c s : S e v e r a l s t u d i e s have been c a r r i e d out on the e f f e c t s of l o c a l a n e s t h e t i c s on the metabolism of i s o l a t e d nervous t i s s u e 108 as w e l l as on t h a t of homogenates. As e a r l y as i n 1919, Niwa showed t h a t c o c a i n e depressed carbon d i o x i d e p r o d u c t i o n i n the 109 s c i a t x c nerve of the f r o g . S h e r i f showed t h a t c o c a i n e and p r o c a i n e i n h i b i t e d r e s p i r a t i o n i n the s c i a t i c nerves of the r a b b i t . These s t u d i e s l e d Watt"1""^ to study the e f f e c t s of l o c a l a n e s t h e t i c s on the r e s p i r a t i o n of b r a i n homogenates. He observed t h a t a number of l o c a l a n e s t h e t i c s such as nuperc a i n e , b u t a c a i n e , t e t r a c a i n e , metycaine, co c a i n e and p r o c a i n e i n h i b i t e d the o x i d a t i o n of s u c c i n a t e and glu c o s e by r a t b r a i n homogenates. Of these drugs, nupercaine was the most e f f e c t i v e . In o r d e r to show t h a t the i n h i b i t i o n of r e s p i r a t i o n was not due to the i n h i b i t i o n o f some o f the g l y c o l y t i c enzymes, Watt^"^ s t u d i e d the e f f e c t of these drugs on the a n a e r o b i c g l y c o l y s i s of r a t b r a i n homogenates and showed t h a t they had no i n h i b i t o r y e f f e c t i n t h i s system; i n c o n t r a s t , t e t r a c a i n e and nupercaine s t i m u l a t e d the a n a e r o b i c g l y c o l y s i s . Watt was unable to g i v e any explanation f o r t h i s and s t a t e d t h a t the i n c r e a s e i n the a n a e r o b i c g l y c o l y s i s m i g h t be due t o t h e m a i n t e n a n c e o f t h e o r i g i n a l h i g h r a t e o f g l y c o l y s i s by some u n e x p l a i n e d mechanism. He c o n c l u d e d t h a t t h e i n h i b i t i o n o f r e s p i r a t i o n by l o c a l a n e s t h e t i c s was due t o i n h i b i t i o n p o s s i b l y a t t h e o x i d a t i o n o f c y t o c h r o m e c - c y t o c h r o m e o x i d a s e s t a g e . Geddes and Q u a s t e l 1 ' ' " 1 o b s e r v e d t h a t t h e l o c a l a n e s t h e t -i c s p r o c a i n e , l i d o c a i n e , t e t r a c a i n e and d i b u c a i n e , a t pharma-c o l o g i c a l l y a c t i v e c o n c e n t r a t i o n s , i n h i b i t t h e K + s t i m u l a t e d r e s p i r a t i o n o f r a t b r a i n c o r t e x s l i c e s i n t h e p r e s e n c e o f g l u c o s e . T h e y h a d l i t t l e o r no e f f e c t a t t h e s e c o n c e n t r a t i o n s on t h e r e s t i n g o r u n s t i m u l a t e d b r a i n c o r t e x r e s p i r a t i o n . R e s u l t s o f t h e s e w o r k e r s f u r t h e r i n d i c a t e d t h a t t h e p o t e n c i e s o f t h e s e d r u g s as i n h i b i t o r s o f K + s t i m u l a t i o n o f b r a i n c o r t e x p a r a l l e l t h e i r a n e s t h e t i c a c t i v i t i e s . 112 F i n k , Kenny and Simp s o n f o u n d t h a t t h e r a t e o f o x y g e n c o n s u m p t i o n o f a s u s p e n s i o n o f mouse h e t e r o p l o i d c e l l s was d e p r e s s e d by v o l a t i l e , l o c a l a nd b a r b i t u r a t e a n e s t h e t i c s . T h e y compared t h e i r r e s u l t s w i t h t h a t o f Geddes and Q u a s t e l 1 1 1 and c o n c l u d e d t h a t t h e s e n s i t i v i t y o f t h e r e s p i r a t i o n o f t h e mouse c e l l s y s t e m t o l o c a l a n e s t h e t i c s i s as g r e a t a s t h a t o f t h e 113 b r a i n s l i c e s . Ryman and W a l s h f i n d t h a t c e r t a i n l o c a l a n e s t h e t i c s ( e . g . , c o c a i n e ) may i n h i b i t t h e c o n d e n s i n g enzyme, b l o c k i n g t h e e n t r y o f a c t i v e a c e t a t e i n t o t h e c i t r i c a c i d c y c l e . The f a c t t h a t l o c a l a n e s t h e t i c s h a v e l i t t l e a c t i o n on t h e m e t a b o l i s m o f t h e r e s t i n g n e r v e makes i t l i k e l y t h a t t h e i r e f f e c t s a r e e x e r t e d by t h e i r b l o c k o f i o n movement i n t h e s t i m -u l a t e d n e r v e . - 26 -1.4 E FFECTS OF TETRODOTOXIN ON THE NERVOUS SYSTEM TTX i s one o f t h e most t o x i c n o n - p r o t e i n s u b s t a n c e s known t o man and has a mechanism o f a c t i o n w h i c h seems v e r y s i m i l a r t o l o c a l a n e s t h e t i c s b u t h a s p o t e n c y o f more t h a n 114 100,0 00 t i m e s t h a t o f c o c a i n e . TTX i s f o u n d i n t h e J a p a n e s e p u f f e r f i s h and i n t h e C a l i f o r n i a n newt. I t s c h e m i s t r y and p h a r m a c o l o g y h ave b e e n reviewed"*""^ . TTX has a v a r i e t y o f p h a r m a c o l o g i c a l a c t i o n s i n v i v o s u c h as t h e d e p r e s s i o n o f r e s p i r a t i o n l e a d i n g t o r e s p i r a t o r y f a i l u r e , p a r a l y s i s o f s k e l e t a l m u s c l e , s u p p r e s s i o n o f s t i m u l u s e v o k e d r e s p o n s e s s u c h as s p i n a l r e f l e x e s and h y p o -t e n s i v e e f f e c t s . T h e s e a s p e c t s have been d i s c u s s e d i n d e t a i l 115 by Kao . - As f a r as t h e e f f e c t o f TTX i n v i t r o i s c o n c e r n e d , t h e r e i s now s u f f i c i e n t e v i d e n c e t o show t h a t i t a c t s on e l e c t r i c a l l y e x c i t a b l e t i s s u e s s u c h as t h e n e r v e f i b e r s and t h e m u s c l e s . I n t h e s e c e l l s , t h e g e n e r a t i o n o f t h e a c t i o n p o t e n t i a l i s a b o l i s h e d by TTX. I n f a c t , b l o c k o f t h e g e n e r a t i o n o f t h e a c t i o n p o t e n t i a l i s c o n s i d e r e d t o be t h e m a i n p r o p e r t y o f TTX. ( i ) A c t i o n o f TTX on t h e N e u r o m u s c u l a r J u n c t i o n and on  t h e I s o l a t e d N e r v e P r e p a r a t i o n s : 117 F u r u k a w a , S a s o o k a an d H o s o y a s t u d i e d t h e e f f e c t s o f TTX on t h e n e u r o m u s c u l a r j u n c t i o n on t h e f r o g n e r v e s a r t o r i u s p r e p a r a t i o n . T h ey showed t h a t r e s p o n s e o f t h e e n d p l a t e r e g i o n t o a c e t y l c h o l i n e i s n o t a f f e c t e d by TTX a t c o n c e n t -r a t i o n s h i g h enough t o b l o c k t h e a c t i o n p o t e n t i a l . T h i s s t u d y -27 -was f o l l o w e d by t h e work o f N a r a h a s i , D e g u c h i , Urakawa and 118 Ohkubo i n w h i c h t h e y a n a l y s e d t h e mode o f a c t i o n o f TTX on f r o g m u s c l e f i b e r membrane. W i t h t h e a i d o f i n t r a c e l l u l a r m i c r o e l e c t r o d e s , t h e y o b s e r v e d t h a t TTX, a t 10 M c o n c e n t -r a t i o n , make t h e a p p l i e d c a t h o d a l c u r r e n t i n e f f e c t i v e i n p r o d u c i n g a c t i o n p o t e n t i a l w h e r e a s t h e r e s t i n g p o t e n t i a l and r e s t i n g membrane r e s i s t a n c e u n d e r g o e s l i t t l e o r no c h a n g e . The a u t h o r s c o n c l u d e d t h a t i n t h e p r e s e n c e o f TTX t h e membrane i s s t a b i l i z e d by i n a c t i v a t i o n o f t h e N a + c a r r y i n g mechanism. 119 However, i t r e m a i n e d f o r N a r a h a s h i , Moore and S c o t t t o do v o l t a g e clamp e x p e r i m e n t s w i t h l o b s t e r g i a n t a x o n s t o v e r i f y t h e a b o v e h y p o t h e s i s . T h e s e s t u d i e s c o n f i r m e d t h e f a c t t h a t TTX, a t a v e r y low c o n c e n t r a t i o n , b l o c k s t h e a c t i o n p o t e n t i a l p r o d u c t i o n t h r o u g h i t s s e l e c t i v e i n h i b i t i o n o f t h e N a + c a r r y -i n g m e c h a n i s m and showed t h a t i t h a s no e f f e c t on t h e K + c a r r y i n g mechanism. T h e s e f i n d i n g s h a v e b e e n f u r t h e r e x t e n d e d 122-125 by a number o f w o r k e r s 120 Kao and Fuhrman had shown t h a t t a r i c h o t o x i n ( w h i c h has now b e e n shown t o be i d e n t i c a l w i t h TTX) e x e r t e d a s t r o n g n e r v e b l o c k i n g a c t i o n on t h e f r o g s c i a t i c n e r v e s . T a k a t a , 121 119 Moore, Kao a n d Fuhrman e x t e n d e d t h e s t u d i e s o f N a r a h a s h i e t a l . w i t h t a r i c h o t o x i n a n d , i n a d d i t i o n , o b s e r v e d t h a t a h i g h c o n c e n t r a t i o n o f C a + + , a p p l i e d c o n c o m i t a n t l y w i t h t h e t o x i n , s i g n i f i c a n t l y i m p r o v e s t h e r e v e r s i b i l i t y o f t h e N a + b l o c k i n g and t h u s g i v e s some p r o t e c t i o n a g a i n s t t h e t o x i n . T h e r e seems t o be l i t t l e d o u b t t h a t TTX a c t s on t h e 124 127 o u t e r r a t h e r t h a n t h e i n t e r n a l s u r f a c e o f t h e a x on ' , i t s e f f e c t b e i n g t o b l o c k t h e i n w a r d movement o f N a + a c c o m p a n y i n g - 28 -t h e g e n e r a t i o n o f a c t i o n p o t e n t i a l . 126 P u l l m a n , L a v e n d e r and Aho s t u d i e d t h e d i r e c t r e n a l e f f e c t s o f TTX i n dog. T e t r o d o t o x i n was i n f u s e d i n d i l u t e s o l u t i o n d i r e c t l y i n t o one r e n a l a r t e r y and c a u s e d h i g h l y s i g n i f i c a n t d i f f e r e n t i a l i n c r e a s e i n K + and M g + + e x c r e t i o n s . U s i n g c r a y f i s h a b d o m i n a l n e r v e f i b e r s , O g u r a and M o r i 128 have shown t h a t i n a l k a l i n e s o l u t i o n TTX i s more e f f e c t -i v e i n t h e s h e a t h e d p r e p a r a t i o n , and i n n e u t r a l s o l u t i o n i t was more e f f e c t i v e i n t h e d e s h e a t h e d o n e , t h e y s u g g e s t e d t h a t t h e c a t i o n i c f o r m , due t o g u a n i d y l g r o u p , i s t h e a c t i v e f o r m o f TTX and t h a t TTX p e n e t r a t e s n e r v o u s t i s s u e more r a p i d l y i n i t s u n c h a r g e d f o r m . ( i i ) E f f e c t s o f TTX on t h e E x c i t a b i l i t y , C a t i o n C o n t e n t  and M e t a b o l i s m o f I s o l a t e d C e r e b r a l T i s s u e s : U n t i l r e c e n t l y most o f t h e e x p e r i m e n t s w i t h TTX were c o n c e r n e d e i t h e r w i t h i n v i v o e f f e c t s o r w i t h e l e c t r o p h y s i o -l o g i c a l e f f e c t s on t h e i s o l a t e d n e r v e s and m u s c l e . I n 1967, a l m o s t s i m u l t a n e o u s l y , r e s u l t s o f two s t u d i e s a p p e a r e d w h i c h o p e n e d t h e p o s s i b i l i t y o f u s i n g TTX as a t o o l t o s t u d y t h e m e t a b o l i s m and t r a n s p o r t phenomenon i n t h e i s o l a t e d c e r e b r a l t i s s u e s . Chan and Quastel"*" 3^ showed t h a t 3 yM TTX i n h i b i t s t h e r e s p i r a t o r y i n c r e a s e o f t h e r a t b r a i n c o r t e x s l i c e s t h a t t a k e s p l a c e upon t h e a p p l i c a t i o n o f e l e c t r i c a l i m p u l s e s b u t has no e f f e c t on t h e K + s t i m u l a t e d r e s p i r a t i o n . T h ey f u r t h e r o b s e r v e d t h a t TTX a l s o i n h i b i t s i n c r e a s e i n t h e r a t e o f r e s p i r a t i o n t h a t - 29 -++ o c c u r s when Ca i s o m i t t e d f r o m t h e i n c u b a t i o n medium. M o r e -o v e r , t h e i r r e s u l t s d e m o n s t r a t e d t h a t t h e i n h i b i t i o n o f a c e t a t e o x i d a t i o n i n b r a i n s l i c e s b y e l e c t r i c a l i m p u l s e s , due t o t h e i n f l u x o f N a + , i s c o m p l e t e l y b l o c k e d by TTX a t s m a l l c o n c e n t -r a t i o n s . Chan a n d Q u a s t e l c o n c l u d e d t h a t TTX b l o c k s t h e i n f l u x o f N a + d u r i n g e l e c t r i c a l s t i m u l a t i o n a n d t h a t t h i s i s r e s p o n s -i b l e f o r t h e p o t e n t m e t a b o l i c e f f e c t s o f TTX on i s o l a t e d b r a i n . 129 M c l l w a m s i m i l a r l y f o u n d t h a t TTX i n h i b i t s t h e m e t a b o l i c r e s p o n s e s i n d u c e d b y e l e c t r i c a l s t i m u l a t i o n i n t h e g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . He a l s o showed t h a t l a r g e i n c r e a s e i n t h e r e s p i r a t i o n due t o e l e c t r i c a l s t i m u l a t i o n a r e g r e a t l y d i m i n i s h e d by 0.4uM TTX b u t i t h a s no e f f e c t o n t h e r e s p i r a t i o n o f t h e u n s t i m u l a t e d t i s s u e ; K + s t i m u l a t e d r e s p i r -a t i o n was f o u n d t o be i n s e n s i t i v e t o TTX. S i m i l a r e f f e c t s w e r e o b s e r v e d w i t h a e r o b i c g l y c o l y s i s . The K + c o n t e n t o f t h e e l e c t r i c a l l y s t i m u l a t e d s l i c e s i s g r e a t e r i n t h e p r e s e n c e o f 129 129 TTX . B e c a u s e TTX i s a g u a n i d i n e d e r i v a t i v e , M c l l w a i n s t u d i e d a number o f g u a n i d i n e d e r i v a t i v e s a n d showed t h a t o n l y some o f t h e m a r e a s e f f e c t i v e a s TTX, b u t a t much h i g h e r c o n c e n t r a t i o n s . 131 Swanson s t u d i e d t h e e f f e c t s o f TTX on t h e e l e c t r i c -a l l y s t i m u l a t e d g u i n e a p i g c e r e b r a l c o r t e x s l i c e s t o s e e i f i t a f f e c t s t h e c a t i o n i c s h i f t s w h i c h n o r m a l l y t a k e p l a c e w i t h e l e c t r i c a l s t i m u l a t i o n . C e r e b r a l c o r t e x s l i c e s l o s e n o n - i n u l i n K + a n d c r e a t i n e p h o s p h a t e , and g a i n n o n - i n u l i n N a + d u r i n g e l e c t r i c a l s t i m u l a t i o n . T h e s e e f f e c t s w e r e shown t o be p r e v e n t e d - 30 -by 1 yM TTX. H i s experiments s t r o n g l y support the c o n c l u s i o n t h a t TTX d i r e c t l y i n t e r f e r e s w i t h the p a s s i v e d o w n h i l l movement of both N a + and K +, which occur upon e l e c t r i c a l s t i m u l a t i o n . I t i s w e l l known t h a t glutamate causes e x c i t a t i o n o f 50 the nervous t i s s u e . E l e c t r i c a l s t i m u l a t i o n or the a d d i t i o n of 5mM L-glutamate causes an i n f l u x of N a + i n t o the i n c u b a t e d 132 134 c e r e b r a l c o r t e x s l i c e s . M c l l w a i n , Harvey and Rodriguez confirmed t h a t TTX almost completely prevents i n c r e a s e i n Na +, induced e l e c t r i c a l l y , and t h a t i t p a r t l y i n h i b i t s , d u r i n g a s h o r t i n i t i a l p e r i o d , the N a + i n f l u x i nduced by glutamate. E x t r u s i o n of Na +, f o l l o w i n g e l e c t r i c a l s t i m u l a t i o n , i s u n a f f e c t -ed by TTX. These s t u d i e s were f u r t h e r extended by P u l l , 133 M c l l w a i n and Ramsay , who c o n s i d e r e d the p o s s i b i l i t y o f g l u t -. . 133 amate a c t i n g i n a way s i m i l a r to c h e l a t i n g agents. They observed t h a t C a + + a c t s y n e r g i s t i c a l l y w i t h TTX i n r e s t r i c t i n g the c a t i o n movements on the a d d i t i o n o f glutamates. 5mM EDTA + + caused an i n c r e a s e i n i n t r a c e l l u l a r Na and a decrease i n K , and these changes were p a r t i a l l y b l o c k e d by TTX. 134 Ramsay and M c l l w a i n s t u d i e d the e f f e c t s o f TTX on C a + + movement i n i n c u b a t e d guinea p i g c e r e b r a l c o r t e x s l i c e s i nduced by glutamate. They found t h a t v e r y low c o n c e n t r a t i o n s 45 of TTX (66-330nM) are capable of i n h i b i t i n g Ca i n f l u x , both i n the presence and absence of L-glutamate. TTX a l s o caused a d e t e c t a b l e d i m i n u t i o n of C a + + e f f l u x . 135 + Okamoto and Q u a s t e l r e p o r t e d t h a t Na i n f l u x and water uptake i n r a t c e r e b r a l c o r t e x s l i c e s i n the presence of 0.1 mM ouabain, or 10 yM p r o t o v e r a t r i n e , or e l e c t r i c a l stimu-- 31 -l a t i o n or the absence of glucose i s p a r t i a l l y or w holly suppressed by 3 yM TTX. On the o t h e r hand, TTX has no e f f e c t on the N a + i n f l u x and water uptake i n the presence of 30 uM d i n i t r o p h e n o l or of 100 mM K +. 136 Itokawa and Cooper showed t h a t p e r f u s i o n w i t h TTX (as w e l l as w i t h ouabain and LSD-25) a t low c o n c e n t r a t i o n s , promoted r e l e a s e of r a d i o a c t i v e thiamine from the s p i n a l cords and the s c i a t i c nerves of b u l l - f r o g s and r a t s which had 35 . been e a r l i e r i n j e c t e d w i t h S - t h i a m i n e . 1.5 EFFECTS OF OTHER NEUROTROPIC DRUGS ON THE NERVOUS SYSTEM (i) Ouabain. (a) I n h i b i t i o n of Na,K-ATPase .by Ouabain; I t has been p r e v i o u s l y s t a t e d t h a t the p r o c e s s by which N a + i s e x t r u d e d from the c e l l and K + i s accumulated i n s i d e the c e l l i s d e s c r i b e d as the "Sodium Pump". I t has been w e l l + + e s t a b l i s h e d t h a t the t r a n s f e r of Na and K , which takes p l a c e through the membrane a g a i n s t a c o n c e n t r a t i o n g r a d i e n t , i s 137 138139 energy dependent . S i n c e Skou ' showed t h a t i n the p e r i p h e r a l nerve of c r a b , an ATPase i s p r e s e n t which r e q u i r e s both Na + and K + f o r a c t i v a t i o n and t h i s enzyme i s i n h i b i t e d by the c a r d i a c g l y c o s i d e ouabain, t h e r e i s overwhelming e v i d -ence i n s u p port of the c o n t r o l l i n g r o l e of t h i s enzyme i n the 137 140-145 t r a n s p o r t p r o c e s s e s ' . T h i s ATPase and the t r a n s p o r t + + mechanism f o r Na and K have a number of f e a t u r e s i n common such as t h e i r l o c a l i z a t i o n , i n the c e l l , t h e i r p r o p e r t i e s of + + energy u t i l i z a t i o n , a c t i v a t i o n by Na and K , and ouabain - 32 -137 i n h i b i t i o n 146 As e a r l y as i n 195 3, S c h a t zman showed t h a t c a r d i a c g l y c o s i d e s a t low c o n c e n t r a t i o n s a r e s p e c i f i c i n h i b i t o r s o f c a t i o n t r a n s p o r t . T h i s was l a t e r c o n f i r m e d b y a number 147-149 o f w o r k e r s . O u a b a i n seems t o a c t on t h e ' p h o s p h o r y -l a t e d f o r m o f t h e N a + , K + - A T P a s e , w h i c h a p p e a r t o a c t as an i n t e r m e d i a t e d u r i n g t h e o p e r a t i o n o f t h i s e n z y m e 1 ^ N a + a c t i v a t e t h e f o r m a t i o n o f t h e p h o s p h o r y l a t e d i n t e r m e d -i a t e w h i l e K + s t i m u l a t e i t s breakdown. The a c t i o n o f K + i i s p r e v e n t e d by o u a b a i n . T h e r e i s a s t o i c h i o m e t r i c r e l a t i o n h b e t w e e n t h e t r a n s p o r t o f N a + and K + . Thus i n e r y t h r o c y t e s 3 N a + i s t r a n s p o r t e d t o t h e o u t s i d e a n d 2 K + i s t r a n s p o r t e d i n s i d e t h e c e l l f o r e a c h m o l e c u l e o f ATP h y d r o l y z e d 1 ^ 162^ + + The Na , K -ATPase a c t i v i t y i n t h e c e r e b r a l c o r t e x i s v e r y h i g h 1 6 3 . Y o s h i d a , Nukada and F u j i s a w a 1 6 ^ showed t h a t 0.0ImM o u a b a i n c a u s e s an a l m o s t c o m p l e t e b l o c k o f u p t a k e + + o f K a n d e x t r u s i o n o f Na f r o m t h e g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . I n r e c e n t y e a r s many s t u d i e s h a v e b e e n c a r r i e d o u t c o n c e r n i n g t h e b i n d i n g o f o u a b a i n and o t h e r c a r d i a c + + g l y c o s i d e s t o t h e Na , K -A T P a s e . S c h w a r t z , M a t s u i and L a u g h t e r 1 6 ^ s t u d i e d t h e b i n d i n g o f t r i t i a t e d d i g o x i n t o t h e h e a r t m u s c l e enzyme and c o n c l u d e d t h a t t h e c o n f o r m a t i o n a l s t a t e o f t h e enzyme i s p r o b a b l y o f p r i m a r y s i g n i f i c a n c e i n g l y c o s i d e b i n d i n g . T h e i r d a t a s u p p o r t t h e c o n c e p t o f an - 33 -" a l l o s t e r i c t y p e " o f enzyme a n d t h e y s t a t e t h a t t h e f o r m a t i o n o f p h o s p h o r y l a t e d enzyme may be one o f a number o f ways i n w h i c h t h e c o n f o r m a t i o n a l n a t u r e o f t h e enzyme may be a l t e r e d . 166 C h a r n o c k a n d P o t t e r u s i n g enzyme f r o m t h e g u i n e a p i g c o r t e x c o n c l u d e d t h a t o u a b a i n may i n h i b i t b o t h p h o s p h o r y -l a t i o n a n d d e p h o s p h o r y l a t i o n o f t h e enzyme d e p e n d i n g on t h e n a t u r e a n d amount o f t h e c a t i o n s p r e s e n t . Y o d a and H o k i n 16 7 o b s e r v e d t h a t t h e b i n d i n g o f c a r d i a c g l y c o s i d e s t o t h e b e e f b r a i n enzyme i s i r r e v e r s i b l e . T h e i r r e s u l t s i n d i c a t e d t h a t t h e s u g a r i n g l y c o s i d e l i n k a g e w i t h t h e 3 p o s i t i o n o f t h e s t e r o i d p l a y s an i m p o r t a n t r o l e i n t h e i r r e v e r s i b l e 156 16 8 b i n d i n g . O t h e r s t u d i e s ' i n d i c a t e t h a t t h e enzyme c a n e x i s t i n s e v e r a l s t a t e s , a n d t h a t w h e t h e r t h e b i n d i n g o f o u a b a i n i s r e v e r s i b l e o r i r r e v e r s i b l e d e p e n d s o n t h e t e m p e r a t u r e a n d o t h e r f a c t o r s . (b) E f f e c t s o f O u a b a i n a n d C a t i o n s on N a + , K + - A T P a s e i n R e l a t i o n t o B r a i n M e t a b o l i s m a n d  T r a n s p o r t : A s e n e r g y d e r i v e d f r o m g l u c o s e m e t a b o l i s m i s u t i l i z e d + + + f o r Na a n d K t r a n s p o r t , a n d a s t h e a c t i v i t y o f Na , K + - A T P a s e i s a f f e c t e d by t h e c a t i o n c o n c e n t r a t i o n s , t h i s h a s l e d t o t h e s u g g e s t i o n t h a t N a + , K + - A T P a s e may be i n v o l v e d i n t h e r e g u l a t i o n o f r e s p i r a t i o n i n t h e c e r e b r a l - 34 -169 c o r t e x s l i c e s . w h i t t a m and B l o n d f o u n d a p a r a l l e l i s m b e t w e e n + + t h e a c t i v i t y o f Na , K -ATPase and a p a r t o f t h e r e s p i r a t i o n o f t h e i n c u b a t e d r a t b r a i n homogenates s u g g e s t i n g t h a t t h e r a t e o f e n e r g y p r o d u c t i o n by r e s p i r a t i o n i s g e a r e d t o i t s r a t e o f 170 u t i l i z a t i o n . Gonda and Q u a s t e l c o u l d f i n d o n l y l i t t l e e f f e c t o f o u a b a i n on t h e r e s p i r a t i o n o f u n s t i m u l a t e d b r a i n c o r t e x s l i c e s b u t i t p a r t i a l l y s u p p r e s s e d t h e K + s t i m u l a t e d r e s p i r a t i o n , t h e amount o f s u p p r e s s i o n d e p e n d i n g on t h e c o n c e n t r a t i o n o f o u a b a i n . T h ey o b s e r v e d a p r o n o u n c e d e f f e c t o f o u a b a i n on t h e r a t e s o f t r a n s f o r m a t i o n o f g l u c o s e i n t o amino a c i d s , and on amino a c i d and c r e a t i n e t r a n s p o r t i n t h e c e r e b r a l c o r t e x s l i c e s . 171 U s i n g f r o g b r a i n , d e P i r a s and Z a d u n a i s k y showed t h a t t h e e f f e c t s o f o u a b a i n on g l u c o s e m e t a b o l i s m d i f f e r a c c o r d i n g + + t o t h e K c o n c e n t r a t i o n s . A t low c o n c e n t r a t i o n s o f K , t h e r e i s a s m a l l b u t c o n s i s t e n t s t i m u l a t i o n o f o x y g e n u p t a k e b u t t h e i n c r e a s e i n r e s p i r a t i o n due t o h i g h K + i s c o m p l e t e l y i n h i b i t e d by o u a b a i n . W i t h g u i n e a p i g c e r e b r a l c o r t e x s l i c e s , Swanson 172 . . and M c l l w a i n o b s e r v e d t h a t o u a b a i n c a u s e s an i n i t i a l i n c r e a s e i n r e s p i r a t i o n b u t a f t e r 40-60 m i n u t e s r e s p i r a t i o n b e g i n t o f a l l . C r e a t i n e p h o s p h a t e and ATP l e v e l s a l s o f a l l a f t e r s h o r t p e r i o d s o f i n c u b a t i o n w i t h g l y c o s i d e s and t h e s e e f f e c t s c o u l d be p a r t i a l l y r e v e r s e d by t r a n s f e r r i n g t h e t i s s u e t o a f r e s h medium. T h e r e was a l s o a g r e a t i n c r e a s e i n t h e N a + and d e c r e a s e i n t h e K + c o n t e n t o f t h e o u a b a i n t r e a t e d s l i c e s 16 4 ( c f . Y o s h i d a e t a l . ) . T h e s e e f f e c t s a r e augmented by e l e c t -r i c a l s t i m u l a t i o n and t h e r e c o v e r y p r o c e s s i s c o m p l e t e l y i n e f f e c t i v e i n t h e p r e s e n c e o f o u a b a i n 1 3 1 . S w a n s o n 1 3 1 n o t e d t h a t i n t h e p r e s e n c e o f o u a b a i n c r e a t i n e p h o s p h a t e l e v e l s - 35 -c o n t i n u e to drop a f t e r e l e c t r i c a l s t i m u l a t i o n has ceased. 173 + Swanson observed t h a t the i n c r e a s e d l o s s o f K and the r i s e i n N a + c o n t e n t of the i n c u b a t e d guinea p i g c e r e b r a l c o r t e x s l i c e s i n the presence of ouabain i s most marked i n a Ca - f r e e medium. T h i s was e x p l a i n e d by the + + s u g g e s t i o n t h a t access of ouabain to the Na, K-ATPase i s more d i f f i c u l t when C a + + i s p r e s e n t i n the m e d i u m 1 3 1 ' 1 7 3 ' 1 7 ^ . The l o s s o f K +, i n the presence of ouabain, i s a l s o slow i n a Na +-175 + f r e e medium (Choline C h l o r i d e was s u b s t i t u t e d f o r Na ). 175 These r e s u l t s l e d Swanson and S t a h l to conclude t h a t i n the presence of N a + t h e r e i s a N a + - i n d u c e d s t r u c t u r a l change i n + + the Na, K-ATPase which a l l o w s a c c e s s i b i l i t y o f ouabain to i t s s i t e of i n h i b i t i o n . 176 Tower showed t h a t ouabain t r e a t e d s l i c e s , and the m i t o c h o n d r i a l f r a c t i o n from such s l i c e s , have h i g h e r c o n t e n t s of C a + + than the c o n t r o l s . S t a h l and S w a n s o n 1 7 7 s t a t e d t h a t the i n c r e a s e d uptake o f C a + + does not appear t o be an a r t i f a c t of p r e p a r a t i o n and might be due to i n c r e a s e d membrane perm-e a b i l i t y or p o s s i b l y to an i n h i b i t i o n of the a c t i v e t r a n s p o r t mechanism f o r e x t r u s i o n of C a + + . 137 Q u a s t e l , w h i l e r e v i e w i n g the c h a r a c t e r i s t i c s o f c a t i o n t r a n s p o r t and r e s p i r a t o r y c o n t r o l i n the b r a i n , concluded t h a t the f l u x e s a t the b r a i n c e l l membrane, as a r e s u l t of v a r i o u s s t i m u l i , may e f f e c t b r a i n metabolism l a r g e l y by t h e i r d i r e c t e f f e c t on the membrane bound ATPase. N e v e r t h e l e s s , i t was p o i n t e d out t h a t i n f l u x of N a + may suppress a c e t a t e o x i d -a t i o n by i t s i n h i b i t i n g a c t i o n on a c e t a t e c o n v e r s i o n to a c e t y l CoA and t h a t K + may a f f e c t the a c t i v i t y of PK. R o l l e s t o n and Newsholme o b s e r v e d t h a t , i n t h e p r e s e n c e o f 0.1 mM o u a b a i n , g l u c o s e u t i l i z a t i o n and a e r o b i c l a c t i c a c i d p r o d u c t i o n by g u i n e a p i g c e r e b r a l c o r t e x s l i c e s i s i n c r e a s e d . T h e s e e f f e c t s a r e n o t o b s e r v e d w i t h .001 mM o u a b a i n . M e a s u r e -ment o f t h e l e v e l o f g l y c o l y t i c i n t e r m e d i a t e s i n t h e p r e s e n c e o f 0.1 mM o u a b a i n l e d t h e s e w o r k e r s t o c o n c l u d e t h a t t h e a d d i t i o n o f 0.1 mM o u a b a i n t o g u i n e a p i g c e r e b r a l c o r t e x s l i c e s c a u s e s i n h i b i t i o n o f e i t h e r G-3-PDH o r p h o s p h o g l y c e r a t e k i n a s e , o r b o t h , i n a manner i n d e p e n d e n t o f t h e known a c t i o n o f o u a b a i n on N a + , K + - A T P a s e . ( i i ) P r o t o v e r a t r i n e (a) S i t e and Mode o f A c t i o n V e r a t r u m a n d r e l a t e d p l a n t s have b e e n u s e d f o r m e d i c i n a l p u r p o s e s f o r many y e a r s . They c o n t a i n a number o f h y p e r t e n s i v e a l k a l o i d s , t h e f i r s t one t o be o b t a i n e d i n c r y s t a l l i n e p r e p -a r a t i o n was p r o t o v e r a t r i n e . The m a j o r e f f e c t o f p r o t o v e r a t r i n e i s a l t e r a t i o n i n t h e p e r m e a b i l i t i e s o f membranes o f a number o f e x c i t a b l e c e l l s . 178 F r a n k o b s e r v e d t h a t i n t h e v e r a t r i n e - t r e a t e d m u s c l e f i b e r s , t h e membrane becomes p e r m e a b l e t o N a + f o l l o w i n g an a c t i o n p o t e n t i a l and t h e e n h a n c e d i n w a r d movement o f N a + c o n s i d e r a b l y d e l a y s r e p o l a r i z a t i o n . U s i n g c r u s t a c e a n a x o n s , W r i g h t and 17 9 T o m i t a c o n c l u d e d t h a t v e r a t r i n e a c t i o n i s t h e r e s u l t o f c h e m i c a l o r m e t a b o l i c r e a c t i o n by t h e a l k a l o i d i n t h e membrane. T h e y s u g g e s t e d t h a t v e r a t r i n e may i n h i b i t t h e N a + e x t r u s i o n m e chanism o r may i t s e l f compete f o r s i t e s i n t h e membrane w i t h Ca o r Na . 73 Shanes s u g g e s t e d t h a t an i n t e r a c t i o n t a k e s p l a c e - 37 -betw e e n t h e a l k a l o i d s and membrane l i p i d s w h i c h a f f e c t t h e i o n i c c h a n n e l s , a f f e c t i n g i n t u r n t h e membrane p e r m e a b i l i t y . However, 180 K u p c h a n and F l a c k e have p o i n t e d o u t t h a t no s p e c i f i c " b i n d i n g s i t e " o r " r e c e p t o r " h a s been r e c o g n i z e d f o r t h e v e r a t r u m a l k a -l o i d s a n d t h a t t h e r e a r e no s p e c i f i c a n t a g o n i s t s known ( c f . K i n i , _ . ,186, and Q u a s t e l ) . (b) B i o c h e m i c a l E f f e c t s o f P r o t o v e r a t r i n e and O t h e r V e r a t r u m A l k a l o i d s 181 W o l l e n b e r g e r o b s e r v e d t h a t v e r a t r u m a l k a l o i d s ( p r o t o v e r a t r i n e and v e r a t r i d i n e ) p r o d u c e s i g n i f i c a n t s t i m u l a t i o n o f r e s p i r a t i o n l a s t i n g f o r s e v e r a l h o u r s i n t h e i n c u b a t e d g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . T h e y i n c r e a s e a e r o b i c g l y c o l y s i s t o t h e n o r m a l a n a e r o b i c l e v e l w h i l e i n h i b i t i n g t h e a n a e r o b i c g l y c o l y s i s . T h e s e e f f e c t s a r e c h a r a c t e r i s t i c o f t h e b r a i n and + 62 r e s e m b l e t h e e f f e c t s o f h i g h K r e p o r t e d by A s h f o r d and D i x o n O m i s s i o n o f K + f r o m t h e medium g i v e s r i s e t o s t r o n g a e r o b i c 181 g l y c o l y s i s w h i c h i s n o t e n h a n c e d f u r t h e r by p r o t o v e r a t r i n e The i n h i b i t i o n o f a n a e r o b i c g l y c o l y s i s may be p a r t i a l l y prevented 182 + by t h e p r e s e n c e o f n i c o t i n a m i d e . The n e t u p t a k e o f K by washed c e r e b r a l c o r t e x s l i c e s i s i n h i b i t e d by p r o t o v e r a t r i n e a t c o n c e n t r a t i o n s w h i c h s t i m u l a t e r e s p i r a t i o n and a e r o b i c g l y c o l y -. 182 s i s 183 W o l l e n b e r g e r s t u d i e d t h e e f f e c t s o f p r o t o v e r a t r i n e on e l e c t r i c a l l y s t i m u l a t e d c e r e b r a l c o r t e x s l i c e s and n o t e d t h a t s i m u l t a n e o u s e x p o s u r e o f c e r e b r a l c o r t e x s l i c e s t o p r o t o -v e r a t r i n e and t o b r i e f c o n d e n s e r p u l s e s b r i n g a b o u t an i n c r e a s e i n t h e r a t e o f r e s p i r a t i o n w h i c h i s g r e a t e r t h a n t h e sum o f t h e i n c r e a s e s c a u s e d by t h e d r u g and by t h e p u l s e s i n d i v i d u a l l y . - 38 -18 4 185 Q u a s t e l ' has noted t h a t p r o t o v e r a t r i n e s t i m u l a t i o n of r a t b r a i n r e s p i r a t i o n i s h i g h l y s e n s i t i v e to n a r c o t i c s and i s d i m i n i s h e d by malonate. K i n i and Q u a s t e l found t h a t the a d d i t i o n of 5 yM p r o t o v e r a t r i n e to a e r o b i c a l l y i n c u b a t e d s l i c e s 14 of r a t b r a i n c o r t e x i n the presence of glucose-U-C g i v e s r i s e t o a r a d i o a c t i v e amino a c i d p a t t e r n which d i f f e r s from t h a t o b t a i n e d i n the absence of p r o t o v e r a t r i n e . These e f f e c t s are 18 0 antagonized by c o c a i n e ( c f . Kupchan and F l a c k e ). ( i i i ) Amphetamines and Nialamide While d i s c u s s i n g the mechanism of r e l e a s e of n o r e p i n e -187 p h r i n e (NE), B r o d i e , Cho, Stephano and Gessa s t a t e d : " I t i s d i s q u i e t i n g t o r e a l i z e t h a t a t p r e s e n t we cannot d e s c r i b e a t a p h y s i o l o g i c a l - b i o c h e m i c a l l e v e l the mechansim of a c t i o n o f any drug t h a t a c t s on the nervous system". I f one knows the mechanism of a c t i o n of even a s i n g l e drug then i t may p r o v i d e a framework on which the study of a c t i o n of o t h e r drugs c o u l d be based. Amphetamine i s a s u i t a b l e c a n d i d a t e f o r a thorough study because of i t s s i m p l i c i t y i n s t r u c t u r e and the m u l t i -p l i c i t y of i t s b i o l o g i c a l e f f e c t s , and i n r e c e n t years i t has been e x t e n s i v e l y s t u d i e d . Amphetamines r e l e a s e catecholamines, from t h e i r n e u r o n a l storage s i t e s but they do not appear to a f f e c t the uptake or r e l e a s e of s e r o t o n i n (5-hydroxytryptamine, 5-HT) . Amphetamine s t r o n g l y a f f e c t s the uptake of NE by the n e u r onal membrane and i s a l s o an i n h i b i t o r of monoamine oxidase (MAO) which i s an important enzyme i n i n a c t i v a t i n g the a c t i o n of monoamines i n CNS-, P a r a l l e l i s m between the s t i m u l a t i n g e f f e c t of amphetamines and 188 18 9 t h e i r e f f e c t s on MAO was f i r s t shown by Mann and Q u a s t e l ' O t h e r e f f e c t s o f amphetamines a r e p r e s u m a b l y l i n k e d up w i t h t h e s e b a s i c e f f e c t s and have r e c e n t l y been d i s c u s s e d i n d e t a i l , 1 9 0 i n a monograph D-Amphetamine i s a t l e a s t 2 0 t i m e s more a c t i v e t h a n 1-amphetamine. A number o f d e r i v a t i v e s o f t h e s e compounds have 1 9 1 b e e n p r e p a r e d w i t h d i f f e r e n t b i o l o g i c a l a c t i v i t y . U s u a l l y h y d r o x y l a t i o n o f p h e n y l g r o u p o r t h e s i d e c h a i n r e d u c e s t h e a c t i v i t y . U n l i k e amphetamine, p - c h l o r o a m p h e t a m i n e c a u s e s a 1 9 2 c e r e b r a l s e r o t o n i n d e p l e t i o n i n r a t s . Amphetamine h a s r e c e n t l y b e en shown t o i n c r e a s e t h e l e v e l s o f 5-HT i n mouse , • 1 9 3 b r a i n N i a l a m i d e i s a MAO i n h i b i t o r and t r e a t m e n t o f t h e animals w i t h t h i s d r u g c a u s e s i n c r e a s e d a c c u m u l a t i o n o f amines i n t h e , . 1 9 4 b r a i n ( i v ) B a r b i t u r a t e s 1 9 5 I n 1 9 3 2 , Q u a s t e l and W h e a t l e y d e m o n s t r a t e d t h a t c e r t a i n b a r b i t u r a t e s as w e l l a s o t h e r a n e s t h e t i c d r u g s s u p p r e s s t h e r e s p i r a t i o n o f b r a i n t i s s u e p r e p a r a t i o n s . T h i s o b s e r v a t i o n was l a t e r c o n f i r m e d by o t h e r w o r k e r s . The c o n c e n t r a t i o n s o f t h e d r u g r e q u i r e d were r e l a t i v e l y l a r g e compared w i t h t h e a n e s t h e t i c d o s e s . B a r b i t u r a t e s h o w e v e r , a t a n e s t h e t i c d o s e s , s u p p r e s s K + as w e l l as e l e c t r i c a l l y s t i m u l a t e d r e s p i r a t i o n 1 9 6 ' 1 9 7 . I t i s now w e l l known, f o l l o w i n g the work o f Mi±aeis and QuasteL 2 8 1 2 8 2 - 2 8 3 a n d E r n s t e r and c o - w o r k e r s , t h a t t h e b a r b i t u r a t e a m y t a l as w e l l as c e r t a i n o t h e r h y p n o t i c s s u p p r e s s t h e o x i d a t i o n o f NADH and h e n c e t h e g e n e r a t i o n o f ATP i n t h e c e l l . Thus a l l t h e p r o c e s s e s w h i c h r e q u i r e ATP a r e a f f e c t e d by t h e s e d r u g s . 1 9 6 1 9 7 T h e s e e f f e c t s have been d i s c u s s e d by Q u a s t e l ' - 40 -B a r b i t u r a t e s do not a f f e c t c e r e b r a l Na +, K +-ATPase a t c o n c e n t r a t i o n s t h a t suppress s t i m u l a t e d b r a i n r e s p i r a t i o n , nor do they i n h i b i t the i n c r e a s e d Na + i n f l u x due to e l e c t r i c a l 198 s t i m u l a t i o n . They a l s o do not show a l l e v i a t i o n o f the 14 14 d e p r e s s i o n of o x i d a t i o n of (1-C ) a c e t a t e to C 0^ brought 198 about by the a p p l i c a t i o n of e l e c t r i c a l p u l s e s . They d i f f e r i n t h i s r e s p e c t to l o c a l a n e s t h e t i c s and TTX. 199 Webb and E l l i o t t found t h a t the i n h i b i t i o n of r e s p i r a t i o n of b r a i n suspensions and s l i c e s by b a r b i t u r a t e s i s accompanied by a c o n s i d e r a b l e i n c r e a s e i n the r a t e of a e r o b i c g l y c o l y s i s . The maximum g l y c o l y t i c r a t e , e q u a l t o or exceeding the normal r a t e of a naerobic g l y c o l y s i s , occurs when the r a t e of oxygen consumption i s i n h i b i t e d about 50 p e r c e n t by the drug. (This i s c o n s i s t e n t w i t h the p r e s e n t i n f o r m a t i o n on the r e l i e f of P a s t e u r e f f e c t i n the presence of r e s p i r a t o r y c h a i n i n h i b i t o r s 199 due to decrease i n ATP.) Webb and E l l i o t t f u r t h e r observed t h a t the r a t e of a n a e r o b i c g l y c o l y s i s of b r a i n suspensions i s u n a f f e c t e d or s l i g h t l y a c c e l e r a t e d by low drug c o n c e n t r a t i o n s (1 mM i n the case o f amytal and p e n t o b a r b i t a l ) , but i s i n h i b i t e d by c o n c e n t r a t i o n s g r e a t e r than those which cause maximum a e r o b i c g l y c o l y s i s . The c o n c e n t r a t i o n s a t which these e f f e c t s occur va r y from drug to drug. (v) Chlorpromazine (CPZ) I n t r o d u c t i o n of CPZ i n the 1950"s was of g r e a t t h e r a -p e u t i c importance and s i n c e then CPZ as w e l l as o t h e r pheno-t h i a z i n e d e r i v a t i v e s have been e x t e n s i v e l y used i n the c l i n i c a l treatment of a number of mental d i s o r d e r s . L i n d a n , Q u a s t e l and S v e d ^ ^ have shown t h a t CPZ a t low 41 -c o n c e n t r a t i o n d e p r e s s e s t h e K + s t i m u l a t i o n o f c e r e b r a l o x y g e n u p t a k e . The e l e c t r i c a l l y s t i m u l a t e d r e s p i r a t i o n i s a l s o r e d u c e d by CPZ. CPZ d i f f e r s f r o m t h e n a r c o t i c s i n i t s h i g h b i n d i n g power w i t h t i s s u e p r o t e i n s b u t r e s e m b l e s t h e n a r c o t i c s i n i t s u n c o u p l i n g o f o x i d a t i v e p h o s p h o r y l a t i o n a t c o n c e n t r a -t i o n s t h a t do n o t a f f e c t t h e r e s p i r a t i o n o f u n s t i m u l a t e d 194 b r a i n c o r t e x s l i c e s . I t has b e e n shown t h a t CPZ u n c o u p l e s t h e o x i d a t i v e p h o s p h o r y l a t i o n i n t h e m i t o c h o n d r i a ^ ^ 204^ CPZ i s an a n t a g o n i s t o f f l a v i n a d e n i n e d i n u c l e o t i d e 205 (FAD) . FAD i s c l a i m e d t o a n t a g o n i z e t h e EEG e f f e c t s o f 206 20V CPZ . D a w k i n s , J u d a h and Rees have shown t h a t CPZ i n h i b i t s e l e c t r o n t r a n s p o r t between NADH and c y t o c h r o m e C. T h i s e f f e c t o f CPZ, t o g e t h e r w i t h i t s s u p p r e s s i o n e f f e c t on membrane 20 8—210 p e r m e a b i l i t y , may a c c o u n t f o r i t s a b i l i t y t o u n c o u p l e • ^ 4 . - v, u 1 4 . - 194,211 o x i d a t i v e p h o s p h o r y l a t i o n 212 CPZ i n h i b i t s t h e r e l e a s e o f a c e t y l c h o l i n e i n c a t b r a i n 194 and s t a b i l i z e s t h e a c e t y l c h o l i n e c o n t a i n i n g s y n a p t i c v e s i c l e s 213 M a i c k e l h a s shown t h a t CPZ i n h i b i t s a c e t y l c h o l i n e s t e r a s e . C o n s i d e r a b l e i n f o r m a t i o n e x i s t s on t h e i n f l u e n c e o f CPZ on t h e l e v e l s o f NE, dopamine and 5-HT i n b r a i n . CPZ d i m i n i s h e s u p t a k e o f NE i n h e a r t and a d r e n a l s and a l s o c a u s e c o m p l e t e r e l e a s e o f 194 196 p a r t i c l e b ound a d r e n a l i n i n a d r e n a l g l a n d p r e p a r a t i o n s ' 214 Magee and R o s s i t e r have shown t h a t low c o n c e n t r a t i o n s 32 o f CPZ b r i n g a b o u t c h a n g e s i n t h e i n c o r p o r a t i o n o f P i n t o v a r i o u s p h o s p h o l i p i d s o f g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . 215 Mule s t u d i e d t h e e f f e c t o f CPZ a n d o t h e r s CNS a c t i n g d r u g s on p h o s p h o l i p d - f a c i l i t a t e d C a + + t r a n s p o r t w h i c h i s i n h i b i t e d , and c o n c l u d e d t h a t an a l t e r a t i o n i n t h e b i n d i n g o f i o n s t o - 42 -p h o s p h o l i p i d s w i t h i n t h e n e u r o n a l membrane may be i n v o l v e d i n t h e p h a r m a c o l o g i c a l a c t i o n o f CNS a c t i n g d r u g s . CPZ d e c r e a s e s 22 42 t h e r a t e s o f p e n e t r a t i o n o f Na a n d K i n t o c e r e b r a l t i s s u e s 216 , a f a c t t h a t m i g h t b e a r on t h e mechanism o f a c t i o n o f t h i s d r u g . 217 B u z a r d has shown t h a t 1 mM CPZ h a s a marked i n h i b i t -o r y e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f r a t b r a i n h o m o g e n a t e s , e s p e c i a l l y i n t h e l a t e r p e r i o d o f i n c u b a t i o n . A t h i g h c o n c e n t r a t i o n s CPZ i s an i n h i b i t o r o f m i c r o s o m a l + + 218 219 Na , K - A T P a s e . A k e r a a n d B r o d y o b s e r v e d t h a t t h e e x p o s u r e o f CPZ s o l u t i o n t o l i g h t e n h a n c e d t h e i n h i b i t i o n o f A T P a s e a c t i v i t y . U l t r a v i o l e t (UV) l i g h t was e v e n more e f f e c t i v e and t h e s e a u t h o r s c o n c l u d e d t h a t a s e m i q u i n o n e f r e e - r a d i c a l o f CPZ r a t h e r t h a n CPZ i t s e l f may be r e s p o n s i b l e f o r t h e i n h i b i t i o n + + 220 o f Na , K -ATPase a c t i v i t y i n v i t r o . I n a l a t e r s t u d y , t h e s e w o r k e r s c o n c l u d e d t h a t t h e CPZ f r e e - r a d i c a l i n h i b i t s enzyme a c t i v i t y by i n t e r a c t i n g w i t h s u l p h y d r y l (-SH) g r o u p s on t h e enzyme i n c o n t r a s t t o o u a b a i n w h i c h i s bound t o a d i f f e r e n t s i t e . ( v i ) R e s e r p i n e P r e p a r a t i o n s f r o m t h e p l a n t R a u w o l f i a s e r p e n t i n a have b e e n u s e d i n I n d i a f o r c e n t u r i e s as a c a l m i n g d r u g . R e s e r p i n e 221 has b een shown t o be t h e p r i n c i p a l a l k a l o i d o f t h i s p l a n t and t h e e f f e c t s o f r e s e r p i n e on CNS h a s b een e x t e n s i v e l y s t u d i e d s i n c e t h e n . The most i m p o r t a n t b i o c h e m i c a l e f f e c t o f r e s e r p i n e i n t h e CNS f o u n d i n v i v o i s t h e r e l e a s e o f NE, 5-HT and dopamine f r o m t h e amine s t o r a g e s i t e s i n b r a i n . P r o l o n g e d t r e a t m e n t by r e s e r p i n e c a u s e d e p l e t i o n o f t h e s e amines f r o m t h e b r a i n . - 43 -Presumably these e f f e c t s are due to changes i n the permeabilities 222 of c e r t a i n n e u r onal membranes. G i a c h e t t i and Shore have shown t h a t r e s e r p i n e g r e a t l y enhances the p e r m e a b i l i t y o f a d r e n e r g i c n e u r o n a l membrane to the outward movement of amines. Reserpine i n t e r f e r e s w i t h the uptake o f the amines by the amine 223 storage g r a n u l e s . Whether r e s e r p i n e a c t s a t the ne u r o n a l 224 membrane or storage granule membrane i s c o n t r o v e r s i a l + + V a s c u l a r t i s s u e Na and K contents have been shown to 225 be changed a f t e r treatment of the animal w i t h r e s e r p i n e The a c t i o n s of r e s e r p i n e have been reviewed i n d e t a i l , ' . . 194,223,226,227 by a number of workers . 1.6 BIOCHEMISTRY OF THE DEVELOPING BRAIN Profound b i o c h e m i c a l changes take p l a c e i n the b r a i n of mammals d u r i n g development. Here we w i l l be concerned o n l y w i t h the problems which are more p e r t i n e n t t o the experiments r e p o r t e d i n t h i s t h e s i s . V a r i o u s aspects of the d e v e l o p i n g 50 22 8 b r a i n have been d i s c u s s e d by M c l l w a i n and Himwich The time a t which the maximum b r a i n growth o c c u r s 229 d i f f e r s from s p e c i e s t o s p e c i e s . In r a t s , the growth r a t e i s maximum a t 5-15 days a f t e r b i r t h , w i t h a peak a t 10 days. In c o n t r a s t to the r a t , i n guinea p i g s the maximum growth occurs b e f o r e b i r t h w i t h a peak a t 17 days b e f o r e b i r t h , w h i l e i n man the growth r a t e o f b r a i n i s maximum a t about the time of , . ,.,229 b i r t h F u n c t i o n a l l y , the major events i n the d e v e l o p i n g nervous system are the formation of synapses and the p r o c e s s of m y e l i n -a t i o n . The m y e l i n a t i o n p e r i o d c o i n c i d e s w i t h the p e r i o d of - 44 -230 g r e a t e s t net increment i n the b r a i n weight . M y e l i n a t i o n f i r s t o c curs c l o s e to the nerve c e l l body and s l o w l y spreads to the t e r m i n a l p o r t i o n of the nerve. However, even i n a d u l t l i f e , nerve f i b e r s are o f t e n not t o t a l l y m y e l i n a t e d , t h e i r t e r m i n a l p o r t i o n s u s u a l l y l a c k i n g myelin (see Davison and 231 P e t e r s f o r d e t a i l s ) . As wit h the pro c e s s o f m y e l i n a t i o n , the time a t which synapses are formed d i f f e r s from animal t o animal. In the guinea p i g c o n s i d e r a b l e synapses are p r e s e n t a t b i r t h , w h i l e i n the r a t a sharp i n c r e a s e i n the number of s y n a p t i c f u n c t i o n s occurs d u r i n g the 3rd and 4th week of 232 development Concomitant w i t h these changes, t h e r e o c c u r s a marked 50 244 change m the enzymic make up of the b r a i n ' . G l y c o l y s i s i s o f major importance i n the i n f a n t r a t h e r than i n the a d u l t b r a i n (except guinea p i g ) . The c i t r i c a c i d c y c l e enzymes i n c r e a s e markedly d u r i n g development and o x i d a t i v e metabolism of b r a i n g r e a t l y i n c r e a s e s d u r i n g m a t u r a t i o n . The hexose monophosphate pathway i s o f l i t t l e importance i n the a d u l t b r a i n although c o n s i d e r a b l e p o r t i o n s o f the glu c o s e u t i l i z e d are channeled through t h i s pathway i n the i n f a n t b r a i n . There i s a decrease i n the q u a n t i t i e s o f e l e c t r o l y t e s 50 t o g e t h e r w i t h a decrease i n water c o n t e n t d u r i n g development However, s e v e r a l important changes occur t h a t are r e l a t e d t o the changes i n the e l e c t r i c a l a c t i v i t y d u r i n g m a t u r a t i o n . K + content i n c r e a s e s w h i l e N a + d i m i n i s h e s . C h l o r i d e c o n t e n t and c h l o r i d e space decrease w i t h development. A c t i v i t y of Na , K -ATPase i s extremely low i n the newborn r a t and i n c r e a s e s r a p i d l y d u r i n g n e o n a t a l development wi t h the maximum a t about 50 days a f t e r - 45 -234 b i r t h . The i n c r e a s e i n the a c t i v i t y of t h i s enzyme may account p a r t l y f o r a l a r g e p o r t i o n of the i n c r e a s e d energy metabolism noted d u r i n g b r a i n m aturation. The i n c r e a s e i n the mitochondrial enzyme system i s e q u a l l y important. In a d d i t i o n , K + i s an e f f i c i e n t i n v i t r o s t i m u l a t o r of the oxygen consumption i n the 235 mature r a t b r a i n but i s i n e f f i c i e n t i n a new born r a t b r a i n T h i s phenomenon may be d i r e c t l y r e l a t e d to the changes i n ATPase .. 233 a c t i v i t y Although Na -K ATPase a c t i v i t y i s very low i n the i n f a n t r a t b r a i n , i t may be s u f f i c i e n t to m a i n t a i n a h i g h + + 236 c e l l u l a r K and low Na 1.7 TRANSPORT OF AMINO ACIDS AND SUGARS ACROSS THE BRAIN CELL  MEMBRANE Although i t has been known f o r a long time t h a t N a + i n f l u e n c e s the t r a n s p o r t of s o l u t e s a c r o s s the c e l l membrane, the i n f l u e n c e of N a + on the t r a n s p o r t of a v a r i e t y of s o l u t e s a c r o s s animal c e l l membranes was f i r s t i d e n t i f i e d i n the f i v e 2 37 2 38 year p e r i o d between 1958 and 1963 . R i k l i s and Q u a s t e l , i n 1958, f i r s t showed t h a t g l u c o s e a b s o r p t i o n by i s o l a t e d guinea p i g s m a l l i n t e s t i n e depends markedly on the presence of Na +. T h i s was soon extended by a number of workers t o i n c l u d e o t h e r compounds such as amino a c i d s , u r a c i l and 3-0-methyl 237 + g l u c o s e . There i s evidence t h a t Na dependent a c t i v e t r a n s -+ 239-241 p o r t systems may be d r i v e n by a p a r a l l e l d o w n h i l l Na f l u x The t r a n s p o r t r e a c t i o n s of amino a c i d s and sugars at 137 the b r a i n c e l l membrane has been reviewed r e c e n t l y by Q u a s t e l ' 242 Although t h e r e i s some exchange d i f f u s i o n , the ammo a c i d s are u s u a l l y accumulated a g a i n s t a c o n c e n t r a t i o n g r a d i e n t . S e v e r a l drugs are known to a f f e c t the t r a n s p o r t of amino a c i d s i n v i v o as w e l l as i n c e r e b r a l c o r t e x s l i c e s . L-amino a c i d s p e n e t r a t e the b r a i n c e l l s more e a s i l y than the c o r r e s -ponding D-isomers. The uptake o f amino a c i d s by c e r e b r a l c o r t e x s l i c e s i s energy dependent and t h e i r accumulation under a e r o b i c 137 c o n d i t i o n s i s b l o c k e d by 2 , 4 - d i n i t r o p h e n o l or i o d o a c e t a t e ' 243 244 ' . Uptake of g l y c i n e by c e r e b r a l c o r t e x s l i c e s under a wide v a r i e t y o f ex p e r i m e n t a l c o n d i t i o n s i s p r o p o r t i o n a l t o 245 the l e v e l o f ATP . There i s a r e d u c t i o n i n c e r e b r a l uptake o f one amino a c i d i n the presence o f another which may be l a r g e l y due t o the mutual c o m p e t i t i o n f o r a common c a r r i e r (or 137 s i t e ) a t the b r a i n c e l l membrane The a c t i v e t r a n s p o r t o f amino a c i d s i n b r a i n i s a l s o + 245 246 247 + Na dependent ' ' . High c o n c e n t r a t i o n s o f K d i m i n i s h the c e r e b r a l uptake o f amino a c i d s by low e r i n g the ATP l e v e l 137 A l s o , t h e r e i s l e s s uptake of amino a c i d s i n the absence ++ ++ of Ca or i n the presence of 10 mM Ca ; the l a t t e r may be + + due t o i n h i b i t i o n o f Na ,K -ATPase. Ouabain i n h i b i t s the uptake of amino a c i d s , which i s c o n s i s t e n t w i t h the r o l e o f 137 t h i s enzyme i n amino a c i d t r a n s p o r t processes . In the presence of ouabain, the amino a c i d s e f f l u x i n t o medium i s i n c r e a s e d 1 7 ^ . A n a e r o b i o s i s , l a c k of Na +, the presence of a r e s p i r a t o r y c h a i n i n h i b i t o r or chlorpromazine a l s o i n c r e a s e s 137 the e f f l u x o f amino a c i d s Although i t i s w e l l e s t a b l i s h e d t h a t g l u c o s e t r a n s p o r t a c r o s s the i n t e s t i n a l w a l l i s Na + and energy dependent, there - 47 -i s no evidence t h a t N a + mediate or i s e s s e n t i a l f o r g l u c o s e 137 t r a n s p o r t xnto the b r a i n c e l l Very l i t t l e i n f o r m a t i o n i s a v a i l a b l e on the s p e c i f i c i t y o f carbohydrate t r a n s p o r t p rocesses and even l e s s on the kinetic 248 p r o p e r t i e s of such p r o c e s s e s i n the b r a i n . S t u d i e s on g l u c o s e t r a n s p o r t have been d i f f i c u l t owing t o the r a p i d metabolism o f g l u c o s e i n the b r a i n under normal c o n d i t i o n s and use of glucose i n suboptimal concentrations renders data from such s t u d i e s o f d o u b t f u l s i g n i f i c a n c e . However, i n the case of pentoses, evidence has emerged t o support the view t h a t a f a c i l i t a t e d or c a r r i e r mediated process r a t h e r than d i f f u s i o n is , ,137,248 i n v o l v e d 248 14 B a c h e l a r d has s t u d i e d the uptake of a number of C l a b e l l e d g l u c o s e analogs i n t o the n o n - r a f f i n o s e space of c e r e -b r a l c o r t e x s l i c e s . K i n e t i c p r o p e r t i e s of the uptake o f 2-deoxyglucose i n d i c a t e t h a t the t r a n s p o r t i s a f a c i l i t a t e d p r o c e s s r a t h e r than d i f f u s i o n . Other experiments i n d i c a t e t h a t deoxyglucose may be regarded as a c o m p e t i t i v e i n h i b i t o r of glucose uptake and the apparent Km f o r glucose t r a n s p o r t has been suggested t o be of the order of 5 mM. C o n s i d e r a t i o n of the f a c t o r s l i k e l y t o operate i n r e g u l a t i n g b r a i n carbohydrate metabolism i n d i c a t e t h a t glucose 2 0 5 1 2 4 9 51 t r a n s p o r t may be a p o s s i b l e c o n t r o l p o i n t ' ' . R o l l e s t o n observed t h a t when the r a t e of glucose metabolism i s r a p i d ( i n the presence of 50 mM K + or 3 mM c y a n i d e ) , i n c r e a s i n g the c o n c e n t r a t i o n o f glucose i n the medium from 3 to 10 mM causes i n c r e a s e d r a t e of g l y c o l y s i s . T h i s was not observed under 51 c o n d i t i o n g i v i n g lower r a t e of metabolism. R o l l e s t o n concluded t h a t t h e r a t e o f e n t r y o f g l u c o s e i n t o t h e t i s s u e may become a l i m i t i n g f a c t o r i n g l u c o s e m e t a b o l i s m . OBJECTIVES The a i m o f t h e p r e s e n t i n v e s t i g a t i o n h as b e e n t o s t u d y t h e e f f e c t o f TTX and o t h e r n e u r o t r o p i c d r u g s on c e r e b r a l a n a e r o b i c g l y c o l y s i s , a n d on t r a n s p o r t p r o c e s s e s o f i n c u b a t e d c e r e b r a l c o r t e x s l i c e s , i n an e f f o r t t o u n d e r s t a n d more f u l l y c e r e b r a l m e t a b o l i c p r o c e s s e s d u r i n g a n o x i a and t h e mode o f a c t i o n o f c e r t a i n n e u r o t r o p i c d r u g s u n d e r t h e s e c o n d i t i o n s . The g e n e r a l a p p r o a c h has been t o i n v e s t i g a t e t h e a c t i o n o f v a r i o u s d r u g s on a n a e r o b i c g l y c o l y s i s o f b r a i n s l i c e s a nd t o s e e w h e t h e r t h e i r a c t i v i t i e s may be a f f e c t e d by a v a r i e t y o f 22 c o n d i t i o n s . The c a t i o n c o n t e n t s , and r a t e s o f Na t r a n s p o r t were s t u d i e d u n d e r v a r i o u s c o n d i t i o n s i n t h e p r e s e n c e o f v a r i o u s d r u g s and t h e s e t h e n r e l a t e d t o t h e a c c o m p a n y i n g c h a n g e s i n t h e b r a i n m e t a b o l i s m . Some e x p e r i m e n t s were a l s o c a r r i e d o u t on t h e t r a n s p o r t o f amino a c i d s and g l u c o s e . P r e l i m i n a r y e x p e r i m e n t s ( C h a p t e r 3) d e a l w i t h t h e r a t e s o f a n a e r o b i c g l y c o l y s i s i n t h e c e r e b r a l c o r t e x s l i c e s . The f o l l o w i n g two c h a p t e r s ( C h a p t e r 4 and 5) d e a l w i t h t h e e f f e c t s o f T T X on t h e t r a n s p o r t p r o c e s s e s and on a n a e r o b i c g l y c o l y s i s o f b r a i n s l i c e s . C h a p t e r s 6 and 7 i n c l u d e e x p e r i m e n t s c a r r i e d o u t w i t h o t h e r d r u g s t o s e e how t h e y r e s e m b l e o r d i f f e r f r o m TTX i n a f f e c t i n g a n o x i c m e t a b o l i s m o f t h e b r a i n s l i c e s . The l a s t c h a p t e r ( C h a p t e r 8) p r e s e n t s a g e n e r a l d i s c u s s i o n o f t h e e x p e r i m e n t s r e p o r t e d i n t h i s t h e s i s a n d o f t h e r e s u l t s and c o n c l u s i o n s . 4 8 a STRUCTURE OF TETRODOTOXIN M o l . Wt. 322 F o r d e t a i l s o f c h e m i s t r y and p h a r m a c o l o g y s e e M o s h e r e t a l 1 1 4 , K a o 1 1 5 and E v a n s 1 1 6 . CHAPTER 2  MATERIALS AND METHODS 2.1 EXPERIMENTAL ANIMALS The animals used i n the experiments were r a t s and guinea p i g s . A d u l t W i s t a r r a t s , weighing about 200-250 g, were o b t a i n e d e i t h e r from the N a t i o n a l L a b o r a t o r y Co., Edmonton or the Department of Zoology V i v a r i u m , The U n i v e r s i t y of B r i t i s h Columbia. I n f a n t r a t s were s u p p l i e d by the Viva r i u m . A d u l t male guinea p i g s , weighing about 300-350 g, and newly born guinea p i g s , o f E n g l i s h s h o r t h a i r s t r a i n , were o b t a i n e d from the Animal U n i t , F a c u l t y o f Medicine, The U n i v e r s i t y o f B r i t i s h Columbia. A l l the a d u l t animals had f r e e access t o food and water u n t i l used, whereas the i n f a n t animals were s e p a r a t e d from the mother s h o r t l y b e f o r e s t a r t o f the experiment. 2.2 CHEMICALS A l l common l a b o r a t o r y chemicals were of "reagent grade" and were used without any f u r t h e r p u r i f i c a t i o n . NAD +, NADH, NADP +, ATP, ADP, AMP, Adenosine, cAMP, phosphoenol p y r u v a t e , l i t h i u m l a c t a t e , e p i n e p h r i n e , n o r e p i n -e p h r i n e , h i s t a m i n e , tyramine, LDH (beef h e a r t ) , LDH ( r a b b i t m u scle), g l u t a m i c dehydrogenase (beef l i v e r ) , pyruvate k i n a s e ( r a b b i t muscle) and phospholipases A and C were ob t a i n e d from Calbiochem, Los Angeles. T r i e t h a n o l a m i n e was the product of Sigma Chemical Co., S t . L o u i s . Hexokinase (HK) and glucose 6-phosphate dehydrogenase (G-6-PDH) were - 50 -purchased e i t h e r from Calbiochem or from Sigma Chemical Co. D i b u t y r y l cAMP was o b t a i n e d from Schwarz BioResearch and a c e t y l c h o l i n e was a product of Matheson Co. Inc., Norwood, Ohio. The r a d i o c h e m i c a l s : sodium-22 (as c h l o r i d e ) , adenine-8-14 14 C s u l f a t e and glucose-2-C were the p r o d u c t s of Radio-14 chem i c a l Centre, Amersham, England. U-C - g l u t a m i c a c i d 14 and g l y c m e - 2 - C were o b t a i n e d from Volk Radiochemxcal Co., Chicago. T e t r o d o t o x i n was purchased from Calbiochem. Ouabain and r e s e r p i n e were o b t a i n e d from N u t r i t i o n a l B i o c h e m i c a l s Co.; p r o t o v e r a t r i n e from K and K L a b o r a t o r i e s , P l a i n v i e w , New York; c h l o r p r o m a z i n e from Poulenc,, M o n t r e a l ; d- and 1-amphetamines from Smith, K l i n e and French L a b o r a t o r i e s , IAC, M o n t r e a l ; p e n t o t h a l from Abbott L a b o r a t o r i e s , M o n t r e a l ; n i a l a m i d e from P f i z e r I n c . , B r o o k l y n , N. Y. and amytal from • ; £ l i L i l l y . - , P r o c a i n e and l i d o c a i n e were o b t a i n e d from Department of Pharmacology, The U n i v e r s i t y of B r i t i s h Columbia. 2.3 PREPARATION OF BRAIN SLICES Rats were stunned by blow a t the back and d e c a p i t a t e d q u i c k l y . The b r a i n s were d i s s e c t e d and kept i n the i n c u b a t i o n medium (without any a d d i t i o n s except glucose) u n t i l s l i c i n g . The c e r e b r a l c o r t e x s l i c e s were prepared w i t h the S t a d i e - R i g g s t i s s u e s l i c e r and a moist r a z o r . The s l i c e s were about 0.4 mm t h i c k and t h i n n e r or t h i c k e r ones d i s c a r d e d . Only one top d o r s a l and one l a t e r a l s l i c e from each hemisphere were used. The s e l e c t e d s l i c e s were kept - 51 -on an i c e - c o l d p e t r i d i s h u n t i l p l a c e d i n t h e i n c u b a t i o n medium i n t h e Warburg v e s s e l . One d o r s a l and one l a t e r a l s l i c e f r o m e a c h h e m i s p h e r e w e i g h i n g a b o u t 60-75 mg, was u s e d i n e a c h v e s s e l . G u i n e a p i g c e r e b r a l c o r t e x s l i c e s were p r e p a r e d i n t h e same way as f o r r a t s , e x c e p t t h a t f o r l a t e r a l s l i c e s p a r i e t a l l o b e s were f i r s t c u t o u t and t h e n t h e s l i c e s were p r e p a r e d f r o m them. When g u i n e a p i g s were u s e d , o n l y one s l i c e weighing a b o u t 40-55 mg was u s e d i n e a c h v e s s e l b e c a u s e o f t h e h i g h r a t e o f g l y c o l y s i s i n t h e g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . I n f a n t r a t b r a i n (1-3 d ay o l d ) s l i c e s were p r e p a r e d by a s l i g h t l y d i f f e r e n t method. A f t e r d e c a p i t a t i o n , b r a i n s were removed, and one s l i c e was c u t f r o m t e m p o r a l - p a r i e t a l p o r t i o n o f e a c h h e m i s p h e r e . Two s l i c e s f r o m e a c h b r a i n , w e i g h i n g a b o u t 70-90 mg, were u s e d f o r e a c h v e s s e l . C e r e b r a l c o r t e x s l i c e s f r o m o l d e r i n f a n t r a t s and n e w l y b o r n g u i n e a p i g s were p r e p a r e d i n t h e same way as f o r a d u l t b r a i n . 2.4 INCUBATION PROCEDURE The s l i c e s were w e i g h e d q u i c k l y on a t o r s i o n b a l a n c e t o t h e n e a r e s t mg and t h e n s u s p e n d e d i n 3 ml p r e c o o l e d i n c u b -a t i o n medium i n t h e Warburg v e s s e l . A d d i t i o n s were e i t h e r made a t t h e b e g i n n i n g o f t h e e x p e r i m e n t o r t i p p e d i n f r o m t h e s i d e arm d u r i n g t h e i n c u b a t i o n p e r i o d . I n c u b a t i o n s were c a r r i e d o u t u s i n g a c o n v e n t i o n a l Warburg m a n o m e t r i c a p p a r a t u s a t 37°C i n an a t m o s p h e r e o f 95 p e r c e n t N 2 : 5 p e r c e n t C 0 2 ( N 2 : C 0 2 ) o r 95 p e r c e n t 0 2 : 5 p e r c e n t C 0 2 - 52 -(C^iCC^) as i n d i c a t e d f o r each experiment. In some e x p e r i -ments, however, the gas phase was changed d u r i n g the i n c u b a t i o n . 2.5 INCUBATION MEDIUM In the experiments d e s c r i b e d i n t h i s t h e s i s , the c e r e b r a l c o r t e x s l i c e s were i n c u b a t e d i n v a r i o u s media depending upon the nature of the experiment. The r e q u i r e d s a l t s o l u t i o n s were made up a t 5 times i s o t o n i c c o n c e n t r a t i o n s and s t o r e d a t 4°C. The f i n a l c omposition of the v a r i o u s media are g i v e n below. (a) Kreb-Ringer Medium Krebs-Ringer b i c a r b o n a t e medium was used f o r an a e r o b i c experiments. I t c o n t a i n e d 119 mM NaCl, 5 mM K C l , 3.6 mM C a C l 2 , 1.2 mM MgS0 4, 1.2 mM KR"2P04 and 25 mM NaHC0 3. Krebs-Ringer phosphate medium c o n t a i n e d K C l , NaCl, C a C l 2 and MgS0 4 as above, but 10 mM Na-phosphate b u f f e r (pH 7.4) was added i n p l a c e of NaHCO-j. Phosphate b u f f e r was added t o the f i n a l i n c u b a t i o n medium a t the end to a v o i d the p r e c i p i t a t i o n o f C a + + and M g + + (as phosphates), t h a t may occur b e f o r e d i l u t i o n . ++ (b) Ca - f r e e Medium In most of the an a e r o b i c experiments the C a + + - f r e e medium used was s i m i l a r t o t h a t d e s c r i b e d by Adams and Q u a s t e l 7 1 . I t c o n t a i n e d 149 mM Na +, 5 mM K +, 125 mM C l ~ — ++ ++ and 29 mM HCO^- Ca and Mg were omitted from the medium as, under these c o n d i t i o n s , a n a e r o b i c g l y c o l y t i c r a t e s showed g r e a t e s t s e n s i t i v i t i e s to v a r i o u s types of neuro-- 53 -t r o p i c drugs and gave the most c o n s i s t e n t r e s u l t s . Omission of phosphate had no apparent e f f e c t i n our experiments. (c) K + - f r e e Medium K + - f r e e medium was prepared by o m i t t i n g K Cl from the C a + + - f r e e medium. I t c o n t a i n e d 154 mM Na +, 125 mM C l ~ and 2 9 mM HCO~. (d) C l - f r e e Medium C l - f r e e medium was prepared by u s i n g s u l f a t e s a l t s o f N a + and K + i n s t e a d of c h l o r i d e s a l t s . I t s composition f o r c a t i o n s was the same as t h a t of C a + + - f r e e medium but the SO^ was used a t h a l f the c o n c e n t r a t i o n of C l . (e) Li" l"-containing Medium The composition o f L i + - c o n t a i n i n g medium was s i m i l a r ++ t o t h a t of Ca - f r e e medium except t h a t some, or a l l , NaCl was r e p l a c e d by L i C l . The c o n c e n t r a t i o n s of L i + are s p e c i f i e d i n the experiments d e s c r i b e d below. (f) High-K + Medium High-K + medium was prepared by r e p l a c i n g some, or a l l , NaCl by K C l , or by r e p l a c i n g NaCl and NaHC0 3 by KCl and ++ + KHCO^, i n the Ca - f r e e medium. In some cas e s , where Na was a l s o t o be reduced i n c o n c e n t r a t i o n (without f u r t h e r i n c r e a s i n g K + a t the same t i m e ) , sucrose (twice the m o l a r i t y of s a l t s o l u t i o n ) was added to m a i n t a i n t o n i c i t y . (g) N a + - f r e e Medium N a + - f r e e medium c o n t a i n e d 26 0 mM suc r o s e , 6 mM KCl and 10 mM t r i s - H C l b u f f e r (pH 7.4). - 54 -2.6 PREPARATION OF KIDNEY MEDULLA K i d n e y m e d u l l a was u s e d as a c o n t r o l t i s s u e i n some o f t h e g l y c o l y s i s e x p e r i m e n t s . The f o l l o w i n g p r o c e d u r e was u s e d f o r t h e p r e p a r a t i o n o f t h e t i s s u e ; r a t s were s t u n n e d by b l o w on t h e h e a d and t h e v i s c e r a o p e n e d ; k i d n e y s were t h e n removed q u i c k l y and e a c h was c u t i n t o two e q u a l h a l v e s . S u b s e q u e n t l y t h e k i d n e y c o r t e x was c u t o f f f r o m t h e m e d u l l a and d i s c a r d e d . Two h a l v e s o f t h e k i d n e y m e d u l l a , e a c h 0 . 5 - 1 mm t h i c k a n d w e i g h i n g a b o u t 120-140 mg, were u s e d i n e a c h v e s s e l . 2.7 PREPARATION OF CAUDATE NUCLEUS The r a t s were s t u n n e d by a b l o w a t t h e b a c k and t h e b r a i n s q u i c k l y r e m oved, e a c h b r a i n was c u t i n t o two h a l v e s a l o n g f i s s u r a l o n g i t u d i n a l i s c e r e b r i and t h e c a u d a t e 250 n u c l e u s was l o c a t e d and d i s s e c t e d o u t . E a c h h a l f o f t h e c a u d a t e n u c l e u s was t h e n c u t i n t o t h r e e p i e c e s , w e i g h e d q u i c k l y and p l a c e d i n t h e W a rburg v e s s e l . 2.8 PREPARATION OF ACETONE POWDER EXTRACTS A c e t o n e powder e x t r a c t s o f t h e r a t c e r e b r a l c o r t e x was 251 252 p r e p a r e d by t h e method o f H a r p u r and Q u a s t e l ' . R a t b r a i n c o r t i c e s were removed and g r o u n d i n i c e - c o l d a c e t o n e . A f t e r f i l t e r i n g and f u r t h e r w a s h i n g w i t h i c e - c o l d a c e t o n e , t h e r e s u l t i n g powder was s t o r e d i n a vacuum d e s s i c a t o r a t 4°C u n t i l u s e d . 100 mg o f t h e a c e t o n e powder was e x t r a c t e d w i t h 10 ml o f a s o l u t i o n , u s u a l l y c o n t a i n i n g 100 mM n i c o t i n a m i d e , 33 mM c y s t e i n e and 156 mM N a C l . The e x t r a c t was c e n t r i f u g e d - 55 -and 1 ml of supernatant was taken i n each v e s s e l . T h i s , on d i l u t i o n t o 3 ml, gave a f i n a l c o n c e n t r a t i o n of 33 mM n i c o t i n a m i d e , 11 mM c y s t e i n e and 52 mM NaCl. Other a d d i t i o n s were made as i n d i c a t e d i n the r e s u l t s . 2.9 PREPARATION OF SYNAPTOSOMES 253 254 The method developed by Whittaker ' as shown i n the Scheme 2 was used f o r the p r e p a r a t i o n of synaptosomes. A f t e r s e p a r a t i o n o f s u b c e l l u l a r p a r t i c l e s on a d e n s i t y g r a d i e n t , the v a r i o u s l a y e r s were c a r e f u l l y removed w i t h the h e l p of a past e u r p i p e t t e . A f t e r c e n t r i f u g a t i o n of the f r a c t i o n having d e n s i t y between 0.8 and 1.2 M of suc r o s e , the r e s i d u e (synaptosomes) were resuspended and used f o r experiments and p r o t e i n d e t e r m i n a t i o n . 2.10 PREPARATION OF MICROSOMES AND ASSAY OF Na +, K +-ATPase Microsomes from guinea p i g b r a i n were pr e p a r e d by the 255 method of Hokin and Yoda . B r a i n s were removed q u i c k l y a f t e r d e c a p i t a t i o n and c e r e b r a l hemispheres were homogenized i n an a l l g l a s s homogenizer i n Sucrose-EDTA (0.25 M sucrose + 2 mM EDTA). The homogenate was f i r s t c e n t r i f u g e d f o r 5 min a t 700 g and the sediment d i s c a r d e d . The supernatant was c e n t r i f u g e d a t 105000 g i n r o t o r 30 i n a Spinco model L p r e p a r a t i v e u l t r a c e n t r i f u g e f o r 10 min. The r e s i d u e was resuspended i n 0.25 M sucrose w i t h hand homogenization. The suspended r e s i d u e was c e n t r i f u g e d a t 8500 g f o r 15 min. The r e s i d u e was d i s c a r d e d and the supernatant was again c e n t r i f u g e d f o r 20 min a t 105000 g. The r e s u l t i n g r e s i d u e (microsomal f r a c t i o n ) was suspended i n su c r o s e -- 56 -Scheme 2 F r a c t i o n a t i o n o f Synaptosomes by Sucrose  G r a d i e n t C e n t r i f u g a t i o n 4 g whole b r a i n (3 r a t s ) + 0.32 M sucrose (36 m l ) , homogenize, 1725 r e v o l u t i o n s per min i n a t e f l o n homogenizer. 5 up and down s t r o k e s . C e n t r i f u g e a t 1000 g f o r 10 min. P r e c i p i t a t e (Ppt) +0.32 M sucrose (32 ml) suspend and c e n t r i f u g e a t 1000 g f o r 10 min 4. 4, Ppt. Sup. Supernatant (Sup) 35 ml x 2 Rotor # 30 Spinco, 15000 g 45 min 4 Ppt. + 0.32 M sucrose 2 ml x 2, hand homogenize (8 ml) 4 Sup. 4 6 ml Layer 1 ml over 0.8 M su c r o s e , 2 ml + 1.2 M su c r o s e , 2 ml and c e n t r i f u g e f o r 45 min a t 100,000 g i n r o t o r # SW39L (without brakes) 4 2 ml A F r a c t i o n B F r a c t i o n C F r a c t i o n E q u a l Volume of 0.3 2 M s u c r o s e , then c e n t r i f u g e a t 100,000 g f o r 30 min 4 Ppt, 4 Supt. SYNAPTOSOMES. Suspend i n 0.32 M sucrose and use. - 57 -EDTA c o r r e s p o n d i n g to 0.5 ml/g of o r i g i n a l b r a i n and was used e i t h e r f o r assay immediately or s t o r e d f r o z e n a t -20°C. The tubes f o r assay of ATPase c o n t a i n e d 0.5 ml of the medium c o n t a i n i n g microsomal suspension (25 yg p r o t e i n ) , 80 mM i m i d a z o l e - H C l b u f f e r (pH 7.1), 2 mM M g C l 2 , 2 mM Na 2ATP, 80 mM NaCl and 60 mM K C l . I n c u b a t i o n p e r i o d was 20 min a t 37°C. A l l assays were c a r r i e d out i n d u p l i c a t e s and o t h e r a d d i t o n s were made as i n d i c a t e d i n the r e s u l t s . The r e a c t i o n was t e r m i n a t e d by the a d d i t i o n of 0.5 ml of 10% t r i c h l o r o a c e t i c a c i d (TCA), the tubes were c o o l e d , c e n t r i f u g e d and phosphate was measured i n the supernatant 256 by the method o f Stanton . From o p t i c a l d e n s i t y (OD) vs time d a t a , the zero time i n t e r c e p t s were determined and a c t u a l amounts of phosphate l i b e r a t e d were then c a l c u l a t e d from these v a l u e s and s t a n d a r d s . 2.11 MEASUREMENT OF GLYCOLYSIS (a) Manometric L a c t a t e p r o d u c t i o n d u r i n g anaerobic g l y c o l y s i s was e s t i m a t e d by measuring carbon d i o x i d e (C0 2) e v o l u t i o n from a b i c a r b o n a t e medium i n the Warburg apparatus i n an atmos-phere of N 2:C0 2. I t i s w e l l known t h a t when th e r e i s e q u i l i b r i u m between C0 2 i n the medium and i n the gaseous phase, each molecule of l a c t i c a c i d produced w i l l r e l e a s e one molecule of C0 2 i n the gas phase. C 0 2 so e v o l v e d can be e a s i l y measured wi t h the h e l p of the manometer. The manometric method i s much more convenient than the a c t u a l l a c t a t e a s s a y s , s i n c e one can measure l a c t a t e p r o d u c t i o n - 58 -c o n t i n u o u s l y o v e r t h e e n t i r e i n c u b a t i o n p e r i o d . U n l e s s o t h e r w i s e s t a t e d , t h e e x p e r i m e n t s were o f 80 min d u r a t i o n . The v e s s e l s were g a s s e d w i t h N 2 : C 0 2 f o r t h e f i r s t 10 min, w h i l e s h a k i n g , i n t h e i n c u b a t i o n b a t h . A f t e r g a s s i n g p e r i o d s t h e s t o p p e r s were c l o s e d and e q u i l i b r a t i o n was c a r r i e d o u t f o r a n o t h e r 10 m i n . M a n o m e t r i c r e a d i n g s were t a k e n a f t e r t h i s 20 min p e r i o d f o r one h o u r . R e s u l t s o b t a i n e d as y l C 0 2 o f l a c t a t e p r o d u c e d (by c a l c u l a t i o n f r o m t h e m a n o m e t r i c r e a d i n g s and f l a s k c o n s t a n t s ) were c o n v e r t e d t o ymoles o f l a c t a t e by d i v i d i n g by 22 .4 (1 mole o f a gas c o r r e s p o n d s t o 22 .4 l i t r e s a t NT P ) . I t has b e e n w e l l e s t a b l i s h e d t h a t t h e m a j o r p r o d u c t d u r i n g a n a e r o b i c g l y c o -l y s i s o f c e r e b r a l c o r t e x s l i c e s i s l a c t a t e a nd t h a t v a l u e s o b t a i n e d by m a n o m e t r i c and e n z y m a t i c a s s a y s c l o s e l y a g r e e , (b) E n z y m a t i c F o r t h e measurement o f l a c t a t e p r o d u c t i o n i n a N a + - f r e e medium, and a e r o b i c a l l y , t h e e n z y m a t i c method was u s e d . I n t h i s method, l a c t a t e i s o x i d i z e d t o p y r u v a t e i n t h e p r e s e n c e o f NAD a n d LDH and t h e p y r u v a t e so f o r m e d i s t r a p p e d by h y d r a z i n e - g l y c i n e b u f f e r . NADH p r o d u c e d c a n be m e a s u r e d 257 s p e c t r o p h o t o m e t r i c a l l y o r f l u o r i m e t r i c a l l y . F o r a s s a y , t h e sample was d e p r o t e i n i z e d w i t h 6 p e r c e n t (v/v) p e r c h l o r i c a c i d a nd t h e s u p e r n a t a n t n e u t r a l i z e d w i t h K2CC>2 u s - " - n 9 p h e n o l r e d as i n d i c a t o r . The r e a c t i o n m i x t u r e , i n a t o t a l volume o f 2.94 m l , c o n t a i n e d 1 ml o f d i l u t e d s a m p l e , 1.3 ml o f h y d r a z i n e - g l y c i n e b u f f e r (0.4 M h y d r a z i n e , 1 M g l y c i n e pH 9 . 5 ) , 0 . 1 1 ml o f NAD + (40 mg/ml) and .03 ml - 59 -o f LDH (1250 I . U . / m l ) . The m i x t u r e was i n c u b a t e d a t room t e m p e r a t u r e i n c u v e t t e s w i t h 1 cm l i g h t p a t h a n d t h e i n c r e a s e i n OD was f o l l o w e d a t 340nm u s i n g a Beckman m o d e l DU s p e c t r o p h o t o m e t e r . A c o n t r o l c u v e t t e , w i t h o u t t h e s a m p l e , was i n c l u d e d e v e r y t i m e . The amount o f l a c t a t e was c a l c u l a t e d f r o m t h e e x t i n c t i o n c o e f f i c i e n t o f NADH. I n some e x p e r i m e n t s l a c t a t e was m e a s u r e d f l u o r i m e t r i c -a l l y u s i n g an Aminco-Bowman s p e c t r o p h o t o f l u o r o m e t e r . I n t h e s e c a s e s , i n 2.1 m l , t h e r e a c t i o n m i x t u r e c o n t a i n e d 2 m l o f h y d r a z i n e - g l y c i n e b u f f e r ( a s a b o v e ) , 25 y l NAD + (2 m g v / m l ) , 20 y l LDH (20 I . U . p e r a s s a y ) , a n d 50 y l o f s a m p l e s o r s t a n d a r d s i n B a u s c h a n d Lomb c o l o r i m e t e r t u b e s . NADH p r o d u c e d a f t e r t h e a d d i t i o n o f enzyme was m e a s u r e d f l u o r i m e t r i c a l l y u n t i l t h e i n c r e a s e i n f l u o r e s c e n c e l e v e l l e d o f f . The e x c i t a t i o n w a v e l e n g t h was 365 nm a n d f l u o r e s c e n t w a v e l e n g t h was 460 nm. The amount o f l a c t a t e was c a l c u l a t e d w i t h t h e h e l p o f t h e s t a n d a r d s . 2.12 MEASUREMENT OF AMINO ACID EFFLUX AND UPTAKE (a) A mino A c i d E f f l u x The c e r e b r a l s l i c e s w e r e i n c u b a t e d f o r 30 m i n i n a C a + + -f r e e medium. A t t h e e n d o f t h e i n c u b a t i o n , t h e s l i c e s w e r e h o m o g e n i z e d w i t h 2.5 ml o f 5% (w/v) c o l d TCA. The ho m o g e n a t e was l e f t i n c o l d f o r 30 m i n a n d t h e n c e n t r i f u g e d ; t h e s u p e r n a t a n t was t r a n s f e r r e d t o a n o t h e r t u b e , t h e p r e c i p i t a t e w a s h e d w i t h 1 m l o f c o l d TCA a n d c e n t r i f u g e d . The two s u p e r n a t a n t s w e r e m i x e d a n d e x t r a c t e d t h r i c e w i t h 3 m l o f d i e t h y l e t h e r t o remove TCA. E t h e r was r e m o v e d - 60 -f r o m t h e w a t e r p h a s e b y b l o w i n g a i r a n d t h e TCA f r e e e x t r a c t , a f t e r s u i t a b l e d i l u t i o n , was u s e d f o r a m i n o a c i d a n a l y s e s . The i n c u b a t i o n medium was t r a n s f e r r e d q u a n t i t a t i v e l y i n a c e n t r i f u g e t u b e , d e p r o t e i n i z e d w i t h TCA a n d t h e s u p e r n a t a n t r e m o v e d . TCA was r e m o v e d f r o m t h e s u p e r n a t a n t f r a c t i o n a s a b o v e a n d t h e e x t r a c t was u s e d a f t e r s u i t a b l e d i l u t i o n f o r a m i n o a c i d a n a l y s e s . The a m i n o a c i d s ( t a u r i n e , a s p a r t i c a c i d , g l u t a m i c a c i d , g l u t a m i n e + s e r i n e , g l y c i n e a n d a l a n i n e ) w e r e s e p a r a t e d o n a Beckman m o d e l 120 B a m i n o a c i d a n a l y z e r , u s i n g a 50 cm s u l f o n a t e d p o l y s t y r e n e - 8% d i v i n y l - b e n z e n e c o p o l y m e r i o n e x c h a n g e r e s i n ( t y p e 50 A - p a r t i c l e s i z e 2 5-31 y) a t 50°C; t h e e l u a n t u s e d was 0.2 N s o d i u m c i t r a t e b u f f e r , pH 3.3. The c o l u m n was r e g e n e r a t e d w i t h 0.2 N NaOH, a n d e q u i l i b r a t e d w i t h t h e b u f f e r , b e f o r e e a c h u s e . (b) A m i n o A c i d I n f l u x C o n d i t i o n s o f i n c u b a t i o n a r e g i v e n w i t h t h e r e s u l t s a n d 22 t h e s a m p l e s w e r e p r e p a r e d a s f o r Na - i n f l u x s t u d i e s . 2.13 MEASUREMENT OF NAD + AND NADH The m e t h o d o f L o w r y a n d c o - w o r k e r s was u s e d f o r t h e e x t r a c t i o n a n d a s s a y o f NAD + and NADH. (a) E x t r a c t i o n o f NADH A t t h e e n d o f i n c u b a t i o n , t h e t i s s u e was h o m o g e n i z e d a t a d i l u t i o n o f a t l e a s t 1:100 i n i c e - c o l d 0.02N NaOH c o n t a i n -i n g 0.5 mM c y s t e i n e . The h o m o g e n a t e was h e a t e d f o r 10 m i n a t 60°C w i t h one h o u r o f h o m o g e n i z a t i o n . C y s t e i n e was a d d e d t o t h e NaOH s o l u t i o n s l i g h t l y b e f o r e u s e . U s u a l l y 10-25 y l - 61 -of the e x t r a c t a f t e r 1:1000 d i l u t i o n o f the t i s s u e was used f o r each assay. (b) E x t r a c t i o n o f NAD + At the end of i n c u b a t i o n , the t i s s u e was homogenized, i n a t l e a s t 1:50 d i l u t i o n , i n a mixture o f 0.01 M H 2 S 0 4 and 0.1 M N a 2 S 0 4 w i t h subsequent h e a t i n g f o r 45 min a t 6 0°C. T h i s reduces NADase a c t i v i t y t o a p o i n t a t which i t i s o r d i n a r i l y not d i s t u r b i n g although some a c t i v i t y may 26 3 p e r s i s t . U s u a l l y 15 y l of the e x t r a c t a f t e r 1:1000 d i l u t i o n was used f o r each assay. (c) Assay of NAD + and NADH To a 200 yl o f the c y c l i n g mixture, c o n t a i n i n g 0.2 M t r i s HC1 pH 8.4, 100 mM L i - l a c t a t e , 0.3 mM ADP, 5 mM a-KG, 0.05 M NH^Ac, 400 yg/ml bovine l i v e r L - g l u t a m i c dehydro-genase and 45 yg/ml beef h e a r t LDH i n a Bausch and Lomb c o l o r i m e t e r tube, 0-30 y l of t i s s u e e x t r a c t was added; c y c l i n g was con t i n u e d f o r 1 hr a t 37°C. A f t e r c y c l i n g , the tubes were t r a n s f e r r e d t o a b o i l i n g water bath f o r 2 min. The tubes were then c o o l e d i n i c e and 200 y l of phosphate:LDH s o l u t i o n (0.65 M Na H 2P0 4, 0.15 M K 2HP0 4 and 1.5 ygi/ of r a b b i t muscle LDH) and 200 yg of NADH was added t o each tube. Subsequently the tubes were i n c u b a t e d f o r 15 min a t room temperature, c o o l e d i n i c e and then 50 y l of 5N HC1 was added. A f t e r a few min a t room temperature, 2 ml of 6 N NaOH was added w i t h immediate mixing. The tubes were then heated a t 60°C f o r 10 min, brought a t room temperature and the f l u o r e s c e n c e was read i n an Aminco-- 62 -Bowman s p e c t r o p h o t o f l u o r o m e t e r . A f t e r t h e a d d i t i o n o f NaOH, t h e t u b e s w e r e k e p t i n d a r k t i l l r e a d i n g s w e r e t a k e n , s i n c e t h e f l u o r e s c e n t f o r m o f NAD + i s s e n s i t i v e t o d e s t r u c t i o n by l i g h t . As p y r u v a t e f o r m a t i o n d u r i n g c y c l i n g i s n o t s t r i c t l y l i n e a r w i t h NAD + o r NADH c o n c e n t r a t i o n s , s t a n d a r d s w e r e i n c l u d e d w i t h e a c h s e t o f d e t e r m i n a t i o n s . 2.14 MEASUREMENT OF CAMP PRODUCTION 264 The m e t h o d o f S h i m i z u , D a l y a n d C r e v e l i n g was u s e d t o m e a s u r e t h e p r o d u c t i o n o f cAMP i n t h e i n c u b a t e d c e r e b r a l c o r t e x s l i c e s . C e r e b r a l c o r t e x s l i c e s w e r e i n c u b a t e d f o r 40 m i n i n a W a r b u r g v e s s e l , u n d e r an a t m o s p h e r e o f O^CO,, i n a K r e b s -14 R i n g e r b i c a r b o n a t e medium c o n t a i n i n g 2 yC o f a d e n i n e - 8 - C s u l f a t e a n d 2 0 mM g l u c o s e . A f t e r a e r o b i c i n c u b a t i o n , t h e s l i c e s w e r e w a s h e d i n a c o l d medium, t r a n s f e r r e d t o a n o t h e r W a r b u r g v e s s e l c o n t a i n i n g d r u g s , a n d w e r e i n c u b a t e d f o r a d e s i r e d p e r i o d , a s d e s c r i b e d w i t h t h e r e s u l t s . A f t e r t h e i n c u b a t i o n , s l i c e s w e r e h o m o g e n i z e d i n 1.5 m l o f 5 p e r c e n t (w/v) i c e - c o l d TCA and c e n t r i f u g e d . The s u p e r n a t a n t was r e m o v e d t o a n o t h e r t u b e a n d 150 y g (100 y l ) o f c o l d cAMP was a d d e d . 50 y l o f t h i s s o l u t i o n was t a k e n f o r d e t e r m i n i n g t o t a l r a d i o a c t i v i t y w h i l e t h e r e s t o f i t was e x t r a c t e d t w i c e w i t h e t h e r t o remove TCA, e x c e s s o f e t h e r was r e m o v e d by b l o w i n g a i r a n d 100 y l was s p o t t e d on a P E I c e l l u l o s e p l a t e ( B r i n k m a n ) . The c h r o m a t o g r a m s w e r e 302 d e v e l o p e d i n 0.25 M L i C l , t h e s p o t s w e r e v i s u a l i z e d by - 63 -UV and the r a d i o a c t i v i t y of cAMP spots determined, a f t e r scrapping, i n 10 ml of s c i n t i l l a t i o n medium, by a Nuclear-Chicago Mark i l i q u i d s c i n t i l l a t i o n counter. The s c i n t i l -lant medium contained, i n 3 l i t r e s , equal volumes of toluene, dioxan and 95% (v/v) ethanol, 15 g 2,5-diphenyloxazole, 150 mg 1,4-bis-(4-methyl-5-phenoxaxol-2-yl) benzene and 240 g naphthalene. 2.15 MEASUREMENT OF ATP LEVELS At the end of the incubation, the s l i c e s were quickly homogenized i n 6% HCIO^. The homogenate was l e f t over i c e for 30 min. The supernatant obtained a f t e r centrifugation of the homogenate was neutralized with a precalculated volume of 5 M I^CO^, the KClO^ p r e c i p i t a t e was centrifuged off and ATP was assayed i n t h i s supernatant e i t h e r by the 265 spectrophotometric method of Lamprecht and Trautschold 266 or by the f l u o r i m e t r i c method of Greengard . Both of these methods u t i l i z e the hexokinase-glucose-6-phosphate dehydrogenase system coupled to NADP+ reduction i n the presence of glucose and other cofactors. The amount of NADP+ reduced i s proportional to the amount of ATP present. 2.16 MEASUREMENT OF N a 2 2 INFLUX 22 0.5 yC Na was tipped i n from the side arm of the Warburg vessel a f t e r a short period of preincubation, as indicated i n the experiments described below. After a desired period of incubation, the s l i c e s were removed and quickly washed twice, i n a non-radioactive incubation medium, and homogenized i n 2.5 ml of 5% (w/v) cold TCA. - 64 -A f t e r one h o u r i n t h e c o l d , t h e homogenate was c e n t r i f u g e d a nd 0.5 m l o f t h e s u p e r n a t a n t was p l a c e d i n 10 m l o f s c i n t -i l l a t i o n medium f o r c o u n t i n g r a d i o a c t i v i t y . A N u c l e a r C h i c a g o M a r k I l i q u i d s c i n t i l l a t i o n c o u n t e r was u s e d . 22 The r e s u l t s o f Na i n f l u x e x p e r i m e n t s a r e e x p r e s s e d a s Na , y e q u i v a l e n t / g j A i n i t i a l w e t w e i g h t , and w e r e c a l -22 c u l a t e d b y m u l t i p l y i n g t h e t i s s u e Na v o l u m e ^ cpm/g t i s s u e ^ , ^ N a + c o n t e n 4 - Qf 4 - n e i n c u b a t i o n c p m / y l o f medium 2 2 g y f l u i d ( y e q u i v / y l ) 2.17 MEASUREMENTS OF N a + AND K + The t i s s u e c o n t e n t s o f u n l a b e l l e d N a + a n d K + w e r e d e t e r m i n e d w i t h an a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e r . S a m p l e s f o r t h e s e a s s a y s w e r e p r e p a r e d a s d e s c r i b e d b e l o w : t h e t i s s u e s l i c e s w e r e r e m o v e d f r o m W a r b u r g v e s s e l s w i t h p o i n t e d f o r c e p s a n d t h e a d h e r i n g f l u i d was r e m o v e d w i t h f i l t e r p a p e r s t r i p s ; t h e s l i c e s w e r e h o m o g e n i z e d i n 3 m l o f 5% c o l d TCA and l e f t i n c o l d f o r an h o u r . The p r e c i p i t a t e was t h e n c e n t r i f u g e d o f f a n d t h e s u p e r n a t a n t s u i t a b l y d i l u t e d (2 mg o f t i s s u e / m l f o r K + and 0.2 mg o f t i s s u e / m l f o r N a + ) . The a t o m i c a b s o r p t i o n was m e a s u r e d a t 294.3 a n d 383.3 nm f o r N a + a n d K + , r e s p e c t i v e l y , w i t h a P e r k i n - E l m e r m o d e l 303 a t o m i c - a b s o r p t i o n s p e c t r o p h o t o m e t e r . The c o n t e n t s o f N a + and K + w e r e c a l c u l a t e d f r o m s t a n d a r d s r u n w i t h e a c h s e t o f d e t e r m i n a t i o n s . 2.18 MISCELLANEOUS METHODS (a) D e t e r m i n a t i o n o f P r o t e i n P r o t e i n was d e t e r m i n e d by t h e m e t h o d o f L o w r y , R o s e b r o u g h , 48 F a r r a n d R a n d a l l - 6 5 -(b) D e t e r m i n a t i o n of Water Uptake At the end of the i n c u b a t i o n , s l i c e s were removed from the medium and excess of f l u i d was completely removed w i t h f i l t e r paper s t r i p s t o u c h i n g the s l i c e s . The s l i c e s were then weighed on a t o r s i o n b a l a n c e ; the d i f f e r e n c e between the i n i t i a l weight and the f i n a l weight p r o v i d e d the measure of water uptake. (c) Use of Cyanide Whenever cyanide was used, i t s s o l u t i o n was n e u t r a l -i z e d w i t h HCl t o pH 7.0-7.5 b e f o r e use, care being taken not to l e t pH go below n e u t r a l i t y . 258 (d) Measurement of A c i d L a b i l e Phosphate The s l i c e s were homogenized q u i c k l y i n i c e - c o l d 5 p e r c e n t (w/v) TCA and the homogenate was l e f t i n i c e f o r 30 min f o r complete e x t r a c t i o n o f a c i d s o l u b l e m a t e r i a l . Adenine n u c l e o t i d e s were absorbed by treatment w i t h a p proximately 50 mg N o r i t A ( p u r i f i e d by s u c c e s s i v e treatments w i t h p y r i d i n e , HCl and d i s t i l l e d water) f o r 10 min a t 2 0°C a c c o r d i n g to the method of Crane and 259 Lipmann The l a b i l e phosphate of the n u c l e o t i d e f r a c t i o n i n the supernatant was assayed a f t e r treatment of the washed c h a r c o a l w i t h 4 ml of N HCl i n a b o i l i n g water bath and subsequent c o o l i n g and c e n t r i f u g i n g , by the 2 6 0 method o f B a r t l e t t (e) Measurement of Phosphoenol pyruvate (PEP) and Pyruvate PEP and pyruvate were determined by f l u o r i m e t r i c - 66 -a d a p t a t i o n o f t h e method o f Czok and E c k e r t 2.19 REPRODUCIBILITY OF THE RESULTS E a c h e x p e r i m e n t was c a r r i e d o u t s e v e r a l t i m e s and were r e p r o d u c i b l e . T h e r e were v a r i a t i o n s i n t h e a b s o l u t e v a l u e s f o u n d i n e x p e r i m e n t s c a r r i e d o u t on d i f f e r e n t d a y s and w i t h d i f f e r e n t b a t c h e s o f a n i m a l s . C o n t r o l e x p e r i m e n t s were, t h e r e f o r e , a l w a y s c a r r i e d o u t . The r e s u l t s g i v e n b e l o w a r e a v e r a g e s o f s e v e r a l v a l u e s o b t a i n e d i n t y p i c a l e x p e r i m e n t s . The d e v i a t i o n s f r o m t h e mean a r e g i v e n . CHAPTER 3 RATE LIMITING FACTORS IN ANAEROBIC GLYCOLYSIS OF CEREBRAL  CORTEX S L I C E S AND ACETONE POWDER EXTRACTS The r a t e l i m i t i n g f a c t o r s o p e r a t i n g i n a n a e r o b i c c e r e b r a l g l u c o s e m e t a b o l i s m , s u c h as NAD +, ATP, AMP, CAMP, c i t r a t e , g l u t a m a t e , a s p a r t a t e a n d c a t i o n s , w i l l now be c o n s i d e r e d . E x p e r i m e n t s were c a r r i e d o u t b o t h w i t h b r a i n s l i c e s and a c e t o n e powder e x t r a c t s o f b r a i n . 3.1 EFFECTS OF CALCIUM ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL  CORTEX S L I C E S The e f f e c t o f C a + + on c e r e b r a l g l y c o l y s i s h a s b e e n d i s c u s s e d i n C h a p t e r 1. R e s u l t s g i v e n i n F i g u r e 1 show t h a t t h e r a t e o f a n a e r o b i c g l y c o l y s i s d e c r e a s e s p r o g r e s s i v e l y ++ ++ w i t h t i m e . F u r t h e r m o r e , t h e a d d i t i o n o f 4 mM Ca t o a Ca - f r e e ' medium h a s a marked s t i m u l a t o r y e f f e c t on t h e r a t e o f a n a e r -o b i c g l y c o l y s i s o f g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . T h i s i s i n a g r e e m e n t w i t h e a r l i e r f i n d i n g s o f Q u a s t e l a n d c o -i 70,71 w o r k e r s The e f f e c t s o f t h e a d d i t i o n o f d i f f e r e n t c o n c e n t r a t i o n s ++ ++ o f Ca t o a Ca - f r e e medium, on a n a e r o b i c g l y c o l y s i s o f r a t o r g u i n e a p i g c e r e b r a l c o r t e x s l i c e s a r e g i v e n i n F i g u r e 2. I t i s c l e a r f r o m t h e s e r e s u l t s t h a t C a + + h a s a g r e a t e r s t i m u l a t o r y e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f g u i n e a p i g c e r e b r a l c o r t e x s l i c e s t h a n on t h a t o f r a t . W i t h g u i n e a p i g b r a i n t h e p r e s e n c e o f e v e n 1 mM C a + + h a s a marked e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s w h e r e a s i n r a t i t - 68 -FIGURE 1 EFFECT OF CALCIUM ON THE ANAEROBIC GLYCOLYSIS OF GUINEA PIG CEREBRAL CORTEX SLICES 150, , Time,in minutes A l l v e s s e l s contained 20 mM gluco s e . A d d i t i o n s were made at zero time and l a c t a t e production was measured m^nometrically as giv<piji i n the m a t e r i a l s and methods.( O ) Ca - f r e e medium; ( @ ) 4 mM Ca added to a Ca - f r e e medium. - 69 -FIGURE 2 EFFECT OF DIFFERENT CONCENTRATION OF CALCIUM ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES 150 •H -P O 0 I I I t I 0 1 2 3 4 Calcium added,in mM In c u b a t i o n s were c a r r i e d out i n a Ca+± f r e e medium c o n t a i n i n g 20 mM g l u c o s e . A d d i t i o n s were made at zero time and l a c t a t e p r o d u c t i o n was measured m a n o m e t r i c a l l y as g i v e n i n the m a t e r i a l s and m e t h o d s . ( B ) Rat; (®)Guinea p i g . - 70 -i s l e s s e f f e c t i v e . The a c c e l e r a t i n g a c t i o n o f Ca on t h e a n a e r o b i c g l y c o l y s i s o f b r a i n s l i c e s i s i n c o n t r a s t t o t h a t 71 on o t h e r t i s s u e s , s u c h as tumour s l i c e s . T h i s e f f e c t o f C a + + i s t h e r e v e r s e o f t h a t on t h e a e r o b i c g l y c o l y s i s o f b r a i n s l i c e s . A d d i t i o n o f C a + + t o a C a + + - f r e e medium r e d u c e s t h e r a t e o f a e r o b i c g l y c o l y s i s i n i n c u b a t e d r a t o r g u i n e a p i g b r a i n s l i c e s . The r e s u l t s o f t h e a d d i t i o n o f d i f f e r e n t c o n c e n t r a t i o n s o f C a + + on t h e a n a e r o b i c g l y c o l y s i s o f immature r a t , and n e w l y b o r n g u i n e a p i g c e r e b r a l c o r t e x s l i c e s a r e shown i n T a b l e 1. R e s u l t s shown i n T a b l e 1 show t h a t t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f 1-3 day o l d r a t b r a i n i s p r a c t i c a l l y i n s e n s i t i v e t o t h e a d d i t i o n o f d i f f e r e n t c o n c e n t r a t i o n s up t o 4 mM o f ++ ++ Ca . Some s t i m u l a t i o n by Ca o c c u r s i n 14-day o l d r a t b r a i n . W i t h n e w l y b o r n g u i n e a p i g c e r e b r a l c o r t e x s l i c e s , t h e p r e s e n c e o f C a + + , however, has a v e r y s t r i k i n g a c c e l e r a t i n g e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s . S t i m u l a t i o n a t 2 mM C a + + i s more t h a n 300%, w i t h v a l u e s a p p r o a c h i n g t h o s e o b t a i n e d f o r t h e a d u l t g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . T h i s f i n d i n g i s c o n s i s t e n t w i t h o u r k n o w l e d g e t h a t t h e b r a i n o f a n e w l y b o r n g u i n e a p i g i s more o r l e s s l i k e a m a t u r e b r a i n . I t i s w e l l known t h a t i n f a n t r a t b r a i n i s v e r y immature i n i t s n e u r o p h y s i o l o g i c a l c h a r a c t e r compared w i t h t h e a d u l t r a t b r a i n , and i t w o u l d be e x p e c t e d t h a t m a j o r d i f f e r e n c e s i n m e t a b o l i s m w o u l d e x i s t between i n f a n t and a d u l t b r a i n . T h i s w i l l be f u r t h e r d i s c u s s e d i n C h a p t e r 8. 71 TABLE 1 EFFECT OF CALCIUM ON THE ANAEROBIC GLYCOLYSIS OF INFANT RAT AND GUINEA P I G CEREBRAL CORTEX S L I C E S A d d i t i o n s L a c t a t e p r o d u c e d , y m o l e s p e r g i n i t i a l w e t wt (20-80 min) 2-day o l d r a t 7-day o l d 1 4 - d a y o l d N e w l y b o r n r a t r a t g u x n e a p i g C o n t r o l C a + + , ImM C a + + , 2mM C a + + , 4mM 24.6 ± 1 .8 22 .8 ± 1.4 24 .5 ± 1.4 32.2 ± 4 .5 2 4 . 1 + 1.4 24 .5 ± 3 .5 30 .5 ± 5 . 3 92 .5 ±12.5 32.2 + 4 .5 25.0 ± 2 .7 42.0 ± 5 .8 137.5 ± 6 .7 28.6 ± 1.7 29 .2 ± 5 .3 33 .5 ± 3 . 5 117.5 ± 4 . 1 I n c u b a t i o n s w e r e c a r r i e d o u t i n a Ca f r e e medium c o n t a i n i n g 20mM g l u c o s e . A d d i t i o n s w e r e made a t z e r o t i m e a n d l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i -c a l l y a s g i v e n i n t h e m a t e r i a l s a n d m e t h o d s . - 72 -3.2 EFFECTS OF EXTERNALLY ADDED NAD+ AND ATP ON THE ANAEROBIC  GLYCOLYSIS OF CEREBRAL CORTEX S L I C E S V a r i o u s c o e n z y m e s , i . e . NAD+, NADH, ATP a n d ADP, a r e n e e d e d f o r t h e o p e r a t i o n o f v a r i o u s s t e p s o f t h e g l y c o l y t i c p a t h w a y . The e s t a b l i s h e d f a c t t h a t t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f i n c u b a t e d b r a i n s l i c e s d e c r e a s e s w i t h t i m e may be due t o t h e l o s s o f s u c h c o e n z y m e s w i t h t i m e o f i n c u b a t i o n o r t o t h e l o s s o r g a i n o f c a t i o n s w h i c h a l s o e x e r t r a t e l i m i t i n g e f f e c t s . The ATP l e v e l i n t h e b r a i n f a l l s r a p i d l y d u r i n g a n o x i a a n d may become a r a t e l i m i t i n g f a c t o r f o r t h e p h o s p h o r y -l a t i o n o f g l u c o s e a n d f r u c t o s e 6 - p h o s p h a t e . M o r e o v e r , 13 + M c l l w a m h a s shown t h a t t h e l e v e l o f NAD + NADH d i m i n i s h e s d u r i n g a n o x i a b u t s t a y s c o n s t a n t u n d e r a e r o b i c c o n d i t i o n s . I n v i e w o f t h e s e f a c t s , t h e e f f e c t o f a d d i t i o n o f NAD + a n d ATP t o t h e i n c u b a t i o n medium, on t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s was s t u d i e d . The e x p e r i m e n t s w e r e c a r r i e d o u t b o t h i n t h e p r e s e n c e a s w e l l a s i n t h e a b s e n c e o f C a + + . R e s u l t s shown i n T a b l e 2 d e m o n s t r a t e t h a t t h e p r e s e n c e o f 0.5 mM NAD + h a s a m a r k e d s t i m u l a t i n g e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f b o t h r a t a n d g u i n e a p i g c e r e b r a l c o r t e x s l i c e s , w h i l e t h e a d d i t i o n o f 2.5 mM ATP h a s l i t t l e o r no e f f e c t . ATP + C a + + , h o w e v e r , t o g e t h e r h a v e a m a r k e d i n h i b i t o r y e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f b o t h r a t a n d g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . 73 TABLE 2 EFFECT OF EXOGENOUS NUCLEOTIDES ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SL I C E S A d d i t i o n s L a c t a t e p r o d u c e d y m o l e s p e r g i n i t i a l wet wt (30-90 min) R a t G u i n e a p i g C o n t r o l ATP, 2.5mM ATP, 2.5mM + C a + + , ImM NAD +, 0.5mM NAD +, 0.5mM + C a + + , ImM 26.4 27.0 1 2 . 1 6 2 . 7 47. 8 45.5 56 .0 36.8 60.0 57.0 I n c u b a t i o n s were c a r r i e d o u t i n a Ca - f r e e medium c o n t a i n i n g 20mM g l u c o s e . A d d i t i o n s were made a t 30 m i n and l a c t a t e p r o d u c t i o n was me a s u r e d e n z y m a t i -c a l l y . R e s u l t s a r e mean o f 2-4 d e t e r m i n a t i o n s w i t h v a l u e s w i t h i n ± 7%. - 74 -3.3 MOVEMENT OF NAD ACROSS THE C E L L MEMBRANE R e s u l t s r e p o r t e d i n C h a p t e r 3.2 show t h a t N A D + c o n c e n t r a t i o n , i n t h e c e r e b r a l c o r t e x s l i c e s , may i n f a c t e x e r t a l i m i t i n g e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o -l y s i s . I t a l s o a p p e a r s t h a t NAD , a d d e d t o t h e i n c u b a t i o n medium, may p e n e t r a t e t h e b r a i n c e l l . A s i t i s b e l i e v e d t h a t n u c l e o t i d e s a r e u n a b l e t o c r o s s t h e c e l l membrane b a r r i e r , e x p e r i m e n t s w e r e c a r r i e d o u t t o o b s e r v e w h e t h e r t h e t r a n s p o r t o f NAD+ i n t o t h e r a t c e r e b r a l c o r t e x s l i c e s c a n t a k e p l a c e u n d e r a n a e r o b i c as w e l l a s a e r o b i c c o n d i t i o n s . R e s u l t s o f t h e s e e x p e r i m e n t s a r e shown i n F i g u r e s 3 a n d 4. The e f f e c t s o f a d d e d 0.5 mM NAD +, on t h e t o t a l N A D + a s w e l l a s NADH c o n t e n t o f t h e r a t c e r e b r a l c o r t e x s l i c e s i n c u b a t e d u n d e r a n a e r o b i c c o n d i t i o n s , a r e shown i n F i g u r e 3. I t c a n be s e e n t h a t , i n b r a i n s l i c e s , i n c u b a t e d i n a b s e n c e o f a d d e d N AD +, t h e r e i s a m a r k e d d e c r e a s e i n t h e N A D + c o n t e n t d u r i n g f i r s t 2 0 m i n o f a n a e r o b i o s i s ; d u r i n g t h e l a t e r p e r i o d i t r e m a i n s r e l a t i v e l y c o n s t a n t . On t h e o t h e r h a n d , t h e NADH c o n t e n t d e c r e a s e s s l i g h t l y b u t s t e a d i l y d u r i n g a n o x i a . I n t h e p r e s e n c e o f 0.5 mM N A D + i n t h e i n c u b a t i o n medium, t h e N A D + c o n c e n t r a t i o n (measured a s t o t a l N AD + p r e s e n t i n t h e s l i c e ) , r e m a i n s a t a r e l a t i v e l y h i g h l e v e l a n d shows o n l y a s l o w d e c l i n e . The NADH l e v e l h o w e v e r r i s e s d u r i n g t h e l a t e r p e r i o d o f i n c u b a t i o n . I t c a n n o t be a s c e r t a i n e d f r o m t h e s e r e s u l t s - 75 -FIGURE 3 EFFECT OF EXOGENOUS NAD+ON THE NAD+ AND NADH CONCENTRATIONS OF RAT CEREBRAL CORTEX SLICES UNDER ANOXIA 200 •H 4J •i-i C •H &> U Q) Ch C n 3 -Q < M O Q 2 160 120 _ 10 20 30 40 50 60 Incubation time,in minutes Incubations were c a r r i e d ou£ under N2~eo_ i n a Ca -free medium containing 20mM glucose.NAD ,when present i n the incubation medium,was added at zero t i m e . ( » ) N A D cone,0.5 mM+NAD was added to the medium;( n ) N A D cone.,no exogenous N A D was added to the medium;( * ) N A D H cone, 0.5 mM N A D was added to the medium; ( O ) N A D H cone,no exogenous N A D was added to the medium. FIGURE 4 EFFECT OF EXOGENOUS NAD + ON THE NAD + AND NADH CONCENTRATIONS OF AE ROBICALLY INCUBATED CEREBRAL CORTEX S L I C E S FROM RAT 240 O I n c u b a t i o n t i m e , i n m i n u t e s I n c u b a t i o n s were c a r r i e d o u £ u n d e r 0 2 : C O „ i n a Ca - f r e e medium c o n t a i n i n g 20mM g l u c o s e . N A D ,when present i n t h e i n c u b a t i o n + medium,was added a t z e r o t i m e . ( +© ) NA^ c o n e , n o e x o g e n o u s NAD was a d d e d t o t h e medium; ( O ) NAD c o n e , 0.5 mM NAD was adde d t o t h e medium;(m )NADH c o n e , 0.5 mM NAD was a d d e d t o t h e medium; ( O ) NADH c o n e , no e x o g e n o u s NAD was added t o t h e medium. - 77 -whether some NAD i s e x t e r n a l l y bound t o the c e r e b r a l c o r t e x s l i c e s o r whether i t i s wholly i n t r a c e l l u l a r 268 (an ATP b i n d i n g p r o t e i n has been i s o l a t e d from b r a i n ). The f a c t s t h a t t h e r e i s a r i s e i n the NADH l e v e l as w e l l as an i n c r e a s e d r a t e of anaerobic g l y c o l y s i s show t h a t some NAD + must e n t e r the c e l l under a n a e r o b i c c o n d i t i o n s . During a e r o b i c c o n d i t i o n s i n the absence o f added NAD + ( F i g u r e 4 ) , the NAD + c o n t e n t of the t i s s u e f a l l s i n the f i r s t 20 min of i n c u b a t i o n . I t then i n c r e a s e s s l o w l y w i t h concomitant decrease i n NADH. In the presence of 0.5 mM exogenous NAD +, the c e r e b r a l l e v e l o f NAD + i s h i g h e r than those o f the c o n t r o l s , w h i l e the NADH l e v e l i s s l i g h t l y lower d u r i n g the i n i t i a l p e r i o d . 26 9 I t has been shown by Weidemann, Hems and Krebs t h a t e x t e r n a l l y added AMP and ATP a f f e c t s the metabolism of r a t kidney. Presumably these n u c l e o t i d e s are a b l e t o pe n e t r a t e the membrane b a r r i e r . P e r m e a b i l i t y of b r a i n c e l l membrane t o ATP has not been i n v e s t i g a t e d by the p r e s e n t author, as s u b s t a n t i a l amounts o f ATP b i n d t o 26 8 the b r a i n membrane p r o t e i n s . Moreover, washing the s l i c e s t o remove the bound ATP may r e s u l t i n a diminution of the content of endogenous ATP and g i v e lower observed v a l u e s . 3.4 EFFECTS OF NAD + ON AEROBIC GLYCOLYSIS I t has been shown i n Chapter 3. 2 t h a t exogenous NAD + s t i m u l a t e s the r a t e of ana e r o b i c g l y c o l y s i s o f - 78 -c e r e b r a l c o r t e x s l i c e s . We a l s o s t u d i e d the e f f e c t of NAD + on the r a t e of a e r o b i c g l y c o l y s i s i n i n c u b a t e d b r a i n s l i c e s . I t i s known t h a t the r a t e o f a e r o b i c g l y c o l y s i s i s reduced under a e r o b i c c o n d i t i o n s due t o o p e r a t i o n of the Pas t e u r e f f e c t (Chapter 1.2). However, i n the presence of r e s p i r a t o r y i n h i b i t o r s or u n c o u p l i n g agents such as 2 , 4 - d i n i t r o p h e n o l (DNP), the r a t e of a e r o b i c g l y c o l y s i s i s c o n s i d e r a b l y i n c r e a s e d . R e s u l t s g i v e n i n F i g u r e 5 show the e f f e c t s o f e x t e r n a l NAD + on DNP-stimulated a e r o b i c g l y c o l y s i s . I t can be seen t h a t , under these c o n d i t i o n s , NAD + has no s t i m u l a t i n g e f f e c t on the r a t e o f g l y c o l y s i s . 3.5 EFFECTS OF CITRATE AND AMP ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES C i t r a t e i s known t o e x e r t i n h i b i t o r y e f f e c t s on the g l y c o l y t i c p r o c e s s and i t has been r e p o r t e d t h a t i t markedly i n h i b i t s the a c t i v i t y of ph o s p h o f r u c t o k i n a s e 35 xn x s o l a t e d systems (Chapter 1.2). The e f f e c t o f 5 mM and 15 mM c i t r a t e on the r a t e o f ana e r o b i c g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s i s shown i n F i g u r e 6. The i n h i b i t i o n o f an a e r o b i c g l y c o l y s i s by c i t r a t e i s c o n s i s t e n t w i t h the known s u p p r e s s i v e a c t i v i t y of c i t r a t e on ph o s p h o f r u c t o k i n a s e a c t i v i t y . The e f f e c t o f AMP on the anaerobic g l y c o l y s i s of the r a t c e r e b r a l c o r t e x s l i c e s , as shown i n F i g u r e 6, i s of importance i n c o n n e c t i o n w i t h i t s s t i m u l a t i o n o f the - 79 -+ FIGURE 5 EFFECT OF NAD IN THE PRESENCE OF VARYING CONCENTRATION OF 2,4-DINITROPHENOL ON THE AEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES 150 0.025 0.05 0.075 0.10 Concentration of 2,4-dinitrophenol,in mM Incubations were c a r r i e d out i n a Krebs-Ringer bicarbonate medium containing 20 mM glucose,under 0„:CO„.Additions were made at 30 min and lac t a t e produced was determined enzymatically at the end of 90 min incubation period.(®)NAD was not added to the medium;(n)0.5 mM NAD was added to the incubation medium. - 80 -FIGURE 6 EFFECTS OF CITRATE AND AMP RAT CEREBRAL ON THE ANAEROBIC GLYCOLYSIS OF CORTEX SLICES 4J 4-> 20 30 40 50 60 70 80 Time, i n minutes ++ Incubations were c a r r i e d out i n a Ca - f r e e medium c o n t a i n i n g 20 mM gl u c o s e . A d d i t i o n s were made at zero time and l a c t a t e p r oduction was measured manometrically,as given i n the m a t e r i a l s and methods.(O )control;(@)5mM c i t r a t e ; ( A ) 1 5 mM c i t r a t e and ( B ) 2 mM AMP. - 81 -3 6 a c t i v i t y of ph o s p h o f r u c t o k i n a s e . The da t a i n d i c a t e an i n h i b i t i o n o f the ana e r o b i c g l y c o l y s i s o f the c e r e b r a l c o r t e x s l i c e s . I t i s e v i d e n t t h a t AMP has o t h e r e f f e c t s t h a t mask i t s s t i m u l a t i o n of p h o s p h o f r u c t o k i n a s e . 3.6 EFFECT OF CYCLIC AMP AND DIBUTYRYL CYCLIC AMP ON THE ANAEROBIC GLYCOLYSIS OF THE CEREBRAL CORTEX SLICES C y c l i c AMP (cAMP) i s known to a f f e c t a number of enzyme systems i n mammalian t i s s u e s , b a c t e r i a 2 7 ^ as w e l l 271 as those i n p l a n t s . There i s evidence t h a t the a c t i o n o f a number of hormones might be mediated through cAMp'72, 273 cAMP i s an a c t i v a t o r o f phosphorylase and i t a l s o d i m i n i s h e s the i n h i b i t o r y e f f e c t o f ATP on pho s p h o f r u c t o -k i n a s e , c a u s i n g a s t i m u l a t i o n of p h o s p h o f r u c t o k i n a s e 35 a c t i v i t y . B r a i n has the h i g h e s t a b i l i t y among mammal-274 i a n t i s s u e s to s y n t h e s i s e cAMP . Si n c e cAMP i s known t o enhance the a c t i v i t y o f p h o s p h o f r u c t o k i n a s e , we s t u d i e d the e f f e c t s o f cAMP on the a n a e r o b i c g l y c o l y s i s of the guinea p i g c e r e b r a l c o r t e x s l i c e s and the r e s u l t s are shown i n F i g u r e 7. I t can be seen t h a t 1 mM cAMP enhances the r a t e o f an a e r o b i c g l y c o l y s i s of the c e r e b r a l c o r t e x s l i c e s . Under s i m i l a r c o n d i t i o n s , d i b u t y r y l cAMP, whose a c t i o n i s s i m i l a r to cAMP i n a number of systems, has no e f f e c t . 275 Dittmann and Herrmann have shown t h a t i n the presence 0.5-1.0 mM cAMP, the r a t e s o f a e r o b i c g l y c o l y s i s and r e s p i r a t i o n of r a b b i t c e r e b r a l c o r t e x s l i c e s are i n -cr e a s e d . - 82 r FIGURE 7 EFFECTS OF CYCLIC AMP AND DIBUTYRYL CYCLIC AMP ON THE ANAEROBIC GLYCOLYSIS OF GUINEA PIG CEREBRAL CORTEX S L I C E S "H 60 i ; ; , B o 0 0 Time , i n m i n u t e s I n c u b a t i o n s were c a r r i e d o u t i n a Ca - f r e e medium c o n t a i n i n g 20 mM g l u c o s e . A d d i t i o n s were made a t z e r o t i m e and l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y , a s g i v e n i n t h e m a t e r i a l s and m e t h o d s . ( O ) c o n t r o l ; ( • )ImM c y c l i c A M P ; ( ® ) 1 mM d i b u t y r y l c y c l i c AMP. - 83 -3.7 EFFECTS OF VARYING CATION CONCENTRATIONS ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES I t has been mentioned i n Chapter 1 t h a t c e r e b r a l metabolism i n v i t r o i s g r e a t l y i n f l u e n c e d by the c o n c e n t r a t i o n of c a t i o n s i n the i n c u b a t i o n medium. K + i s 36 301 r e q u i r e d f o r the a c t i v i t y of pyruvate k i n a s e ' . The a c t i v i t y o f pyruvate k i n a s e i n c r e a s e s g r e a t l y w i t h + 59 .increase i n K c o n c e n t r a t i o n up t o 50 mM but a h i g h c o n c e n t r a t i o n of K + has been shown t o have an i n h i b i t o r y e f f e c t on the an a e r o b i c g l y c o l y s i s of c e r e b r a l c o r t e x 6 2 s l i c e s . However, i n the l a t t e r study, the concent-r a t i o n o f N a + i n the i n c u b a t i o n medium was not changed when the K + c o n t e n t was i n c r e a s e d . Sodium i o n s are 5 3a known to i n h i b i t the a c t i v i t y of hexokinase and pyruvate k i n a s e 3 7 ' 3 ^ ' 3 ^ ' 3 ^ 1 ^ Hence the e f f e c t s o f v a r y i n g c o n c e n t r a t i o n s of N a + and K + on the r a t e s o f anaerobic g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s were + + s t u d i e d . The e f f e c t o f r e p l a c i n g Na w i t h L i was a l s o s t u d i e d . The r e s u l t s o f these experiments are shown i n F i g u r e 8. The evidence i n d i c a t e s t h a t i n c r e a s i n g the concent-r a t i o n of K , w h i l e a t the same time d e c r e a s i n g the c o n c e n t r a t i o n o f Na +, has a marked s t i m u l a t i n g e f f e c t on the r a t e o f c e r e b r a l a n a e r o b i c g l y c o l y s i s . At ve r y h i g h concentrations o f K +, some i n h i b i t i o n was observed. N a + has marked i n h i b i t o r y e f f e c t on the g l y c o l y s i s of b r a i n s l i c e s . L i + a c t s i n a manner s i m i l a r t o Na +. These data - 84 -FIGURE 8 EFFECT OF VARYING CONCENTRATIONS OF K + , L i + AND Na+ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES 150 (0 "3 ui i i i i i 0 30 + 60 90 120 150 K + , L i or Na C o n e , i n mM Incubations were c a r r i e d out i n the presence of 20 mM g l u c o s e . The medium contained e i t h e r Na ,K++29 mM Na Li ++29 mM Na . When req u i r e d , t h e medium was made i s o t o n i c w i t h s u c r o s e . L a c t a t e production was measured m a n o m e t r i c a l l y f ( o )Na cone.; ( ®)K cone.+ 29 mM Na ; ( • ) L i cone. +29 mM Na . - 85 -show t h a t w h i l e c o n s i d e r i n g e f f e c t s o f a p a r t i c u l a r c a t i o n on c e r e b r a l a n a e r o b i c g l y c o l y s i s , the concent-r a t i o n o f o t h e r c a t i o n s p r e s e n t i n the i n c u b a t i o n medium must a l s o be taken i n t o account. 3.8 EFFECTS OF L-GLUTAMATE AND OF AMMONIUM IONS ON THE  ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES. 277 During h i s s t u d i e s on glutamine f o r m a t i o n , Krebs observed t h a t L-glutamate i n h i b i t s the a n a e r o b i c form-a t i o n o f l a c t i c a c i d . T h i s phenomenon was f u r t h e r 27 8 i n v e s t i g a t e d i n g r e a t e r d e t a i l by Weil-Malherbe who showed t h a t both D- and L-glutamate are a c t i v e i n i n h i b i t i n g a n a e r o b i c g l y c o l y s i s . T h i s e f f e c t i s r e v e r s e d on a d d i t i o n of py r u v a t e . Because o f the p o s s i b l e r o l e o f glutamate as an e x c i t a t o r y amino a c i d i n the c e n t r a l nervous system, and i t s p o s s i b l e e f f e c t s on c a t i o n movements, i t s e f f e c t on the r a t e of an a e r o b i c g l y c o l y s i s was r e i n v e s t i g a t e d . R e s u l t s , which are shown i n F i g u r e 9, demonstrate t h a t glutamate i n h i b i t s the r a t e of anaerobic g l y c o l y s i s of c e r e b r a l c o r t e x s l i c e s i n a Krebs-Ringer b i c a r b o n a t e ++ medium as w e l l as i n a Ca - f r e e medium. However, the e f f e c t of glutamate i n a Ca - f r e e medium i s l e s s than t h a t i n a C a + + - c o n t a i n i n g (Krebs-Ringer bicarbonate) medium. D-glutamate was a l s o e f f e c t i v e i n d i m i n i s h i n g the r a t e o f anae r o b i c g l y c o l y s i s but to a l e s s e r e x t e n t . 278 + Weil-Malherbe noted t h a t NH^ i n h i b i t s the an a e r o b i c g l y c o l y s i s o f guinea p i g c e r e b r a l c o r t e x s l i c e s . FIGURE 9 EFFECTS OF GLUTAMATE AND NH* ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX S L I C E S I n c u b a t i o n s were c a r r i e d o u t i n a medium c o n t a i n i n g 20 mM g l u c o s e . A d d i t i o n s were made a t z e r o t i m e and l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o p p t r i c a l l y as g i v e n i n t h e m a t e r i a l s and m e t h o d s . ( O ) C a - f r e e m e d i u m , c o n t r o l ; ( © ) K r e b s - R i n g e r b i c a r b o n a t e m e d i u m , c o n t r o l ; ( A ) K r e ^ - R i n g e r b i c a r b o n a t e medium,with 5 mM L - g l u t a m a t e ; ( A ) C a - f r e e m edium,with 5 mM L - g l u t a m a t e ; ( B ) K r e ^ s - R i n g e r b i c a r b o n a t e m edium.with 5 mM D - g l u t a m a t e ; ( a ) C a - f r e e medium,with 5mM NH.. - 86 -- 87 -H o w e v e r , h i s r e s u l t s w e r e n o t c o n s i s t e n t ; i n some e x p e r i m e n t s .1-3 mM NH^"1" h a d l i t t l e o r no e f f e c t w h i l e , i n some e x p e r i m e n t s , c o n s i d e r a b l e i n h i b i t i o n o f a n a e r o b i c g l y c o l y s i s was o b s e r v e d a t 1 mM NH^ +. The e f f e c t s o f a d d i t i o n o f 5mM NH^"1" t o a C a + + - f r e e medium on c e r e b r a l a n a e r o b i c g l y c o l y s i s i s shown i n Figure 9 U n d e r t h e g i v e n e x p e r i m e n t a l c o n d i t i o n s , i t h a s l i t t l e o r no e f f e c t o n t h e r a t e o f a n a e r o b i c g l y c o l y s i s . I t w i l l be shown l a t e r , i n C h a p t e r 4, t h a t NH* h a s a m a r k e d e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s when t h e l a t t e r i s s t i m u l a t e d b y c e r t a i n d r u g s , s u c h a s t e t r o d o t o x i n . 3.9 EFFECT OF NAD + AND ATP, I N A SODIUM-FREE MEDIUM, ON THE  ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX S L I C E S The e f f e c t s o f NAD + a n d ATP on t h e a n a e r o b i c g l y c o -l y s i s o f c e r e b r a l c o r t e x s l i c e s i n a N a + - f r e e medium a r e shown i n T a b l e 3. I t c a n be s e e n t h a t t h e s e n u c l e o t i d e s h a v e no e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s i n a N a + - f r e e medium, e i t h e r i n t h e p r e s e n c e o r i n t h e a b s e n c e o f C a + + . F u r t h e r , t h e r a t e o f g l y c o l y s i s , i n c o n t r o l s l i c e s , i s i t s e l f g r e a t l y r e d u c e d as c o m p a r e d t o a medium c o n t a i n i n g n o r m a l c o n c e n t r a t i o n o f N a + a n d K + . Thus t h e a n a e r o b i c g l y c o l y s i s shows a m a r k e d d e p e n d e n c e on t h e p r e s e n c e o f c a t i o n s i n t h e i n c u b a t i o n medium. As h a s b e e n shown e a r l i e r ( C h a p t e r 3.7), r e p l a c i n g N a + by K + i n t h e i n c u b a t i o n medium h a s a s t i m u l a t o r y e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s . 88 TABLE 3 EFFECTS OF CALCIUM AND NAD + ON THE ANAEROBIC GLYCOLYSIS OF GUINEA PIG CEREBRAL CORTEX SLICES IN A SODIUM FREE MEDIUM In c u b a t i o n L a c t a t e produced, A d d i t i o n p e r i o d ymoles per g i n i t i a l wet wt C o n t r o l 0-30 min 18.1 C o n t r o l 0-90 min 20.8 C a + + , ImM 0-90 min 23.0 C a + + , 4mM 0-90 min 20 .2 NAD +, 0.5mM 0-90 min 24.5 NAD +, 0.5mM + C a + + , ImM 0-90 min 23.5 I n c u b a t i o n s were c a r r i e d out i n a medium c o n t a i n i n g 260mM s u c r o s e , 6mM K C l , 20mM gl u c o s e and lOmM T r i s - H C l b u f f e r (pH 7.4). L a c t a t e p r o d u c t i o n was measured e n z y m a t i c a l l y . A d d i t i o n s were made a t 30 min. R e s u l t s are averages of 2-4 d e t e r m i n a t i o n s with v a l u e s w i t h i n ± 7%. - 89 -3.10 RATE L I M I T I N G FACTORS OF GLYCOLYSIS IN THE ACETONE POWDER EXTRACTS To m a i n t a i n o p t i m a l r a t e s o f g l y c o l y s i s b y a c e t o n e p o w d e r e x t r a c t s , o r g r o u n d c e r e b r a l t i s s u e s , i t i s n e c e s s a r y t o a d d v a r i o u s c o e n z y m e s a n d c o - f a c t o r s t o t h e s u s p e n s i o n s . + + T h x s i s n e c e s s a r y b e c a u s e NAD , ATP, K and p h o s p h a t e a r e l o s t q u i c k l y f r o m t h e t i s s u e d u r i n g p r e p a r a t i o n o f t h e b r a i n a c e t o n e p o w d e r . A l t h o u g h A T P a s e i s v e r y l o w i n t h e 279 28 0 a c e t o n e p o w d e r s , t h e r e i s a h i g h NADase a c t i v i t y i n t h e b r a i n t i s s u e w h i c h p e r s i s t s e v e n i n t h e a c e t o n e p o w d e r s . F o r t h i s r e a s o n , i t i s n e c e s s a r y t o a d d n i c o t i n a m i d e , w h i c h 28 0 i s a s t r o n g i n h i b i t o r o f NADase , i n t h e e x t r a c t s . R e s u l t s g i v e n i n F i g u r e 10 show t h e t i m e c o u r s e o f g l y c o l y s i s b y a c e t o n e p o w d e r e x t r a c t s f o r t i f i e d w i t h N A D +, ATP, M g + + a n d K + . U n d e r t h e g i v e n e x p e r i m e n t a l c o n d i t i o n s , t h e r a t e o f a n a e r o b i c g l y c o l y s i s by t h e e x t r a c t s i s a p p r o x -i m a t e l y c o n s t a n t a n d i t i s p r o p o r t i o n a l t o t h e q u a n t i t y o f e x t r a c t t a k e n . M o r e o v e r , t h e m a g n i t u d e o f t h e r a t e o f g l y c o l y s i s by t h e a c e t o n e p o w d e r e x t r a c t s i s much h i g h e r t h a n t h a t o f a c o r r e s p o n d i n g q u a n t i t y o f t h e b r a i n s l i c e s . T h i s w i l l be f u r t h e r d i s c u s s e d i n C h a p t e r 8. The e f f e c t s o f c h a n g i n g c o n c e n t r a t i o n o f t h e d i f f e r e n t c o f a c t o r s a n d s u c h r a t e l i m i t i n g c o m p o n e n e t s a s K + , N a + , N A D + a n d ATP a r e shown i n F i g u r e 11. When t h e c o n c e n t r a t i o n o f ATP, o r NAD + i s i n c r e a s e d , w i t h o u t c h a n g e o f t h e N a + c o n c e n t r a t i o n , t h e r a t e o f g l y c o l y s i s i s i n c r e a s e d . - 90 -FIGURE 10 ANAEROBIC GLYCOLYSIS BY ACETONE POWDER EXTRACTS OF RAT BRAIN 12.5 Time, i n m i n u t e s I n c ; u b a t i o n | were c a r r i e d o u t i n a medium c o n t a i n i n g 52 mM Na ,28 mMK+,4 mM Mg ,33 mM c y s t e i n e , 3 3 mM n i c o t i n a m i d e , 0.5 mM NAD and 0.8 mM A T P . L a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y . A l l t h e v e s s e l s c o n t a i n e d 20 mM g l u c o s e . ( O ) 2 0 mg a c e t o n e powder e x t r a c t p e r v e s s e l ; ( © ) 4 0 mg a c e t o n e powder e x t r a c t p e r v e s s e l . - 91 -I n c r e a s e o f N a + has an i n h i b i t o r y e f f e c t and i n c r e a s i n g K + c o n c e n t r a t i o n has a s l i g h t s t i m u l a t i n g e f f e c t . The e f f e c t o f i n c r e a s e d K + i s more marked when t h e c o n c e n t r a t i o n o f N a + i s low. T h e s e e x p e r i m e n t s d e m o n s t r a t e t h a t d i f f e r e n t c o f a c t o r s may e x e r t r a t e l i m i t i n g e f f e c t s on t h e s p e e d o f a n a e r o b i c g l y c o l y s i s by t h e a c e t o n e powder e x t r a c t s . SUMMARY OF CHAPTER 3 R e s u l t s g i v e n i n t h i s c h a p t e r d e s c r i b e t h e a c t i o n o f v a r i o u s compounds t h a t have r a t e l i m i t i n g e f f e c t s on t h e p r o c e s s o f g l y c o l y s i s on t h e r a t e s o f a n a e r o b i c g l y c o l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s a nd o f e x t r a c t s o f a c e t o n e powder o f b r a i n . (1) I n c o n f i r m a t i o n o f t h e e a r l i e r work o f Q u a s t e l and h i s c o w o r k e r s ' ^ ' 7 1 , i t i s f o u n d t h a t C a + + h a s a ma r k e d s t i m u l a t o r y e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f a d u l t r a t a n d g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . (2) C a + + has l i t t l e e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f i n f a n t r a t b r a i n , w h e r e a s t h e marked r e s p o n s e o f i n f a n t g u i n e a p i g b r a i n t o C a + + i s s i m i l a r t o t h a t o f t h e a d u l t b r a i n . (3) E x t e r n a l l y added NAD + s t i m u l a t e s t h e r a t e o f c e r e b r a l a n a e r o b i c g l y c o l y s i s w h i l e a d d i t o n o f ATP has l i t t l e o r no e f f e c t i n a C a + + - f r e e i n c u b a t i o n medium. The a d d i t i o n o f ATP i n p r e s e n c e o f C a + + i n h i b i t s t h e r a t e o f a n a e r o b i c g l y c o l y s i s . I t i s t h o u g h t t h a t t h i s i s due t o ATP c h e l a t i o n o f C a + + . (4) Movement o f NAD + a c r o s s t h e b r a i n c e l l membrane h a s been s t u d i e d . I t a p p e a r s t h a t NAD + c r o s s t h e b r a i n c e l l - 92 -FIGURE 11 EFFECT OF VARYING CONCENTRATIONS OF RATE LIMITING FACTORS ON THE ANAEROBIC GLYCOLYSIS OF BRAIN ACETONE POWDER EXTRACTS 1500 "g 1250 in i L D U 1 o 04 1000 c o •p o (0 cn 750 u w O rH O 500 n O •O O U Ch <D +J nJ -P O 250 ATP,0.8 mM;NAD+,0.5 mM;Na+,52 mM. ATP, 0.8 mM;NAD+,0.5mM;Na+,106 mM. ATP,0.8,mM;NAD ,0.2 mM, Na ,52 mM. ATP.0.4 mM;Na+,52 mM;NAD+,0.5 mM 10 20 30 40 50 60 K c o n c e n t r a t i o n , i n mM Incu b a t i o n s were c a r r i e d o u t + a s c j i v e n + w i t h F i g u r e 10 except t h a t the c o n c e n t r a t i o n s o f K , Na, NAD and ATP were v a r i e d . - 93 -membrane under both a n a e r o b i c and a e r o b i c c o n d i t i o n s . (5) NAD + has no e f f e c t on the a e r o b i c g l y c o l y s i s t h a t has been s t i m u l a t e d by 2 , 4 - d i n i t r o p h e n o l . (6) C i t r a t e and AMP are i n h i b i t o r s o f the r a t e of anae r o b i c g l y c o l y s i s of c e r e b r a l c o r t e x s l i c e s . C y c l i c AMP has a s t i m u l a t o r y e f f e c t . (7) I t i s known t h a t a hig h c o n c e n t r a t i o n (100 mM) of K +, when added t o a Ringer medium, i n h i b i t s the r a t e o f c e r e b r a l anaerobic g l y c o l y s i s . I t i s shown t h a t when the c o n c e n t r a t i o n o f N a + i n the i n c u b a t i o n medium i s decreased a t the time when K + i s r a i s e d , K + has a marked s t i m u l a t o r y a c t i o n on the r a t e o f ana e r o b i c g l y c o l y s i s . (8) The e f f e c t s o f N a + and K + are a l s o apparent d u r i n g the g l y c o l y s i s brought about by the acetone powder e x t r a c t s of b r a i n . The e f f e c t s of these i o n s are not as l a r g e as those shown i n an a e r o b i c g l y c o l y s i s by b r a i n t i s s u e s l i c e s . (9) W h i l s t i t i s confirmed t h a t L-glutamate i s an i n h i b i t o r o f the r a t e of ana e r o b i c g l y c o l y s i s o f the c e r e b r a l c o r t e x s l i c e s , NH^ "1" a t e q u i v a l e n t c o n c e n t r a t i o n s has no e f f e c t . (10) I t i s e v i d e n t t h a t a v a r i e t y o f f a c t o r s e x e r t r a t e l i m i t i n g e f f e c t s on ana e r o b i c g l y c o l y s i s of c e r e b r a l c o r t e x s l i c e s . In p a r t i c u l a r , a t t e n t i o n i s drawn t o the marked + + ++ e f f e c t s of Na , K and Ca CHAPTER 4 EFFECTS OF TETRODOTOXIN ON CEREBRAL METABOLISM AND TRANSPORT IN ANOXIA I t h a s b e e n m e n t i o n e d i n C h a p t e r 1 t h a t TTX, t h e p u f f e r f i s h n e u r o t o x i n , i n low c o n c e n t r a t i o n s , s u p p r e s s e s t h e g e n e r a t i o n o f a c t i o n p o t e n t i a l s i n a v a r i e t y o f e x c i t -a b l e t i s s u e s . I t h a s a l s o b e e n shown t h a t TTX b l o c k s t h e i n c r e a s e i n r e s p i r a t i o n b r o u g h t a b o u t by a p p l i c a t i o n o f e l e c t r i c i m p u l s e s as w e l l as by o m i s s i o n o f C a + + f r o m t h e i n c u b a t i o n m e d i u m 1 3 ^ . M o r e o v e r , C a + + i n c r e a s e s t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f g u i n e a p i g , as w e l l a s o f r a t c e r e b r a l c o r t e x s l i c e s ( C h a p t e r 3 ) . I n v i e w o f t h e p o s s i b i l i t y t h a t C a + + and TTX m i g h t a c t s i m i l a r l y on t h e b r a i n t i s s u e , t h e e f f e c t o f TTX on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s was i n v e s t i g a t e d . I n a d d i t i o n , k i d n e y m e d u l l a and a c e t o n e powder e x t r a c t s o f b r a i n h a v e b e e n u s e d t o a s c e r t a i n w h e t h e r t h e e f f e c t o f TTX i s a s p e c i f i c p r o p e r t y o f e x c i t a b l e t i s s u e and i f t h e i n t e g r i t y o f t h e b r a i n c e l l membrane i s a l s o r e q u i r e d f o r a c t i o n . R e s u l t s o f e x p e r i m e n t s c a r r i e d o u t , t o t h r o w l i g h t on t h e s e p r o b l e m s , w i l l be d e s c r i b e d b e l o w . 4.1 EF F E C T S OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF GUINEA PIG AND RAT CEREBRAL CORTEX S L I C E S R e s u l t s o f a t y p i c a l e x p e r i m e n t , s h o w i n g t h e e f f e c t o f 2 yM TTX on t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l - 95 -c o r t e x s l i c e s i n a Ca - f r e e , as w e l l as i n a K r e b s - R i n g e r b i c a r b o n a t e medium, a r e g i v e n i n F i g u r e 12. I t c a n be s e e n t h a t TTX i n c r e a s e s t h e r a t e o f a n a e r o b i c g l y c o l y s i s i n b o t h t h e m e d i a . However, t h e p e r c e n t a g e i n c r e a s e i s g r e a t e r i n t h e ++ ++ Ca - f r e e medium t h a n m t h a t c o n t a i n i n g Ca . T h i s i s p a r t l y due t o t h e f a c t t h a t t h e c o n t r o l v a l u e s o f g l y c o l y s i s ( w i t h o u t TTX) a r e g r e a t e r i n a K r e b s - R i n g e r b i c a r b o n a t e medium t h a n i n t h e C a + + - f r e e medium ( F i g u r e 1 2 ) . The same e f f e c t s o f TTX on a n a e r o b i c g l y c o l y s i s a r e o b s e r v e d w i t h g u i n e a p i g c e r e b r a l c o r -t e x s l i c e s . The e f f e c t s o f t h r e e d i f f e r e n t c o n c e n t r a t i o n s o f TTX on t h e r a t e s o f a n a e r o b i c g l y c o l y s i s o f g u i n e a p i g and r a t c e r e -b r a l c o r t e x s l i c e s a r e shown i n F i g u r e 13. I t i s e v i d e n t t h a t t h e r a t e o f a n a e r o b i c g l y c o l y s i s i n c r e a s e s w i t h i n c r e a s e d c o n -c e n t r a t i o n o f TTX i n b o t h t h e a n i m a l s , a l t h o u g h t h e r a t e o f g l y c o l y s i s i n t h e a b s e n c e o f TTX i s g r e a t e r i n g u i n e a p i g c e r -e b r a l c o r t e x s l i c e s t h a n i n r a t c o r t e x s l i c e s ; t h e maximum r a t e s o b t a i n e d f o r t h e two a n i m a l s , i n t h e p r e s e n c e o f TTX, a r e a b o u t t h e same. 4.2 EFFECTS OF CALCIUM ON TETRODOTOXIN STIMULATED  ANAEROBIC GLYCOLYSIS The e f f e c t s o f a d d i t i o n o f d i f f e r e n t c o n c e n t r a t i o n s o f c a l c i u m , t o a C a + + - f r e e medium i n t h e p r e s e n c e o f 2yM TTX, on t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l - 96 -FIGURE 12 EFFECTS OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SL I C E S 80 . 1 T i m e , i n m i n u t e s I n c u b a t i o n s were c a r r i e d o u t i n t h e p r e s e n c e o f 20 mM g l u c o s e . T T X ( 2 uM),when p r e s e n t , w a s a d d e d a t z e r o t i m e and l a c t a t e p r o -d u c t i o n was m e a s u r e d m a n o m e ^ r i c a l l y a s g i v e n i n t h e m a t e r i a l s and m e t h o d s . ( O ) c o n t r o l , C a - f r e e m e d i u m ; ( A ) c o n t r o l , K r e b s - R i n g e r b i c a r b o n a t e m e d i u m ; ( ® ) 2 uM TTX p r e s e n t , C a - f r e e medium; ( S ) 2 uM TTX p r e s e n t , K r e b s - R i n g e r b i c a r b o n a t e medium. - 97 -FIGURE 13 EFFECTS OF DIFFERENT CONCENTRATIONS OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SL I C E S TTX c o n c e n t r a t i o n , i n uM ++ I n c u b a t i o n s were c a r r i e d o u t i n a C a " ' - f r e e medium c o n t a i n i n g 20 mM g l u c o s e . T T X was p r e s e n t f r o m t h e s t a r t o f t h e e x p e r i m e n t and l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y a s g i v e n i n t h e m a t e r i a l s and m e t h o d s . ( O ) G u i n e a p i g c e r e b r a l c o r t e x s l i c e s ; ( 9 ) r a t c e r e b r a l c o r t e x s l i c e s . - 98 -c o r t e x s l i c e s a r e shown i n F i g u r e 14. W i t h g u i n e a p i g s , ++ t h e p r e s e n c e o f Ca , u n d e r t h e s e c o n d i t i o n s , c a u s e s f u r t h e r i n c r e a s e i n t h e r a t e o f a n a e r o b i c g l y c o l y s i s w i t h v a l u e s a p p r o a c h i n g t h a t o b t a i n e d w i t h 4 mM C a + + a l o n e . However, w i t h r a t s , no s u c h i n c r e a s e i s o b s e r v e d ; t h e r a t e s o f g l y c o l y s i s e i t h e r r e m a i n t h e same (as o b t a i n e d w i t h TTX a l o n e ) o r a r e s l i g h t l y d e c r e a s e d . U n d e r t h e same e x p e r i m e n t a l c o n d i t i o n s , t h e amount o f a c i d l a b i l e p h o s p h a t e was a l s o m e a s u r e d ( n o t shown), s i n c e t h i s g i v e s some i n d i c a t i o n o f t h e e n e r g y s t a t u s o f t h e c e l l . The v a l u e s o b t a i n e d i n t h e p r e s e n c e o f TTX were h i g h e r t h a n t h e c o r r e s p o n d i n g c o n t r o l s . ATP c o n c e n t -r a t i o n s i n t h e s e t i s s u e s were a l s o m e a s u r e d a n d t h e r e s u l t s w i l l be d i s c u s s e d i n C h a p t e r 5.6. 4.3 E F F E C T OF TETRODOTOXIN ON NAD + MOVEMENTS ACROSS THE  CELL MEMBRANE I t h a s b e e n shown i n C h a p t e r 3 t h a t t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s d e c r e a s e s w i t h t i m e o f i n c u b a t i o n . A t t h e same t i m e t h e r e i s d e c r e a s e i n t h e NAD + c o n c e n t r a t i o n u n d e r a n o x i a . As NAD + i s one o f t h e c o f a c t o r s r e q u i r e d f o r g l y c o l y s i s , i t seemed t o us t h a t t h e d e c l i n e o f t h e r a t e o f g l y c o l y s i s m i g h t be due t o l o s s o f NAD + f r o m t h e t i s s u e i n t o t h e medium. I t seemed p o s s i b l e t h a t TTX m i g h t i n c r e a s e t h e r a t e o f g l y c o l y s i s by b l o c k i n g t h e e f f l u x o f NAD + d u r i n g t h e i n c u b a t i o n . A l t h o u g h t h e d e c l i n e o f NAD + l e v e l o b s e r v e d by us was o n l y 20% d u r i n g t h e - 99 -FIGURE 14 EFFECTS OF DIFFERENT CONCENTRATIONS OF CALCIUM ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES IN THE PRESENCE OF TETRODOTOXIN Ca c o n e . , i n mM ++ I n c u b a t i o n s were c a r r i e d o u t jj-iji a Ca - f r e e medium c o n t a i n i n g 2 0 mM g l u c o s e . 2 uM TTX and Ca ,when p r e s e n t , w e r e added a t z e r o t i m e a n d l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y as g i v e n i n t h e m a t e r i a l s and methods. - 100 -20-60 min incubation period, t h i s loss may s t i l l be s i g -n i f i c a n t , i f i t occurs only from the extramitochondrial compartment (cytoplasm) where g l y c o l y s i s i s taking place. However, i n the presence of 2 yM TTX, contrary to the above assumption, no increase i n the NAD+ l e v e l of the cerebral cortex s l i c e s was observed. We also assayed the NAD+ l e v e l i n the medium at the end of the incubation 2 61 ~ 2 6 3 H~ period, by the c y c l i n g method of Lowry , but no NAD could be detected i n the incubation medium (results not shown). The presence of NAD+ i n the incubation medium was 284 also tested by the method of Quastel and Wheatley but attempts to show i t s presence were not successful. However, these experiments led to the finding that some l a c t i c dehydrogenase appears i n the medium i n which the cerebral cortex s l i c e s are incubated. Stern, Eggleston, Hems and 2 85 Krebs have shown that glutamic-aspartic transaminase di f f u s e s from incubated guinea pig cortex s l i c e s i n t o the medium. I t thus appears that c e r t a i n enzymes may leak in t o the medium from incubated brain s l i c e s but i t i s uncertain whether t h i s i s simply due to the breakdown of damaged c e l l s . 4.4 EFFECTS OF TETRODOTOXIN ON THE AMINO ACID  EFFLUX FROM THE CEREBRAL CORTEX SLICES As has been shown i n Chapter 4.3, the p o s s i b i l i t y that the e f f e c t of TTX on the anaerobic g l y c o l y s i s might be due to - 101 -b l o c k i n g o f NAD e f f l u x h a s b e en r u l e d o u t . However, u n d e r c o n d i t i o n s o f a n o x i a , o w i n g t o t h e f a l l i n c e l l c o n c e n t -r a t i o n o f ATP, a c t i v e t r a n s p o r t mechanisms d i m i n i s h i n a c t i v i t y . Hence, t h e p o s s i b i l i t i e s e x i s t t h a t some o f t h e i n t e r m e d i a t e s o f g l y c o l y s i s m i g h t l e a k o u t i n t o t h e i n c u b a t i o n medium and t h u s r e d u c e t h e r a t e o f a n a e r o b i c g l y c o l y s i s . F o r e x a m p l e , compounds l i k e p y r u v a t e a r e known t o i n c r e a s e t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f 71 c e r e b r a l c o r t e x s l i c e s . I f TTX b l o c k s t h e e f f l u x o f s u c h compounds f r o m t h e s l i c e s , t h e n i t may e x e r t , i n d i r e c t l y , an a c c e l e r a t i n g e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s . E n e r g y d e p e n d e n t t r a n s p o r t p r o c e s s e s d i m i n i s h i n a c t i v i t y d u r i n g a n o x i a and a s a r e s u l t amino a c i d s c o n c e n t -r a t e d a g a i n s t a c o n c e n t r a t i o n g r a d i e n t i n t h e b r a i n t e n d t o l e a k i n t o t h e i n c u b a t i o n medium. The a c t i v e t r a n s p o r t o f . amino a c i d s i s a s s o c i a t e d w i t h t h e c a t i o n movements a t t h e b r a i n c e l l membrane and b e c a u s e t h e s e movement a r e a f f e c t e d by TTX, i t i s p o s s i b l e t h a t TTX may a l s o i n f l u e n c e t h e c e r e b r a l e f f l u x o f amino a c i d s . T h i s e f f e c t may be more p r o n o u n c e d i n t h e c a s e o f a s p a r t i c a c i d and g l u t a m i c a c i d as t h e s e a r e p r e s e n t i n b r a i n s l i c e s i n l a r g e amounts. The r e s u l t s r e p o r t e d i n t h i s s e c t i o n show t h a t t h i s c o n c l u s i o n i s c o r r e c t . The amino a c i d c o n c e n t r a t i o n s i n t h e t i s s u e , as w e l l as i n t h e i n c u b a t i o n medium, i n t h e p r e s e n c e and a b s e n c e o f - 102 -2 yM TTX are given i n Table 4. I t can be seen that the concentrations of a number of amino acids are higher i n the incubated s l i c e s i n the presence of TTX than i n i t s absence. The most marked e f f e c t occurs with glutamic and aspartic acids. Thus, for these acids, the t i s s u e / medium r a t i o s , i n the presence of glucose are 1, whilst i n the ad d i t i o n a l presence of TTX the r a t i o s are 2.5 and 2.0 respectively. The same e f f e c t of TTX was observed when glucose i s absent from the incubation medium although to a le s s e r extent. This w i l l be discussed further i n Chapter 8. 4.5 EFFECT OF TETRODOTOXIN ON THE UPTAKE OF AMINO ACIDS UNDER ANAEROBIC CONDITIONS As TTX diminishes the e f f l u x of amino acids from incubated cerebral cortex s l i c e s under anaerobic conditions (previous s e c t i o n ) , i t was decided to study the rates of uptake of amino acids (glutamic ac i d and glycine) under the 14 same conditions. C - l a b e l l e d amino acids were used for these experiments, r e s u l t s of which are shown i n Table 5. When no c a r r i e r (unlabelled) amino acid i s present i n the incubation medium, the r a d i o a c t i v i t y taken up by the s l i c e s i s more than doubled by the presence of TTX. A s i m i l a r e f f e c t i s obtained i n the presence of c a r r i e r amino acids. Glutamic acid i s metabolized i n the brain tis s u e but the increased uptake of radioactive glutamic acid i n these experiments can not r e s u l t wholly from the metabolism. 10 3 TABLE EFFECTS OF TETRODOTOXIN ON AMINO ACID CONTENT OF RAT CEREBRAL CORTEX SLICES UNDER ANOXIA Additions Amino Acids +Glucose -TTX +Glucose +TTX -Glucose -TTX -Glucose +TTX TAURINE Tissue 2.25 + .01 2.3 ± .05 Medium 3.21 ± .01 3.76 ± .34 2.34 ± .03 3.26 ± .34 2.39 ± .03 2.97 ± .20 ASPARTIC Tissue 0.96 ± .05 1.65 ± .02 ACID Medium 1.24 + .2 0.82 + .17 1.11 ± .04 1.47 ± .20 1.55 ± .20 1.14 GLUTAMINE + SERINE Tissue 1.31 ± .07 1.60 ± .10 1.01 Medium 3.07 + .22 2.52 ± .10 3.27 ± .07 1.16 ± 2.8 ± 1 5 GLUTAMIC Tissue 6.67 ± .5 9.1 ± .20 ACID Medium 6.10 ± .34 3.54 ± .44 5.63 ± .06 7.25 ± .65 7.68 ± .80 4.60 ±1.2 GLYCINE Tissue 0.41 + .05 0.57 ± .02 0.41 ± .07 Medium 0.62 ± .04 .46 ± .04 0.72 ± .18 0.42 ± .02 0.56 ± .02 ALANINE Tissue 0.36 ± .05 0.56 ± .02 Medium 0.64 ± .02 0.61 ± .01 0.36 ± .23 0.83 ± .17 0.33 ± .11 0.54 ± .10 Incubations were c a r r i e d out for 30 min i n a C a + ~ free medium under N 2:C02- 2uM TTX or 20mM glucose, when present, were added at zero time. At the end of incubation, tissue as well as medium was analyzed for amino acids, as given i n the materials and methods. Results are expressed as ymoles of amino acid per g i n i t i a l wet wt of the s l i c e s . 104 TABLE 5 EFFECTS OF TETRODOTOXIN ON THE UPTAKE OF AMINO ACIDS UNDER ANAEROBIC CONDITIONS BY RAT CEREBRAL CORTEX SLICES A. Uptake of U-C 1 4 L-Glutamate 15 min Incubation 30 min Incubation Amino acid No with 5mM No with 5mM Additions uptake c a r r i e r L-Glutamate c a r r i e r L-Glutamate NO TTX cpm per 3 7 7 000 178,750 466,000 221,500 r wet wt ' ' ' umoles per 0 > 0 Q 1 9 g wet wt 4.18 0.0024 5.16 WITH 2yM TTX cpm per 815,000 241,700 1080,000 293,000 r wet wt ' ' ' ymoles per > Q 0 4 2 g wet wt 5.56 ,0055 6.85 B. Uptake of 2-Clh Glycine 15 min Incubation Additions Amino acid uptake No c a r r i e r with 2mM Glycine NO TTX WITH 2yM TTX cpm per g wet wt ymoles per g wet wt cpm per g wet wt ymoles per g wet wt 186,500 0.022 377,500 0.044 133,750 1.85 207,000 2. 86 10 4A TABLE 5 (Continued) Incubations were c a r r i e d out for 15 min i n a Ca free medium containing 20mM glucose under N2:C02« Labelled amino acids, with or without c a r r i e r were tipped i n from the side arm of the Warburg vessel. TTX, when present, was added at zero time. T o t a l cpm per ml of the medium were 214000 for Clh-glutamate and 145000 for C 1 4 - g l y c i n e . Amino acid uptake was calculated by d i v i d i n g cpm/g by s p e c i f i c a c t i v i t i e s and were not corrected for swelling or i n t r a c e l l u l a r space. Each value represent averages of duplicate determinations within ± 7%. - 105 -because s i m i l a r r e s u l t s are obtained with glycine which metabolizes at a much slower rate than an equivalent quantity of glutamate. Moreover, the metabolism of glutamic acid (e.g., conversion to glutamine) i s greatly reduced under anaerobic conditions, and there i s normally l i t t l e or no uptake of amino acids against a concentration gradient under such conditions. 4.6 EFFECT OF TETRODOTOXIN AT DIFFERENT GLUCOSE  CONCENTRATIONS ON THE ANAEROBIC GLYCOLYSIS OF  CEREBRAL CORTEX SLICES When the rate of g l y c o l y s i s i s high, the rate of glucose entry i n the c e l l may become rate l i m i t i n g . This can be true under anaerobic conditions, as a considerable amount of glucose i s almost excl u s i v e l y metabolized through the g l y c o l y t i c pathway. The e f f e c t of TTX on the anaerobic g l y c o l y s i s of cerebral cortex s l i c e s was, therefore, investigated at d i f f e r e n t concentrations of glucose. The r e s u l t s of these experiments are shown i n Figure 15. I t can be seen that, when the concentration of glucose i s increased, the rate of anaerobic g l y c o l y s i s i n the presence of TTX progressively increases u n t i l a maximum i s obtained at about 50 mM. The rates of anaerobic g l y c o l y s i s , i n the control s l i c e s ( i . e . without TTX) do not show any s i g n i f i c a n t increase with varying glucose concent-rations above about 5 mM. 4.7 GLUCOSE TRANSPORT IN CEREBRAL CORTEX SLICES 14 The e f f e c t s of 2 yM TTX on the glucose-2-C transport - 1 0 6 -FIGURE 15 EFFECT OF VARYING GLUCOSE CONCENTRATION ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES IN THE PRESENCE AND ABSENCE OF TETRODOTOXIN 100 -•P 0 20 40 60 80 100 Glucose c o n c e n t r a t i o n , i n mM Incubations were c a r r i e d out i n a C a + + - f r e e medium.TTX( 2 uM) and glucose,when present,were added at zero time and l a c t a t e p r oduction was measured manometrically,as given i n the m a t e r i a l s and methods.(©)TTX absent;(O) 2 uM TTX present. - 107 -i n t h e r a t c e r e b r a l c o r t e x s l i c e s a r e g i v e n i n T a b l e 6. I t i s e v i d e n t t h a t t h e amount o f r a d i o a c t i v i t y p r e s e n t i s g r e a t e r i n s l i c e s w h i c h h ave b e e n i n c u b a t e d w i t h TTX. How-e v e r , i n t h e p r e s e n c e o f i o d e c e t a t e w h i c h b l o c k e d g l y c o l y -s i s , t h e p r e s e n c e o f TTX b r i n g s a b o u t no i n c r e a s e d r a d i o -a c t i v i t y i n t h e s l i c e s . I t may be c o n c l u d e d , t h e r e f o r e , t h a t t h e i n c r e a s e d r a d i o a c t i v i t y , i n t h e a b s e n c e o f i o d o a c e t a t e , i n t h e TTX t r e a t e d s l i c e s i s due t o t h e p r e s e n c e o f g l y c o l y -t i c m e t a b o l i t e s i n t h e t i s s u e . T h e s e e x p e r i m e n t s d e m o n s t r a t e t h e f a c t t h a t t h e g r e a t e r r a t e o f g l y c o l y s i s o b t a i n e d i n t h e p r e s e n c e o f TTX a t h i g h g l u c o s e c o n c e n t r a t i o n s i s n o t due t o u n s p e c i f i c f a c i l i t a t i o n o f g l u c o s e e n t r y i n t o t h e s l i c e s . I t i s p r o b a b l y due t o t h e f a c t t h a t w i t h h i g h r a t e s o f g l y -c o l y s i s , t h e i n c r e a s e d g l u c o s e g r a d i e n t w i t h i n c r e a s e d e x t e r n a l c o n c e n t r a t i o n s o f g l u c o s e r e s u l t s i n a g r e a t e r s a t u r a t i o n o f t h e g l y c o l y t i c enzymes and t h e r e f o r e i n an o p t i m a l r a t e o f g l y c o l y s i s . 4.8 EFFECTS OF TETRODOTOXIN, IN THE PRESENCE OF SOME AMINO  ACIDS, ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX  S L I C E S I t i s w e l l known t h a t , o f a l l t h e amino a c i d s p r e s e n t i n t h e b r a i n , g l u t a m a t e and i t s i m m e d i a t e m e t a b o l i t e s a r e o f 28 6 o u t s t a n d i n g i m p o r t a n c e t o t h e b r a i n . The e f f e c t o f g l u t -amate on t h e a n a e r o b i c g l y c o l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s h as b e e n d i s c u s s e d e a r l i e r i n C h a p t e r 3. L - g l u t a m a t e i s known t o c a u s e e x c i t a t i o n and t h u s d e p o l a r i z a t i o n o f t h e 286 n e r v e c e l l w h i l e i t h as no s u c h e f f e c t on t h e g l i a l c e l l s . 108 TABLE 6 EFFECTS OF TETRODOTOXIN ON THE GLUCOSE TRANSPORT IN RAT CEREBRAL CORTEX SLICES UNDER ANOXIA Additions 5mM glucose cpm per g. wet wt Glucose uptake ymoles/g 20mM glucose Glucose cpm per g wet wt uptake ymoles/g None 2yM TTX Iodoacetate, 0: 2mM Iodoacetate, 0. 2mM + TTX, 2yM 183,000 201,000 183,000 173,000 3.2 3.5 3.2 3.0 174,000 198,000 185,000 166 ,000 12.1 13.7 12.8 11.9 Incubations were ca r r i e d out anaerobically i n a » Ca+~t_ free medium for 15 min. TTX, iodoacetate or cold glucose, when present, were added at zero time. 0.5yC of glucose-2-C 1 h was added and incubation was ca r r i e d out for another 5 min. Tot a l cpm i n the incubation medium was 289000/ml. Glucose uptake was calculated by d i v i d i n g cpm/g by s p e c i f i c a c t i v i -t i e s and were not corrected f o r swelling or i n t r a -c e l l u l a r space. Each value represent averages of duplicate determinations within + 5%. - 109 -M c l l w a i n and h i s c o w o r k e r s s t u d i e d t h e e f f e c t s o f TTX on t h e c a t i o n i c c h a n g e s o f t h e i n c u b a t e d c e r e b r a l c o r t e x s l i c e s i n d u c e d by g l u t a m a t e . T h e s e a u t h o r s showed t h a t TTX p a r t l y i n h i b i t s t h e i n c r e a s e i n N a + c a u s e d by 5 mM g l u t a m a t e . I t was t h o u g h t d e s i r a b l e , t h e r e f o r e , t o e xamine t h e e f f e c t s o f g l u t a m a t e and o t h e r amino a c i d s on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s i n t h e p r e s e n c e o f TTX. The r e s u l t s a r e shown i n T a b l e 7. I t i s e v i d e n t t h a t i n t h e p r e s e n c e o f 5 mM L - g l u t a m a t e , w h i c h i n h i b i t s a n a e r o b i c g l y c o l y s i s , t h e a c c e l e r a t i n g e f f e c t o f TTX on t h e r a t e o f a n a e r o b i c g l y c o l y s i s i s d i m i n i s h e d , b o t h i n a C a + + - f r e e a s w e l l a s i n a K r e b s -R i n g e r b i c a r b o n a t e medium. D - G l u t a m a t e a l s o i n h i b i t s t h e a c c e l e r a t i n g a c t i o n o f TTX, w h i l e 5 mM L - a s p a r t a t e has no e f f e c t . 4.9 E FFECTS OF CITRATE, AMP AND NH*^ ON THE TETRODOTOXIN STIMULATION OF GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES As p o i n t e d o u t i n C h a p t e r 3, c i t r a t e , AMP and NH^ h a v e i n h i b i t o r y e f f e c t s on t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . E x p e r i m e n t s were c a r r i e d o u t , t h e r e f o r e , t o a s c e r t a i n t h e e f f e c t s o f TTX on t h e r a t e o f a n a e r o b i c g l y c o l y s i s i n t h e p r e s e n c e o f t h e s e compounds. The r e s u l t s a r e g i v e n i n T a b l e 8. I t c a n be s e e n t h a t 15 mM c i t r a t e and 5 mM NH* have marked i n h i b i t o r y e f f e c t s on t h e TTX s t i m u l a t e d a n a e r o b i c g l y c o l y s i s , w h e r e a s AMP, u n d e r t h e s e c o n d i t i o n s , h a d o n l y a s l i g h t e f f e c t . 110 TABLE 7 EFFECTS OF TETRODOTOXIN, IN THE PRESENCE OF SOME AMINO ACIDS, ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX S L I C E S FROM RAT A d d i t i o n s Lactate produced ymoles per g i n i t i a l wet wt (20-80 min) C a + i free medium Krebs-Ringer bicarbonate medium None 5mM L-Glutamate 5mM D-Glutamate 5mM L-Aspartate 2yM TTX 5mM L-Glutamate + 2yM TTX 5mM D-Glutamate + 2uM TTX 5mM L-Aspartate + 2yM TTX 31.7 ± 4.3 20.9 + 2.3 38.4 ± 2.0 72.8 ±17.0 22.4 ± 2.5 38.0 ± 4.4 28.6 ± 3.7 34.4 ± 3.8 65.0 38.4 ± 4.4 43.5 ± 2.4 76.5 ±15.0 A l l vessels contained 20mM glucose. Additions were made at zero time and la c t a t e production was measured manometrically, as given i n the materials and methods. I l l TABLE 8 EFFECTS OF CITRATE, AMP AND NH\+ IN THE PRESENCE OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES Additions None 15mM c i t r a t e 2mM AMP 5mM NH4CI Lactate produced ymoles per g i n i t i a l wet wt (20-80 min) No TTX with 2yM TTX 25.0 + 4.9 63.0 + 6.7 17. 8 + 3.5 16.9 + 2.0 15.5 + 5.0 50.5 + 3.1 29.9 + 5.8 37.0 + 4.3 Incubations were c a r r i e d out i n a Ca i free medium containing 20mM glucose. Additions were made at zero time and la c t a t e production was measured manometri-c a l l y , as given i n the materials and methods. - 112 -4.10 EFFECTS OF PHOSPHOLIPASES ON THE TETRODOTOXIN STIMULATED GLYCOLYSIS OF THE RAT CEREBRAL CORTEX  SLICES The s i t e of action of TTX appears to be located at the 124 127 outer surface of the c e l l membrane ' . Phospholipids are known to be major and important membrane constituents. 287 Cuthbert has shown the importance of membrane l i p i d s 288 i n some aspects of drug action. Heilbronn studied the e f f e c t of phospholipases on the uptake of atropine and acet y l choline by mouse brain cortex s l i c e s . His r e s u l t s demonstrate that the phospholipases decrease the uptake of atropine and p a r t i c u l a r l y that of acetylcholine by the s l i c e s . The e f f e c t of the enzyme was time-dependent, and, up to a c e r t a i n l i m i t , concentration-dependent. In view of the f a c t that s i t e of action of TTX might be at the c e l l membrane of cerebral cortex c e l l s , and that TTX might be acting by i n t e r a c t i o n with the membrane constituents, the e f f e c t of TTX on the anaerobic g l y c o l y s i s of the phospholipase treated cerebral cortex s l i c e s was studied. Results of these experiments are shown i n Table 9. I t can be seen that, with progressively higher concentrations of phospholipase A, the percentage stimulation of anaerobic g l y c o l y s i s by TTX i s decreased. TTX i s , however, s t i l l e f f e c t i v e during the early period (20-50 minutes) of the experiment. This i s true, even at phospholipase A concent-r a t i o n of 40 units/vessel (3 ml), present from zero time up 113 TABLE 9 EFFECTS OF TETRODOTOXIN IN THE PRESENCE OF PHOSPHOLIPASES ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX S L I C E S A d d i t i o n s Amount o f P h o s p h o l i p a s e , I.U. p e r v e s s e l (3 ml) L a c t a t e p r o d u c e d y m o l e s p e r g i n i t i a l wet wt 20-50 min 50-80 m i n None 2yM TTX P h o s p h o l i p a s e A P h o s p h o l i p a s e A + 2yM TTX P h o s p h o l i p a s e C P h o s p h o l i p a s e C + 2yM TTX P h o s p h o l i p a s e A P h o s p h o l i p a s e A + 2yM TTX P h o s p h o l i p a s e A P h o s p h o l i p a s e A + 2yM TTX P h o s p h o l i p a s e A P h o s p h o l i p a s e A + 2yM TTX P h o s p h o l i p a s e A P h o s p h o l i p a s e A + 2uM TTX 1 1 1 1 5 5 20 20 40 40 60 60 22.5 44.9 12.1 51.8 20.9 33.5 13.6 37.5 25.0 45.1 22.3 50.5 20.3 22.1 ± 4.8 ±13 ± 3.1 ± 1.4 ± 0.4 ± 1.3 ± 0.5 ± 2.7 ± 1.7 ± 4.0 ± 1.3 ± 1.4 ± 0.7 ±11. 8 10.5 ± 1.4 30.8 ±10 5.8 ± 2.6 33.9 ± 2.3 7.4 ± 1.5 11.8 ± 2.9 7.8 ± 1.1 28.0 ± 2.7 12.9 ± 0.5 12.3 ± 1.6 8.5 ± 1.3 14.3 ± 1.8 11.2 ± 0.5 17.9 ± 1.0 I n c u b a t i o n s were c a r r i e d o u t i n a C a + + f r e e medium c o n t a i n i n g 20mM g l u c o s e . A d d i t i o n s were made a t z e r o t i m e and l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i -c a l l y , a s g i v e n i n t h e m a t e r i a l s and m e t h o d s . - 114 -to the end of experiment. Phospholipase C, at the concent-r a t i o n tested, i s more potent than phospholipase A i n a f f e c t i n g the TTX stimulated g l y c o l y s i s of cerebral cortex s l i c e s . These r e s u l t s lead to the conclusion that TTX may act by combining with phospholipid constituents of the c e l l membrane which are slowly attacked by phospholipases. However, i t i s possible that the products of phospholipase a c t i v i t y may i n h i b i t the action of TTX. This i s yet to be resolved. 4.11 EFFECTS OF TETRODOTOXIN ON ANAEROBIC GLYCOLYSIS OF  KIDNEY MEDULLA SLICES AND ACETONE POWDER EXTRACTS Experiments were c a r r i e d out to see i f the accelerating e f f e c t of TTX on anaerobic g l y c o l y s i s of cerebral cortex s l i c e s i s s p e c i f i c f or the brain t i s s u e . I t i s known that kidney medulla s l i c e s have high g l y c o l y t i c rates and hence i t was selected for examination. The e f f e c t s of TTX on the anaerobic g l y c o l y s i s of kidney medulla s l i c e s are given i n Figure 16. From t h i s data i t can be concluded that TTX has absolutely no e f f e c t on the anaerobic g l y c o l y s i s of kidney medulla. This observation demonstrates that the e f f e c t of TTX on cerebral cortex s l i c e s i s s p e c i f i c , and that the anaerobic g l y c o l y s i s of a l l tissues i s not se n s i t i v e to i t . The e f f e c t of TTX and ouabain on the anaerobic glyco-l y s i s of acetone powder extracts i s shown i n Figure 17. The re s u l t s prove that TTX has no e f f e c t on the process of c e l l free anaerobic g l y c o l y s i s and that a membrane phenomenon i s - 115 -FIGURE 16 EFFECTS OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF RAT KIDNEY MEDULLA SLICES Incubation conditions were same as i n Figure 1 3 . ( O ) c o n t r o l ; ( • )with 2 uM TTX. - 116 -FIGURE 17 EFFECTS OF TETRODOTOXIN AND OUABAIN ON THE ANAEROBIC GLYCOLYSIS OF ACETONE POWDER EXTRACTS FROM RAT BRAIN M 1000 20 30 40 50 60 70 81) T i m e , i n m i n u t e s I n c u b a t i o n c o n d i t i o n s w e r e same a s i n F i g u r e 1 0 . ( ± ) c o n t r o l ; ( O ) w i t h 2 jaM TTX; ( « ) w i t h 10 pM o u a b a i n . - 117 -i n v o l v e d i n t h e mechanism o f a c t i o n o f TTX. I n t a c t c e l l s a r e c l e a r l y n e c e s s a r y f o r t h e a c t i o n o f TTX. 4.12 EFFECTS OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS  OF DEVELOPING BRAIN CORTEX SLICES E f f e c t s o f TTX on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s f r o m r a t s o f d i f f e r e n t a g e s a r e g i v e n i n T a b l e 10. I t i s e v i d e n t t h a t w i t h 2 o r 7-day o l d r a t b r a i n s l i c e s t h e g l y c o l y t i c b e h a v i o u r i s u n a f f e c t e d by TTX. However, s l i c e s f r o m 14-day o l d a n i m a l s show an a p p r e c i a b l e r e s p o n s e . The e f f e c t s o f d i f f e r e n t c o n c e n t r a t i o n s o f TTX on t h e a n a e r o b i c g l y c o l y s i s o f n e w l y b o r n g u i n e a p i g c e r e b r a l c o r t e x s l i c e s a r e a-lso- shown i n F i g u r e 18. The a n a e r o b i c g l y c o l y s i s o f i n f a n t g u i n e a p i g c e r e b r a l c o r t e x s l i c e s a r e e x t r e m e l y s e n s i t i v e t o TTX and e v e n 0.2 uM TTX i s as e f f e c t i v e as 10 yM TTX. M o r e o v e r , t h e r a t e o f g l y c o l y s i s i n t h e p r e s e n c e o f TTX i s c o n s t a n t d u r i n g t h e t i m e p e r i o d t e s t e d . E x p e r i m e n t s w i t h some o t h e r d r u g s , w h i c h w i l l be d i s c u s s e d l a t e r , show t h a t t h e s e n s i t i v i t y o f t h e i n f a n t g u i n e a p i g s t o d r u g s i s much g r e a t e r t h a n t h e a d u l t b r a i n t i s s u e s t e s t e d . I t i s i m p o r t a n t i n c o n s i d e r i n g t h e s e r e s u l t s t o a p p r e c i a t e t h e w e l l known f a c t t h a t n e w l y b o r n g u i n e a p i g s a r e v e r y m a t u r e compared w i t h n e w l y b o r n r a t s . 4.13 EFFECT OF TETRODOTOXIN IN PRESENCE OF GLUTAMATE,  ASPARTATE AND NH* ON THE ANAEROBIC GLYCOLYSIS OF  DEVELOPING CORTEX S L I C E S We have shown e a r l i e r i n t h i s c h a p t e r (4.8 and 4.9) 118 TABLE 10 EFFECT OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF INFANT RAT CEREBRAL CORTEX SLICES Additions Lactate produced ymoles per g i n i t i a l wet wt (20-80 min) 2-day old r a t 7-day old ra t 14-day old ra t None 25 + 1.5 21.2 + 0.45 25.4 + 3.1 0. 2yM TTX 28.6 + 1.8 26.0 + 2.2 42.5 + 1.3 2yM TTX 29.9 + 5.9 27.7 + 1.3 48.3 + 9.9 lOyM TTX 29. 8 + 0.9 30.0 + 0.5 51.4 + 6.6 2yM + TTX ImM C a + + 35.8 + 3.5 31.3 + • 1.3 45.3 + 4.4 2yM + TTX 2mM C a + + 31.7 + 0.9 34.4 + 0.4 54.5 + 6.2 2yM + TTX 4mM C a + + 29.5 + 3.5 29.0 55.8 + 6.7 Incubations were c a r r i e d out i n a Ca t free medium containing 20mM glucose. Additions were made at zero time and lactate production was measured manometrically, as given i n the materials and methods. - 119 -150 Time,in minutes FIGURE 18 EFFECTS OF DIFFERENT CONCENTRATIONS OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF NEWLY BORN GUINEA PIG CEREBRAL CORTEX SLICES ++ Incubations were c a r r i e d out i n a Ca -free medium containing 2 0 mM glucose.TTX,when present,was added at zero time and lact a t e production was measured manometrically,as given i n the materials and methods.( • ) control; ( • )0.2 uM TTX; (• ) 2 uM TTX;(A)10 pM TTX. - 120 -t h a t t h e TTX s t i m u l a t e d g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s shows s e n s i t i v i t y t o g l u t a m a t e as w e l l a s t o NH*. I t was d e s i r a b l e , t h e r e f o r e , t o s t u d y t h e e f f e c t s o f t h e s e and a l l i e d s u b s t a n c e s on TTX s t i m u l a t e d g l y c o l y s i s o f 2-week o l d r a t and n e w l y - b o r n g u i n e a p i g c e r e b r a l c o r t e x s l i c e s t o t h r o w more l i g h t on t h e mechanism o f a c t i o n o f TTX. R e s u l t s o f t h e s e e x p e r i m e n t s a r e shown i n T a b l e 11. I t i s c l e a r t h a t t h e s e compounds have i n h i b i t o r y e f f e c t s on t h e r a t e o f TTX a c c e l e r a t e d g l y c o l y s i s . The 2-week o l d r a t b r a i n i s more s e n s i t i v e t o NH* t h a n t h a t o f t h e a d u l t . 4.14 EFFECTS OF TETRODOTOXIN ON THE AEROBIC GLYCOLYSIS OF  ADULT RAT CEREBRAL CORTEX S L I C E S The e f f e c t s o f TTX on t h e a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s a r e shown i n F i g u r e 19. TTX h a s no e f f e c t on t h e r a t e o f a e r o b i c g l y c o l y s i s o f t h e r a t c e r e b r a l c o r t e x s l i c e s when t h e l a t t e r a r e i n c u b a t e d i n a ++ K r e b s - R i n g e r b i c a r b o n a t e medium. M o r e o v e r , i n a Ca - f r e e medium, TTX h a s an i n h i b i t o r y e f f e c t on t h e a e r o b i c g l y c o -l y s i s . I n a Ca - f r e e medium, u n d e r a e r o b i c c o n d i t i o n s t h e r a t e o f l a c t a t e p r o d u c t i o n , i n t h e a b s e n c e o f t h e d r u g , i s g r e a t e r t h a n t h a t i n a K r e b s - R i n g e r medium. The i n h i b i t o r y e f f e c t o f TTX on t h e r a t e o f a e r o b i c g l y c o l y s i s ++ ++ i n a Ca - f r e e medium i s s i m i l a r t o t h a t o f Ca a n d a n a l o g o u s t o t h a t on t h e r e s p i r a t i o n o f r a t b r a i n c o r t e x s l i c e s i n c u b a t e d u n d e r s i m i l a r c o n d i t i o n s (Chan and Q u a s t e l ) . R e s u l t s g i v e n i n T a b l e 11A show t h e e f f e c t s o f TTX on t h e a e r o b i c g l y c o l y s i s i n t h e p r e s e n c e o f c y a n i d e . I n - 121 -FIGURE 19 EFFECTS OF TETRODOTOXIN AND OUABAIN ON THE AEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES 4J •H +J •H U CD CU W CD i H O 1 'O o o u cu 0) +J rO 4J O fO 20 40 60 Time,in minutes 80 A l l vessels +contained 20 mM glucose.A:Krebs-Ringer bicarbonate medium.B:Ca -free medium.Additions were made at zero time and lac t a t e production was measured e n z y m a t i c a l l y . ( v ) c o n t r o l ; (• )2 pM TTX; ( • ) 10 pM ouabain. 122 Additions TABLE 11 EFFECTS OF TETRODOTOXIN IN THE PRESENCE OF SOME AMINO ACIDS OR NHi*"1" ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES FROM INFANT ANIMALS Lactate produced ymoles per g i n i t i a l wet wt (20-80 min) 2-week old newly born rat guinea pig None 25.5 + 3.1 34.4 + 2.7 5mM L-glutamate 19.0 + 3.1 22.3 + 0.9 5mM D-glutamate 19.2 + 1.8 32.2 + 2.3 2yM TTX 48.3 + 9.9 129.5 + 6.7 5mM L-Aspartate 17.4 + 1.3 25.9 + 3.1 5mM NH4CI 13.9 + 2.2 -2yM TTX + 5mM L-glutamate 25.5 + 0.5 51.5 + 1.8 + 5mM D-glutamate 29. 5 + 4.0 98.1 + 3.1 + 5mM L-Aspartate 28.6 + 4.0 113.7 + 3.5 + 5mM NH4CI 17.0 + 3.1 -Incubation conditions were same as i n Table 10. 123 TABLE 11A EFFECTS OF TETRODOTOXIN ON THE AEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES IN THE PRESENCE OF CYANIDE L a c t a t e produced ymoles per g i n i t i a l wet wt (20-80 min) Medium No TTX w i t h 2yM TTX Krebs-Ringer 112.9 + 6.7 117.2 +9.6 bicarbonate C a + + - f r e e 63.8 ± 8.9 103.1 ± 2.5 A l l v e s s e l s contained 20mM glucose. A d d i t i o n s were made a t zero time and l a c t a t e p roduction was measured manometrically, as given i n the m a t e r i a l s and methods, (gaseous phase was O 2 : C 0 2 i n these experiments) - 124 -a Ca - f r e e medium, u n d e r a e r o b i c c o n d i t i o n s , when t h e e l e c t r o n t r a n s p o r t c h a i n i s b l o c k e d by c y a n i d e , TTX i s e f f e c t i v e i n f u r t h e r e n h a n c i n g t h e r a t e o f g l y c o l y s i s . However, i n a K r e b s - R i n g e r b i c a r b o n a t e medium, TTX i s n o t e f f e c t i v e i n f u r t h e r e n h a n c i n g t h e g l y c o l y s i s , p o s s i b l y due t o o p t i m a l r a t e s o f g l y c o l y s i s i n t h e c o n t r o l s i t s e l f . SUMMARY OF CHAPTER 4 1. T e t r o d o t o x i n (TTX) m a r k e d l y a c c e l e r a t e s t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f r a t and g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . T h e s e e f f e c t s o f TTX a r e s i m i l a r t o t h o s e 71 ++ r e p o r t e d by Q u a s t e l and c o w o r k e r s on Ca w i t h t h e g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . 2. The r a t e o f a n a e r o b i c g l y c o l y s i s i n p r e s e n c e o f 2 yM TTX i s f u r t h e r e n h a n c e d by t h e a d d i t i o n o f C a + + i n t h e g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . I n r a t s t h e a d d i t i o n o f C a + + has e i t h e r no e f f e c t or i t has a s l i g h t l y i n h i b i t o r y e f f e c t . 3. The a c c e l e r a t i o n o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s , by t h e p r e s e n c e o f TTX, i s n o t due t o suppression o f t h e e f f l u x o f NAD + u n d e r a n o x i c c o n d i t i o n s . 4. The e f f l u x o f g l u t a m i c a c i d o r o f a s p a r t i c a c i d f r o m t h e i n c u b a t e d c e r e b r a l c o r t e x s l i c e s , due t o a n a e r o b i o s i s , i s m a r k e d l y s u p p r e s s e d by 2 yM TTX. TTX a l s o c a u s e s i n c r e a s e d u p t a k e o f r a d i o a c t i v e g l u t a m i c a c i d a nd g l y c i n e , f r o m t h e i n c u b a t i o n medium, by t h e c e r e b r a l c o r t e x s l i c e s u n d e r a n o x i a . 5. I n t h e p r e s e n c e o f i n c r e a s i n g c o n c e n t r a t i o n s o f - 125 -g l u c o s e , t h e s t i m u l a t i o n o f a n a e r o b i c g l y c o l y s i s , due t o t h e p r e s e n c e o f TTX, i s e n h a n c e d , i n d i c a t i n g t h a t when t h e r a t e o f a n a e r o b i c g l y c o l y s i s i s h i g h , t h e g l u c o s e c o n c e n t -r a t i o n may become r a t e l i m i t i n g . The above e f f e c t i s n o t due however, t o an u n s p e c i f i c f a c i l i t a t i o n o f g l u c o s e e n t r y i n t o t h e c e r e b r a l c o r t e x s l i c e s by t h e p r e s e n c e o f TTX a s shown by e x p e r i m e n t s w i t h l a b e l l e d g l u c o s e . 6. The TTX s t i m u l a t i o n o f g l y c o l y s i s i s p a r t i a l l y , o r c o m p l e t e l y r e v e r s e d by t h e a d d i t i o n o f L - g l u t a m a t e , D - g l u t a m a t e , c i t r a t e o r NH*. 7. TTX i s e f f e c t i v e i n a c c e l e r a t i n g t h e r a t e o f a n a e r o b i c g l y c o l y s i s e v e n i n t h e p r e s e n c e o f p h o s p h o l i p a s e s , a l t h o u g h t h e p e r c e n t a g e s t i m u l a t i o n d u r i n g t h e l a t e r p e r i o d i s c o n s i d e r a b l y d e c r e a s e d . 8. TTX has no e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f k i d n e y m e d u l l a s l i c e s o r o f a c e t o n e powder e x t r a c t s o f b r a i n . 9. TTX h a s l i t t l e o r no e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f 2- o r 7-day o l d r a t b r a i n b u t i t s e f f e c t i v e n e s s i n c r e a s e s m a r k e d l y a t a b o u t 1 4 t h d a y . T h i s p e r i o d c o i n c i d e s w i t h t h a t o f maximum b r a i n g r o w t h and m y e l i n a t i o n . The a n a e r o b i c g l y c o l y s i s by s l i c e s o f n e w l y b o r n g u i n e a p i g s , w h i c h shows many m a t u r e c h a r a c t e r i s t i c s , i s v e r y s e n s i t i v e t o TTX. The TTX s t i m u l a t e d g l y c o l y s i s o f i n f a n t b r a i n i s i n h i b i t e d l i k e t h a t o f t h e a d u l t b r a i n , by L - g l u t a m a t e , D - g l u t a m a t e o r 10. The r a t e o f a e r o b i c g l y c o l y s i s o f t h e c e r e b r a l c o r t e x - 126 -s l i c e s i n a K r e b s - R i n g e r b i c a r b o n a t e medium i s u n a f f e c t e d ++ by TTX, w h i l s t i n a Ca - f r e e medium i t i s s l i g h t l y d e p r e s s e d by TTX. The r a t e o f a e r o b i c g l y c o l y s i s i n t h e p r e s e n c e o f c y a n i d e i n a C a + + - f r e e medium i s i n c r e a s e d by TTX. However, i n a K r e b s - R i n g e r medium u n d e r t h e same c o n d i t i o n s , TTX has no e f f e c t . CHAPTER 5 FURTHER STUDIES ON THE MECHANISM OF ACTION OF  TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS  OF CEREBRAL CORTEX S L I C E S I t h a s b e e n shown t h a t t h e p r e s e n c e o f TTX, a t l o w c o n c e n t r a t i o n s , g r e a t l y e n h a n c e s t h e r a t e o f a n a e r o b i c g l y c o -l y s i s o f c e r e b r a l c o r t e x s l i c e s and, m o r e o v e r , t h a t t h i s p h e n o -menon i s c h a r a c t e r i s t i c o f m a t u r e c e r e b r a l t i s s u e . The r e -s u l t s o f e x p e r i m e n t s , c a r r i e d o u t t o t h r o w f u r t h e r l i g h t on t h e mode o f a c t i o n o f TTX on t h e b r a i n t i s s u e , w i l l now be d e s c r i b e d . 5.1 EFFECTS OF PRE-INCUBATION I N OXYGEN ON THE TETRODOTOXIN STIMULATED GLYCOLYSIS OF CEREBRAL CORTEX S L I C E S A s e a r l y a s 1928, R o s e n t h a l a n d L a s n i t z k i 2 8 9 showed t h a t a b r i e f p e r i o d o f i n c u b a t i o n i n p r e s e n c e o f o x y g e n r e s u l t s i n a marked i n c r e a s e i n t h e s u b s e q u e n t r a t e o f a n a e r o b i c g l y c o l y s i s . T h i s phenomenon was f u r t h e r s t u d i e d i n l i v e r , t u m o u r s a n d o t h e r n o r m a l t i s s u e s , i n c l u d i n g b r a i n , 290-294 a n ( j f r o m t h e s e s t u d i e s i t was c o n c l u d e d t h a t t h e s t i m u l a t i o n o f a n a e r o b i c g l y c o l y s i s b y p r e v i o u s i n c u b a t i o n i n o x y g e n i s a phenomenon o f g e n e r a l i m p o r t a n c e a n d o c c u r s i n a l l n o r m a l a d u l t ? 91 t i s s u e s . x However, i t h a s n o t b e e n p o s s i b l e t o d e f i n e d e f i n i t e l y t h e c h a n g e s a s s o c i a t e d w i t h t h e a e r o b i c p r e - i n c u b a -- 128 t i o n of the tissues.292 In view of the above, i t was considered desirable to see i f the anaerobic g l y c o l y s i s , which has been stimulated by previous oxygenation, can be further enhanced by TTX. The r e s u l t s of these experiments are shown i n Figure 20. I t can be seen that when the s l i c e s are pre-incubated a e r o b i c a l l y , i n the absence of TTX, the drug i s s t i l l e f f e c t i v e i n enhancing the rate of anaerobic g l y c o l y s i s . Longer periods of aerobic pre-incubation (40 minutes) lower the t o t a l amount of the l a c t a t e produced during the subsequent anaerobic period, both i n the control as well as i n the TTX-treated s l i c e s . However, the stimulation of anaerobic g l y c o l y s i s i n the presence of TTX w^ s t i l l very apparent. 5.2 EFFECTS OF TETRODOTOXIN AFTER VARIOUS PERIODS OF ANAEROBIOSIS ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES The r e s u l t s of experiment shown i n Figure 20 demon-stra t e that TTX i s e f f e c t i v e a f t e r the preliminary pre-incuba-t i o n of the s l i c e s i n oxygen. The rate of anaerobic g l y c o l y s i s attained with the a e r o b i c a l l y incubated s l i c e s i s greater than that found with the corresponding s l i c e s which were not exposed to oxygen. Hence i t was thought desirable to carry out experi-ments, i n which the s l i c e s have been pre-incubated for varying periods i n nitrogen, to see i f TTX i s s t i l l e f f e c t i v e i n enhan-cing the rate of anaerobic g l y c o l y s i s . Results of a t y p i c a l - 129 -FIGURE 20 EFFECT OF AEROBIC PREINCUBATION ON THE TETRODOTOXIN STIMULATION OF ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES 125 . . 20 30 40 50 60 70 80 Time,in minutes Cerebral cortex s l i c e s were preincubated i n a Krebs-Ringer bicarbonate medium containing 20 mM glucose for 20 min under 02:00-.They were then transferred to another set of vessels containing,in addition,2 pM TTX.Lactate production was measured manometrically under subsequent anaerobic period, as given i n the materials and methods.(O)control;(•)2 pM TTX. - 130 -e x p e r i m e n t , i n w h i c h TTX h a s b e e n " t i p p e d i n " a f t e r v a r y i n g p e r i o d s o f a n a e r o b i o s i s , a r e shown i n F i g u r e 21. T h i s e x p e r i -ment shows t h a t t h e a b i l i t y o f TTX i n e n h a n c i n g t h e a n a e r o b i c g l y c o l y s i s , o f t h e c e r e b r a l c o r t e x s l i c e s , d e c r e a s e s p r o g r e s -s i v e l y a s t h e p e r i o d o f a n a e r o b i o s i s , b e f o r e t h e a d d i t i o n o f TTX, i s i n c r e a s e d . T h u s , a l t h o u g h 2 min o f a n o x i a h a d no e f f e c t on t h e s t i m u l a t i o n o f a n a e r o b i c g l y c o l y s i s b y TTX, t h e 5 m i n p e r i o d c o n s i d e r a b l y r e d u c e d t h e a b i l i t y o f c e r e b r a l c o r t e x s l i c e s t o r e s p o n d t o TTX a n d a f t e r a 10 m i n " p e r i o d , i t was i n e f f e c t i v e . T h i s e x p e r i m e n t d e m o n s t r a t e s t h a t c h a n g e s i n t h e c e r e b r a l c o r t e x s l i c e s , d u r i n g t h e f i r s t few m i n u t e s o f a n o x i a , a r e v e r y i m p o r t a n t f o r t h e e n h a n c i n g e f f e c t o f TTX on t h e r a t e o f a n a e r o b i c g l y c o l y s i s . 5.3 EFFECTS OF TETRODOTOXIN I N THE PRESENCE OF PYRUVATE  ON THE ANAEROBIC GLYCOLYSIS OF THE CEREBRAL  CORTEX S L I C E S The f a c t t h a t T TX i s n o t e f f e c t i v e i n s t i m u l a t i n g t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s a f t e r b r i e f p e r i o d s o f a n o x i a r a i s e s t h e q u e s t i o n a s t o t h e c h a n g e s t h a t o c c u r i n t h e c e r e b r a l c o r t e x s l i c e s u n d e r a n a e r o b i c c o n d i t i o n s . The m a j o r e f f e c t o f a n o x i a on a l i v i n g t i s s u e i s t h a t t h e o x i d a t i v e mechanisms d e p e n d e n t on t h e p r e s e n c e o f o x y g e n a r e no l o n g e r o p e r a t i v e . I n b r a i n , t h e c e l l ATP l e v e l d r o p s q u i c k l y . A number o f ATP d e p e n d e n t p r o c e s s e s a r e i m p a i r e d - 131 -FIGURE 21 EFFECT OF ADDITION OF TETRODOTOXIN AFTER VARYING TIME PERIODS OF ANOXIA ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES 80 Time,in minutes ++ Incubations were c a r r i e d out i n a Ca -free medium containing 20 mM glu c o s e . ( B ) N o addition; ( O ) 2 pM TTX,from zero time; ( • ) 2 pM TTX added at 2min; ( A )2 pM TTX added at 5 min; ( • )2 pM TTX added at 10 min .The gas phase was anaerobic throughout the experiment , and lactate production was measured manometrically as given in the materials and methods. - 132 -a n d t h i s r e s u l t s , f o r e x ample, i n c h a n g e s i n t h e c e r e b r a l c a t i o n c o n t e n t s , b e c a u s e o f t h e i n a b i l i t y o f t h e s o d i u m pump t o m a i n t a i n c a t i o n g r a d i e n t s a n d s o l u t e c o n c e n t r a t i o n s . W!e h a v e shown i n C h a p t e r 4.4 t h a t t h e p r e s e n c e o f TTX m a r k e d l y s u p p r e s s e s t h e e f f l u x o f amino a c i d s f r o m t h e i n c u b a t e d c e r e b r a l c o r t e x s l i c e s t h a t o c c u r s u n d e r a n a e r o b i c c o n d i t i o n s . E f f l u x o f v a r i o u s m e t a b o l i t e s a n d c a t i o n s , as a r e s u l t o f a n o x i a , may r e d u c e t h e r a t e o f g l y c o l y s i s . T e t r o d o -t o x i n was, t h e r e f o r e , t h o u g h t t o h a v e s t i m u l a t i n g e f f e c t on a n a e r o b i c g l y c o l y s i s , b y p r e v e n t i n g t h e e f f l u x o f s u c h r a t e r e g u l a t i n g s u b s t a n c e s , t h e r e b y r a i s i n g t h e i r c o n c e n t r a t i o n i n t h e c e l l . One s u c h compound m i g h t be p y r u v a t e , as t h e a d d i t i o n o f p y r u v a t e t o t h e i n c u b a t i o n medium g r e a t l y e n h a n c e s t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s . M o r e -o v e r , i t i s t h o u g h t t h a t a e r o b i c p r e - i n c u b a t i o n r e s u l t s i n a c c u m u l a t i o n o f p y r u v a t e i n t h e t i s s u e ^ l and, h e n c e , i n s u b s e -q u e n t l y h i g h e r r a t e s o f a n a e r o b i c g l y c o l y s i s . T h e r e f o r e , e x p e r i m e n t s w e r e c a r r i e d o u t t o s e e i f TTX c o u l d a c t b y p r e -v e n t i n g t h e e f f l u x o f p y r u v a t e f r o m i n c u b a t e d c e r e b r a l c o r t e x s l i c e s . R e s u l t s g i v e n i n T a b l e 12 show t h e e f f e c t s o f TTX on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s i n t h e p r e -s e n c e o f p y r u v a t e ; t h e r a t e o f a n a e r o b i c g l y c o l y s i s i s f u r t h e r i n c r e a s e d b y TTX, t h e s t i m u l a t o r y e f f e c t i n a C a + + - f r e e medium 133 TABLE 12 EFFECTS OF TETRODOTOXIN IN THE PRESENCE OF PYRUVATE ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES Additions Lactate produced ymoles per g i n i t i a l wet wt (20-80 min) Krebs-Ringer bicarbonate medium Ca ++ free medium None 2yM TTX ImM Pyruvate ImM Pyruvate + 2yM TTX lOmM Pyruvate lOmM Pyruvate + 2yM TTX 38.0 ± 4.4 65 113.4 ± 6.3 124.1 ± 5.4 31.7 ± 4.3 76 75 ± 14.9 117.9 ± 14.3 70.1 ± 12.7 128.1 ± 6.5 A l l vessel contained 20mM glucose. Additions were made at zero time and lactate production was measured manometrically, as given i n the materials and methods. - 134 -being almost a d d i t i v e . These r e s u l t s show t h a t the e f f e c t of TTX on the anaerobic g l y c o l y s i s can not be due t o i n h i b i t i o n of the e f f l u x of pyruvate from the incubated c e r e b r a l c o r t e x s l i c e s , s i n c e i n t h a t case, an a d d i t i v e e f f e c t on anaerobic g l y c o l y s i s would not have been observed. 5.4 EFFECTS OF GLUCOSE ADDITION UNDER VARIOUS CONDITIONS  ON THE STIMULATION OF ANAEROBIC GLYCOLYSIS BY  TETRODOTOXIN Experiments were c a r r i e d out t o o b t a i n more i n f o r m a t i o n concerning the c o n d i t i o n s t h a t might a f f e c t the TTX s t i m u l a t i o n of anaerobic g l y c o l y s i s i n c e r e b r a l c o r t e x s l i c e s . I t i s w e l l known t h a t very l i t t l e endogenous energy reserves are present i n the b r a i n t i s s u e and t h a t exogenous glucose i s the major source of energy (see Chapter 1 ) . I t i s p o s s i b l e t o deplete s l i c e s of ATP by i n c u b a t i n g them i n the absence of glucose or i n the presence of uncoupling agents. Experiments were c a r r i e d out t o see i f TTX i s e f f e c t i v e , under such c o n d i t i o n s , i n enhancing the anaerobic g l y c o l y s i s of such s l i c e s . R e s u l t s i n Table 13 show the e f f e c t s of a d d i t i o n of glucose a f t e r p eriods of a n a e r o b i o s i s and a e r o b i o s i s on the TTX s t i m u l a t i o n of g l y c o l y s i s . I t can be seen from these e x p e r i -ments t h a t glucose should be present before the s t a r t of a n a e r o b i o s i s t o o b t a i n an e f f e c t of TTX (Table 13A). Once the s l i c e s have been exposed t o anoxia, the g l y c o l y s i s of the c e r e -135 TABLE 13 EFFECTS OF ADDITION OF GLUCOSE UNDER VARIOUS CONDITIONS ON THE TETRODOTOXIN STIMULATION OF ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES A. 2yM TTX from s t a r t Time of addition of Glucose Lactate produced ymoles per g i n i t i a l wet wt (20-80 min) ImM Pyruvate No present from s t a r t Pyruvate Absent Present Absent Present 0 min 0 min 15 min 15 min 60.3 ± 4.5 129.5 ± 5.0 18. 8 22.3 29.0 ± 4.9 83.5 ± 10.7 17.6 ± 1.8 16.5 ± 4.0 B. 2yM TTX from s t a r t Time of addition of Glucose ImM Pyruvate from s t a r t Lactate produced ymoles per g i n i t i a l wet wt (30-90 min) Absent Present Absent Present 0 min 0 min 10 min 10 min Present Present Present Present 68.8 ± 4.5 190.2 ± 0.9 54.9 ± 2.7 180.8 ± 4.4 135A TABLE 13 (Continued) 2yM TTX from s t a r t Time of addition of Glucose ImM Pyruvate from s t a r t Lactate produced ymoles per g i n i t i a wet wt (40-100 min) Absent Present Absent Present 0 min 0 min 25 min 25 min Present Present Present Present 79.0 + 2.7 174.6 ± 3 .6 85.7 ± 1 .3 1 5 3 . 1 + 1.0 When present, f i n a l concentration of glucose was 20mM. Incubations were c a r r i e d out i n a Ca+"t- free medium. (A) Incubation was anaerobic. (B) F i r s t 10 min period was aerobic followed by anaerobiosis. (C) F i r s t 15 min period was anaerobic followed by 10 min aerobic period and subsequent anaerobiosis. Lactate production was measured manometrioally. - 136 -b r a l cortex s l i c e s i s not affected by TTX, i r r e s p e c t i v e of whether TTX i s present from the very beginning or i s added to the medium l a t e r on. However, when the preliminary incubation, without glucose i s aerobic, and i s i n the presence of pyruvate, and then glucose i s added to the incubation medium, a stimulation of g l y c o l y s i s by TTX takes place (Table 13B). The loss of response of anaerobic g l y c o l y s i s to TTX, a f t e r a period of anaerobiosis i n the absence of glucose, does not appear to be due to a permanent damage to the t i s s u e . Thus, a f t e r a period of anaerobiosis, i f the s l i c e s are exposed to oxygen b r i e f l y , then the a b i l i t y of the s l i c e s to have increased rate of g l y -c o l y s i s , i n the presence of TTX, during subsequent anaerobic periods i s regained (Table 13C). The s i g n i f i c a n c e of these experiments w i l l be discussed i n Chapter 8. 5.5 EFFECT OF AEROBIC INCUBATION WITH DINITROPHENOL ON THE  TETRODOTOXIN STIMULATION OF ANAEROBIC GLYCOLYSIS, AND  ON THE ATP LEVEL OF THE RAT CEREBRAL CORTEX SLICES From the experiments just reported i t appears that the ATP concentration i n the s l i c e s might be important for the a c t i v i t y of TTX, since t h e i r a b i l i t y to respond to TTX (after a b r i e f period of anaerobiosis) i s regained by aerobic incu-bation. Hence experiments were c a r r i e d out to define more c l e a r l y the exact r o l e of ATP i n the stimulation of anaerobic g l y c o l y s i s of cerebral cortex s l i c e s by TTX. In these experi-- 137 -merits, ATP l e v e l s were measured under a v a r i e t y of c o n d i t i o n s and these were then r e l a t e d t o the r a t e s of anaerobic g l y c o l y s i s . Table 14A shows t h a t the ATP content of a e r o b i c a l l y incubated c e r e b r a l c o r t e x s l i c e s decreases i n the presence of 2, 4 - d i n i t r o p h e n a l (DNP). When the s l i c e s are incubated anaero-b i c a l l y f o r 15 min f o l l o w e d by 10 min aerobic i n c u b a t i o n , the subsequent ATP l e v e l i s r e l a t i v e l y h i g h (Table 14B). A f u r t h e r anaerobic p e r i o d reduces the ATP l e v e l s i g n i f i c a n t l y (Table 14C). However, the ATP l e v e l i n the s l i c e s exposed t o TTX, i s h i g h e r both i n the presence or absence of DNP than i n those not so exposed. From the data on subsequent r a t e s of anaerobic g l y c o l y s i s under these as w e l l as under s i i g h t l y d i f f e r e n t c o n d i t i o n s (see Table 14C), i t can be seen t h a t the r a t e of anaerobic g l y c o l y s i s of the DNP-treated s l i c e s i s s l i g h t l y lower than those which were not exposed t o i t . The r a t e s of g l y c o l y -s i s are h i g h e r when pyruvate i s present from the beginning of the experiment b e f o r e the a d d i t i o n of glucose. When glucose i s added a f t e r the aer o b i c i n c u b a t i o n p e r i o d , the r a t e s of g l y -c o l y s i s are lower than t h a t of those s l i c e s , incubated i n a medium, t o which glucose had been added before the aer o b i c p e r i o d . The s i g n i f i c a n c e of these r e s u l t s w i l l be d i s c u s s e d i n Chapter 8. 138 TABLE 14 EFFECTS OF INCUBATION WITH 2,4-DINITROPHENOL (DNP) ON THE TETRODOTOXIN STIMULATION OF ANAEROBIC GLYCOLYSIS AND THE ATP CONTENTS OF RAT CEREBRAL CORTEX SLICES A. E f f e c t of DNP on the ATP concentration of cerebral cortex s l i c e s incubated a e r o b i c a l l y for one hour i n a Krebs-Ringer phosphate medium. DNP was added at zero time and the medium contained 20mM glucose. ATP Additions . . . , . , . . ymoles per g m i t r a l wet wt None 1.57 O.lmM DNP 0.66 B. E f f e c t s of DNP on the ATP concentration i n the presence of TTX. Incubations were c a r r i e d out for 25 minutes i n a C a + + free medium containing 20mM glucose. F i r s t 15 min period was anaerobic followed by 10 min aerobic period. Additions were made at zero time. Additions ATP ymoles per g i n i t i a l wet wt None 1.48 O.lmM DNP 1.16 2 yM TTX 1.42 O.lmM DNP 1 7 + 2yM TTX 138A TABLE 14 (Continued) C. E f f e c t s of DNP and TTX on the ATP content and anaerobic g l y c o l y s i s of cerebral cortex s l i c e s . Incubations were c a r r i e d out i n a C a + - free medium. F i r s t 15 min period was anaerobic followed by 10 min aerobic period and subsequent anaerobiosis. Lactate production was measured manometrically from 40-10 0 min. TTX and DNP were present from zero time. Lactate produced i s expressed as ymoles of lactate per g i n i t i a l wet wt of the s l i c e s . Addition No Pyruvate ATP content at 35 min ymoles per g i n i t i a l wet wt Lactate produced Glucose at zero time ImM Pyruvate from s t a r t Lactate produced Glucose added at 15 min Lactate produced Glucose added at 25 min None DNP, O.lmM TTX, 2yM TTX, 2uM + DNP, O.lmM 0.45 0. 43 0.64 0.59 37.9 ± 1.78 26.8 ± 1.3 146 ± 1.3 117 ± 6.3 114.7 ± 3.1 86.6 ± 9.8 79.5 ± 8.0 49.6 ± 7.1 178.6 ± 7.0 159.8 ± 6.7 136.2 ± 2.2 101 ±16.9 - 139 -5 .6 EFFECTS OF TETRODOTOXIN AND OUABAIN ON THE ATP  CONTENT OF GUINEA PIG CEREBRAL CORTEX SLICES As has been mentioned i n Chapter 4, the concentrations of a c i d l a b i l e phosphates i n the incubated cerebral cortex s l i c e s are r a i s e d i n the presence of both TTX and ouabain. Under these conditions the rates of g l y c o l y s i s are also higher. Table 1 5 shows the e f f e c t of ouabain and TTX on the ATP contents of the incubated cerebral cortex s l i c e s . I t can be seen that i n the presence of both these drugs the l e v e l of ATP i n the s l i c e s i s increased. These r e s u l t s w i l l be further discussed i n Chap-te r s 6 and 8 . 5 . 7 EFFECTS OF RAISING ATP LEVEL BY AEROBIC INCUBATION  WITH ADENOSINE ON THE TETRODOTOXIN STIMULATED  GLYCOLYSIS OF THE RAT CEREBRAL CORTEX SLICES Abadom and S c h o l e f i e l d and o t h e r s 2 4 5 , 2 9 5 have reported an increase i n the ATP (7 min phosphate) content when the cerebral cortex s l i c e s are incubated a e r o b i c a l l y i n the presence of adenosine. Experiments were c a r r i e d out, therefore, to see i f s l i c e s treated i n such a manner show more responsiveness to the e f f e c t s of TTX on the anaerobic g l y c o l y s i s . In these experiments (see Table 1 6 ) , the s l i c e s were pre-incubated a e r o b i c a l l y f o r 40 min i n a Krebs-Ringer bicarbonate s o l u t i o n containing 1 RM adenosine and 20niM glucose; subsequently the s l i c e s were trans f e r r e d to another set of vessels containing TTX. 140 TABLE 15 EFFECTS OF TETRODOTOXIN AND OUABAIN ON THE ATP CONTENT OF GUINEA PIG CEREBRAL CORTEX SLICES UNDER ANOXIA A d d i t i o n , .. .. . y m o l e s p e r g i n i t i a l wet wt. None 0.46 ± 0.06 o u a b a i n , lOyM 0.71 + 0.05 TTX, 2yM 0.6 8 ± 0.02 o u a b a i n , lOyM n K n _ n Q + ImM C a + + ° - 5 0 1 ° ' 0 9 TTX, 2yM n en + n n + ImM C a + + ° - 6 0 1 I n c u b a t i o n s were c a r r i e d o u t f o r 9 0 m i n u n d e r N 2:C02 i n a Ca ++- f r e e medium c o n t a i n i n g 20mM g l u c o s e . ATP was m e a s u r e d e n z y m a t i c a l l y , a s d e s c r i b e d i n t h e m a t e r i a l s and m e t h o d s . 141 TABLE 16 EFFECT OF AEROBIC PREINCUBATION IN ADENOSINE ON THE TETRODOTOXIN STIMULATION OF ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES Addition Lactate produced ymoles per g i n i t i a l wet wt (20-80 min) s l i c e s not incubated with adenosine s l i c e s incubated with adenosine None 80.4 ± 6.7 102.1 ± 4.5 2yM TTX, present from zero time 132 ± 1.5 136.1 ± 4.0 TTX added a f t e r 15 min anaerobiosis, 2yM f i n a l concentration 84. 8 92.2 Cerebral cortex s l i c e s were incubated for 40 min under 02:C0 2 i n a Krebs-Ringer bicarbonate medium containing 20mM glucose. They were then transferred to a C a + i free medium containing 20mM glucose, with or without TTX. Anaerobic g l y c o l y s i s was then measured manometrically during subsequent 20-80 min anaerobic period, as described i n the materials and methods. - 142 -The anaerobic g l y c o l y s i s was then measured. These r e s u l t s with the adenosine treated s l i c e s sr>w that there i s no increase i n response to TTX, although the g l y c o l y t i c rates are higher than that of the non-treated s l i c e s . When TTX i s added, a f t e r 15 min anaerobiosis to the adenosine treated s l i c e s , there i s no accel e r a t i n g e f f e c t on the rate of anaerobic g l y c o l y s i s . 5.8 EFFECTS OF TETRODOTOXIN ON cAMP PRODUCTION IN CEREBRAL  CORTEX SLICES C y c l i c AMP has been shown to increase the rate of aerobic as we l l as anaerobic g l y c o l y s i s i n brain (Chapter 3). Further, Mansour and Stone have shown that some drugs such as LSD-25 increase the production of cAMP i n l i v e r flukes, and t h i s i n turn increases the rate of l a c t a t e production, presumably by a c t i v a t i o n of phosphofructokinase a c t i v i t y . Mcllwain and h i s coworkers^ 0^ established that when the cerebral cortex s l i c e s are e l e c t r i c a l l y stimulated, formation of cAMP i n the s l i c e s i s considerably increased. As has been mentioned i n Chapter 1, e l e c t r i c a l stimulation also increases the rate of aerobic g l y c o l y s i s . Hence experiments were c a r r i e d out to see i f the stimulation of anaerobic g l y c o l y s i s of cerebral cortex s l i c e s by TTX i s due to increased formation of cAMP. I f TTX increases the formation of cAMP then i t may f a c i l i t a t e the phosphofruc-tokinase step, e s p e c i a l l y at the beginning of the experimsnt, by decreasing the degree of i n h i b i t i o n of phosphofructokinase - 143 -by ATP; at a l a t e r stage, when ATP l e v e l decreases, i t should not have any e f f e c t . The increased rate of g l y c o l y s i s at the s t a r t of the experiment w i l l tend to keep the rate of decline of ATP content slow, and t h i s may r e s u l t i n an increased rate of g l y c o l y s i s . O C^. A We used the method of Shimizu et.aJL. to measure the production of cAMP. i n t h i s method, as described i n Chapter 2, the cerebral cortex s l i c e s are f i r s t pre-incubated i n oxygen i n the presence of C14 -adenine to get a pool of C 1 4-ATP. The e f f e c t of drugs on cAMP can be e a s i l y measured by observing the rates of conversion of C 1 4-ATP to C14-cAMP under various condi-t i o n s . Results i n Table 17A show that the rates of incorpora-t i o n of adenine-8-C 1 4 i n the cerebral cortex s l i c e s as w e l l as i n i t s ATP pool increases with time. The observation of Shimizu e t . a l . 2 ^ 4 that histamine greatly increases the production of cAMP, was confirmed. However, any increased production of cAMP i n the presence of TTX could not be observed (Table 17B). This shows that the e f f e c t of TTX on the anaerobic g l y c o l y s i s of the cerebral cortex s l i c e s i s not due to an increase i n the concen-t r a t i o n of cAMP i n the b r a i n c e l l s . 5.9 EFFECT OF PROTOVERATRINE ON THE TETRODOTOXIN  STIMULATED GLYCOLYSIS I t i s known that protoveratrine increases the i n f l u x of Na + i n t o excitable c e l l s . 7 2 ' 1 3 5 On the other hand, TTX i s 144 TABLE 17 EFFECTS OF TETRODOTOXIN ON THE cAMP FORMATION IN THE CEREBRAL CORTEX SLICES A. Incorporation of adenine-8-C 1 4 sulfate into r a t cerebral cortex s l i c e s and i t s ATP pool. Cerebral cortex s l i c e s were incubated i n Krebs-Ringer bicarbonate containing 20mM glucose and 2yc.of adenine-8-C 1 h s u l f a t e (51.5 mc/mM). At the end of the incubation s l i c e s were homogenized i n 5% TCA. After deproteinization, TCA was removed with ether and a portion was counted for t o t a l r a d i o a c t i v i t y while another portion was spotted with c a r r i e r ATP on a PEI c e l l u l o s e plate and developed i n IM L i C l ; r a d i o a c t i v i t y i n ATP spots were determined^after scrapping, i n a Mark I l i q u i d s c i n t i l l a t i o n counter. Incubation time i n min Adenine-8-C 1 1 4 incorporated, x l O 4 c.p.m. per g i n i t i a l wet wt Total ATP 5 171 5.0 10 288 16.9 20 471 42.7 40 1043 144A TABLE 17 ( C o n t i n u e d ) B. C o n v e r s i o n o f C 1 1 ATP t o cAMP i n g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d as i n A. A f t e r 40 rain, t h e p u l s e l a b e l l e d s l i c e s were t r a n s f e r r e d t o a Ca +t_ f r e e medium c o n t a i n i n g ImM c a f f e i n e a n d 20mM g l u c o s e , w i t h and w i t h o u t 2yM TTX. I n c u b a t i o n s were c a r r i e d o u t f o r a n o t h e r 10 m i n i n N 2 : C 0 2 and t o t a l r a d i o a c t i v i t y i n t h e s l i c e s as w e l l as t h a t i n cAMP was d e t e r m i n e d as g i v e n i n t h e m a t e r i a l s and m e t h o d s . c.p.m. p e r g i n i t i a l wet wt A d d i t i o n s T o t a l , x l O 5 cAMP, x l O 3 ImM c a f f e i n e 103 ± 7 159 ± 26 ImM c a f f e i n e + 2yM TTX 102 ± 8 95 ± 45 - 145 -known to block the generation of the action p o t e n t i a l s i n such c e l l s (Chapter 1). As noted by Wollenberger, protovera-t r i n e i n h i b i t s the rate of anaerobic g l y c o l y s i s of cerebral cortex s l i c e s . In view of t h e i r opposite e f f e c t s on the anaerobic g l y c o l y s i s of cerebral cortex s l i c e s , i t was decided to study t h e i r e f f e c t s together. Results of such an experiment are shown i n Figure 22. I t i s evident that the e f f e c t s of these drugs are antogonistic. Thus 5 uM TTX protoveratrine i n h i b i t s the enhanced g l y c o l y s i s caused by 2 yiM TTX to a con-siderable extent. When the concentration of protoveratrine was further increased, up to 20 uM, the e f f e c t of TTX i s com-p l e t e l y abolished (results not shown). 5.10 EFFECTS OF TETRODOTOXIN ON THE Na-22 TRANSPORT IN  THE RAT CEREBRAL CORTEX SLICES I t has been well established that the movement of Na + during the generation of an action p o t e n t i a l i s blocked by TTX (Chapter 1). Further, both glutamate and protoveratrine reverse the stimulating e f f e c t of TTX on the anaerobic g l y c o l y -s i s (Chapters 4.8 and 5.9). These two agents are knownto increase the i n f l u x of Na + i n the cerebral cortex s l i c e s . More-over, Na + has i n h i b i t o r y e f f e c t on the rate of anaerobic g l y c o l y s i s (Chapters 1.2 and 3.7). Hence, i t appeared to us that TTX might act by preventing the i n f l u x of Na +, i n the - 146 -FIGURE 22 EFFECTS OF TETRODOTOXIN,IN THE PRESENCE OF PROTOVERATRINE,ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES 60 ' , Time, i n minutes Incubations were c a r r i e d out i n a C a + + - f r e e medium containing 20 mM glucose.Additions were made at zero time and lactate production was measured manomtrically as given i n the materials and methods.(O)control; ( A ) 5 uM protoveratrine;( • )2 uM TTX; ( B ) 5 uM protoveratrine and 2 uM TTX. - 147 -c e r e b r a l c o r t e x s l i c e s , a t t h e o n s e t o f a n o x i a . I t was d e c i d e d , t h e r e f o r e , t o s t u d y t h e i n f l u x o f N a 2 2 i n t h e p r e s e n c e and a b s e n c e o f TTX. The r e s u l t s o f t h e s e e x p e r i m e n t s a r e s u m m a r i z e d i n Table 18. T h e s e r e s u l t s show t h a t TTX h a s v e r y l i t t l e e f f e c t pp on t h e Na"" i n f l u x u n d e r t h e g i v e n e x p e r i m e n t a l c o n d i t i o n s . I f t h e e f f e c t o f TTX i s due o n l y t o t h e s u p p r e s s i o n o f N a + - i n f l u x , t h e n r e p l a c i n g t h e c h l o r i d e o f t h e i n c u b a t i o n medium b y s u l f a t e s h o u l d r e s u l t i n g r e a t e r r a t e o f a n a e r o b i c g l y c o l y s i s ( i t i s known t h a t t h e i n f l u x o f N a + due t o a n a e r o -b i o s i s i s s u p p r e s s e d when C l " i s r e p l a c e d b y S 0 4 2 9 8 ) ; TTX s h o u l d h a v e l e s s e f f e c t u n d e r t h e s e c o n d i t i o n s . The r e s u l t s o f t h e s e e x p e r i m e n t s a r e g i v e n i n T a b l e 19. I t i s s e e n t h a t T T X i s s t i l l e f f e c t i v e i n i n c r e a s i n g t h e r a t e o f a n a e r o b i c g l y c o l y s i s . M o r e o v e r t h e r a t e o f a n a e r o b i c g l y c o l y s i s i n t h e c o n t r o l s l i c e s i s n o t v e r y l a r g e a s compared t o t h a t i n t h e C l ~ medium. T h i s e x p e r i m e n t t h u s r u l e s o u t t h e p o s s i b i l i t y t h a t t h e e f f e c t o f TTX on c e r e b r a l a n a e r o b i c g l y c o l y s i s i s s o l e l y due t o s u p p r e s s i o n o f t h e N a + - i n f l u x . F u r t h e r m o r e , t h e e f f e c t o f TTX on N a 2 ^ t r a n s p o r t i n a s u l f a t e medium i s n e g l i g i b l e . T h e r e i s v e r y l i t t l e w a t e r u p t a k e b y t h e c e r e b r a l c o r t e x s l i c e s , u n d e r t h e s e c o n d i t i o n s , i n d i c a t i n g t h e s u p p r e s s i o n o f N a + i n f l u x ( n o t s h o w n ) . 148 TABLE 18 EFFECT OF SOME NEUROTROPIC DRUGS ON Na 2 2 INFLUX IN RAT CEREBRAL CORTEX SLICES Additions yequivalents Na +, corresponding to Na 2 2 i n s l i c e s , per g i n i t i a l wet wt 10 min 15 min 60 min None 0.2yM TTX 2yM TTX lOyM TTX O.lmM Lidocaine 0.25mM Amytal lOyM ouabain 170 + 13 162 ± 10 151 ± 7 177 ± 4 151 ± 8 149 ± 5 143 208 ± 12 189 ± 2 186 ± 2 179 ± Cerebral cortex s l i c e s were incubated i n a Ca free medium containing 20mM glucose under N 2:C0 2. Additions were made at zero time. After 15 min, 0.5 yc of Na 2 2 was added from the side arm and Na 2 2 i n f l u x was determined a f t e r various i n t e r v a l s , (10 min, 15 min or 60 min), as given i n the materials and methods. The above values have not been corrected for swelling. 149 TABLE 19 EFFECTS OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS AND N a 2 2 TRANSPORT IN RAT CEREBRAL CORTEX S L I C E S IN A CHLORIDE FREE MEDIUM L a c t a t e p r o d u c e d y e q u i v a l e n t N a + A d d i t i o n s y m oles p e r g i n i t i a l c o r r e s p o n d i n g t o N a 2 2 , wet wt (20-80 min) p e r g i n i t i a l wet wt None 14.7 ± 1 . 0 6 1 . 5 ± 1 . 5 2yM TTX 38.0 ± 1.8 60.0 ± 1.7 C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d i n a C l ~ f r e e medium c o n t a i n i n g 20mM g l u c o s e . L a c t a t e p r o d u c t i o n was d e t e r m i n e d m a n o m e t r i c a l l y as g i v e n i n t h e m a t e r i a l s and methods. F o r N a 2 2 i n f l u x e x p e r i m e n t s , c o n d i t i o n s were same as T a b l e 18 e x c e p t t h a t i n c u b a t i o n t i m e i n N a 2 2 was 15 m i n . - 150 -5.11 EFFECTS OF TETRODOTOXIN, AT VARIOUS CATION CONCEN-TRATIONS OF THE MEDIUM, ON THE ANAEROBIC GLYCOLYSIS  OF CEREBRAL CORTEX SLICES Ashford and D i x o n 6 2 showed that, i n the presence of 100 mM K +, both r e s p i r a t i o n and aerobic g l y c o l y s i s of the cere-b r a l cortex s l i c e s are increased. However, i n contrast to aerobic g l y c o l y s i s , anaerobic g l y c o l y s i s i s depressed (see Chapter 1.2). We have seen i n Chapter 3 that when K + concen-t r a t i o n i s increased with a corresponding decrease i n Na + con-centration, there i s a considerable increase i n the rate of anaerobic g l y c o l y s i s . Experiments were c a r r i e d out, therefore, to observe whether under these varying cation concentrations, TTX i s s t i l l e f f e c t i v e i n stimulating the rate of anaerobic g l y c o l y s i s of the cerebral cortex s l i c e s . Table 20 shows the e f f e c t of TTX on the anaerobic g l y c o l y s i s of r a t cerebral cortex s l i c e s i n a C a + + - f r e e medium, to which a d d i t i o n a l K + has been added. I t i s evident from these r e s u l t s that, when the K + concentration i s increased without decreasing the corresponding Na + concentration, TTX i s not e f f e c t i v e i n increasing the rate of anaerobic g l y c o l y s i s . However, when Na + concentration i s decreased i n proportion to the increase of K + concentration, then the rate of g l y c o l y s i s i s increased by TTX (Table 21). Under these conditions, TTX has progressively less e f f e c t as Na + i s decreased u n t i l a 151 TABLE 20 EFFECT OF PRESENCE OF DIFFERENT CONCENTRATION OF K + ON THE TETRODOTOXIN STIMULATION OF ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES K +, added to Lactate produced C a + + free ymoles per g i n i t i a l wet wt (20-80 min) medium, i n mM No TTX 2yM TTX 0 26.3 ± 0.4 77.7 ± 1.8 33.3 19.7 ± 1.3 21.0 ± 1.3 66.6 20.1 ± 3.6 24.5 ± 2.4 100 21.0 ± 0.9 23.2 ± 2.7 Cerebral cortex s l i c e s were incubated i n a Ca -free medium containing 20mM glucose. Additions were made at zero time and lactate production was measured manometrically as given i n the materials and methods. 152 TABLE 21 EFFECTS OF TETRODOTOXIN AT VARYING CATION CONCENTRATION OF THE MEDIUM ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES L a c t a t e p r o d u c e d N a + K + 2yM y m o l e s p e r g i n i t i a l wet wt TTX (20-80 min) 149 5 0 21 149 5 + 72 89 60 0 59 89 60 + 70 29 60 0 127 29 60 + 144 29 125 0 107 29 125 . + 130 0 154 0 115 0 154 + • 114 H i g h p o t a s s i u m medium was p r e p a r e d by r e p l a c i n g some o r a l l N a + by K + as d e s c r i b e d i n C h a p t e r 2.5 f . Medium c o n t a i n e d 20mM g l u c o s e , TTX was add e d a t z e r o t i m e and l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i -c a l l y . V a l u e s a r e a v e r a g e s o f two d e t e r m i n a t i o n s w i t h i n ± 7%. - 153 -maximum r a t e of g l y c o l y s i s i s obtained when most of the Na"1" was re p l a c e d by K + ( T a b l e 21). When the r a t e of g l y c o l y s i s i s very h i g h ( i . e . , N a + i s completely r e p l a c e d by K + ) , then other agents such as C a + + showed no s t i m u l a t o r y e f f e c t . As the r a t e of anaerobic g l y c o l y s i s i n the presence of h i g h c o n c e n t r a t i o n s of K + ( i n presence of 149 111M Na +) i s not i n f l u e n c e d by TTX, i t was decided t o study t h e i r e f f e c t s at a l a t e r stage i . e . , when the e f f e c t of TTX has a l r e a d y been e s t a b l i s h e d . The r e s u l t s of these experiments are shown i n Fi g u r e 23. I t i s evident t h a t , when s o l u t i o n s of K +, N a + or L i + are t i p p e d i n from the s i d e arm of the Warburg v e s s e l g i v i n g a f i n a l c o n c e n t r a t i o n of 100 mM ( i n a d d i t i o n t o t h a t a l r e a d y p r e s e n t ) , the r a t e of g l y c o l y s i s , which i s h i g h due t o the presence of TTX, i s reduced s i g n i f i c a n t l y . Of the c a t i o n s t e s t e d , K + i s the most e f f e c t i v e , f o l l o w e d by L i + and Na +, i n depressing the TTX-stimulated g l y c o l y s i s . Under same c o n d i t i o n s t h e r e i s an in c r e a s e d i n f l u x of N a 2 2 ( p r e l i m i n a r y r e s u l t s ) . The e f f e c t of c a t i o n s on anaerobic g l y c o l y s i s does not appear t o be due t o changes i n the t o n i c i t y s i n c e a d d i t i o n s of sucrose, a t 66, 133, or 200 mM f i n a l c o n c e n t r a t i o n s , have l i t t l e or no e f f e c t on the r a t e of anaerobic g l y c o l y s i s of b r a i n s l i c e s (pre-l i m i n a r y experiments). The l a c k of e f f e c t of TTX i n the presence of h i g h K + and 149 mM N a + may be due t o inc r e a s e d i n f l u x of Na +. This w i l l be f u r t h e r d i s c u s s e d i n Chapter 8. - 154 -FIGURE 23 EFFECTS OF ADDITION OF HIGH CONCENTRATIONS OF CATIONS ON THE TETRODOTOXIN STIMULATED ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES Time , i n minutes Incubations were c a r r i e d out i n a C a + + - f r e e medium containing 20 mM glucose.Arrow points out time at which 1 M solution of KCl,NaCl or L i C l was tipped i n so as to give a f i n a l concentration of 100 mM.Lactate production was measured manometrically,as given i n the materials and methods.(—•) control, ( )2 uM TTX from zero time,no cation was added; ( ) 2 uM TTX from zero- time, Na was tipped i n ; ( )2 uM TTX from z<pro time,Li was tipped i n ; ( )2 p.M TTX from zero time,K was tipped i n . - 155 -5.12 EFFECTS OF TETRODOTOXIN ON THE Na + and K+ LEVELS OF  INCUBATED CEREBRAL CORTEX SLICES UNDER ANOXIA I t i s we l l known that the e f f e c t s of e l e c t r i c a l stimu-l a t i o n on cation movements i n incubated brain s l i c e s are blocked by TTX, as shown by i t s metabolic e f f e c t s ^ 2 ^ ' l3<-> a n G : i t s e f f e c t s on the i n f l u x of N a + 1 3 2 and on the e f f l u x of K + 1 2 9 ' 1 3 1 . Also, as noted (see Chapter 1), K + stimulate pyruvate k i n a s e 6 7 , 3 ® ^ while Na + i n h i b i t s both h e x o k i n a s e 5 3 a and pyruvate kinase.37-39. In view of these marked e f f e c t s of these cations on the metabolism of cerebral t i s s u e , i t seemed quite possible that these might play an important r o l e , under the conditions of anoxia, when action p o t e n t i a l s might be generated. TTX could g r e a t l y influence the K +/Na + r a t i o of the cerebral cortex s l i c e s by blocking the movements of these cations. An increased K +/Na + r a t i o i n the c e l l w i l l r e s u l t i n a greater rate of anaerobic g l y c o l y s i s . However, as already mentioned (see 5.9), we have been unable to detect large changes i n the Na 2 2 i n f l u x under anoxia i n the presence of TTX. In view of t h i s , i t was considered necessary to measure both the K + and Na + contents of incubated cerebral cortex s l i c e s under anoxia. The r e s u l t s of these experiments are described below. The e f f e c t s of 2 uM TTX on the Na + and K + l e v e l i n the guinea p i g cerebral cortex s l i c e s , incubated under anoxic conditions, are shown i n Figure 24. I t w i l l be seen that, i n - 156 -FIGURE 24 EFFECTS OF TETRODOTOXIN ON THE SODIUM AND POTASSIUM CONCENTRA-TIONS OF GUINEA PIG CEREBRAL CORTEX SLICES UNDER ANOXIA % 3 0 0 -0 10 20 30 40 50 60 70 80 Time,in minutes Incubations were c a r r i e d out i n a C a + + - f r e e medium containing 2Q_ mM glucose.TTX,when present,was added at zero time.Na and K contents were determined as given in the materials and methods. ( "j-Na, con t r o l ; ( O )Na ,with 2 pM TTX; ( • )K ,control; ( • ) K,with 2 uM TTX. - 157 -the presence of TTX, the amount of K + r e t a i n e d i n the s l i c e s i s t w i c e the c o n c e n t r a t i o n i n the c o n t r o l s . At the same time, there i s a r e d u c t i o n i n the amount of Na +. S i m i l a r r e s u l t s on the K + content are obtained w i t h r a t c e r e b r a l c o r t e x s l i c e s . These r e s u l t s make i t reasonable t o conclude t h a t the e f f e c t of TTX on the anaerobic g l y c o l y s i s of the c e r e b r a l c o r t e x s l i c e s may be due t o an i n c r e a s e i n the K +/Na + r a t i o . 5.13 EFFECTS OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS  AND N a 2 2 TRANSPORT IN THE CAUDATE NUCLEUS OF RAT As TTX i s b e l i e v e d t o act o n l y on e x c i t a b l e c e l l s , i t i s l i k e l y t h a t the e f f e c t of TTX w i l l be c o n f i n e d t o the neurons. Caudate nucleus i s a p a r t of b r a i n which has r e l a -t i v e l y few g l i a l c e l l s . Experiments were c a r r i e d out, t h e r e f o r e , on the e f f e c t s of TTX on the r a t e of anaerobic g l y c o l y s i s and on N a 2 2 t r a n s p o r t i n the caudate nucleus. R e s u l t s of these experiments are shown i n Table 22. The anaerobic g l y c o l y s i s of caudate nucleus i s in c r e a s e d by the presence of TTX but the magnitude of s t i m u l a t i o n i s not g r e a t e r than t h a t observed w i t h the c e r e b r a l c o r t e x s l i c e s . Moreover, TTX has no e f f e c t on the N a 2 2 i n f l u x . These r e s u l t s w i l l be f u r t h e r d i s c u s s e d i n Chapter 8. 5.14 EFFECTS OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF  SYNAPTOSOMAL PREPARATIONS OF RAT BRAIN As mentioned e a r l i e r , 2 pM TTX a c c e l e r a t e s the r a t e of anaerobic g l y c o l y s i s of a d u l t r a t s and guinea pigs but not 158 TABLE 22 EFFECTS OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS AND Na22.TRANSPORT IN CAUDATE NUCLEUS OF RAT Additions Lactate produced ymoles per g i n i t i a l wet wt (20-80 min) yequivalent Na, corresponding to Na 2 2, per g i n i t i a l wet wt None 26.8 ± 1.0 1 0 1 + 2 2yM TTX 62.5 + 5.8 104 ± 2 Incubations were ca r r i e d out i n a medium containing 20mM glucose. TTX was added at zero time and lactate production was measured manometrically, as given i n the materials and methods. For Na 2 2 experiments, conditions were same as i n Table 18 except that incu-bation time i n Na 2 2 was 15 min. - 159 -of 2-day o l d r a t b r a i n . The s e n s i t i v i t y of the r a t b r a i n s l i c e s t o TTX i n c r e a s e s c o n s i d e r a b l y at about 14th day a f t e r b i r t h , which c o i n c i d e s w i t h the time of maximum b r a i n growth and m y e l i n a t i o n . I t has a l s o been mentioned (Chapter 1.6) t h a t the O c: O O C A nerve endings are not myelinated. W h i t t a k e r ^ h a s developed techniques by which i t i s p o s s i b l e t o separate the nerve endings from other s u b c e l l u l a r p a r t i c l e s . As the nerve endings and r e c e p t o r s are developed i n the b r a i n d u r i n g maturation, e x p e r i -ments were c a r r i e d out t o see whether the anaerobic g l y c o l y s i s of the nerve ending p a r t i c l e s i s a f f e c t e d by TTX and other drugs. R e s u l t s of these experiments are shown i n F i g u r e 25. These experiments showed t h a t TTX and ouabain have l i t t l e or no e f f e c t on the r a t e of anaerobic g l y c o l y s i s of synaptosomes, w h i l e 4 ~-M C a + + has an i n h i b i t o r y a c t i o n . 5.15 EFFECTS OF PRE-INCUBATION IN OXYGEN ON THE K + AND Na +  CONTENTS OF CEREBRAL CORTEX SLICES UNDER ANOXIA IN  THE PRESENCE OF TETRODOTOXIN B r i e f a e r o b i c p r e - i n c u b a t i o n of b r a i n s l i c e s i n c r e a s e s the subsequent r a t e of anaerobic g l y c o l y s i s , both i n the presence and absence of TTX, as compared t o t h a t obtained w i t h the non-oxygenated s l i c e s ( S e c t i o n 5.1). Experiments were c a r r i e d out, t h e r e f o r e , t o evaluate the e f f e c t s of short (10 min) a e r o b i c p r e - i n c u b a t i o n on the Na + and K + content of the c e r e b r a l c o r t e x s l i c e s i n the presence of TTX. I t i s evident from the r e s u l t s FIGURE 25 EFFECTS OF TETRODOTOXIN,CALCIUM AND OUABAIN ON THE ANAEROBIC GLYCOLYSIS OF RAT SYNAPTOSOMES Incubations were c a r r i e d out i n a Ca -free medium containing 20 mM glucose.Additions were made at zero time and lactate production was measured manometrically as given i n the materials and methods.( • )control; (A)10 uM ouabain; (O) 2 uM TTX ; ( B ) 4 mM Ca4".4" - 160 -FIGURE 25 30 40 50 60 70 80 Time,in minutes - 161 -shown i n F i g u r e 26 t h a t the r e t e n t i o n of K i n the oxygenated s l i c e s d u r i n g the subsequent p e r i o d of anoxia i s much g r e a t e r . I n the presence of 2 uM of TTX, there i s ve r y l i t t l e l o s s of K + and l e s s g a i n of N a + d u r i n g the anaerobic i n c u b a t i o n . There i s an i n i t i a l drop i n the K + content d u r i n g the p r e l i m i -nary a e r o b i c p e r i o d . The e f f e c t of C a + + on the K + l e v e l i s a l s o shown i n the same f i g u r e . I t i s c l e a r t h a t there i s some in c r e a s e d r e t e n t i o n of K + by the s l i c e s i n the presence of C a + + , but i t was not as e f f e c t i v e as TTX. Because the r e s u l t s w i t h c a t i o n contents are more c l e a r cut i n the oxygenated s l i c e s , i n a l l subsequent experiments r e p o r t e d below the technique of p r i o r oxygenation was used t o study the r e t e n t i o n o f K + and uptake of Na + by incubated b r a i n s l i c e s . 5.16 EFFECTS OF TETRODOTOXIN ON THE N a + AND K + LEVELS OF  INFANT RAT AND GUINEA PIG CEREBRAL CORTEX SLICES R e s u l t s of experiments on the e f f e c t of TTX on the anaerobic g l y c o l y s i s of developing b r a i n c o r t e x s l i c e s have been g i v e n i n Chapter 4.12. I t was of i n t e r e s t , t h e r e f o r e , t o observe whether TTX a f f e c t s N a + and K + contents of the i n f a n t r a t , as w e l l as i n f a n t guinea p i g , c e r e b r a l c o r t e x s l i c e s . The r e s u l t s of these experiments are shown i n Fig u r e s 27 and 28. I t i s evident t h a t TTX has l i t t l e or no e f f e c t on the c a t i o n content of i n f a n t (2-day old) r a t b r a i n s l i c e s but the i n f a n t - 162 -FIGURE 26 EFFECTS OF TETRODOTOXIN AND CALCIUM ON THE SODIUM AND POTASSIUM CONCENTRATIONS OF RAT' CEREBRAL CORTEX SLICES 300 250 . 4 J tj 200 i H (0 •H -P •H C • H 150 U <u Ch cn +» g 100 H > •H d (D 3_ O 50 -0 5 ( AEROBIC 10 5 10 ) ( A N A E R O B I C Time, i n m i n u t e s 30 ++ I n c u b a t i o n s were c a r r i e d o u t i n a Ca - f r e e medium c o n t a i n i n g 20 mM g l u c o s e . I n i t i a l 10 min p e r i o d was a e r o b i c ( 0 _ : C O _ ) f o l l o w e d by a n a e r o b i c p e r i o d ( N 2 : C 0 2 ^ - A d d i t i o n s were made a t + z e r o t i m e . V e r t i c a l b a r s r e p r e s e n t s t a n d a r d d e v i a t i o n s . ( • ) N a , c o n t r o l ; (• ]Na, . w i t h 2 uM TTX; ( O ) K +, c o n t r o l ; ( • ) K + , w i t h 2 uM TTX. (A ) 4mM Ca ,K . r - 163 -175 150 -4-» +j 1 2 5 CD f0 •H +J •H 100 n CD to +J c CD > •H 3 cr CD 3 . 75 -r 50 O f 2 25 0 5 (AEROBIC B ) 10 5 10 ) (A N A E R 0 Time,in minutes FIGURE 27 : EFFECTS OF TETRODOTOXIN ON THE SODIUM AND POTASSIUM CONCENTRATIONS OF NEWLY BORN GUINEA PIG CEREBRAL CORTEX SLICES. + I n c u b a t i o n c o n d i t i o n s were same as i n F i g u r e 2 6 . ( A ) N a , c o n t r o l ; ( A ) Na ,with 2 uM TTX; .(©) K + , c o n t r o l ; ( O ) K + , w i t h 2 uM TTX. - 164 -FIGURE 28 EFFECTS OF TETRODOTOXIN ON THE SODIUM AND POTASSIUM CONCENTRA-TIONS OF TWO DAY OLD RAT CEREBRAL CORTEX SLICES 0 5 10 5 10 30 ( AEROBIC )( A N A E R O B I C ) Time,in minutes Incubation conditions were same as i n Figure 26 . ( • )Na*. control; (•)Na ,with 2 uM TTX; ( A ) K , control; K ,with 2 pM TTX ( A )• - 165 -guinea p i g shows a marked i n c r e a s e i n the K + content, and a decrease i n N a + content, i n the presence of TTX. 5.17 EFFECTS OF TETRODOTOXIN ON THE Na + and K + LEVELS OF  KIDNEY MEDULLA SLICES . Kidney medulla was used as a c o n t r o l t i s s u e (Chapter 4.11). As has been shown i n F i g u r e 29, TTX has no e f f e c t on the K + and Na + contents of the kidney medulla s l i c e s . This r e s u l t i s c o n s i s t e n t w i t h the c o n c l u s i o n t h a t the e f f e c t of TTX i s s p e c i f i c t o the nervous t i s s u e . 5.18 EFFECTS OF TETRODOTOXIN IN THE PRESENCE OF CHELATING  AGENTS ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL  CORTEX SLICES Chan and Quastel-^O have shown t h a t the i n c r e a s e i n r e s p i r a t i o n brought about by the absence of C a + + i s prevented by TTX. We have a l s o seen, i n Chapter 4, t h a t TTX a c t s l i k e C a + + i n i n c r e a s i n g the anaerobic g l y c o l y s i s and suppressing the enhanced a e r o b i c g l y c o l y s i s caused by the absence of C a + + . I t has been known f o r a long time, t h a t Ca s t a b i l i z e s the b i o l o g i c a l membranes. I t i s p o s s i b l e t h a t TTX may a c t by i n t e r - a c t i n g w i t h membrane-bound C a + + and thus change i t s per-m e a b i l i t y . To t e s t t h i s p o s s i b i l i t y , i t was considered d e s i r a b l e t o study the e f f e c t s of TTX on anaerobic g l y c o l y s i s i n the presence of c h e l a t i n g agents EDTA and EGTA (EDTA c h e l a t e s both C a + + and M g + + w h i l e EGTA i s more s p e c i f i c i n b i n d i n g C a + + ) . - 166 -FIGURE 29 EFFECTS OF TETRODOTOXIN ON THE SODIUM AND POTASSIUM CONCENTRATIONS OF RAT KIDNEY MEDULLA SLICES 150 01 I l I J I 0 5 1 0 5 10 313 ( AEROBIC) ( A N A E R O B I C ) Time, i n minutes Incubation conditions were same as i n Figure. 26. ( • )Na +,control; ( • ) Na , with 2 uM TTX; ( • ) K ,control ; ( 0 ) K ,with 2 pM TTX. - 167 -R e s u l t s o f e x p e r i m e n t s , g i v e n i n T a b l e 23, show t h a t TTX i s i n e f f e c t i v e i n t h e p r e s e n c e o f t h e s e c h e l a t i n g a g e n t s . I t i s t h o u g h t t h a t t h i s i s due t o t h e v e r y l a r g e i n f l u x o f N a + b r o u g h t a b o u t b y t h e c o m p l e t e c h e l a t i o n o f C a + + w h i c h masks a n y e f f e c t o f TTX o r t h a t t h e r e o c c u r s e x c e s s i v e d e p o l a r i z a t i o n i n w h i c h c o n d i t i o n TTX i s i n e f f e c t i v e . 5.19 EFFECTS OF AEROBIC PRE-INCUBATION WITH ETHANOL ON THE TETRODOTOXIN STIMULATION OF ANAEROBIC GLYCOLYSIS  OF CEREBRAL CORTEX S L I C E S O QQ I s r a e l , K a l a n t a n d L e B l a n c ^ ^ h a v e shown t h a t , i n t h e p r e s e n c e o f e t h a n o l , c e r e b r a l s l i c e s w h i c h h a v e l o s t K + do n o t r e g a i n i t t o t h e same e x t e n t a s t h a t o b t a i n e d w i t h t h e s l i c e s w h i c h h a v e n o t b e e n e x p o s e d t o e t h a n o l . T h i s a c t i o n was s t a t e d t o b e m e d i a t e d t h r o u g h p a r t i a l i n h i b i t i o n o f N a + , K + - A T P a s e b y e t h a n o l . I f t h e e f f e c t s o f TTX on a n a e r o b i c g l y c o l y s i s a r e due t o t h e r e t e n t i o n o f K + , t h e n t h e s l i c e s w h i c h h a v e b e e n i n c u b a t e d , u n d e r o x y g e n , i n t h e p r e s e n c e o f e t h a n o l s h o u l d show l e s s r e s p o n s e t o TTX. R e s u l t s o f t y p i c a l e x p e r i m e n t s a r e shown i n T a b l e 24; t h e y show t h a t t h i s i s i n d e e d t h e c a s e . However, t h i s d o e s n o t r u l e o u t t h e p o s s i b i l i t y t h a t e t h a n o l may d i r e c t l y i n t e r f e r e w i t h t h e mode o f a c t i o n o f TTX. F u r t h e r work i s n e c e s s a r y t o s e t t l e t h i s . 168 TABLE 23 EFFECTS OF EDTA AND EGTA ON THE TETRODOTOXIN STIMULATION OF ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES A d d i t i o n s L a c t a t e p r o d u c e d y m o l e s p e r g i n i t i a l wet wt (20-80 min) R a t G u i n e a P i g None 2yM TTX ImM EDTA ImM EGTA ImM EDTA + 2yM TTX ImM EGTA + 2yM TTX 24.4 ± 2.8 81.1 ± 7.6 11.3 24.9 ± 3.5 17.8 + 4.3 27.3 ± 1.9 30.7 ± 7.6 141.8 ±14.5 11.8 ± 0.8 17.7 ± 2.9 14.9 ± 4.6 37.5 ± 8.0 C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d i n a C a + + f r e e medium c o n t a i n i n g 20mM g l u c o s e . A d d i t i o n s were made a t z e r o t i m e and l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y a s g i v e n i n t h e m a t e r i a l s and m ethods. 169 TABLE 24 EFFECT OF ETHANOL ON THE TETRODOTOXIN STIMULATION OF ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES A d d i t i o n s None 200mM E t h a n o l 2yM TTX 2uM TTX + 200mM E t h a n o l umoles p e r L a c t a t e p r o d u c e d g i n i t i a l wet wt (30-90 min) 23.7 ± 0.5 36.2 ± 4.5 107.2 ± 2.0 82.5 ± 3.1 I n c u b a t i o n s were c a r r i e d o u t i n a Ca - f r e e medium c o n t a i n i n g 20mM g l u c o s e . F i r s t 10 m i n i n c u b a t i o n was a e r o b i c (02:C02) f o l l o w e d by a n a e r o b i o s i s . A d d i t i o n s were made a t z e r o t i m e and l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i -c a l l y as g i v e n i n t h e m a t e r i a l s and methods. - 170 -5.20 EFFECTS OF TETRODOTOXIN ON THE Na + AND K + CONTENT OF  THE CEREBRAL CORTEX SLICES IN THE PRESENCE OF  GLUTAMATE, ASPARTATE, HOMOCYSTEIATE AND N H A + We have shown i n Chapter 4 . 8 that i n the presence of glutamate and N H 4 + , TTX i s much less e f f e c t i v e i n increasing the anaerobic g l y c o l y s i s of the cerebral cortex s l i c e s . Under the same conditions aspartate was found to have no e f f e c t . Hence experiments were c a r r i e d out to see i f the aation concen-t r a t i o n of the s l i c e s , i n the presence of TTX, i s affected by glutamate, N H 4 and other amino acids. The r e s u l t s of these experiments are given i n Table 25 0 I t w i l l be seen that when L- or D-glutamate i s present, the retention of K + i s les s i n the s l i c e s than i n those incubated i n the absence of the amino acids. In the presence of NH^ "1", TTX had a s i g n i f i c a n t but smaller e f f e c t on the K + l e v e l s . Under the same conditions, when L-aspartate or DL-homocysteate i s present, increased r e -tenti o n of K + i n the presence of TTX i s evident. These r e s u l t s demonstrate that the e f f e c t s of glutamate and NH^* on the TTX stimulation of anaerobic g l y c o l y s i s may be mediated through changes i n the cerebral concentrations of Na + and K +. 5 021 EFFECTS OF TETRODOTOXIN ON THE PYRUVATE AND  PHOSPHOENOL-PYRUVATE CONTENTS OF CEREBRAL  CORTEX SLICES UNDER ANOXIA We have seen, i n sections 5.12 and 5.15, that i n the 171 TABLE 25 EFFECTS OF TETRODOTOXIN ON THE SODIUM AND POTASSIUM CONCENTRATIONS OF RAT CEREBRAL CORTEX SLICES IN THE PRESENCE OF SOME AMINO ACIDS AND NHi*"1" Addition Cation Aerobic Anaerobic 10 min 5 min 10 min 30 min 5mM L-Glutamate Na1 K + 160 42 170 25 195 21 220 16 5mM L-Glutamate + 2yM TTX K Na + + 130 48 155 42 175 37 220 23 5mM D-Glutamate Na"1 K"1 147 46 175 29 200 20 232 15 5mM D- Na Glutamate + 2yM TTX K + + 150 49 155 43 165 36 215 23 5mM L-Aspartate Na" K 145 40 180 27 190 21 220 15 5mM L- Na Aspartate + 2yM TTX K + + 140 48 135 42 170 38 210 30 5mM DL-Homocisteiate Na1 K 153 48 200 27 200 20 223 14 5mM DL- Na Homocysteiate + 2yM TTX K + + 150 47 160 46 175 38 235 45 171A TABLE 25 ( C o n t i n u e d ) A d d i t i o n C a t i o n 5mM NH^Cl A e r o b i c A n a e r o b i c 10 min 5 min 10 min 30 min N a + 125 145 170 210 K + 38 24 16 15 rc M MII m N a + 1 2 0 1 2 5 1 4 0 1 8 5 5mM N H 4 C I + 2yM TTX K + 4 Q 3 5 3 Q 1 8 I n c u b a t i o n c o n d i t i o n s were same as i n F i g u r e 26. E a c h v a l u e r e p r e s e n t a v e r a g e s o f two e x p e r i m e n t s w i t h i n ± 7%. F o r c o n t r o l s , s e e F i g u r e 26. R e s u l t s a r e e x p r e s s e d as y e q u i v a l e n t s p e r g i n i t i a l wet wt. - 172 -presence of 2 uM TTX, there i s an increase i n the K +/Na + r a t i o of the incubated cerebral cortex s l i c e s a f t e r the onset of anoxia. I f the e f f e c t of TTX on the anaerobic g l y c o l y s i s i s due to the f a c i l i t a t i o n of pyruvate kinase step because of an increase i n the K +/Na + ratio.., then i n the presence of TTX there should be a decrease i n the phosphoenol-pyruvate (PEP) content and an increase i n the pyruvate content. Experiments were c a r r i e d out therefore to t e s t the above hypothesis. For these experiments, conditions under which maximum e f f e c t on the K +/Na + r a t i o i s obtained, were selected. As shown i n Table 26, i n the presence of TTX the concentration of pyruvate i n the cerebral cortex s l i c e s i s indeed increased. Phosphoenol-pyruvate contents i n the incubated s l i c e s were extremely low and accurate determination was therefore not pos s i b l e . The increase i n pyruvate contents i n the presence of TTX thus supports the view that the e f f e c t s of TTX on the anaerobic g l y c o l y s i s i s due to the f a c i l i t a t i o n of the pyruvate kinase step; presumably i t i s not due to block of pyruvate e f f l u x from the s l i c e s during incubation, as TTX i s e f f e c t i v e i n increasing the rate of anaerobic g l y c o l y s i s even i n the presence of pyruvate (Section 5.3). 173 TABLE 26 EFFECTS OF TETRODOTOXIN ON THE PYRUVATE AND PHOSPHOENOL PYRUVATE CONTENT OF RAT CEREBRAL CORTEX SLICES mymoles p e r g i n i t i a l wet wt A d d i t i o n s P y r u v a t e PEP Z e r o t i m e 107 ± 6 6 2 + 5 None 52 ± 8 LOW 2 yM TTX 79 ± 6 LOW I n c u b a t i o n s were c a r r i e d o u t i n a C a + + f r e e medium c o n t a i n i n g 20mM g l u c o s e f o r 10 m i n a e r o b i c a l l y f o l l o w e d by 20 min a n a e r o b i o s i s . P y r u v a t e and PEP were m e a s u r e d a t t h e b e g i n n i n g and e n d o f t h e i n c u b a t i o n p e r i o d , as g i v e n i n t h e m a t e r i a l s and me t h o d s . t - 174 -SUMMARY OF CHAPTER 5 1. P r i o r o x y g e n a t i o n o f t h e c e r e b r a l c o r t e x s l i c e s i n c r e a s e s t h e s u b s e q u e n t r a t e o f a n a e r o b i c g l y c o l y s i s , b o t h i n t h e a b s e n c e and p r e s e n c e o f TTX, t o a v a l u e g r e a t e r t h a n t h a t f o u n d i n t h e n o n - o x y g e n a t e d s l i c e s . 2. TTX i s n o t e f f e c t i v e i n i n c r e a s i n g t h e a n a e r o b i c g l y c o l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s i f i t i s a d d e d 10 min, o r l a t e r , a f t e r t h e o n s e t o f a n o x i a . 3. The e f f e c t s o f TTX on t h e a n a e r o b i c g l y c o l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s do n o t seem t o b e m e d i a t e d t h r o u g h t h e r e t e n t i o n o f p y r u v a t e . I t s e f f e c t on a n a e r o b i c g l y c o l y s i s b y d i r e c t l y i n f l u e n c i n g ATP l e v e l s , i s u n l i k e l y . (See C h a p t e r 8 ) . The ATP c o n t e n t s o f t h e c e r e b r a l c o r t e x s l i c e s a r e h i g h e r i n t h e p r e s e n c e o f TTX u n d e r a n o x i a t h a n i n t h e a b s e n c e o f TTX. 4. D u r i n g a e r o b i c i n c u b a t i o n , u p t a i e o f a d e n i n e i n t o t h e c e r e b r a l c o r t e x s l i c e s a nd i t s i n c o r p o r a t i o n i n t o ATP i n c r e a s e s w i t h t i m e . The f o r m a t i o n o f cAMP, f r o m t h e p u l s e l a b e l l e d ATP o f t h e i n c u b a t e d s l i c e s , i s n o t i n c r e a s e d b y TTX, ." . u n d e r a n a e r o b i c c o n d i t i o n s . 5. P r o t o v e r a t r i n e r e v e r s e s t h e e f f e c t s o f TTX on t h e a n a e r o b i c g l y c o l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s . M o r e o v e r , t h e i n h i b i t o r y e f f e c t o f p r o t o v e r a t r i n e on c e r e b r a l a n a e r o b i c g l y c o l y s i s i s b l o c k e d b y TTX. - 175 -6 0 TTX has l i t t l e e f f e c t on the Na^ z i n f l u x i n t o the c e r e b r a l c o r t e x s l i c e s under anoxia. 7. TTX has no a c c e l e r a t i n g e f f e c t on anaerobic g l y c o -l y s i s when h i g h K + i s present i n the i n c u b a t i o n medium con-t a i n i n g normal N a + c o n c e n t r a t i o n . I f the Na + i s reduced at the same time t h a t K + i s in c r e a s e d , the r a t e of anaerobic g l y c o l y s i s i n c r e a s e s and TTX has p r o g r e s s i v e l y l e s s e f f e c t w i t h i n c r e a s i n g K + c o n c e n t r a t i o n . + + + 8. K , L i or Na (100 mJM f i n a l concentration) when added t o the i n c u b a t i o n medium, i n which the anaerobic g l y c o -l y s i s of c e r e b r a l c o r t e x s l i c e s has been a c c e l e r a t e d by TTX, has an i n h i b i t o r y e f f e c t on the TTX s t i m u l a t e d g l y c o l y s i s . 9. K + content of the a n a e r o b i c a l l y incubated c e r e b r a l c o r t e x s l i c e s i s i n c r e a s e d i n the presence of TTX. There i s decrease i n the N a + content at the same time. 10. TTX i n c r e a s e s the anaerobic g l y c o l y s i s of caudate 22 nucleus but Na i n f l u x i s not suppressed. 11. TTX and ouabain have no e f f e c t on the anaerobic g l y c o l y s i s of synaptosomes but C a + + has an i n h i b i t o r y a c t i o n . 12. The e f f e c t s of TTX on the K + and N a + content i s much more apparent i f , a f t e r the a d d i t i o n of TTX, the s l i c e s are incubated f o r a short p e r i o d i n oxygen p r e l i m i n a r y t o the onset of anoxia. - 176 -13. TTX has l i t t l e or no e f f e c t on the Na + and K + contents of 2-day o l d r a t c e r e b r a l c o r t e x s l i c e s but i n f a n t guinea p i g s are s i m i l a r t o a d u l t animals i n r e t a i n i n g more K + and t a k i n g up l e s s N a + i n the presence of TTX. 14. TTX has no e f f e c t on the anaerobic g l y c o l y s i s of c e r e b r a l c o r t e x s l i c e s i n the presence of c h e l a t i n g agents, EDTA and EGTA. 15. Aerobic p r e - i n c u b a t i o n of the s l i c e s i n the presence of ethanol decreases the subsequent r a t e of anaerobic g l y c o l y -s i s i n the presence of TTX. 16. Ammonium i o n s , L-glutamate and D-glutamate reduce the e f f e c t of TTX on the r e t e n t i o n of K + by the c e r e b r a l c o r t e x s l i c e s under anoxia (see Table 25), w h i l e L-aspartate and DL-homocysteiate have no e f f e c t . 17. I n the presence of TTX, there i s an in c r e a s e i n the pyruvate content of the c e r e b r a l c o r t e x s l i c e s under anoxia. This supports the hypothesis t h a t the e f f e c t s of TTX on anaerobic g l y c o l y s i s might be due i n d i r e c t l y t o the f a c i l i t a t i o n of the pyruvate k i n a s e s t e p . CHAPTER 6 EFFECTS OF OUABAIN AND LOCAL ANESTHETICS ON THE CEREBRAL  METABOLISM AND TRANSPORT UNDER ANOXIA I t was shown i n C h a p t e r s 4 and 5 t h a t TTX g r e a t l y i n c r e a s e s t h e r a t e o f a n a e r o b i c g l y c o l y s i s a s w e l l a s K + / Na r a t i o o f t h e i n c u b a t e d c e r e b r a l t i s s u e . I t was c o n c l u d e d t h a t t h i s i n c r e a s e i n t h e K + / N a + r a t i o i s r e s p o n s i b l e f o r t h e s t i m u l a t i n g a c t i o n o f TTX on t h e a n a e r o b i c g l y c o l y s i s . I n v i e w o f t h e f a c t t h a t some o f t h e e f f e c t s o f l o c a l a n e s t h e t i c s on c e r e b r a l p r o c e s s e s r e s e m b l e t h o s e o f TTX ( C h a p e r 1^ t h e e f f e c t s o f l o c a l anesthetics on a n a e r o b i c g l y c o l y s i s were i n v e s t i g a t e d . + + O u a b a i n i s a s t r o n g i n h i b i t o r o f Na , K - A T P a s e , w h i c h i s r e s p o n s i b l e f o r m a i n t a i n i n g i o n g r a d i e n t s a c r o s s t h e b r a i n c e l l membrane. As n o t e d , o u a b a i n has marked e f f e c t s on t h e m e t a b o l i s m o f a e r o b i c a l l y i n c u b a t e d c e r e b r a l t i s s u e . M o s t o f t h e s e e f f e c t s a r e due t o i n h i b -i t i o n o f membrane bound A T P a s e . R o l l e s t o n and Newsholme 52 . showed t h a t lOOyM o u a b a i n i n c r e a s e s t h e f o r m a t i o n o f l a c t i c a c i d i n t h e a e r o b i c a l l y i n c u b a t e d c e r e b r a l c o r t e x s l i c e s . F u r t h e r m o r e , t h e y c o n c l u d e d t h a t o u a b a i n may be an i n h i b i t o r o f g l y c e r a l d e h y d e 3 - p h o s p h a t e d e h y d r o g e n a s e . 181 W o l l e n b e r g e r f o u n d t h a t some d i g i t a l i s a l k a l o i d s i n h i b i t t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s i n a manner s i m i l a r t o t h a t o f p r o t o v e r a t r i n e . The e f f e c t o f o u a b a i n on t h e a n a e r o b i c g l y c o l y s i s was i n v e s t i g a t e d i n t h e l i g h t o f p r e s e n t k n o w l e d g e as t o i t s mode o f a c t i o n . The - 178 -r e s u l t s o f e x p e r i m e n t s c a r r i e d o u t w i t h o u a b a i n a n d l o c a l a n e s t h e t i c s w i l l be c o n s i d e r e d i n t h i s C h a p t e r . 6.1 EFFECTS OF OUABAIN ON THE ANAEROBIC GLYCOLYSIS OF  CEREBRAL CORTEX S L I C E S E x p e r i m e n t s c a r r i e d o u t w i t h 10 uM o u a b a i n showed t h a t i t g r e a t l y e n h a n c e s t h e r a t e o f a n a e r o b i c g l y c o l y s i s i n a C a + + - f r e e medium. A d o s e r e s p o n s e c u r v e f o r d i f f e r e n t c o n c e n t r a -t i o n s o f o u a b a i n i s shown i n F i g u r e 30. I t i s e v i d e n t t h a t , w i t h r a t c e r e b r a l c o r t e x s l i c e s , t h e r a t e o f a n a e r o b i c g l y c o -l y s i s i n c r e a s e s p r o g r e s s i v e l y w i t h i n c r e a s i n g o u a b a i n c o n c e n t -r a t i o n s i n a C a + + - f r e e medium. However, w i t h g u i n e a p i g b r a i n a h i g h r a t e o f g l y c o l y s i s i s o b t a i n e d e v e n w i t h 1 yM o u a b a i n , and f u r t h e r i n c r e a s e i n i t s c o n c e n t r a t i o n had no a d d i t i o n a l s t i m u l a t o r y e f f e c t . T h e s e r e s u l t s a r e c o n t r a r y t o t h o s e r e p o r t e d by W o l l e n b e r g e r u s i n g o t h e r d i g i t a l i s a l k a l o i d s and a C a + + - c o n -t a i n i n g medium f o r i n c u b a t i o n o f t h e b r a i n s l i c e s . I t was d e c i d e d , t h e r e f o r e , t o c a r r y o u t e x p e r i m e n t s u s i n g a K r e b s -R i n g e r b i c a r b o n a t e i n c u b a t i o n medium i n p l a c e o f a C a + + - f r e e medium. T h e s e r e s u l t s show t h a t i n a n o r m a l b a l a n c e d K r e b s -R i n g e r b i c a r b o n a t e medium, o u a b a i n has much l e s s s t i m u l a t o r y e f f e c t t h a n t h a t t a k i n g p l a c e i n a C a + + - f r e e medium. T h i s s t a n d s i n c o n t r a s t t o t h e e f f e c t o f TTX on t h e a n a e r o b i c g l y c o l y s i s i n a K r e b s - R i n g e r b i c a r b o n a t e medium, where c o n -s i d e r a b l e s t i m u l a t i o n i s o b s e r v e d ( C h a p t e r 4.1). I n v i e w o f t h e s e r e s u l t s , i t was d e c i d e d t o s t u d y t h e e f f e c t s o f o u a b a i n , i n t h e p r e s e n c e o f d i f f e r e n t c o n c e n t r a -- 179 -FIGURE 30 EFFECTS OF DIFFERENT CONCENTRATIONS OF OUABAIN ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES 150 C •H e o CO I o CN •P rd •H +1 •H c U 0) Ou CO T3 CD O 3 tJ O U Ou CD •p ra -p o rd 125 -100 -20 40 60 80 Ouabain concentration,in uM 100 Cerebral cortex s l i c e s were incubated i n a medium containing 2 0 mM glucose.Additions were made at zero time and lactate production was measured manome^rically as given i n the +materials and methods.(•)Guinea pig,Ca -free medium;(•)rat,Ca -free medium;(O)rat,Krebs-Ringer bicarbonate medium. - 180 -t i o n s o f C a + + , on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . I n t h e s e e x p e r i m e n t s , r e s u l t s o f w h i c h a r e shown i n F i g u r e 3 1 , d i f f e r e n t c o n c e n t r a t i o n s o f C a + + (1-4 mM) were a d d e d t o a C a + + - f r e e medium f r o m t h e s t a r t o f t h e e x p e r i m e n t ( s e e ++ F i g u r e 2 f o r r e s u l t s w i t h Ca a l o n e ) . T h e s e r e s u l t s demon-s t r a t e t h a t when C a + + i s p r e s e n t i n a d d i t i o n t o o u a b a i n , t h e g l y c o l y t i c r a t e i s n o t a s h i g h a s w i t h o u a b a i n a l o n e . T h u s , when b o t h t h e s e s u b s t a n c e s a r e p r e s e n t t o g e t h e r t h e y have an a n t a g o n i s t i c a c t i o n on t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s . 6.2 EFFECTS OF OUABAIN ON THE ANAEROBIC GLYCOLYSIS OF INFANT  RAT BRAIN CORTEX S L I C E S The p r i m a r y a c t i o n o f o u a b a i n i s known t o be on t h e N a + , K + - A T P a s e . I n i n f a n t r a t s , t h e N a + , K + - A T P a s e a c t i v i t y i s v e r y low b u t i n c r e a s e s r a p i d l y d u r i n g t h e 2-3 week p e r i o d a f t e r b i r t h . The e f f e c t s o f o u a b a i n on t h e a n a e r o b i c g l y c o l y s i s o f d e v e l o p i n g b r a i n was, t h e r e f o r e , i n v e s t i g a t e d a n d t h e r e s u l t s a r e g i v e n i n T a b l e 27. T h e s e r e s u l t s i n d i c a t e t h a t o u a b a i n h a s l i t t l e o r no e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f 2-day o l d r a t b r a i n . However, t h e a n a e r o b i c g l y c o l y s i s o f 2-week o l d r a t b r a i n i s c o n s i d e r a b l y s t i m u l a t e d i n t h e p r e s e n c e o f o u a b a i n . The e f f e c t o f o u a b a i n i n t h e p r e s e n c e o f C a + + i s v e r y s i m i l a r t o t h a t f o r a d u l t b r a i n , i . e . t h e a n a e r o b i c g l y c o -l y s i s o f i n f a n t r a t b r a i n i n t h e p r e s e n c e o f o u a b a i n i s a l s o ++ d i m i n i s h e d i n t h e p r e s e n c e o f Ca - 181 -FIGURE 31 EF F E C T OF VA-RYING CALCIUM CONCENTRATION IN THE PRESENCE OF OUABAIN ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX S L I C E S C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d i n a Ca - f r e e medium c o n t a i n i n g 10 uM o u a b a i n , 2 0 mM g l u c o s e and d i f f e r e n t c o n c e n t r a t i o n s o f Ca . A d d i t i o n s were made a t z e r o t i m e and l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y as g i v e n i n t h e m a t e r i a l s and methods. 182 TABLE 27 EFFECT OF OUABAIN ON THE ANAEROBIC GLYCOLYSIS OF DEVELOPING RAT CEREBRAL CORTEX SL I C E S Lactate produced ymoles per g i n i t i a l wet wt (20-80 min) Addition 2 day old 7 day old 14 day old None 25 .0 + 1.5 21. 2 + 0.45 25 .4 + 3.1 lyM ouabain 38 .4 + 4.4 30. 8 + 1.7 31 .7 + 1.9 lOyM ouabain 41 .1 + 1.8 38. 8 + 5.8 64 .7 + 2.3 lOOyM ouabain 32 .6 + 1.4 46. 1 + 8.5 87 .0 + 8.0 lOyM ouabain + ImM C a + + 36 .1 + 1.9 29. 4 + 1.3 39 .7 + 3.6 lOyM ouabain + 2mM C a + + 36 .0 + 3.1 35. 3 + 6.2 46 .2 + 7.1 lOyM ouabain + 4mM C a + + 40 .1 + 2.3 35. 7 + 3.0 46 .0 + 2.7 Cerebral cortex s l i c e s were incubated i n a Ca-free medium containing 20mM glucose. Additions were made at zero time and lactate production was measured manometrically as given i n the materials and methods. - 183 -6.3 EFFECTS OF OUABAIN AND TETRODOTOXIN ON THE ANAEROBIC  GLYCOLYSIS OF RAT CEREBRAL CORTEX S L I C E S AT DIFFERENT  CONCENTRATIONS OF N a + IN THE INCUBATION MEDIUM Sodiu m i o n s a r e known t o i n f l u e n c e t h e a c t i v i t y o f p y r u v a t e k i n a s e . As shown i n C h a p t e r 3.7, an i n c r e a s e i n t h e c o n c e n t r a t i o n o f N a + d e c r e a s e s t h e r a t e o f a n a e r o b i c g l y c o -l y s i s o f c e r e b r a l c o r t e x s l i c e s . As o u a b a i n i n d u c e s an i n f l u x o f N a + i n t o c e r e b r a l c o r t e x s l i c e s , i n v e s t i g a t i o n s were made o f t h e e f f e c t s o f o u a b a i n a t d i f f e r e n t c o n c e n t r a -t i o n s o f N a + (30-150 mM) on r a t e s o f c e r e b r a l a n a e r o b i c g l y c o l y s i s . The r e s u l t s o f t h e s e i n v e s t i g a t i o n s a s w e l l a s o f t h o s e c a r r i e d o u t w i t h TTX a r e shown i n F i g u r e 3 2 . I t i s e v i d e n t t h a t i n t h e p r e s e n c e o f o u a b a i n , o r o f TTX, a t low c o n c e n t r a t i o n s o f N a + , t h e r a t e s o f a n a e r o b i c g l y c o l y s i s a t t a i n e d a r e h i g h e r t h a n t h o s e o b t a i n e d w i t h n o r m a l N a + c o n c e n t r a t i o n s (See C h a p t e r 8 ) . 6.4 E F F E C T S OF OUABAIN ON ANAEROBIC GLYCOLYSIS OF ACETONE  POWDER OF BRAIN R e s u l t s o f e x p e r i m e n t s c a r r i e d o u t on t h e e f f e c t s o f o u a b a i n on t h e a n a e r o b i c g l y c o l y s i s o f b r a i n a c e t o n e powder e x t r a c t a r e shown i n F i g u r e 17. T h e s e r e s u l t s show t h a t , as f o r TTX, o u a b a i n i s a l s o n o t e f f e c t i v e i n i n c r e a s i n g t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f b r a i n a c e t o n e powder e x t r a c t s . Hence t h e e f f e c t o f o u a b a i n seems t o r e q u i r e t h e i n t e g r i t y o f t h e c e l l . - 184 -FIGURE 32 EFFECTS OF OUABAIN AND TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES AT VARYING CONCENTRATIONS OF SODIUM 125 ^ 0I I I I 1 1— 0 30 + 60 90 120 150 Na concentration,in mM Incubation medium contained 5 K ,20'mM glucose and varying concentrations of Na .29 mM Na was present as HCO^.When Na concentration was below 149 mM,sucrose was used to replace Na .Additions were made at zero time and lactate production was measure manometrically as given i n the materials and methods . ( • ) control; ( n ) 2 uM TTX and (O)10 pM ouabain. - 185 -6.5 EFFECTS OF OUABAIN IN THE PRESENCE OF L-GLUTAMATE,  CITRATE, AMP AND NH* ON THE ANAEROBIC GLYCOLYSIS OF THE  CEREBRAL CORTEX S L I C E S We have n o t e d e a r l i e r , i n C h a p t e r s 4 and 5, t h a t t h e r a t e o f TTX s t i m u l a t e d g l y c o l y s i s i s s u p p r e s s e d i n t h e p r e s e n c e o f NH*, c i t r a t e o r o f g l u t a m a t e . E x p e r i m e n t s w ere, t h e r e f o r e , c a r r i e d o u t t o o b s e r v e w h e t h e r i n t h e p r e s e n c e o f t h e s e s u b s t a n c e s o u a b a i n i s e f f e c t i v e i n i n c r e a s i n g t h e a n a e r o b i c g l y c o l y s i s . R e s u l t s o f t h e s e e x p e r i m e n t s ( T a b l e 2 8) i n d i c a t e t h a t c i t r a t e h as a s t r o n g i n h i b i t o r y e f f e c t on t h e o u a b a i n s t i m u l a t e d g l y c o l y s i s and t h i s i s e x p l a i n e d by t h e i n h i b i t i o n o f p h o s p h o f r u c t o k i n a s e by c i t r a t e w h i l e g l u t a m a t e i s l e s s i n h i b i t o r y . The i n h i b i t o r y e f f e c t o f g l u t a m a t e may be due t o t h e l o w e r i n g o f ATP c o n c e n t r a t i o n u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s . AMP and NH +^ have l i t t l e o r no i n h i b i t o r y e f f e c t . T h i s i s i n c o n t r a s t t o t h a t o b t a i n e d w i t h TTX, w h i c h h a s v e r y l i t t l e s t i m u l a t i n g a c t i o n i n t h e + + p r e s e n c e o f NH ^. I n t h e p r e s e n c e o f o u a b a i n t h e Na i n f l u x and K + e f f l u x i s a l r e a d y h i g h , a n d t h u s u n d e r t h e s e c o n d i t i o n s N H + 4 h a s no e f f e c t on t h e g l y c o l y s i s (See C h a p t e r 4 . 9 ) . 6.6 EFFECTS OF OUABAIN ON THE AEROBIC GLYCOLYSIS OF CEREBRAL  CORTEX S L I C E S The e f f e c t s o f 10 yM o u a b a i n on t h e a e r o b i c g l y c o l y s i s o f ++ r a t c e r e b r a l c o r t e x s l i c e s i n c u b a t e d i n a Ca - f r e e a s w e l l as i n a K r e b s - R i n g e r b i c a r b o n a t e medium was i n v e s t i g a t e d ( F i g u r e 1 9 ) . I t i s e v i d e n t t h a t a t 10 yM c o n c e n t r a t i o n , 186 TABLE 2 8 EFFECTS OF OUABAIN I N THE PRESENCE OF GLUTAMATE, C I T R A T E , NHi+ + AND AMP ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX S L I C E S A d d i t i o n s Lactate produced ymoles per g i n i t i a l wet wt (20-80 min) C a + + free medium Krebs-Ringer bicarbonate medium None 10yM ouabain lOuM ouabain + 5mM NHi+ + 5mM L-Glutamate lOuM ouabain + 5mM L-Glutamate 5mM D-Glutamate 10yM ouabain + 5mM D-Glutamate lOOyM ouabain lOOyM ouabain + 5mM L-Glutamate 5mM c i t r a t e 15mM c i t r a t e lOyM ouabain + 5mM c i t r a t e lOyM ouabain +15mM c i t r a t e AMP 2mM lOyM ouabain + AMP 2mM 31.7 ± 4.3 95.9 + 3.6 99.1 ± 3.1 20.9 ± 2.3 63.8 ± 3.0 38.0 ± 4.4 58.5 ± 6.3 102.6 ± 3.0 82.6 ± 1.8 13.8 ± 2.3 12.5 ± 0.9 52.2 ± 3.1 22.3 ± 4.0 15.5 ± 5.0 83.9 ±16.5 28.6 ± 3.7 41.7 ± 4.5 34.4 ± 3.8 56.7 ± 2.7 Incubation conditions were same as i n Table 27. - 187 -o u a b a i n h a s l i t t l e o r no e f f e c t on t h e r a t e o f a e r o b i c g l y c o l y s i s . I n a C a + + - f r e e medium, h o w e v e r , a e r o b i c g l y c o -l y s i s i s s l i g h t l y s u p p r e s s e d by TTX w h i c h i s i n c o n t r a s t t o t h e e f f e c t s o f o u a b a i n (See C h a p t e r 8 ) . 6.7 EFFECTS OF OUABAIN ON THE ATP CONTENTS OF CEREBRAL CORTEX  S L I C E S M e a s u r e m e n t o f ATP c o n t e n t s show t h a t u n d e r a n a e r o b i c c o n d i t i o n s t h e r e i s i n f a c t a n i n c r e a s e i n i t s c o n c e n t r a t i o n ( T a b l e 15) i n t h e p r e s e n c e o f o u a b a i n . As t h e ATP c o n t e n t o f t h e t i s s u e i s v e r y much r e d u c e d u n d e r a n o x i a , t h e t i s s u e c o n c e n t r a t i o n o f ATP may become r a t e l i m i t i n g f o r t h e p h o s p h o r y l a t i o n o f g l u c o s e a n d f r u c t o s e 6- p h o s p h a t e . I t may t h u s e x e r t a r a t e l i m i t i n g e f f e c t on t h e s p e e d o f a n a e r o b i c g l y c o l y s i s . A s a c o n s i d e r a b l e + + amount o f ATP i s consumed by Na , K - A T P a s e , t h e e f f e c t o f o u a b a i n on t h e a n a e r o b i c g l y c o l y s i s may be t h e r e s u l t o f t h e i n c r e a s e d c o n c e n t r a t i o n o f ATP due t o i t s d e c r e a s e d u t i l i z a t i o n . H o w e v e r , t h e p o s s i b l i l i t y c a n n o t be r u l e d o u t t h a t i n c r e a s e i n t h e ATP c o n t e n t may be due t o a g r e a t e r r a t e o f a n a e r o b i c g l y c o l y s i s i n t h e p r e s e n c e o f o u a b a i n . T h i s w i l l be d i s c u s s e d f u r t h e r i n C h a p t e r 8. 6.8 EFFECTS OF ADDITION OF OUABAIN AFTER VARIOUS PERIODS  OF ANAEROBIOSIS ON THE ANAEROBIC GLYCOLYSIS OF RAT  CEREBRAL CORTEX S L I C E S I t h a s b e e n shown e a r l i e r t h a t a d d i t i o n o f TTX, 10 m i n o r - 188 -more a f t e r t h e o n s e t o f a n o x i a , h a s no s t i m u l a t i n g a c t i o n on t h e r a t e o f a n a e r o b i c g l y c o l y s i s . T h i s e f f e c t was a t t r i b u t e d t o t h e l o s s o f K + f r o m t h e b r a i n c o r t e x s l i c e s d u r i n g t h e f i r s t few min o f a n o x i a . I n v e s t i g a t i o n s were t h e r e f o r e made on t h e e f f e c t s o f t h e a d d i t i o n o f o u a b a i n , a t v a r i o u s t i m e i n t e r v a l s o f a n o x i a , i n o r d e r t o compare i t s a c t i o n w i t h TTX. T h e s e r e s u l t s show t h a t o u a b a i n ( F i g u r e 33) has p r o g r e s s i v e l y l e s s e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s i f i t i s add e d t o t h e i n c u b a t i o n medium a f t e r t h e o n s e t o f a n o x i a . T h i s shows t h a t c h a n g e s o c c u r r i n g i n t h e c e r e b r a l c o r t e x s l i c e s d u r i n g t h e e a r l y p e r i o d o f a n o x i a a r e i m p o r t a n t f o r t h e e n h a n c i n g e f f e c t o f o u a b a i n on t h e a n a e r o b i c g l y c o l y s i s . 6.9 EFFECTS OF ADDITION OF OUABAIN AND TETRODOTOXIN  TOGETHER ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX  SLI C E S S i n c e b o t h o u a b a i n and TTX were f o u n d t o i n c r e a s e t h e r a t e o f a n a e r o b i c g l y c o l y s i s i n a C a + + - f r e e medium, t h e c o m b i n e d e f f e c t s o f t h e s e d r u g s were s t u d i e d . T h e s e r e s u l t s ( F i g u r e 34) show t h a t , t o g e t h e r , t h e s e d r u g s i n c r e a s e t h e r a t e o f g l y c o l y s i s t o a v a l u e h i g h e r t h a n t h a t o b t a i n e d when e i t h e r d r u g i s p r e s e n t a l o n e . 6.10 EFFECTS OF OUABAIN ON THE N a + a n d K + CONTENTS OF RAT  CEREBRAL CORTEX S L I C E S UNDER ANOXIA I t h as been shown t h a t i n t h e p r e s e n c e o f TTX, t h e r e i s - 18 9 -FIGURE 33 EFFECT OF ADDITION OF OUABAIN AFTER VARYING TIME PERIODS OF ANOXIA ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES 125 20 30 40 50 60 70 80 Time,in minutes Incubations were c a r r i e d out i n a Ca -free medium containing 20 mM glucose.Ouabain was added afte r varying periods of anaerobiosis so as to give a f i n a l concentration of 10 uM.Lactate production was measured manometrically as given i n the materials and methods.Ouabain added at (•)0 t i m e , ( _ ) 5 min or ( 9 ) 10 min after onset of anoxia. ( O ) c o n t r o l . - 190 -FIGURE 34 EFFECTS OF OUABAIN AND TETRODOTOXIN TOGETHER ON THE ANAEROBIC GLYCOLYSIS OF RAT' CEREBRAL CORTEX SL I C E S 150 •P St •P rH t0 •H -P •H C •H tn QJ Cu CO QJ -a o 3 o M Ou 0) •p fO •p o (0 125 -100 30 40 50 60 Time, i n m i n u t e s 70 80 I n c u b a t i o n c o n d i t i o n s were same as i n F i g u r e 2 2 . ( • ) c o n t r o l ; ( O ) 2 uM TTX* A ) 10 uM o u a b a i n ; ( B ) 2 pM TTX and 10 uM o u a b a i n . - 191 -a n i n c r e a s e i n K b u t a d e c r e a s e i n Na c o n t e n t o f t h e c e r e b r a l c o r t e x s l i c e s u n d e r a n o x i a . O u a b a i n i s n o t known + + t o d i m i n i s h e i t h e r t h e e f f l u x o f K o r t h e i n f l u x o f Na ; i t h a s i n f a c t , j u s t t h e o p p o s i t e e f f e c t s s i n c e N a + , K + -A T P a s e i s b l o c k e d b y o u a b a i n . S t u d i e s w e r e , t h e r e f o r e , made o f t h e c a t i o n c o n t e n t s o f c e r e b r a l c o r t e x s l i c e s u n d e r a n o x i a i n t h e p r e s e n c e o f o u a b a i n . The r e s u l t s shown i n F i g u r e 35 i n d i c a t e t h a t , i n t h e p r e s e n c e o f o u a b a i n u n d e r a n o x i a , t h e r e i s no i n c r e a s e d r e t e n t i o n o f K + and t h e r e i s e i t h e r no e f f e c t , o r o n l y a s l i g h t i n c r e a s e i n t h e N a + c o n t e n t . On t h e o t h e r hand,-when o u a b a i n a n d TTX a r e p r e s e n t t o g e t h e r , t h e r e i s a n + + i n c r e a s e i n K a n d a s l i g h t d r o p i n t h e Na c o n t e n t . U n d e r a n o x i a , t h e i n f l u x o f N a + i n c e r e b r a l t i s s u e i s v e r y l a r g e , 22 and any e f f e c t on Na - i n f l u x c o u l d n o t be s e e n ( T a b l e 1 8 ) . I t i s e v i d e n t f r o m t h e s e e x p e r i m e n t s t h a t t h e e f f e c t o f o u a b a i n on a n a e r o b i c g l y c o l y s i s i s n o t m e d i a t e d t h r o u g h + + i t s a c t i o n on t h e Na a n d K c o n t e n t s o f t h e b r a i n c e l l s . I n t h i s r e s p e c t , i t s mode o f a c t i o n d i f f e r s f r o m t h a t o f TTX. 6.11 EFFECTS OF OUABAIN, G a + + AND N A D + ON THE MICROSOMAL  N a + , K + - A T P a s e R e s u l t s r e p o r t e d i n S e c t i o n 6.1 show t h a t , i n t h e ++ p r e s e n c e o f Ca , o u a b a i n i s n o t v e r y e f f e c t i v e i n i n c r e a s i n g t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . F u r t h e r m o r e , o u a b a i n a n d C a + + seem t o - 192 -I FIGURE 35 EFFECTS OF OUABAIN AND TETRODOTOXIN AND OUABAIN TOGETHER ON THE SODIUM AND POTASSIUM CONCENTRATIONS OF RAT CEREBRAL CORTEX SL I C E S 0 5 10 5 10 30 ( AEROBIC ) ( A N A E R O B I C ) Time , i n m i n u t e s I n c u b a t i o n c o n d i t i o n s were same as i n F i g u r e 26. A d d i t i o n s we^e made a t z e r o t i m e . ( B ) N a f w i t h 10 pM o u a b a i n ; ( • ) Na , w i t h 10 pip o u a b a i n and 2 uM T T X ; ( C ) K , w i t h 10 uM o u a b a i n ; ( O ) K , w i t h 10 uM o u a b a i n and 2 pM T T X . C o n t r o l s a r e same as i n F i g u r e 26. - 193 -a n t a g o n i z e e a c h o t h e r . I t i s w e l l e s t a b l i s h e d t h a t t h e m a j o r e f f e c t o f o u a b a i n i s t h e i n h i b i t i o n o f N a + , K + -A T P a s e ( s e e C h a p t e r 1 . 5 ) . Under a n a e r o b i c c o n d i t i o n s , a s a l r e a d y p o i n t e d o u t ( S e c t i o n 6 . 7 ) , t h e ATP c o n c e n t r a t i o n f a l l s a nd i t may become r a t e l i m i t i n g f o r a n a e r o b i c g l y c o -l y s i s . I t was f u r t h e r s u g g e s t e d t h a t o u a b a i n m i g h t a c t by d e c r e a s i n g t h e r a t e o f u t i l i z a t i o n o f ATP by N a + , K + -A T P a s e . Our r e s u l t s on t h e a n t a g o n i s m between o u a b a i n and ++ ++ Ca w o u l d be e x p l a i n e d i f , i n t h e p r e s e n c e o f Ca , t h e i n h i b i t o r y e f f e c t o f o u a b a i n on t h e membrane bound Nat K + - A T P a s e i s d i m i n i s h e d . W h i l e s t u d y i n g t h e o u a b a i n i n d u c e d c h a n g e s i n t h e N a + and K + c o n t e n t , a s w e l l a s on 131 t h e r e s p i r a t i o n o f t h e c e r e b r a l c o r t e x s l i c e s , Swanson ' 173 came t o a s i m i l a r c o n c l u s i o n a n d s t a t e d t h a t i n t h e ++ + + p r e s e n c e o f Ca , a l l t h e Na , K -ATPase o f t h e membrane i s n o t a c c e s s i b l e t o o u a b a i n . I n v i e w o f t h e s e c o n s i d e r a t i o n s , i t was d e c i d e d t o s t u d y t h e e f f e c t s o f C a + + and o u a b a i n when p r e s e n t t o g e t h e r on + + t h e a c t i v i t y o f Na , K - A T P a s e . The r e s u l t s o b t a i n e d i n d i c a t e t h a t , u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s i n b r a i n m i c r o s o m a l p r e p a r a t i o n s , t h e e f f e c t s o f o u a b a i n and C a + + a r e n o t a n t a g -o n i s t i c ( T a b l e 2 9 ) . T h i s was t r u e e v e n when m i c r o s o m a l p r e p -19 4 TABLE 29 EFFECTS OF OUABAIN, C a + + AND NAD+ ON THE MICROSOMAL Na +, K +-ATPase A c t i v i t y of Na +, K -ATPase Additions . . , ymoles P i per mg protein per hour Experiment 1 None 76.8 ImM C a + + 31.9 2mM C a + + 30.8 4mM C a + + 27.5 lOyM ouabain 49.6 lOyM ouabain O D + ImM C a + + 2 8 ' 6 lOyM ouabain o c T + 2mM C a + + 2 6 * 7lOyM ouabain ot- q + 4mT4 C a + + Experiment 2 None 57.6 lOOyM ouabain 30.1 0.2mM NAD+ 60.4 0.4mM NAD+ 72.5 0.6mM NAD+ 72.5 0.8mM NAD+ 63.2 Microsomal preparations were prepared and ATPase a c t i v i t y assayed as given i n the materials and methods. Additions were made before the addition of the enzyme to the incubation mixture. Each value represent averages of duplicate determination, - 195 -a r a t i o n s a r e p r e i n c u b a t e d i n t h e p r e s e n c e o f e i t h e r o u a b a i n o r C a + + . O t h e r r e s u l t s g i v e n i n T a b l e 2 9 show t h a t NAD + d o e s n o t i n h i b i t t h e N a + , K + - A T P a s e a c t i v i t y . T h i s a s p e c t w i l l be d i s c u s s e d f u r t h e r i n C h a p t e r 8 . 6 .12 EFFECTS OF OUABAIN ON AMINO ACID EFFLUXES KRQM THE  CEREBRAL CORTEX S L I C E S UNDER ANOXIA TTX b l o c k s t h e e f f l u x o f amino a c i d s f r o m t h e c e r e b r a l c o r t e x s l i c e s u n d e r a n o x i a ( C h a p t e r 4 . 4 ) . E x p e r i m e n t s were, t h e r e f o r e , c a r r i e d o u t t o s e e i f o u a b a i n a l s o a f f e c t s t h e e f f l u x o f amino a c i d s f r o m t h e i n c u b a t e d s l i c e s . R e s u l t s o f t h e s e e x p e r i m e n t s a r e shown i n T a b l e 30. I t i s e v i d e n t t h a t i n t h e p r e s e n c e o f o u a b a i n , c o n s i d e r a b l e amounts o f amino a c i d s a r e r e l e a s e d f r o m t h e i n c u b a t e d b r a i n t i s s u e i n t o t h e i n c u b a t i o n medium. However, t h e e f f l u x o f amino a c i d s i n t h e p r e s e n c e o f o u a b a i n i s c o n s i d e r a b l y r e d u c e d by t h e p r e s e n c e o f TTX. The above f i n d i n g s i n d i c a t e t h a t t h e i n h i b i t i o n o f amino a c i d e f f l u x by TTX c a n n o t be due t o an i n c r e a s e i n t h e r a t e o f a n a e r o b i c g l y c o l y s i s . TTX p r e s u m a b l y a c t s d i r e c t l y on t h e b r a i n c e l l membrane by a b o l i s h i n g t h e g e n e r a t i o n o f t h e a c t i o n p o t e n t i a l s w h i c h r e s u l t s i n i n c r e a s e d r e l e a s e o f t i s s u e amino a c i d s . 6.13 EFFECTS OF PROCAINE AND LIDOCAINE ON THE ANAEROBIC GLYCO- LYS I S OF RAT CEREBRAL CORTEX SLICES I t has b e en p o i n t e d o u t e a r l i e r ( C h a p t e r 1.3) t h a t i n many r e s p e c t s , t h e mode o f a c t i o n o f l o c a l a n e s t h e t i c s a r e s i m i l a r 196 TABLE 30 EFFECTS OF OUABAIN AND OUABAIN + TETRODOTOXIN TOGETHER ON THE AMINO ACID EFFLUX FROM RAT CEREBRAL CORTEX SLICES UNDER ANOXIA Amino A c i d s A d d i t i o n s - G l u c o s e + o u a b a i n + G l u c o s e + o u a b a i n - G l u c o s e + o u a b a i n +TTX + G l u c o s e + o u a b a i n +TTX TAURINE T i s s u e Medium 2. 0 0 ± 0 • 0 1 3.55+0.11 2.47±0.25 3.30+0.08 1.58±0 .36 3.6810.34 2, 3, 1110.11 0 0 ± 0 . 0 3 ASPARTIC T i s s u e ACID Medium 0.94+0.08 1.88±0.36 0.98±0.19 1.6510.07 0.8610.22 2 . 1 8 ± 0 . 2 7 1.5310.13 0.97± 0 . 1 5 GLUTAMINE T i s s u e +• SERINE Medium 0.7010.06 3.3710.13 1.0310.11 2.8010.08 0 . 4 2 ± 0 . 0 7 3.54±0.37 1.4910.28 2.7410.18 GLUTAMIC T i s s u e ACID Medium 3.8210.40 8.2 ±0.40 5.4111.05 6.9510.10 3.1010.46 8.0510.60 8.2410.61 3.5910.15 GLYCINE T i s s u e Medium 0.2310.04 0.9310.21 0.3710.05 0.8510.05 1.0810-08 0.4510.03 0.4710.04 ALANINE T i s s u e Medium 0. 1, 1710 2810 02 21 0.2510.05 0 . 8810.03 1.0510-16 0.4810.04 0 .5610.02 I n c u b a t i o n c o n d i t i o n s were same as i n T a b l e 4. F o r c o n t r o l s w i t h o u t any d r u g , s e e T a b l e 4. Con-c e n t r a t i o n o f o u a b a i n was lOyM and o f TTX was 2yM. - 197 -t o t h a t o f TTX. T h e s e d r u g s b l o c k t h e g e n e r a t i o n o f a c t i o n p o t e n t i a l and i n d o i n g so t h e y a f f e c t t h e movements o f N a + and K + . E x p e r i m e n t s were, t h e r e f o r e , c a r r i e d o u t t o s e e i f l o c a l a n e s t h e t i c s a f f e c t t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s i n a manner s i m i l a r t o t h a t o f TTX. The r e s u l t s g i v e n i n T a b l e 31 show t h a t , i n t h e p r e s e n c e o f l o c a l a n e s t h e t i c s p r o c a i n e o r l i d o c a i n e , t h e r a t e o f a n a e r -o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s i s i n c r e a s e d . However, a h i g h e r c o n c e n t r a t i o n o f l o c a l a n e s t h e t i c i s r e q u i r e d t o o b t a i n t h e same s t i m u l a t i o n as t h a t o b t a i n e d w i t h TTX. The p r e s e n c e o f C a + + d i m i n i s h e s t h e s t i m u l a t o r y e f f e c t s o f l o c a l a n e s t h e t i c s , as i n t h e c a s e o f TTX. A d d i t i o n a l s i m i l a r i t i e s between TTX and l o c a l a n e s t h e t i c s may be s e e n when t h e l a t t e r a r e added t o t h e i n c u b a t i o n medium a f t e r 15 min o f a n o x i a . Under t h e s e c o n d i t i o n s , t h e s e d r u g s have no e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s . 6.14 EFFECTS OF LIDOCAINE ON THE ANAEROBIC GLYCOLYSIS OF INFANT  RAT AND GUINEA PIG CEREBRAL CORTEX SL I C E S F u r t h e r e x p e r i m e n t s were c a r r i e d o u t t o o b s e r v e w h e t h e r t h e l o c a l a n e s t h e t i c l i d o c a i n e w i l l i n c r e a s e t h e a n a e r o b i c g l y c o l y s i s o f b r a i n s l i c e s o b t a i n e d f r o m i n f a n t a n i m a l s ( F i g -u r e 3 6 ) . I t w i l l be s e e n t h a t l i d o c a i n e has no e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f 2-day o l d r a t b r a i n s l i c e s b u t i t m a r k e d l y i n c r e a s e s t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f n e w l y b o r n g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . As w i t h t h e r e s u l t s f o u n d w i t h TTX, t h i s must be r e l a t e d t o t h e m a t u r i t y o f t h e 198 TABLE 31 EFFECTS OF PROCAINE AND LIDOCAINE ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES Additions Lactate produced ymoles per g i n i t i a l wet wt (20-80 min) C a + + free medium Krebs-Ringer bicarbonate medium None O.OlmM Procaine O.OlmM Lidocaine 0.1 mM Procaine 0.1 mM Lidocaine 24.6 ± 1.4 40.6 ± 6.8 45.6 ± 2.0 66.0 ± 1.0 93.7 ±17.0 42.4 ± 6.7 58.0 ± 8.9 70.5 ± 3.1 A l l vessels contained 20mM glucose. Additions were made at zero time and lactate production was measured manometrically, as given i n the materials and methods. x - 199 -FIGURE 36 EFFECT OF LIDOCAINE ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES 150 Newly born guinea p i g 0.25 0.5 0.75 Lidocaine concentration,in mM 1.0 ++ Cerebral cortex s l i c e s were incubated in a Ca -free medium containing 20 mil glucose .Additions were made at zero time and lactate production was measured manometrically as given i n the materials and methods. - 200 -b r a i n t i s s u e . 6.15 EFFECTS OF LIDOCAINE ON THE N a + AND K + CONTENTS OF  CEREBRAL CORTEX SL I C E S S i n c e t h e a c t i o n o f l o c a l a n e s t h e t i c s on a n a e r o b i c g l y c o l y s i s i s s i m i l a r t o t h a t o f TTX and s i n c e i n t h e p r e s e n c e + + o f t h e l a t t e r t h e r e i s an i n c r e a s e i n t h e K /Na r a t i o , i t was t h o u g h t d e s i r a b l e t o o b s e r v e t h e e f f e c t s o f l o c a l a n e s t h e t i c s + + on t h e Na and K c o n t e n t s o f i n c u b a t e d c e r e b r a l c o r t e x s l i c e s . A r e l a t i v e l y h i g h c o n c e n t r a t i o n (0.5 mM) o f l i d o c a i n e was u s e d i n t h e s e e x p e r i m e n t s (See T a b l e 3 2 ) . I t i s + + e v i d e n t t h a t t h e r e i s an i n c r e a s e i n K /Na r a t i o o f b r a i n s l i c e s i n t h e p r e s e n c e o f l i d o c a i n e . The r e s u l t s w i t h a d u l t r a t c e r e b r a l c o r t e x s l i c e s a r e t o be e x p e c t e d a s t h e l o c a l a n e s t h e t i c s b l o c k t h e g e n e r a t i o n o f a c t i o n p o t e n t i a l s t h a t d e v e l o p on t h e o n s e t o f a n o x i a . However, t h e a n a e r o b i c g l y c o l y s i s o f 2-day o l d r a t i s n o t a f f e c t e d by l i d o c a i n e b u t i n i t s p r e s e n c e t h e r e i s s t i l l an i n c r e a s e i n t i s s u e K + / N a + r a t i o . P o s s i b l y , t h i s i s an u n s p e c i f i c e f f e c t o f t h e h i g h c o n c e n t r a t i o n o f l o c a l a n e s t h e t i c , o r i n i n f a n t r a t b r a i n s l i c e s t h e r a t e l i m i t i n g s t e p i n g l y c o l y s i s i s n o t ^ - d e p e n d -e n t , a s a p p e a r s t o be t h e c a s e i n t h e b r a i n s o f m a t u r e a n i m a l s . T h i s w i l l be f u r t h e r d i s c u s s e d i n C h a p t e r 8. SUMMARY OF CHAPTER 6 1. O u a b a i n , a t low c o n c e n t r a t i o n s (1-100 yM) g r e a t l y increases t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f b o t h r a t a n d g u i n e a p i g c e r e b r a l c o r t e x s l i c e s i n a C a + + - f r e e medium. 201 TABLE 32 EFFECTS OF LIDOCAINE ON THE SODIUM AND POTASSIUM CONTENTS OF CEREBRAL CORTEX SLICES Age o f R a t s 2-day A d u l t C a t i o n A e r o b i c A n a e r o b i c 10 min 5 min 10 min 30 m i n N a + 100 111 120 142 o l d K + 56 57 49 43 N a + 127 130 132 155 K + 54 48 40 35 I n c u b a t i o n c o n d i t i o n s were same as i n F i g u r e 26. E a c h v a l u e r e p r e s e n t a v e r a g e s o f two e x p e r i m e n t s w i t h i n ± 7%. F o r c o n t r o l s s e e F i g u r e 26 and 28. R e s u l t s a r e e x p r e s s e d as y e q u i v a l e n t s p e r g i n i t i a l w et wt. 0.5mM l i d o c a i n e was p r e s e n t f r o m t h e s t a r t o f t h e e x p e r i m e n t . - 202 -2. O u a b a i n h a s v e r y l i t t l e s t i m u l a t i n g e f f e c t on t h e a n a e r -o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s i n c u b a t e d i n a K r e b s - R i n g e r b i c a r b o n a t e medium. M o r e o v e r , when C a + + i s adde d t o a C a + + - f r e e medium, t h e e n h a n c i n g e f f e c t o f o u a b a i n on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s i s c o n s i d e r a b l y r e d u c e d . T h u s , Ca and o u a b a i n a r e a n t a g o n i s t -i c i n t h e i r e f f e c t s on t h e c e r e b r a l a n a e r o b i c g l y c o l y s i s . 3. O u a b a i n has l i t t l e o r no e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f 2-day o l d r a t b r a i n s l i c e s i n a C a + + - f r e e medium. I t s e f f e c t i v e n e s s i n e n h a n c i n g t h e a n a e r o b i c g l y c o l y s i s o f i n f a n t r a t b r a i n i n c r e a s e s c o n s i d e r a b l y a t a b o u t t h e s e c o n d week. 4. B o t h o u a b a i n and TTX s i g n i f i c a n t l y i n c r e a s e t h e r a t e o f a n a e r o b i c g l y c o l y s i s , e v e n when most o f t h e N a + i s r e p l a c e d by s u c r o s e . The d e g r e e o f s t i m u l a t i o n d e c r e a s e s w i t h i n c r e a s i n g N a + c o n c e n t r a t i o n . 5. O u a b a i n h a s no e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f a c e t o n e powder e x t r a c t s . 6. C i t r a t e (15 mM) i n h i b i t s t h e o u a b a i n s t i m u l a t e d g l y c o l y s w h i l e g l u t a m a t e h a s some i n h i b i t o r y e f f e c t , p o s s i b l y due t o l o w e r i n g o f ATP. AMP and NH* have l i t t l e o r no e f f e c t on t h e s t i m u l a t i o n o f a n a e r o b i c g l y c o l y s i s by o u a b a i n . 7. O u a b a i n (10 yM) h a s l i t t l e o r no e f f e c t on t h e a e r o b i c g l y c o l y s i s o f i n c u b a t e d b r a i n s l i c e s . ++ 8. I n t h e p r e s e n c e o f ouabari i n a Ca - f r e e medium, u n d e r a n o x i a , t h e c e l l c o n t e n t o f ATP i s i n c r e a s e d . - 203 -9. I f o u a b a i n i s ad d e d t o t h e i n c u b a t i o n medium a f t e r i n c r e a s i n g p e r i o d s o f a n o x i a , i t has p r o g r e s s i v e l y l e s s e r e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s i n a C a + + - f r e e medium. ++ 10. When o u a b a i n and TTX a r e b o t h p r e s e n t i n a Ca - f r e e medium, t h e r a t e o f a n a e r o b i c g l y c o l y s i s i s h i g h e r t h a n t h a t o b t a i n e d when e i t h e r d r u g i s p r e s e n t a l o n e . + + 11. O u a b a i n h a s no e f f e c t on t h e K o r Na c o n t e n t o f t h e c e r e b r a l c o r t e x s l i c e s i n a C a + + - f r e e medium u n d e r a n o x i a . TTX, however, r e d u c e s t h e K + l o s s and t h e N a + g a i n by t h e t i s s u e i n p r e s e n c e o f o u a b a i n i n a C a + + - f r e e medium. T h u s TTX a c t s i n d e p e n d e n t l y o f o u a b a i n . ++ + + 12. O u a b a i n a n d Ca b o t h i n h i b i t t h e Na , K -ATPa s e i n m i c r o s o m a l p r e p a r a t i o n s . When b o t h a r e p r e s e n t t o g e t h e r , no a n t a g o n i s m on t h e i n h i b i t i o n o f N a + , K + - A T P a s e i s o b s e r v e d . + + + NAD d o e s n o t i n h i b i t t h e Na , K - A T P a s e . 13. O u a b a i n does n o t a f f e c t t h e e f f l u x o f amino a c i d s f r o m t h e c e r e b r a l c o r t e x s l i c e s u n d e r a n o x i a . However, TTX s t i l l b l o c k s t h e e f f l u x o f amino a c i d s i n t h e p r e s e n c e o f o u a b a i n , i n d i c a t i n g t h a t t h e t h e i n c r e a s e d amino a c i d c o n t e n t o f t h e c e r e b r a l c o r t e x s l i c e s i n t h e p r e s e n c e o f TTX i s n o t due t o g r e a t e r o p e r a t i o n o f t h e Na +-pump^as a r e s u l t o f an i n c r e a s e d r a t e o f a n a e r o b i c g l y c o l y s i s . TTX i s p r e s u m a b l y a c t i n g by b l o c k i n g t h e g e n e r a t i o n o f a c t i o n p o t e n t i a l s i n d u c e d by a n o x i a , t h a t r e s u l t i n an i n c r e a s e d e f f l u x o f amino a c i d s . - 204 -14. P r o c a i n e and l i d o c a i n e a c t l i k e TTX i n i n c r e a s i n g t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s f r o m m a t u r e b r a i n . I n t h e p r e s e n c e o f 0.5 mM l i d o c a i n e , t h e c e l l u l a r r a t i o o f K + / N a + i s i n c r e a s e d . Thus t h e l o c a l a n e s t h e t i c s r e s e m b l e TTX i n t h e i r m e t a b o l i c e f f e c t s on b r a i n d u r i n g a n o x i a . CHAPTER 7 EFFECTS OF VARIOUS NEUROTROPIC DRUGS ON THE ANAEROBIC  GLYCOLYSIS OF CEREBRAL CORTEX S L I C E S I t was p o i n t e d o u t i n C h a p t e r 1 t h a t C a + + and a number o f b a s e s , s u c h as p y r r o l e and p y r i d i n e , g r e a t l y e n h a n c e t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f g u i n e a p i g c e r e b r a l c o r t e x 71 s l i c e s (Adams and Q u a s t e l ) . The f i n d i n g s o f Adams and Q u a s t e l on t h e e f f e c t s o f C a + + on a n a e r o b i c g l y c o l y s i s , were c o n f i r m e d a n d f u r t h e r e x t e n d e d t o i n f a n t c e r e b r a l t i s s u e s ( C h a p t e r 3 ) . I n a d d i t i o n , e f f e c t s o f v a r i o u s d r u g s s u c h as TTX, l o c a l a n e s t h e t i c s and o u a b a i n were r e p o r t e d ( C h a p t e r s 4 - 6 ) . I t became c l e a r f r o m t h e s e s t u d i e s t h a t TTX and l o c a l a n e s t h e t i c s may a c t on a n a e r o b i c g l y c o l y s i s by a f f e c t i n g t h e K + / N a + r a t i o i n t h e b r a i n c e l l s . I n v i e w o f t h e s e r e s u l t s , i t was d e c i d e d t o s t u d y t h e e f f e c t s o f v a r i o u s o r g a n i c b a s e s on c e r e b r a l a n a e r o b i c g l y c o l y s i s , t o s e e w h e t h e r t h e i r a c t i o n t o o i s m e d i a t e d t h r o u g h c h a n g e s i n t h e c a t i o n c o n c e n t r a t i o n s i n t h e b r a i n c e l l . I n a d d i t i o n , e f f e c t s o f o t h e r d r u g s s u c h a s b a r b i t u r a t e s , r e s e r p i n e and amphetamine as w e l l as b i o g e n i c a m i n e s were a l s o s t u d i e d . The r e s u l t s o f t h e s e e x p e r i m e n t s a r e d e s c r i b e d b elow. 7.1 ACTION OF PYRROLE AND PYRIDINE ON THE ANAEROBIC GLYCOLYSIS  OF CEREBRAL CORTEX S L I C E S The e f f e c t s o f some o r g a n i c b a s e s on t h e a n a e r o b i c g l y c o l y s i s o f g u i n e a p i g and r a t c e r e b r a l s l i c e s a r e shown i n F i g u r e 37. I t i s s e e n t h a t t h e r a t e o f a n a e r o b i c g l y c o l y s i s - 206 -FIGURE 37 EFFECTS OF PYRROLE AND PYRIDINE ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SL I C E S 125 P y r i d i n e , G u i n e a p i g P y r r o l e , G u i n e a p i g P y r i d i n e , R a t JL 0 10 20 30 C o n c e n t r a t i o n o f p y r r o l e and p y r i d i n e , i n mM I n c u b a t i o n s were c a r r i e d o u t i n a C a + + - f r e e medium c o n t a i n i n g 20 mM g l u c o s e . A d d i t i o n s were made a t z e r o t i m e and l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y as g i v e n i n t h e m a t e r i a l s and methods. - 207 -o f g u i n e a p i g c e r e b r a l c o r t e x s l i c e s i s e n h a n c e d i n t h e p r e s e n c e o f p y r r o l e and p y r i d i n e , c o n f i r m i n g t h e f i n d i n g s 71 o f Adams and Q u a s t e l . I t i s a l s o e v i d e n t t h a t p y r i d i n e h a s no e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s i n c o n t r a s t t o i t s e f f e c t on t h a t o f g u i n e a p i g . I n g e n e r a l , i t a p p e a r s t h a t r a t c e r e b r a l c o r t e x s l i c e s a r e l e s s r e s p o n s i v e t h a n g u i n e a p i g c e r e b r a l c o r t e s s l i c e s t o d r u g s , a t e q u i v a l e n t c o n c e n t r a t i o n s . The e f f e c t o f 30 mM p y r r o l e , i n t h e p r e s e n c e o f 5 mM L - g l u t a m a t e , D - g l u t a m a t e o r L - a s p a r t a t e , i s shown i n T a b l e 33. I t i s e v i d e n t f r o m t h e s e r e s u l t s t h a t s t i m u l a t i o n o f g l y c o l y s i s by p y r r o l e i s s i m i l a r t o t h a t o f TTX i n s h o w i n g r e s p o n s e t o d i f f e r e n t amino a c i d s . T h u s , i n t h e p r e s e n c e o f 5 mM L - g l u t a m a t e , 30 mM p y r r o l e h a s l i t t l e o r no s t i m u l a t o r y e f f e c t on t h e a n a e r o b i c g l y c o l y s i s . D - g l u t a m a t e i s p a r t i a l -l y e f f e c t i v e w h i l e L - a s p a r t a t e h as no e f f e c t i n p r e v e n t i n g t h e s t i m u l a t i o n o f g l y c o l y s i s by p y r r o l e . M o r e o v e r , i f p y r r o l e i s a d d e d t o t h e i n c u b a t i o n medium a f t e r 15 min a n a e r o -b i o s i s , i t h a s no s t i m u l a t o r y e f f e c t . 7.2 EFFECTS OF PYRROLE ON THE N a + AND K + CONTENTS OF GUINEA  PIG CEREBRAL CORTEX S L I C E S UNDER ANOXIA I n t h e p r e v i o u s s e c t i o n , i t has been shown t h a t , i n s e v e r a l r e s p e c t s , t h e a c t i o n o f p y r r o l e on a n a e r o b i c g l y c o l y -s i s i s s i m i l a r t o t h a t o f TTX. As t h e a c t i o n o f TTX on a n a e r o b i c g l y c o l y s i s i s p r e s u m a b l y m e d i a t e d t h r o u g h i t s e f f e c t on t h e N a + and K + c o n t e n t s o f c e r e b r a l c o r t e x s l i c e s , 208 TABLE 33 EFFECT OF PYRROLE ON THE ANAEROBIC GLYCOLYSIS OF GUINEA PIG CEREBRAL CORTEX SLICES IN THE PRESENCE OF SOME AMINO ACIDS . Lactate produced Additions ymoles per g i n i t i a l wet wt (20-80 mi None 48.4 ± 12.5 30mM Pyrrole 94.3 ± 16.7 5mM L-Glutamate 25.5 ± 4.0 47.4 ± 5.7 30mM P y r r o l e + 5mM L - G l u t a m a t e 5mM L - A s p a r t a t e 51.7 ± 5.0 5mM L - A s p a r t a t e pc q + o o +30mM P y r r o l e " 5mM D-Glutamate 31.5 ± 6.9 66.5 ± 1.5 5mM D - G l u t a m a t e +30mM P y r r o l e 30mM Pyrrole, tipped i n a f t e r 43.3 ± 1.0 15 min anoxia Cerebral cortex s l i c e s were incubated i n a Ca-free medium containing 20mM glucose. Additions were made at zero time (except one case, as shown) and lactate production was measured manometrically as given i n the materials and methods. - 209 -i t was desirable to study the e f f e c t s of pyrrole on the cat-i o n i c concentrations. Results of these experiments are given i n Figure 38. I t can be seen that, i n the presence of 30 mM pyrrole, there i s an increase i n the retention of K + but a decrease i n the uptake of Na . These r e s u l t s indicate that the action of pyrrole on anaerobic g l y c o l y s i s may also be mediated through changes i n the cation concentration. 7.3 EFFECTS OF AMYTAL AND PENTOTHAL ON THE ANAEROBIC  GLYCOLYSIS OF CEREBRAL CORTEX SLICES Barbiturates have long been shown to diminish the brain 195 tiss u e r e s p i r a t i o n . As noted, i n Chapter L5 both the barbiturates (amytal and pentothal) as well as c e r t a i n hypnot-i c s , suppress the oxidation of NADH, and hence the generation 281—283 of ATP i n the c e l l . Using suspensions of cerebral 199 t i s s u e , Webb and E l l i o t t showed that, amytal enhances the rate of aerobic g l y c o l y s i s and suppresses the r e s p i r a t i o n . This i s i n agreement with present knowledge on the e f f e c t s of amytal on oxidative processes. However, these workers could only observe very l i t t l e e f f e c t of amytal on the anaerobic g l y c o l y s i s of the c e l l suspensions. The mechanism of barbiturate action on the nerve c e l l i s s t i l l not f u l l y understood. They are known to suppress e l e c t -r i c a l a c t i v i t y of excited nerve c e l l s but so far no significant e f f e c t of the barbiturates, at anesthetic concentration, on Na + movement i n the cerebral cortex s l i c e s have been observed 198 As i t was found that TTX, and l o c a l anesthetics, FIGURE 38 EFFECT OF PYRROLE ON THE SODIUM AND POTASSIUM CONCENTRATIONS OF GUINEA PIG CEREBRAL CORTEX SLICES UNDER ANOXIA 300 r 1 Time,in minutes Incubation conditions were same as i n Ficjure 24.(»)Na control; (O)Na. ,with 30 mM pyrrole;( • )K ,with 30 mM pyrrole;( B ) K ,control. - 211 -g r e a t l y i n c r e a s e t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s , i t was t h o u g h t d e s i r a b l e t o s t u d y t h e e f f e c t s o f b a r b i t u r a t e a n e s t h e t i c s on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l t i s s u e . The e f f e c t s o f d i f f e r e n t c o n c e n t r a t i o n s o f s o d i u m a m y t a l on t h e a n a e r o b i c g l y c o l y s i s o f a d u l t r a t and i n f a n t r a t , a s w e l l a s on i n f a n t g u i n e a p i g s a r e g i v e n i n F i g u r e 39. I t i s s e e n t h a t a m y t a l , a t c o n c e n t r a t i o n s e x c e e d i n g t h e a n e s t h e t i c , g r e a t l y e n h a n c e s t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s f r o m a d u l t r a t and i n f a n t g u i n e a p i g b u t i t h a s no e f f e c t on s l i c e s f r o m i n f a n t r a t b r a i n . I n a d u l t r a t b r a i n s l i c e s , a t a n e s t h e t i c d o s e s (0.25 mM) a m y t a l has a r e l a t i v e l y s m a l l e f f e c t b u t i n f a n t g u i n e a p i g s ( t h a t h a v e m a t u r e c h a r a c t e r i s t i c s ) show v e r y c o n s i d e r a b l e s e n s i t i v i t y t o t h e d r u g . The p r e s e n c e o f t r a c e s o f o x y g e n i n t h e ^ r C C ^ m i x t u r e u s e d may be enough t o o x i d i z e NADH f o r m e d and t h u s s u p p r e s s g l y c o l y s i s by i n c r e a s i n g t h e ATP l e v e l . A m y t a l u n d e r t h e s e c o n d i t i o n s c a n i n c r e a s e t h e g l y c o l y t i c r a t e by b l o c k i n g t h e o x i d a t i o n o f NADH. However, t h i s p o s s i b i l i t y i s u n l i k e l y s i n c e p r e l i m i n a r y e x p e r i m e n t s have shown t h a t t h e p r e s e n c e o f 0.5 mM a z i d e , w h i c h b l o c k s t h e r e s p i r a t o r y c h a i n , h a s no e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . M o r e o v e r , when a m y t a l i s adde d 15 min a f t e r t h e o n s e t o f a n o x i a , i t h a s no e f f e c t on t h e a n a e r o b i c g l y c o l y s i s . T h i s r u l e s o u t t h e p o s s i b i l i t y t h a t a m y t a l m i g h t a c t by b l o c k i n g t h e o x i d a t i o n o f NADH o r any u n i d e n t i f i e d m e t a b o l i t e . O t h e r e x p e r i m e n t s showed t h a t a m y t a l i s l e s s e f f e c t i v e i n - 212 -FIGURE 39 EFFECT OF AMYTAL ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES 175 Newly born guinea p i g Two day o l d r a t J . JL JL 0.25 0.5 0.75 Amytal c o n c e n t r a t i o n , i n mM 1.0 Incubation c o n d i t i o n s were same as i n Figure 37 - 213 -a Krebs-Ringer bicarbonate medium when compared with i t s action i n a C a + + - f r e e medium. E f f e c t s of pentothal on anaerobic g l y c o l y s i s resembles that of amytal (preliminary experiments). From the above i t appears that the e f f e c t s of amytal on anaerobic g l y c o l y s i s might be mediated through changes i n c a t i o n i c concentrations. Further experiments were c a r r i e d out to examine t h i s p o s s i b i l i t y . The r e s u l t s (Table 34) showed that at anesthetic doses, amytal (or pentothal) has no e f f e c t on the K + contents. However, there i s some reduction i n the Na + contents of the s l i c e s under our experimental conditions. With in f a n t guinea pigs, a s l i g h t increase i n the K + contents i n the presence of amytal was observed (Table 35). I t appears that while anesthetic concentrations of amytal (0.25 mM) have but l i t t l e e f f e c t on anaerobic g l y c o l y s i s of r a t brain - e s p e c i a l l y when compared with the r e l a t i v e l y large e f f e c t s due to the l o c a l anesthetics or TTX - increasing concentrations of the barbiturates do bring about i o n i c changes that r e s u l t i n increased rates of anaerobic g l y c o l y s i s . These doubtless are re l a t e d to the modification by barbitur-ates but more work i s required to e s t a b l i s h the nature of these changes. 7.4 EFFECTS OF CHLORPROMAZINE ON THE ANAEROBIC GLYCOLYSIS OF  CEREBRAL CORTEX SLICES Chlorpromazine i s known to suppress the anaerobic glyco-217' l y s i s i n the c e l l free extracts from brain . The e f f e c t s of 214 TABLE 34 EFFECTS OF SOME NEUROTROPIC DRUGS ON THE SODIUM AND POTASSIUM CONTENTS OF RAT CEREBRAL CORTEX SLICES Addition Cation Aerobic Anaerobic 10 min 5 min 10 min 30 min 0.25mM Amytal Na + K + 125 44 140 31 145 26 175 20 O.lmM Na+ Pentothal K+ 120 42 140 30 147 24 170 17 0.25mM Na1 Chlorpro-mazine K + 132 45 125 31 145 25 175 16 i ^ Na 12uM R e s e r p i n e K + + 115 46 140 40 135 32 147 34 Incubation conditions are same as i n Figure 26. Each value represent averages of two experiments within ± 7%. For controls see Figure 26. Results are expressed as uequivalents per g i n i t i a l wet wt. - 215 -d i f f e r e n t c o n c e n t r a t i o n s o f c h l o r p r o m a z i n e on t h e a n a e r o b i c g l y c o l y s i s o f a d u l t r a t c e r e b r a l c o r t e x s l i c e s a r e shown i n F i g u r e 40. I t i s e v i d e n t t h a t c h l o r p r o m a z i n e d o e s n o t have any s i g n i f i c a n t s t i m u l a t i n g e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s . E f f e c t s o f c h l o r p r o m a z i n e on t h e N a + and K + c o n t e n t s o f a d u l t r a t and i n f a n t g u i n e a p i g c e r e b r a l c o r t e x s l i c e s a r e g i v e n i n T a b l e s 34 a n d 35. U n l i k e t h e a n e s t h e t i c s , c h l o r -p r o m a z i n e , a t t h e c o n c e n t r a t i o n t e s t e d , h a s no e f f e c t on t h e N a + and K + c o n t e n t s o f i n c u b a t e d c e r e b r a l c o r t e x s l i c e s . 7.5 EFFECTS OF AMPHETAMINE AND NIALAMIDE ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX S L I C E S Amphetamine i s one o f t h e most p o t e n t o f t h e c e n t r a l n e r -v o u s s y s t e m s t i m u l a t i n g d r u g s . I t i n h i b i t s t h e u p t a k e o f n o r -e p i n e p h r i n e by t h e s t o r a g e g r a n u l e s a n d r e l e a s e s t h e amines f r o m t h e i r s t o r a g e s i t e s . As n o t e d i n C h a p t e r 1.5, i t a l s o i n h i b i t s monoamine o x i d a s e (MAO). N i a l a m i d e i s an i n h i b i t o r o f MAO. The n e t r e s u l t o f a p p l i c a t i o n o f t h e s e d r u g s t o t h e c e n t r a l n e r v o u s s y s t e m i s an i n c r e a s e i n t h e amount o f f r e e b i o g e n i c a m i n e s . The e f f e c t s o f b o t h d - and 1-amphetamine as w e l l a s n i a l -amide on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s was s t u d i e d ( F i g u r e 41) . As i s e v i d e n t ^ t h e s e d r u g s have r e l a t i v e l y s m a l l o r no e f f e c t s on t h e r a t e o f a n a e r o b i c g l y c o -l y s i s o f c e r e b r a l c o r t e x s l i c e s . 216 TABLE 35 EFFECTS OF SOME NEUROTROPIC DRUGS ON THE SODIUM AND POTASSIUM CONTENTS OF NEWLY BORN GUINEA PIG CEREBRAL CORTEX SLICES A d d i t i o n C a t i o n A e r o b i c A n a e r o b i c 10 min 5 min 10 min 30 min 0.25mM Amytal Na K 125 47 140 42 145 38 175 36 0.25mM Na i C h l o r p r o -mazme 127 44 145 33 167 29 175 21 I n c u b a t i o n c o n d i t i o n s are same as i n F i g u r e 27. Each v a l u e r e p r e s e n t averages o f two experiments w i t h i n ± 7%. For c o n t r o l s , see F i g u r e 27. R e s u l t s are expressed as y e q u i v a l e n t s per g i n i t i a l wet wt. - 217 -FIGURE 40 EF F E C T OF CHLORPROMAZINE ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX S L I C E S 100 0 0.25 0.5 0.75 1.0 C h l o r p r o m a z i n e c o n c e n t r a t i o n f i n mM I n c u b a t i o n c o n d i t i o n s were same as i n F i g u r e 37. - 218 -FIGURE 41 EFFECTS OF AMPHETAMINE AND NIALAMIDE ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES I n c u b a t i o n c o n d i t i o n s were same as i n F i g u r e 37.(9)1-Amphet-a m i n e ; ( A ) d - a m p h e t a m i n e ; ( O ) n i a l a m i d e . - 219 -7.6 E F F E C T OF RESERPINE ON THE ANAEROBIC GLYCOLYSIS OF  CEREBRAL CORTEX SLICES R e s e r p i n e c a u s e s r e l e a s e o f amines f r o m t h e i r s t o r a g e s i t e s s o t h a t t h e y a r e more s u s c e p t i b l e t o d e g r a d a t i o n by monoamine o x i d a s e ( C h a p t e r 1.5). However, i t has no i n h i b i t o r y e f f e c t on MAO. P r o l o n g e d t r e a t m e n t w i t h r e s e r -p i n e may t h e r e f o r e c a u s e d e p l e t i o n o f a mines f r o m c e r e b r a l t i s s u e . I t i s u n c e r t a i n w h e t h e r r e s e r p i n e a f f e c t s N a + a n d K + f l u x e s a c r o s s t h e b r a i n c e l l membrane. The e f f e c t s o f r e s e r p i n e on t h e r a t e o f a n a e r o b i c g l y c o -l y s i s were i n v e s t i g a t e d ( F i g u r e 4 2). I t was f o u n d t h a t low c o n c e n t r a t i o n s o f r e s e r p i n e m a r k e d l y e n h a n c e t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f a d u l t r a t and i n f a n t g u i n e a p i g c e r e -b r a l c o r t e x s l i c e s . S i n c e a number o f d r u g s a f f e c t a n a e r o b i c g l y c o l y s i s by m o d i f y i n g t h e c a t i o n i c f l u x e s , t h e e f f e c t s o f r e s e r p i n e on + + t h e Na a n d K c o n t e n t s o f t h e c e r e b r a l c o r t e x s l i c e s u n d e r a n o x i a were i n v e s t i g a t e d . T h e s e r e s u l t s ( T a b l e 34) showed t h a t i n t h e p r e s e n c e o f 12 uM r e s e r p i n e , t h e r e i s an i n c r e a s e i n t h e K + / N a + r a t i o o f t h e i n c u b a t e d r a t c e r e b r a l t i s s u e . I t was t h o u g h t p o s s i b l e t h a t r e s e r p i n e may e x e r t t h i s e f f e c t i n d i r e c t l y b y amine r e l e a s e . I n v e s t i g a t i o n s w ere, t h e r e f o r e , c a r r i e d o u t as t o t h e e f f e c t s o f b i o g e n i c a m i n e s on t h e a n a e r o b i c g l y c o l y s i s . The r e s u l t s i n d i c a t e t h a t u n l i k e r e s e r p i n e , t h e s e a m i n e s have no l a r g e e f f e c t s on t h e r a t e o f a n a e r o b i c g l y c o l y s i s ( T a b l e 3 6 ) . T h e s e e x p e r i m e n t s show t h a t t h e a c t i o n o f r e s e r p i n e i s u n l i k e l y t o be m e d i a t e d t h r o u g h t h e r e l e a s e o f a m i n e s . I t i s t h e r e f o r e c o n c l u d e d t h a t r e s e r p i n e - 220 -FIGURE 42 EFFECTS OF RESERPINE ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SL I C E S 221 TABLE 36 EFFECTS OF SOME AMINES ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES Lactate produced ymoles per g i n i t i a l wet wt (20-80 mm) None 25.6 ± 2.3 0.0ImM Epinephrine 21.0 0.1 mM Epinephrine 29.0 + 2.4 1 mM Tyramine 30.6 ± 1.0 0.1 mM Norepinephrine 2 8.6 ± 4.5 1 mM Norepinephrine 30.8 ± 5.0 1 mM Histamine 22.8 ± 2.4 50 yM Paraxon 26.8 ± 1.3 50 yM Paraxon + ImM Acetylcholine 29.0 ± 3.6 Incubations were ca r r i e d out i n a Ca free medium containing 20 mM glucose. Additions were made at zero time and lactat e production was measured manometrically as given i n the materials and methods. - 222 -may e x e r t e f f e c t s on c a t i o n t r a n s p o r t , a s w e l l as on i t s w e l l - k n o w n e f f e c t s on amine s t o r a g e . SUMMARY OF CHAPTER 7 1. O b s e r v a t i o n s o f Adams and Q u a s t e l , t h a t p y r r o l e i n c r e a s e s t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f g u i n e a p i g c e r e b r a l c o r t e x s l i c e s i n a C a + + - f r e e medium, have been c o n f i r m e d . 2. I n t h e p r e s e n c e o f 30 mM p y r r o l e , t h e i n c u b a t e d g u i n e a p i g c e r e b r a l c o r t e x s l i c e s l o s e l e s s K + and g a i n l e s s N a + u n d e r a n o x i a . I t i s c o n c l u d e d t h a t t h e a c t i o n o f p y r r o l e on a n a e r o b i c g l y c o l y s i s i s m e d i a t e d t h r o u g h i n c r e a s e i n t h e K + / N a + r a t i o o f t h e b r a i n c e l l . 3. P y r r o l e s t i m u l a t e d g l y c o l y s i s r e s e m b l e s TTX s t i m u l a t e d g l y c o l y s i s by b e i n g i n h i b i t e d by 5 mM g l u t a m a t e b u t n o t by 5 mM a s p a r t a t e . 4. I n a C a + + - f r e e medium a m y t a l i n c r e a s e s t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f a d u l t r a t a s w e l l as i n f a n t g u i n e a p i g c e r e b r a l c o r t e x s l i c e s w h i l e i t has no e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f i n f a n t r a t s . I t s e f f e c t s , a t a n e s t h e t i c c o n c e n t -r a t i o n s , a r e l e s s t h a n t h o s e o f TTX o r l o c a l a n e s t h e t i c s . I n f a n t g u i n e a p i g c e r e b r a l c o r t e x s l i c e s were f o u n d t o be v e r y s e n s i t i v e t o t h e p r e s e n c e o f a m y t a l . 5. A m y t a l h a s no e f f e c t on t h e K + c o n t e n t o f t h e i n c u b a t e d r a t c e r e b r a l c o r t e x s l i c e s u n d e r a n o x i a , w h i l e t h e N a + c o n t e n t i s s l i g h t l y d e c r e a s e d . 6. The e f f e c t o f a m y t a l on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s seems t o be i n d e p e n d e n t o f i t s a c t i o n as a r e s p i r a t o r y i n h i b i t o r . 7. C h l o r p r o m a z i n e h a s l i t t l e o r no e f f e c t on t h e a n a e r o b i c + + g l y c o l y s i s o r Na a n d K c o n t e n t o f t h e i n c u b a t e d c e r e b r a l c o r t e x s l i c e s u n d e r a n o x i a . 8. D- and L-amphetamine, as w e l l as n i a l a m i d e , h a s l i t t l e o r no e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . 9. R e s e r p i n e , a t low c o n c e n t r a t i o n s , i n c r e a s e s t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . The K + / N a + r a t i o o f t h e i n c u b a t e d c e r e b r a l c o r t e x s l i c e s , i n t h e p r e s e n c e o f 12 yM r e s e r p i n e , i s i n c r e a s e d u n d e r a n o x i a . 10. E p i n e p h r i n e , n o r e p i n e p h r i n e , h i s t a m i n e , t y r a m i n e and a c e t y l c h o l i n e have l i t t l e o r no s t i m u l a t o r y a c t i o n on a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . P r e s u m a b l y t h e a c t i o n o f r e s e r p i n e i s n o t m e d i a t e d t h r o u g h t h e r e l e a s e o f amines f r o m s t o r a g e s i t e s . CHAPTER 8 DISCUSSION 8.1. E FFECT OF CALCIUM IONS ON CEREBRAL ANAEROBIC GLYCOLYSIS R e s u l t s d e s c r i b e d i n C h a p t e r 3 show t h a t a v a r i e t y o f com p o u n d s , e.g. c a t i o n s , n u c l e o t i d e s e t c . , e x e r t r a t e l i m i t i n g a c t i o n s on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s a s w e l l as o f a c e t o n e powder e x t r a c t s f r o m b r a i n . Thus t h e ++ p r e s e n c e o f Ca an t h e i n c u b a t i o n medium g r e a t l y e n h a n c e s t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s ( F i g -++ u r e s 1 and 2 ) . The e f f e c t o f Ca on t h e c e r e b r a l c o r t e x s l i c e s d i f f e r s m a r k e d l y f r o m t h a t on E h r l i c h a s c i t e s tumour e x t r a c t s , where i t i n h i b i t s g l y c o l y s i s 6 ^ . On t h e o t h e r hand, C a + + h a s no e f f e c t on t h e g l y c o l y s i s o f i n t a c t tumour c e l l s 6 ^ ' 7 . The e f f e c t o f C a + + on t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f g u i n e a p i g c e r e b r a l c o r t e x s l i c e s has b e e n s t u d i e d i n some 71 d e t a i l by Adams & Q u a s t e l b u t t h e y c o u l d n o t o f f e r a s a t i s -f a c t o r y e x p l a n a t i o n f o r t h i s phenomenon a t t h a t t i m e . T h e s e w o r k e r s , however, f o u n d t h a t a number o f o r g a n i c b a s e s a c t i n a s i m i l a r way t o C a + + , and c o r r e l a t e d t h e s e e f f e c t s w i t h t h e i r d i s s o c i a t i o n c o n s t a n t s . I t was a l s o f o u n d t h a t a m i x t u r e o f b a s e s w i t h C a + + g a v e no a d d i t i v e s t i m u l a t o r y e f f e c t s u g g e s t i n g a common s i t e o f a c t i o n . I t i s p o s s i b l e , as p o i n t e d o u t by Adams & Q u a s t e l , t h a t t h e e f f e c t s a r e a s s o c i a t e d w i t h c h a n g e s i n membrane p e r m e a b i l -i t y b r o u g h t a b o u t by t h e i o n s . I n t h e p r e s e n c e o f C a + + , t h e r e i s some i n c r e a s e d r e t e n t i o n o f K + by t h e i n c u b a t e d c e r e b r a l c o r - 225 -t e x s l i c e s ( F i g u r e 26) and t h i s may be one o f t h e r e a s o n s why C a + + has a s t i m u l a t o r y e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . A v i e w t h a t C a + + a c t s by b e c o m i n g p a r t o f t h e s t r u c t u r a l p a t t e r n o f t h e c e l l membrane i n s u c h a way t h a t a c t i v e t r a n s -71 p o r t o f g l u c o s e i n t o t h e n e u r o n i s f a c i l i t a t e d i s u n l i k e l y t o be t r u e as t h e r e i s no e v i d e n c e t h a t t h e r e i s a c t i v e ( i . e . e n e r g y a s s i s t e d ) t r a n s p o r t o f g l u c o s e i n t o b r a i n i n v i t r o , and as t h e r a t e o f c e r e b r a l a n a e r o b i c g l y c o l y s i s i s n o t a f f e c t e d by i n c r e a s i n g t h e g l u c o s e c o n c e n t r a t i o n f r o m 5 mM t o 50 mM. Un d e r a n a e r o b i c c o n d i t i o n s , t h e ATP c o n t e n t o f t h e c e l l i s much d i m i n i s h e d and may become a r a t e l i m i t i n g f a c t o r f o r t h e p h o s p h o r y l a t i o n o f g l u c o s e and f r u c t o s e 6 - p h o s p h a t e . C a + + i s a s t r o n g i n h i b i t o r o f m i c r o s o m a l N a + , K + - A T P a s e , and i t may t h e r e f o r e s t i m u l a t e t h e a n a e r o b i c g l y c o l y s i s by d e c r e a s i n g t h e + + l o s s o f ATP b y Na ,K -ATPas e a c t i v i t y . However, i t i s u n c e r t a i n as t o w h e t h e r e x t e r n a l C a + + i n h i b i t s t h e enzyme i n b r a i n s l i c e s . • i i « , , , . . _ _ ++ f r o m t h e i n c u b -I t i s now w e l l known t h a t o m i s s i o n o f Ca a t i o n medium r e s u l t s i n an i n f l u x o f N a + + i n t h e n e r v e c e l l w i t h c o n c o m i t a n t d e p o l a r i z a t i o n 3 ^ . I n t h e p r e s e n c e o f C a + + t h i s ( Na +) i n f l u x i s p r e v e n t e d and may, a l o n g w i t h t h e r e t e n t i o n o f K + , and p o s s i b l e i n c r e a s e i n t h e c e l l ATP c o n c e n -t r a t i o n , be r e s p o n s i b l e f o r t h e i n c r e a s e d r a t e o f g l y c o l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s . The e f f e c t s o f C a + + on t h e g l y c o l y s i s o f d e v e l o p i n g b r a i n ( T a b l e 1) p r o v i d e some i n t e r e s t i n g f e a t u r e s . I n f a n t r a t b r a i n i s n o t known t o r e s p o n d t o t h e p r e s e n c e o f h i g h K + o r e l e c t r i c a l - 226 -235 s t i m u l a t i o n . R e s u l t s r e p o r t e d h e r e i n show t h a t t h e a n -a e r o b i c g l y c o l y s i s o f 2-day o l d r a t b r a i n d o e s n o t r e s p o n d t o C a + + . The r e s p o n s i v e n e s s i n c r e a s e s d u r i n g t h e p e r i o d o f maximum b r a i n g r o w t h a n d m y e l i n a t i o n ( i e . d u r i n g t h e 2nd w e e k ) , I n f a n t g u i n e a p i g b r a i n was f o u n d t o b e e x t r e m e l y s e n s i t i v e t o t h e p r e s e n c e o f Ca i n t h e i n c u b a t i o n medium; t h i s i s n o t u n e x p e c t e d , a s i s w e l l known t h a t t h e b r a i n o f i n f a n t g u i n e a p i g i s more m a t u r e t h a n t h a t o f i n f a n t r a t b r a i n a n d b e h a v e s l i k e a n a d u l t b r a i n ( C h a p t e r 1 ) . ++ A l t h o u g h t h e r e i s e v i d e n c e t h a t C a i n h i b i t s some o f t h e g l y c o l y t i c enzymes i n c e l l f r e e e x t r a c t s , t h i s i s n o t t h e c a s e w i t h t h e b r a i n s l i c e s a s i n t h a t e v e n t r e m o v i n g C a + + f r o m t h e i n c u b a t i o n medium w i l l r e s u l t i n d e c l i n e i n t h e i n t r a c e l -l u l a r C a + + , a n d t h i s w o u l d r e s u l t i n g r e a t e r r a t e o f g l y c o -l y s i s . H o w e v e r , t h i s i s n o t t r u e . The g l y c o l y s i s o f c e l l - f r e e e x t r a c t s a n d s y n a p t o s o m e s b e h a v e s i m i l a r l y i n t h e s e n s e t h a t b o t h a r e i n h i b i t e d b y C a + + . 8.2. EFFECTS OF EXOGENOUS NUCLEOTIDES ON THE ANAEROBIC GLYCO- L Y S I S I t i s known t h a t t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s d e c r e a s e s p r o g r e s s i v e l y w i t h t i m e . As h a s b e e n p o i n t e d o u t i n C h a p t e r 3, t h i s d e c r e a s e may be due t o t h e l o s s o f c o - e n z y m e s o r due t o a c h a n g e i n t h e c a t i o n i c com-p o s i t i o n o f t h e c e r e b r a l c o r t e x s l i c e s . M c l l w a i n h a s f o u n d + 15 t h a t t i s s u e NAD + NADH l e v e l d e c r e a s e s d u r i n g a n o x i a . I n v i e w o f t h i s f a c t , e x p e r i m e n t s w e r e c a r r i e d o u t t o e x a m i n e - 227 -w h e t h e r t h e NAD c o n c e n t r a t i o n o f t h e c e r e b r a l c o r t e x s l i c e s becomes r a t e l i m i t i n g f o r a n a e r o b i c g l y c o l y s i s . R e s u l t s g i v e n i n T a b l e 2 i n d i c a t e t h a t t h e a d d i t i o n o f NAD + t o t h e i n c u b a t i o n medium i n c r e a s e s t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s . ATP, u n d e r t h e same c o n d i t i o n s , has no e f f e c t b u t p o s s i b l y i t i s h y d r o l y z e d b e f o r e r e a c h i n g t h e s i t e o f g l y c o l y s i s . T r a n s p o r t o f NAD + h a s b e e n i n v e s t i -g a t e d ( F i g u r e s 3 and 4) and i t has b e e n f o u n d t h a t c o n s i d e r -a b l e e x o g e n o u s NAD + c a n p e n e t r a t e t h e c e l l . The p o s s i b i l i t y t h a t t h e s e r e s u l t s a r e an a r t i f a c t due t o mere b i n d i n g o f NAD + t o t h e o u t e r membrane, i s r u l e d o u t b e c a u s e , u n d e r t h e same c o n d i t i o n s , t h e r e i s an i n c r e a s e i n t h e c o n c e n t r a t i o n o f NADH w i t h i n t h e c e l l w i t h c o n c o m i t a n t i n c r e a s e i n t h e r a t e o f a n a e r o b i c g l y c o l y s i s . T h i s o c c u r s i n s p i t e o f t h e f a c t t h a t c o n s i d e r a b l e NADase i s p r e s e n t i n t h e b r a i n c e l l and no i n h i b i t o r o f NADase was u s e d d u r i n g t h e s e e x p e r i m e n t s . P o s -s i b l y i n t h e i n t a c t c e l l , NAD + i s n o t d e g r a d e d as r a p i d l y a s i t i s i n homogenates o r a c e t o n e powder e x t r a c t s . T h i s m i g h t be due t o t h e l o c a l i z a t i o n o f NADase i n s u b c e l l u l a r s t r u c -t u r e s s u c h a s l y s o s o m e s . NAD + has no e f f e c t on t h e r a t e o f a e r o b i c g l y c o l y s i s i n p r e s e n c e o f DNP. T h i s l a c k o f e f f e c t o f NAD + i n t h e p r e s e n c e o f DNP may be due t o t h e f a c t t h a t an o p t i m a l r a t e o f g l y c o l y s i s i s o b t a i n e d i n t h e p r e s e n c e o f t h i s s u b s t a n c e . The e f f e c t o f NAD + on t h e a n a e r o b i c g l y c o l y s i s o f c e r e -b r a l c o r t e x s l i c e s i s n o t due t o i n h i b i t i o n o f A T P a s e b e c a u s e - 228 -NAD + has no e f f e c t on t h e a c t i v i t y o f t h i s enzyme. T h e s e r e s u l t s p o i n t t o t h e p o s s i b i l i t y t h a t u n d e r a n a e r o b i c c o n -d i t i o n s t h e c e l l NAD + c o n c e n t r a t i o n may i n f a c t be r a t e -l i m i t i n g f o r a n a e r o b i c g l y c o l y s i s . I n t h e p r e s e n c e o f C a + + , 2.5 mM ATP has an i n h i b i t o r y e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s ( T a b l e 2 ) . T h i s i s p o s s i b l y due t o c h e l a t i o n o f C a + + i n t h e i n c u b a t i o n med-ium by ATP, w h i c h i s w e l l known t o combine w i t h C a + + . A l t e r -n a t i v e l y , i t i s p o s s i b l e ( b u t l e s s l i k e l y ) t h a t i n t h e p r e -s e n c e o f C a + + , ATP may p e n e t r a t e t h e t i s s u e u n h y d r o l y z e d and i n h i b i t t h e p h o s p h o f r u c t o k i n a s e a c t i v i t y . 8.3. EFFECTS OF CATIONS ON THE ANAEROBIC GLYCOLYSIS The r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s shows s t r o n g d e p e n d e n c e f o r t h e p r e s e n c e o f c a t i o n s i n t h e i n c u b a t i o n medium ( T a b l e 3 ) . A d d i t i o n o f a h i g h c o n c e n t r a t i o n o f K + t o a R i n g e r med-ium i n h i b i t s t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r -t e x s l i c e s . However, r e s u l t s , shown i n F i g u r e 8, i n d i c a t e t h a t when t h e c o n c e n t r a t i o n o f N a + i s r e d u c e d and K + i s i n c r e a s e d a t t h e same t i m e , t h e r a t e o f a n a e r o b i c g l y c o l y s i s i s e n h a n c e d . T h i s i s e x p l a i n e d by t h e f a c t t h a t an i n c r e a s e d c o n c e n t r a t i o n s o f K + i n t h e i n c u b a t i o n medium r e s u l t i n c o n s i d e r a b l e i n f l u x + 135 o f Na i n t o t h e b r a i n c e l l and t h e i n h i b i t o r y e f f e c t o f t h e i n c r e a s e d c e l l c o n c e n t r a t i o n o f N a + may t h e n o u t w e i g h t h e s t i m -u l a t o r y a c t i o n o f K + . When t h e N a + c o n c e n t r a t i o n i n t h e i n c u b -a t i o n medium i s a l s o d e c r e a s e d a t t h e t i m e when K + i s i n c r e a s e d , -22q-l e s s Na e n t e r s t h e b r a i n c e l l i n t h e p r e s e n c e o f a h i g h c o n c e n t r a t i o n o f K + . T h e r e r e s u l t s l i t t l e i n c r e a s e i n t h e i n t r a c e l l u l a r N a + compared w i t h t h e i n c r e a s e i n c e l l u l a r K + and t h i s p r e s u m a b l y r e s u l t s i n a g r e a t e r r a t e o f a n a e r o b i c g l y c o l y s i s . D e c r e a s i n g t h e e x t r a c e l l u l a r c o n c e n t r a t i o n o f N a + , w i t h o u t i n c r e a s i n g t h e K + c o n c e n t r a t i o n , i t s e l f r e s u l t s i n g r e a t e r r a t e o f a n a e r o b i c g l y c o l y s i s ( F i g u r e 3 2 ) . Thus t h e K + / N a + r a t i o has a c o n t r o l l i n g e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . The i n c r e a s e d r a t e o f a n a e r o b i c g l y c o l y s i s i n t h e p r e s e n c e o f h i g h K + / N a + r a t i o i s p r e s u m a b l y m e d i a t e d t h r o u g h c h a n g e s i n t h e a c t i v i t y o f p y r u v a t e k i n a s e . O v e r 0-50 mM r a n g e , d o u b l i n g t h e K + c o n -c e n t r a t i o n r e s u l t s i n a t w o - f o l d i n c r e a s e i n t h e p y r u v a t e 59 + +301 k i n a s e a c t i v i t y . Na i n h i b i t s t h e a c t i v a t i o n by K I t i s c o n f i r m e d t h a t L - g l u t a m a t e i n h i b i t s t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . ' I t i s now w e l l known t h a t L - g l u t a m a t e c a u s e s a l a r g e N a + - i n f l u x and t h i s i s d o u b t l e s s r e s p o n s i b l e f o r i t s i n h i b i t o r y a c t i o n on t h e a n a e r o b i c g l y c o -l y s i s . 8.4. ANAEROBIC GLYCOLYSIS OF ACETONE POWDER EXTRACTS OF BRAIN From t h e r a t e s o f a n a e r o b i c g l y c o l y s i s i n t h e a c e t o n e powder e x t r a c t s , i t c a n be o b s e r v e d t h a t w i t h s u c h e x t r a c t s , a much h i g h e r r a t e o f g l y c o l y s i s i s o b t a i n e d as compared t o t h e c o r r e s p o n d i n g amount o f t h e b r a i n s l i c e s ( F i g u r e s 10 & 1 1 ) . T h i s may be due t o t h e f a c t t h a t i n t h e c e l l - f r e e e x t r a c t s t h e g l y c o l y t i c i n t e r m e d i a t e s and coenzymes may be r e a d i l y - 230 ~ a v a i l a b l e to the enzymes as compared to the brain s l i c e s , where the s p e c i f i c l o c a l i z a t i o n of the enzymes and compart-mentalization of the metabolites must be playing an import-ant r o l e i n determining the o v e r a l l rate of g l y c o l y s i s . When the rate of g l y c o l y s i s i s high, as i n the acetone pow-der extracts, K + does not have very large e f f e c t s on the rate of anaerobic g l y c o l y s i s . 8.5. EFFECTS OF TETRODOTOXIN ON ANAEROBIC GLYCOLYSIS OF BRAIN TTX exerts marked e f f e c t s on the metabolism of brain cortex s l i c e s incubated a e r o b i c a l l y under a v a r i e t y of con-130 d i t i o n s . I t was shown by Chan and Quastel , and by M c l l -129 warn , independently and at about the same time, i n 1967, that the drug suppresses the increased r e s p i r a t i o n induced by ap p l i c a t i o n of e l e c t r i c a l impulses. Moreover, the drug at small concentrations, suppresses the e l e c t r i c a l l y induced i n -f l u x of Na + as shown d i r e c t l y by measurements of the i n f l u x of l a b e l l e d Na + and i n d i r e c t l y by the stimulatory action of the drug on the cerebral oxidation of acetate which i s inhib-i t e d by the i n f l u x of Na + due to e l e c t r i c a l stimulation. 135 Recently, i t has been shown by Okamoto and Quastel that tetrodotoxin i n h i b i t s both the increased water uptake and i n f l u x of Na + that occurs under a va r i e t y of aerobic incu-bation conditions, for example i n the presence of protovera-t r i n e or of ouabain or i n the absence of glucose. It was con-cluded that under such conditions action potentials are gen-erated i n the incubated brain s l i c e s , or that there i s some - 2 3 i -a c t i v a t i o n o f a s p e c i f i c s o d i u m c u r r e n t s y s t e m , t o a c c o u n t f o r t h e marked e f f e c t s o f t e t r o d o t o x i n i n v i t r o . R e s u l t s shown i n F i g u r e 12 show t h a t t h e p r e s e n c e o f low c o n c e n t r a t i o n s o f TTX m a r k e d l y s t i m u l a t e s t h e r a t e o f a n a e r -o b i c g l y c o l y s i s o f t h e i n c u b a t e d c e r e b r a l c o r t e x s l i c e s , b o t h i n a C a + + - c o n t a i n i n g as w e l l as i n a C a + + - f r e e medium. T h i s phenomenon i s o f c o n s i d e r a b l e i n t e r e s t . The a n o x i c c o n d i t i o n c a n be r e g a r d e d as one l e a d i n g t o t h e g e n e r a t i o n o f a c t i o n p o t e n t i a l s , a s u n d e r t h e s e c o n d i t i o n s t h e r e i s s t i m u l a t i o n o f t h e c a t i o n c a r r y i n g s y s t e m r e s u l t i n g i n d e p o l a r i z a t i o n o f t h e c e l l . The e f f e c t o f TTX on a n a e r o b i c g l y c o l y s i s d i f f e r s i n r a t and g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . T h u s , w i t h r a t t h e v a l u e s o b t a i n e d i n t h e p r e s e n c e o f a m i x t u r e o f C a + + and TTX b o t h t o g e t h e r a r e e i t h e r t h e same o r s l i g h t l y r e d u c e d w h i l e i n t h e g u i n e a p i g t h e r a t e i s f u r t h e r i n c r e a s e d ( F i g u r e 1 4 ) . T h i s i s p o s s i b l y due t o t h e f a c t t h a t C a + + e x e r t s q u a l i -t a t i v e l y d i f f e r e n t e f f e c t s on c e r e b r a l a n a e r o b i c g l y c o l y s i s a c c o r d i n g t o t h e a n i m a l s p e c i e s . Na"1" I n f l u x : B e c a u s e TTX i s a s p e c i f i c i n h i b i t o r o f t h e N a + - c a r r y i n g s y s t e m d u r i n g an a c t i o n p o t e n t i a l , i t was t h o u g h t t h a t i t may a c t on a n a e r o b i c g l y c o l y s i s by a b o l i s h i n g t h e g e n e r a t i o n o f a c t i o n p o t e n t i a l s and h e n c e t h e c o n c o m i t a n t movement o f N a + a t t h e o n s e t o f a n o x i a . However, l a r g e r e f f e c t s o f TTX on t h e i n f l u x o f l a b e l l e d N a + d u r i n g a n o x i a were n o t o b s e r v e d - 232 - t ( T a b l e 1 8 ) . T h u s , when t h e i n f l u x o f Na was s t u d i e d , t h e p e r c e n t a g e s u p p r e s s i o n o f N a + i n f l u x i s n o t l a r g e enough t o a s s i g n i t t h e m a j o r r o l e i n e n h a n c i n g t h e p y r u v a t e k i n a s e a c t i v i t y by i t s d e c r e a s e d c o n c e n t r a t i o n . C l - f r e e M e d i a S u b s t i t u t i o n o f C l by SO^ d o e s n o t r e s u l t i n i n c r e a s e d r a t e s o f a n a e r o b i c g l y c o l y s i s - e i t h e r i n t h e a b s e n c e as w e l l as i n t h e p r e s e n c e o f TTX ( T a b l e 1 9 ) . I t i s w e l l known t h a t when C l i s r e p l a c e d by t h e r e l a t i v e l y i m p e r m e a b l e SO^ , t h e r e i s r e d u c e d i n f l u x o f N a + i n t o t h e i n c u b a t e d c e r e b r a l c o r t e x s l i c e s u n d e r a v a r i e t y o f c o n d i t i o n s . TTX i s f o u n d t o be e f f e c t i v e i n i n c r e a s i n g t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f t h e b r a i n s l i c e s i n a C l - f r e e medium i n s p i t e o f t h e r e d u c e d i n f l u x o f N a + . M o r e o v e r , t h e r a t e o f g l y c o l y s i s i n a C l - f r e e medium i s n o t g r e a t e r t h a n i n a medium i n w h i c h n o r m a l c o n -c e n t r a t i o n o f C l i s p r e s e n t . I t i s d i f f i c u l t t o c o n c l u d e f r o m t h e s e r e s u l t s t h a t c h a n g e s i n t h e c e l l N a + a r e s o l e l y r e s p o n s i b l e f o r t h e i n c r e a s e i n t h e r a t e o f a n a e r o b i c g l y c o -l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s i n t h e p r e s e n c e o f TTX. A number o f e x p e r i m e n t s were c a r r i e d o u t t o t e s t o t h e r p o s s i b i l i t i e s as t o t h e mode o f a c t i o n o f TTX. As i t has b e e n m e n t i o n e d e a r l i e r i n t h i s c h a p t e r , a number o f f a c t o r s may become r a t e l i m i t i n g f o r t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s . C hanges i n t h e c o n c e n t r a t i o n o f e a c h o f t h e s e f a c t o r s may r e s u l t i n an i n h i b i t i o n o r a c t i -v a t i o n o f t h e r a t e o f g l y c o l y s i s , and hence t h e s e were i n v e s t i -- 23 3 -gated i n an e f f o r t to throw more l i g h t on the mechanism of enhancement of anaerobic g l y c o l y s i s by the presence of TTX. These experiments w i l l now be discussed. Role of Nucleotides. As has been mentioned, there i s a marked decline i n the concentration of NAD+ + NADH of the cerebral cortex s l i c e s under anoxia and i t may have a rate-l i m i t i n g e f f e c t on the anaerobic g l y c o l y s i s . The exact reason for the decline i n the NAD+ + NADH l e v e l i s not known but possibly i t may be due to a va r i e t y of causes such as (i) rapid + + breakdown of NAD under anoxia, ( i i ) loss of NAD from the tissue during anaerobic incubation. The second p o s s i b i l i t y has been ruled out by us as there i s no evidence i f there i s a leakage of NAD+ (or NADH) into the incubation medium. If i t does leak out i n the medium under the given experimental conditions then i t i s degraded so quickly that i t can not be detected i n our experiments. Furthermore, there i s no increase i n the NAD l e v e l of the cerebral cortex s l i c e s i n the presence of TTX (Chapter 4.3). Hence, the p o s s i b i l i t y that the e f f e c t of TTX on anaerobic g l y c o l y s i s i s due to an increase i n the c e l l NAD+ l e v e l due to reduced e f f l u x i s considered most u n l i k e l y . As early as 1928 i t was demonstrated that a short pre-incubation i n oxygen markedly increases the subsequent rate 289 of anaerobic g l y c o l y s i s . This has been shown f o r a number of adult tissues, including brain, but the reason for t h i s i s 290-294 not clear . It has been stated that the increased rate of anaerobic g l y c o l y s i s of the cerebral cortex s l i c e s a f t e r an, - 234 -a e r o b i c p r e i n c u b a t i o n m i g h t be t h e r e s u l t o f a c c u m u l a t i o n o f 71 p y r u v a t e d u r i n g t h e a e r o b i c p e r i o d . However, t h e r o l e o f ATP c o n c e n t r a t i o n , w h i c h c a n e x e r t a r a t e l i m i t i n g e f f e c t on a n a e r o b i c g l y c o l y s i s , has n o t b e e n d i s c u s s e d . One o f t h e m a j o r c o n s e q u e n c e s o f a n o x i a i s t h e l o s s o f t h e c a p a c i t y o f t h e t i s s u e t o c a r r y o u t m i t o c h o n d r i a l p h o s -p h o r y l a t i o n r e s u l t i n g i n d e c r e a s e d ATP c o n c e n t r a t i o n , and many o f t h e o t h e r e f f e c t s o f a n o x i a a r e i n d i r e c t l y due t o a d e -c r e a s e i n t h e ATP c o n t e n t . Under a n a e r o b i c c o n d i t i o n s , t h e ATP c o n t e n t f a l l s t o a v e r y low l e v e l a n d, as p o i n t e d o u t e a r l i e r , i t may e x e r t a r a t e l i m i t i n g e f f e c t on t h e g l y c o -l y s i s d u r i n g s u b s e q u e n t a n a e r o b i o s i s . I t was o b s e r v e d by us t h a t t h e r e i s an i n c r e a s e i n t h e a c i d l a b i l e p h o s p h a t e and t h e ATP c o n t e n t ( T a b l e 15) o f t h e c e r e b r a l c o r t e x s l i c e s i n t h e p r e s e n c e o f TTX u n d e r a n a e r o b i c c o n d i t i o n s , and h e n c e t h e p o s s i b i l i t y e x i s t e d t h a t TTX may a c t by i n c r e a s i n g t h e ATP c o n t e n t o f t h e i n c u b a t e d s l i c e s . E x p e r i m e n t s were c a r r i e d o u t , t h e r e f o r e , t o a s c e r t a i n t h e e x t e n t t o w h i c h t h e c e l l l e v e l o f ATP i s r e s p o n s i b l e f o r t h e s t i m u l a t i n g a c t i o n o f TTX on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . I n t h e s e e x p e r i m e n t s , c o n d i t i o n s were made s u c h t h a t t h e r e was i n d u c e d e i t h e r a d e c r e a s e o r an i n c r e a s e i n t h e ATP c o n t e n t o f t h e c e r e b r a l c o r t e x s l i c e s i n t h e p r e s e n c e o f TTX i n t h e i n c u b a t i o n medium ( T a b l e s 13 and 1 4 ) . When TTX i s added t o t h e i n c u b a t i o n medium a f t e r 10 o r - 235 -15 m i n u t e s o f a n o x i a , t h e n i t has no s t i m u l a t i n g e f f e c t on t h e a n a e r o b i c g l y c o l y s i s ( F i g u r e 2 1 ) . T h i s shows t h a t some c h a n g e s , t a k i n g p l a c e d u r i n g t h e f i r s t few m i n o f a n o x i a , s u c h as d e c r e a s e i n ATP c o n t e n t a n d / o r l o s s o f K + , a r e r e -s p o n s i b l e f o r t h e e f f e c t s o f t e t r o d o t o x i n on t h e a n a e r o b i c g l y c o l y s i s . I f a n a e r o b i o s i s i s c a r r i e d o u t f o r 15 m i n i n t h e a b s e n c e o f g l u c o s e b u t i n t h e p r e s e n c e o f TTX, and t h e n g l u c o s e i s ad d e d t o t h e i n c u b a t i o n medium, TTX i s n o t e f f e c t -i v e i n i n c r e a s i n g t h e r a t e o f a n a e r o b i c g l y c o l y s i s . However, i f t h e f i r s t 10 m i n i n c u b a t i o n p e r i o d i s made a e r o b i c , i n t h e a b s e n c e o f g l u c o s e b u t i n t h e p r e s e n c e o f p y r u v a t e , t h e n TTX i s s t i l l e f f e c t i v e i n i n c r e a s i n g t h e s u b s e q u e n t r a t e o f a n a e r o b i c g l y c o l y s i s ( T a b l e 1 3 ) . A s i m i l a r r e s u l t i s o b t a i n e d i f t h e p r e l i m i n a r y a n o x i c p e r i o d (15 min) i s f o l l o w e d by a 10 m i n a e r o b i c p e r i o d and t h e n by t h e a d d i t i o n o f g l u c o s e . When p y r u v a t e i s p r e s e n t f r o m z e r o t i m e b u t g l u c o s e i s a d d e d i m m e d i a t e l y a f t e r a p e r i o d o f a n o x i a , t h e n TTX i s n o t e f f e c t -i v e i n i n c r e a s i n g t h e r a t e o f a n a e r o b i c g l y c o l y s i s . T h e s e r e s u l t s show t h a t e i t h e r t h e p r e s e n c e o f g l u c o s e f r o m t h e b e g i n n i n g o f t h e e x p e r i m e n t o r an a e r o b i c p r e i n c u -b a t i o n i s n e c e s s a r y f o r t h e s l i c e s t o show any r e s p o n s e t o TTX. One t h a t w o u l d be g r e a t l y i n f l u e n c e d u n d e r t h e s e c o n -d i t i o n s s h o u l d be t h e ATP c o n c e n t r a t i o n o f t h e c e r e b r a l c o r -t e x s l i c e s . E x p e r i m e n t s c a r r i e d o u t , i n t h e p r e s e n c e o f DNP, w h i c h u n c o u p l e s t h e o x i d a t i v e p h o s p h o r y l a t i o n and h e n c e d e -c r e a s e s t h e ATP c o n t e n t s ( T a b l e 1 4 ) , show t h a t when t h e a e r o -- 2 3 6 -b i c p r e i n c u b a t i o n i s c a r r i e d o u t i n t h e p r e s e n c e o f DNP, t h e n t h e p e r c e n t s t i m u l a t i o n o f a n a e r o b i c g l y c o l y s i s i n t h e p r e s e n c e o f TTX i s n o t a f f e c t e d . T h e s e e x p e r i m e n t s l e a d t o t h e c o n c l u s i o n t h a t t h e c e l l ATP i s n o t r e s p o n s i b l e f o r t h e e f f e c t o f TTX on t h e s p e e d o f a n a e r o b i c g l y c o l y s i s . E x p e r i m e n t s c a r r i e d o u t w i t h a d e n o s i n e - p r e i n c u b a t e d s l i c e s show t h a t a l t h o u g h w i t h s u c h s l i c e s , t h e r e i s an i n -c r e a s e i n t h e r a t e o f l a c t a t e p r o d u c t i o n i n c o n t r o l s , p o s -s i b l y due t o a r i s e i n ATP, t h e e f f e c t o f TTX i s u n a f f e c t e d . T h e s e s l i c e s a l s o r e s p o n d i n a manner s i m i l a r t o t h e non-a d e n o s i n e p r e i n c u b a t e d s l i c e s i n s h o w i n g t h e e f f e c t s o f TTX when i t i s adde d a f t e r 15 m i n o f a n o x i a . The h i g h e r c o n t e n t o f ATP i n t h e i n c u b a t e d c e r e b r a l c o r -t e x s l i c e s , i n t h e p r e s e n c e o f TTX, may be t h e r e s u l t o f a h i g h e r r a t e o f g l y c o l y s i s i t s e l f r a t h e r t h a n t h a t o f an i n t e r -f e r e n c e o f TTX w i t h t h e e n e r g y u t i l i z a t i o n p r o c e s s e s o f t h e b r a i n c e l l s . R o l e o f P y r u v a t e P y r u v a t e i s known t o i n c r e a s e c o n s i d e r a b l y t h e r a t e o f a n a e r o b i c g l y c o l y s i s and i t has b e e n s t a t e d t h a t i t m i g h t a c t by a f f e c t i n g t h e NAD +/NADH r a t i o . As NAD +/NADH r a t i o i s i m p o r t a n t f o r t h e r e g u l a t i o n o f t h e r a t e o f a n a e r o b i c g l y c o -l y s i s , TTX may a c t by p r e v e n t i n g t h e e f f l u x o f p y r u v a t e and h e n c e a f f e c t i n g t h e NAD +/NADH r a t i o . However, when p y r u v a t e and TTX a r e p r e s e n t t o g e t h e r , t h e i r e f f e c t o n a n a e r o b i c g l y -c o l y s i s i s a d d i t i v e . T h i s shows t h a t b o t h t h e s e a g e n t s must - 237 -be a f f e c t i n g t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s by d i f f e r e n t m e c h a n i s m s . T h e s e e x p e r i m e n t s r u l e o u t t h e p o s s i b i l i t y t h a t t h e e f f e c t o f TTX i s due t o t h e s u p p r e s s i o n o f p y r u v a t e e f f l u x f r o m t h e i n c u b a t e d s l i c e s . R o l e o f G l u c o s e C o n c e n t r a t i o n The p e r c e n t a g e s t i m u l a t i o n o f a n a e r o b i c g l y c o l y s i s by TTX f a l l s when t h e e x t e r n a l g l u c o s e c o n c e n t r a t i o n i s d e -c r e a s e d . The r a t e o f a n a e r o b i c g l y c o l y s i s , i n t h e p r e s e n c e o f TTX, p r o g r e s s i v e l y i n c r e a s e s u n t i l a b o u t 50 mM g l u c o s e c o n c e n t r a t i o n , when a maximum r a t e o f g l y c o l y s i s i s o b t a i n e d . T h i s e f f e c t i s n o t due t o f a c i l i t a t i o n o f g l u c o s e e n t r y i n t o t h e s l i c e s by TTX ( T a b l e 6 ) . T h e s e r e s u l t s d e m o n s t r a t e t h a t , u n d e r c o n d i t i o n s when t h e r a t e o f a n a e r o b i c g l y c o l y s i s i s h i g h , t h e c o n c e n t r a t i o n o f g l u c o s e becomes a r a t e l i m i t i n g f a c t o r . T h i s i s d o u b t l e s s due t o t h e f a c t t h a t w i t h h i g h r a t e s o f g l y c o l y s i s , h i g h e x t e r n a l c o n c e n t r a t i o n o f g l u c o s e a r e r e q u i r e d t o s a t u r a t e t h e g l u c o s e u t i l i z a t i o n s y s t e m i n t h e b r a i n c e l l o r c e l l c o m p a r t m e n t s . R o l e o f cAMP As shown i n C h a p t e r 5.8, t h e cAMP f o r m a t i o n i n t h e c e r e b r a l c o r t e x s l i c e s i n t h e p r e s e n c e o f TTX i s n o t i n -c r e a s e d ; i n s t e a d a d e c r e a s e i s o b s e r v e d . T h e s e r e s u l t s d e m o n s t r a t e t h a t t h e e f f e c t o f TTX on t h e r a t e o f a n a e r o b i c g l y c o l y s i s i s n o t due t o i n c r e a s e d f o r m a t i o n o f cAMP. R e c e n t l y , i t has b e e n shown t h a t u n d e r a v a r i e t y o f c o n -d i t i o n s , when a c t i o n p o t e n t i a l s a r e g e n e r a t e d and d e p o l a r i z -- 238 ^ a t i o n o c c u r s , s u c h as i n t h e p r e s e n c e o f h i g h K , p r o t o v e r a -t r i n e , b a t r a c h o t o x i n o r e l e c t r i c a l s t i m u l a t i o n , t h e p r o d u c t i o n o f cAMP i n t h e c e r e b r a l c o r t e x s l i c e s i s i n c r e a s e d (297 ,300); t h e i n c r e a s e c a u s e d by b a t r a c h o t o x i n i s b l o c k e d by TTX. D i r -e c t e f f e c t s o f TTX on t h e cAMP c o n t e n t s o f c e r e b r a l c o r t e x s l i c e s h a v e n o t b e e n r e p o r t e d . E f f e c t s o f TTX i n t h e P r e s e n c e o f L - g l u t a m a t e , NH^ + and P r o t o -v e r a t r i n e A number o f e x p e r i m e n t s show, i n d i r e c t l y , t h a t t h e e f f e c t s o f TTX on t h e a n a e r o b i c g l y c o l y s i s m i g h t be due t o e f f e c t s on t h e movements o f c a t i o n s r a t h e r t h a n t o d i r e c t e f f e c t s on t h e t r a n s p o r t a n d / o r c e l l u l a r l e v e l o f m e t a b o l i t e s . T h u s , p r o t o -v e r a t r i n e , known t o g e n e r a t e a c t i o n p o t e n t i a l s and t o s t i m u l a t e i n f l u x o f N a + and e f f l u x o f K + , a l s o i n h i b i t s t h e r a t e o f a n a e r -o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . E x p e r i m e n t s c a r r i e d o u t on t h e e f f e c t s o f TTX on t h e a n a e r o b i c g l y c o l y s i s , i n t h e p r e s e n c e o f p r o t o v e r a t r i n e , show t h a t t h e s e d r u g s a r e a n t a g o -n i s t i c t o e a c h o t h e r ( F i g u r e 2 2 ) . T h u s , i n t h e p r e s e n c e o f 5 yM p r o t o v e r a t r i n e , 2 yM TTX has l e s s s t i m u l a t o r y e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s and t h e e f f e c t o f TTX p r o g r e s -s i v e l y d e c r e a s e s w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f p r o t o v e r a -t r i n e . S i m i l a r l y , L - g l u t a m a t e i s known t o s t i m u l a t e t h e e n t r y + 286 o f Na i n t o b r a i n c e l l s * The e f f e c t o f TTX on t h e a n a e r o -b i c g l y c o l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s i s a b o l i s h e d i f g l u t a m a t e i s a l s o p r e s e n t i n t h e i n c u b a t i o n medium f r o m t h e s t a r t o f t h e e x p e r i m e n t . I n t h e p r e s e n c e o f 5mM NH^"1", TTX ha s no e f f e c t on a n a e r o b i c g l y c o l y s i s . NH^ "*" i s known t o i n c r e a s e t h e e f f l u x o f K + f r o m t h e c e r e b r a l c o r t e x s l i c e s 3 ^ 5 . T h e s e e x p e r i m e n t s p o i n t o u t t h a t t h e e f f e c t o f TTX on t h e a n a e r o b i c g l y c o l y s i s i s p r e s u m a b l y due t o i t s e f f e c t o n c e r -e b r a l c a t i o n i c movements a t t h e o n s e t o f a n o x i a . E f f e c t s o f TTX on C o n t e n t s o f N a + and K + The e f f e c t s o f TTX on t h e N a + and K + c o n t e n t o f t h e c e r e b r a l c o r t e x s l i c e s were d i r e c t l y i n v e s t i g a t e d . T h e s e r e s u l t s ( F i g u r e s 24,26,27) show t h a t i n t h e p r e s e n c e o f TTX, t h e c e l l u l a r l e v e l o f c a t i o n s a r e d e f i n i t e l y a f f e c t e d ; t h u s i n t h e p r e s e n c e o f TTX t h e K + / N a + r a t i o o f t h e c e r e b r a l c o r t e x s l i c e s i s i n c r e a s e d . B e c a u s e N a + i s an i n h i b i t o r o f a n a e r o b i c g l y c o l y s i s w h i l e K + i s an a c t i v a t o r 3 7 ' ^ , i t i s t h e r e f o r e l i k e l y t h a t TTX s t i m u l a t e s t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s by i n c r e a s i n g t h e K c o n t e n t and d e c r e a s i n g t h e N a + c o n t e n t . As h as b e e n m e n t i o n e d e a r l i e r , t h e m e t a b o l i c e f f e c t s o f TTX a r e b e s t s e e n i f t h e s l i c e s a r e p r e i n c u b a t e d i n o x y g e n be-f o r e t h e o n s e t o f a n o x i a . When t h e K + l e v e l o f t h e c e r e b r a l c o r t e x s l i c e s i s m e a s u r e d u n d e r s u c h c o n d i t i o n s , t h e r e s u l t s show t h a t t h e r e i s v e r y l i t t l e l o s s o f K + i n t h e p r e s e n c e o f TTX d u r i n g t h e s u b s e q u e n t a n o x i c p e r i o d ( F i g u r e s 2 6 , 2 7 ) . T h i s i s p r o b a b l y due t o t h e f a c t s (a) t h a t t h e r e i s d e c r e a s e d l o s s o f K d u r i n g p r e l i m i n a r y a e r o b i c i n c u b a t i n g p e r i o d , and (b) t h a t TTX t a k e s some t i m e t o b i n d t o t h e s i t e s a t t h e b r a i n c e l l membrane b e f o r e i t may a f f e c t t h e c a t i o n movements. T h u s , i f - 240 -t h e i n i t i a l p e r i o d i s w h o l l y a n o x i c , t h e r e i s c o n s i d e r a b l e l o s s o f K + and g a i n p f N a + by t h e s l i c e s b e f o r e TTX c o u l d be v e r y e f f e c t i v e i n a f f e c t i n g t h e c a t i o n movements. The c o n -c e n t r a t i o n o f c a t i o n s , m e a s u r e d i n t h e p r e s e n c e o f g l u t a -mate o r NH^ +, showed t h a t t h e r e i s l e s s i n c r e a s e i n t h e r e -t e n t i o n o f K + and l e s s d e c r e a s e i n t h e g a i n o f N a + by t h e s l i c e s u n d e r t h e s e c o n d i t i o n s ( T a b l e 25). T h e s e r e s u l t s a r e f u r t h e r e v i d e n c e t h a t c a t i o n movements a r e i n v o l v e d when t h e r a t e o f a n a e r o b i c g l y c o l y s i s i n t h e p r e s e n c e o f TTX i s a f f e c t -ed by a v a r i e t y o f a g e n t s . + + Q u a l i t a t i v e l y , t h e i n c r e a s e i n t h e K /Na r a t i o o f t h e c e r e b r a l c o r t e x s l i c e s i n t h e p r e s e n c e o f TTX, u n d e r a n o x i a , i s s u f f i c i e n t t o e x p l a i n t h e i n c r e a s e d r a t e o f a n a e r o b i c g l y -c o l y s i s . Can t h e c h a n g e s i n t h e N a + and K + c o n c e n t r a t i o n s o b s e r v e d e x p l a i n t h e r e s u l t s q u a n t i t a t i v e l y ? I f i t i s a s s -umed t h a t t h e m a j o r s i t e o f g l y c o l y s i s i s i n t h e n e u r o n s and s i n c e TTX a c t s o n l y on t h e e l e c t r i c a l l y e x c i t a b l e n e u r o n s , i t f o l l o w s t h a t t h e c h a n g e s i n t h e Na and K f o u n d i n t h e w h o l e t i s s u e a r e p r o p o r t i o n a t e l y much g r e a t e r i f t h e y o c c u r o n l y i n t h e n e u r o n s . As i n o u r e x p e r i m e n t s , t h e Na and K c o n c e n -t r a t i o n s were m e a s u r e d i n t h e w h o l e t i s s u e , t h e y g i v e t h e a v e r -age c o m p o s i t i o n o f t h e s e c a t i o n s and t h e a c t u a l c o n c e n t r a t i o n o f N a + and K + may v a r y c o n s i d e r a b l y i n t h e n e u r o n s and g l i a l c e l l s . The l a r g e e f f e c t s o f TTX on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s may t h e n be r e a d i l y e x p l a i n e d on t h i s b a s i s . R e s u l t s o f e x p e r i m e n t s on t h e c a u d a t e n u c l e u s ( T a b l e 22) - 241 -i n d i c a t e t h a t i t s a n a e r o b i c g l y c o l y s i s i s a l s o i n c r e a s e d i n t h e p r e s e n c e o f TTX, b u t t h e m a g n i t u d e o f s t i m u l a t i o n i s a b o u t t h e same as t h a t o b s e r v e d w i t h t h e c e r e b r a l c o r t e x s l i c e s . The c o n c l u s i o n t h a t t h e e f f e c t o f TTX on t h e a n a e r o b i c g l y c o l y s i s i s m e d i a t e d t h r o u g h i n c r e a s e i n t h e K + / N a + r a t i o i s f u r t h e r s u p p o r t e d by t h e f a c t t h a t i n t h e p r e s e n c e o f TTX t h e r e i s an i n c r e a s e i n t h e p y r u v a t e c o n t e n t w h i c h w o u l d be e x p e c t e d i f t h e r e i s f a c i l i t a t i o n o f t h e p y r u v a t e k i n a s e s t e p due t o i n c r e a s e d K + i n t h e b r a i n c e l l ( T a b l e 2 6 ) . R e s u l t s o f e x p e r i m e n t s c a r r i e d o u t , t o e x p l a i n t h e e f -f e c t s o f TTX on t h e a n a e r o b i c g l y c o l y s i s a t d i f f e r e n t N a + and K + c o n c e n t r a t i o n s i n d i c a t e t h a t t h e c o n c e n t r a t i o n o f b o t h c a t -i o n s a r e i m p o r t a n t f o r t h e r e g u l a t i o n o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . T h u s , when K + c o n c e n t r a t i o n i s r a i s e d w i t h a c o r r e s p o n d i n g d e c r e a s e i n t h e N a + c o n t e n t t h e n a h i g h e r r a t e o f g l y c o l y s i s i s o b t a i n e d and t h e d e g r e e o f s t i m u l a t i o n i n t h e p r e s e n c e o f TTX p r o g r e s s i v e l y d e c r e a s e s u n t i l a n o p t i m a l r a t e o f g l y c o l y s i s i s o b t a i n e d . U n d e r t h e s e c o n d i t i o n s , i n h i b i t i o n by N a + i s n o t o b s e r v e d s i n c e i t s c o n -c e n t r a t i o n i n t h e i n c u b a t i o n medium i s low. When t h e c o n c e n -t r a t i o n o f N a + i s d e c r e a s e d w i t h o u t i n c r e a s e i n t h e K + c o n -c e n t r a t i o n ( N a + i s r e p l a c e d by s u c r o s e ) , t h e n t h e r a t e o f g l y -c o l y s i s i s h i g h e r t h a n t h e c o n t r o l s h a v i n g n o r m a l (149 mM) c o n -c e n t r a t i o n s o f N a + ( F i g u r e 3 2 ) . The r a t e o f g l y c o l y s i s u n d e r t h e s e c o n d i t i o n s i s m a r k e d l y s t i m u l a t e d by t h e p r e s e n c e o f TTX i n t h e i n c u b a t i o n medium. Under t h e s e c o n d i t i o n s , p r e s u m a b l y , - 242 -prevention of the e f f l u x of c e l l u l a r K plays a very import-ant part. We have seen (Table 2 0) that when the concen-t r a t i o n of K + i n the incubation medium i s high, i n addition to 149 mM Na +, then TTX i s not e f f e c t i v e i n increasing the rate of anaerobic g l y c o l y s i s of the cerebral cortex s l i c e s . I t i s known that high K + causes increased i n f l u x of Na + into 135 + the cerebral t i s s u e and when 149 mM Na i s present i n the incubation medium, there i s no stimulation of g l y c o l y s i s be-cause of increased Na + i n f l u x . This increase i n the i n f l u x of Na + i s presumably not i n h i b i t e d by TTX and thus, i n t h i s respect, i t d i f f e r s from the Na + i n f l u x caused by protovera-t r i n e , e l e c t r i c a l stimulation or anaerobiosis. Results obtained with kidney medulla s l i c e s on the ef-fects of TTX on anaerobic g l y c o l y s i s and Na + and K + contents •ehow that TTX had no e f f e c t on ei t h e r of them (Figures 16 and 29). This shows that the e f f e c t of TTX on anaerobic gly-c o l y s i s i s not an unspe c i f i c phenomenon and perhaps i s con-fined only to cerebral t i s s u e . The r e s u l t s with acetone pow-der extracts of brain demonstrate that the i n t e g r i t y of brain c e l l membrane i s required for TTX to show any e f f e c t on the rate of anaerobic g l y c o l y s i s and TTX does not act by a f f e c t -ing any of the enzymes of the g l y c o l y t i c pathway. E f f e c t s on Amino Acid Transport Under Anaerobic Conditions TTX markedly suppresses the e f f l u x of amino acids from the incubated cerebral cortex s l i c e s . This e f f e c t i s very pronounced i n the case of glutamic and aspartic acids which are present i n r e l a t i v e l y large amounts (Table 4). Abadom - 24 3 -and Scholefie have shown that when the ATP content of the cerebral cortex s l i c e s i s higher, there i s greater uptake of glycine from the incubation medium. One can argue that i n the presence of TTX, greater amounts of ATP are present i n the s l i c e s due to a higher rate of anaerobic g l y c o l y s i s and as a r e s u l t the transport system works more e f f i c i e n t l y , r e s u l t i n g i n greater uptake of amino acids. However, such a p o s s i b i l i t y i s ruled out by the f a c t that there i s an increased reten-t i o n of amino acids even i n the presence of a mixture of ouabain and TTX (Table 30). Ouabain i s known to block the active uptake of amino acids and i t s action i s not antagonized by TTX. Thus TTX must be acting independently of the ouabain s e n s i t i v e amino acid transport system. The increased tissue contents of amino acids i n the presence of TTX i s even apparent, i n the absence of glucose, although to a lesser extent. Experiments with l a b e l l e d amino acids (Table 5) showed that there i s a s i g n i f i c a n t increase i n the uptake by the an-ae r o b i c a l l y incubated s l i c e s i n the presence of TTX. Presum-ably t h i s i s due to two reasons: (i) increased uptake of amino acids by the s l i c e s because of the greater rate of anaerobic g l y c o l y s i s and hence the higher ATP l e v e l and ( i i ) because of i n h i b i t o r y e f f e c t of TTX on the e f f l u x of amino acids from the s l i c e s . E f f e c t s of TTX i n the Presence of Chelating Agents and Phospho- lipases . TTX has l i t t l e or no e f f e c t on the rate of anaerobic g l y c o l y s i s i n the presence of EDTA and EGTA (Table 23). In t h e p r e s e n c e o f t h e s e s u b s t a n c e s t h e r e i s a r a p i d i n f l u x o f N a + i n t o and a r a p i d e f f l u x o f K + f r o m - t h e c e r e b r a l t i s s u e . + 133 T h i s i n f l u x o f Na i s o n l y p a r t l y i n h i b i t e d by TTX . Thus i t a p p e a r s t h a t t h e a c t i o n o f TTX, i n t h e p r e s e n c e o f c h e -l a t i n g a g e n t s , must be m e d i a t e d t h r o u g h t h e i r a c t i o n s on t h e N a + and K + movements. T h i s c o n c l u s i o n f u r t h e r s u p p o r t s o u r v i e w t h a t t h e a c t i o n o f TTX on t h e c e r e b r a l a n a e r o b i c g l y c o -l y s i s i s m e d i a t e d t h r o u g h i t s e f f e c t s on t h e K + and N a + c o n -t e n t s . R e s u l t s on t h e e f f e c t s o f TTX i n t h e p r e s e n c e o f p h o s -p h o l i p a s e s ( T a b l e 9) show t h a t d u r i n g t h e e a r l y p e r i o d o f i n -c u b a t i o n , p h o s p h o l i p a s e A h a s no marked e f f e c t on t h e a n a e r -o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . However, d u r i n g t h e l a t e r p a r t o f t h e i n c u b a t i o n (50-80 m i n ) , t h e a n a e r o b i c g l y c o l y s i s i s c o n s i d e r a b l y r e d u c e d i n t h e p r e s e n c e o f 20 o r more I n t e r n a t i o n a l U n i t s o f p h o s p h o l i p a s e p e r v e s s e l . T h e s e r e s u l t s i n d i c a t e t h a t p o s s i b l y TTX a c t s a t t h e membrane and b i n d s t o t h e r e c e p t o r s w h i c h h a s p h o s p h o l i p i d c o n s t i t u e n t s , and w h i c h i s s l o w l y a t t a c k e d by t h e p h o s p h o l i p a s e s . T h i s f i n d i n g f u r t h e r p o i n t s o u t t h e i m p o r t a n c e o f membrane c o n -s t i t u e n t s i n t h e a c t i o n o f TTX on t h e c e r e b r a l m e t a b o l i s m . E f f e c t s o f TTX on D e v e l o p i n g B r a i n R e s u l t s o f e x p e r i m e n t s c a r r i e d o u t on t h e d e v e l o p i n g r a t b r a i n shows t h a t TTX has l i t t l e o r no e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f 2-day o l d r a t b r a i n . However, t h e b r a i n f r o m 2-week o l d r a t i s s e n s i t i v e t o TTX. I n v e s t i g a t i o n s on t h e Na and K + c o n t e n t s o f 2-day o l d r a t c e r e b r a l c o r t e x s l i c e s show - 245 -t h a t t h e r e i s no s i g n i f i c a n t i n c r e a s e i n t h e K + / N a + r a t i o i n t h e p r e s e n c e o f TTX. T h e s e r e s u l t s show t h a t m a t u r i t y o f t h e i b r a i n i s r e q u i r e d f o r TTX t o have any e f f e c t on i t s m e t a b o l i s m . The r e c e p t o r s f o r TTX a r e n o t d e v e l o p e d i n t h e 2-day o l d r a t b r a i n and t h e y a r e f o r m e d d u r i n g t h e l a t e r p e r i o d o f l i f e . The i n f a n t g u i n e a p i g b r a i n s l i c e s a r e e x t r e m e l y s e n s i -t i v e t o TTX and 0.2 yM TTX i s as e f f e c t i v e i n i n c r e a s i n g a n -a e r o b i c g l y c o l y s i s a s 10 yM. F u r t h e r m o r e , i n t h e p r e s e n c e o f TTX, t h e r e i s an i n c r e a s e d r e t e n t i o n o f K + and l e s s u p t a k e o f N a + by t h e s l i c e s . T h i s shows t h a t w i t h t h e i n f a n t g u i n e a p i g b r a i n , t o o , t h e i n c r e a s e i n a n a e r o b i c g l y c o l y s i s i n t h e p r e -s e n c e o f TTX i s m e d i a t e d t h r o u g h i n c r e a s e i n t h e K + / N a + r a t i o . E f f e c t s o f TTX on A e r o b i c G l y c o l y s i s The r a t e s o f a e r o b i c g l y c o l y s i s o f b r a i n s l i c e s a r e i n -c r e a s e d when C a + + i s o m i t t e d f r o m t h e i n c u b a t i o n medium. T h i s i s p r e s u m a b l y due t o g r e a t e r N a + i n f l u x and t h e c o r r e s p o n d i n g i n c r e a s e i n t h e a c t i v i t y o f N a + , K + - A T P a s e . T h i s r e s u l t s i n a d e c r e a s e i n ATP c o n c e n t r a t i o n o f t h e c e l l and t h e i n h i b i t o r y e f f e c t o f ATP on g l y c o l y s i s i s d i m i n i s h e d . I n a K r e b s - R i n g e r medium ( c o n t a i n i n g C a + + ) 2 yM TTX h a s no e f f e c t on t h e a e r o b i c g l y c o l y s i s , b u t i n a Ca - f r e e medium i t i s s u p p r e s s e d by TTX, t h e r e s u l t i n g v a l u e s a p p r o a c h i n g t h o s e o b t a i n e d i n a K r e b s -R i n g e r medium. T h u s , i n t h i s r e g a r d , TTX a c t s l i k e C a + + and t h i s i s a n a l o g o u s t o t h e r e s u l t s o b t a i n e d by Chan and Q u a s t e l ^ who showed t h a t t h e i n c r e a s e i n r e s p i r a t i o n , b r o u g h t a b o u t by e m i s s i o n o f C a + + f r o m t h e i n c u b a t i o n medium, i s s u p p r e s s e d by TTX. - 246 -8.6 EFFECTS OF OUABAIN ON CEREBRAL METABOLISM AND TRANSPORT As h a s b e e n d i s c u s s e d e a r l i e r , t h e e f f e c t s o f TTX on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s i s m e d i -a t e d t h r o u g h c h a n g e s i n t h e N a + and K + c o n c e n t r a t i o n s . How-e v e r , t h e e f f e c t o f o u a b a i n on t h e a n a e r o b i c g l y c o l y s i s c a n -n o t be due t o i t s e f f e c t s on t h e c a t i o n f l u x e s s i n c e o u a b a i n i s an i n h i b i t o r o f Na , K -ATPase and, u n d e r a e r o b i c c o n d i -t i o n s , t h e p r e s e n c e o f t h i s d r u g l e a d s t o a g r e a t i n f l u x o f N a + i n t o t h e c e r e b r a l c o r t e x s l i c e s and an e f f l u x o f K + . U n d e r a n a e r o b i c c o n d i t i o n s , t h e Na , K -ATPase i s n o t o p e r -a t i v e t o t h e same e x t e n t as u n d e r a e r o b i c c o n d i t i o n s due t o a f a l l i n ATP c o n c e n t r a t i o n and a c o n s i d e r a b l e amount o f N a + e n t e r s i n and K + comes o u t o f t h e b r a i n s l i c e s . F u r t h e r , u n d e r a n a e r o b i c c o n d i t i o n s o u a b a i n h a s l i t t l e o r no e f f e c t on t h e Na and K c o n t e n t s ( F i g u r e 3 5 ) , a r e s u l t w h i c h f a v o u r s t h e v i e w t h a t u n d e r t h e s e c o n d i t i o n s , t h e o p e r a t i o n o f N a + , K + -A T P a s e i s l i m i t e d . T h e s e r e s u l t s i n d i c a t e t h a t t h e s t i m u -l a t i o n o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s i n a Ca f r e e medium by o u a b a i n c a n n o t be due t o i t s e f f e c t s on t h e c a t i o n i c c o n t e n t s , and t h e r e f o r e , u n d e r t h e s e c o n d i t i o n s , + + t h e Na and K c o n t e n t s do n o t p l a y a r a t e l i m i t i n g r o l e on t h e s p e e d o f a n a e r o b i c g l y c o l y s i s . U n d er a n a e r o b i c c o n d i t i o n s , t h e ATP c o n c e n t r a t i o n f a l l s t o a v e r y low l e v e l ( T a b l e 15) and i t may become r a t e l i m i t -i n g f o r t h e p h o s p h o r y l a t i o n o f g l u c o s e and f r u c t o s e 6-phos-p h a t e . S i n c e a c o n s i d e r a b l e amount o f ATP i s consumed by + + Na , K - A T P a s e i n t h e b r a i n c e l l , t h e i n h i b i t i o n o f t h i s e n -- 2 4 7 -zyme i n t h e p r e s e n c e o f o u a b a i n w i l l r e s u l t i n a g r e a t e r c e l l c o n c e n t r a t i o n p f ATP due t o a d e c r e a s e i n i t s u t i l i z a t i o n . T h i s may i n t u r n make a g r e a t e r amount o f ATP a v a i l a b l e f o r s u g a r p h o s p h o r y l a t i o n s r e s u l t i n g i n g r e a t e r r a t e o f a n a e r o b i c g l y c o l y s i s . An i n c r e a s e i n t h e ATP c o n t e n t i n t h e p r e s e n c e o f o u a b a i n i s i n f a c t o b s e r v e d ( T a b l e 15) b u t i t c a n n o t be a s c e r t a i n e d w h e t h e r t h i s i n c r e a s e i s due t o t h e i n h i b i t i o n o f N a + , K + - A T P a s e o r due t o a h i g h e r r a t e o f g l y c o l y s i s i t s e l f , a s n o t e d w i t h TTX. P o s s i b l y w i t h o u a b a i n , an i n c r e a s e d r a t e o f g l y c o l y s i s , due t o i n h i b i t i o n o f N a + , K + - A T P a s e , l e a d s t o d i m i n i s h e d f a l l i n t h e ATP c o n c e n t r a t i o n , and t h i s r e s u l t s i n h i g h e r o b s e r v e d v a l u e s f o r ATP. The e f f e c t s o f o u a b a i n i n t h e p r e s e n c e o f C a + + show t h a t when C a + + i s p r e s e n t i n t h e i n c u b a t i o n medium, i t i s n o t as e f f e c t i v e i n i n c r e a s i n g t h e a n a e r o b i c g l y c o l y s i s a s i t i s i n t h e a b s e n c e o f C a + + . I f t h e a c t i o n o f o u a b a i n on t h e a n a e r -o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s i s due t o t h e i n h i b -+ + l t i o n o f Na , K - A T P a s e , t h e n t h e s e o b s e r v a t i o n s l e a d t o t h e c o n c l u s i o n t h a t i n t h e p r e s e n c e o f C a + + , o u a b a i n d o e s n o t i n -h i b i t N a + , K + - A T P a s e c o m p l e t e l y . T h i s c o n c l u s i o n i s i n a g r e e -ment w i t h t h e d a t a o f Swanson and h i s c o - w o r k e r s who o b s e r v e d t h a t when o u a b a i n i s added t o g u i n e a p i g c e r e b r a l c o r t e x s l i c e s i n c u b a t i n g i n a medium i n t h e p r e s e n c e o f C a + + , t h e r e i s an i n i t i a l r i s e i n r e s p i r a t i o n f o l l o w e d a f t e r 45 o r 60 m i n by a s l i g h t d e p r e s s i o n . When Ca i s o m i t t e d f r o m t h e i n c u b a t i o n medium, a d e c r e a s e i n r e s p i r a t i o n i n t h e p r e s e n c e o f o u a b a i n was - 24 8 -172 ' o b s e r v e d . T h e s e r e s u l t s were e x p l a i n e d as r e s u l t i n g f r o m + + t h e i n c o m p l e t e i n h i b i t i o n o f Na , K,-ATPase i n t h e p r e s e n c e o f C a + + by o u a b a i n , w h i c h a l l o w e d t h e u n i n h i b i t e d p o r t i o n o f t h i s enzyme t o r e s p o n d t o c a t i o n s h i f t s by c o n s u m i n g h i g h e n e r g y p h o s p h a t e s and t h e r e b y s t i m u l a t i n g r e s p i r a t i o n ' ' + + The e f f e c t s o f o u a b a i n on t h e i n h i b i t i o n o f Na ,K - A T P a s e i n t h e p r e s e n c e o f C a + + were s t u d i e d ( T a b l e 2 9 ) , b u t t h e r e s u l t s show t h a t , i n m i c r o s o m a l p r e p a r a t i o n s , C a + + and o u a b a i n do n o t show any a n t a g o n i s m . P e r h a p s i n b r a i n t i s s u e s l i c e s , t h e a c c e s -++ + + s i b i l i t i e s o f Ca and o u a b a i n t o Na , K -ATPase i s d i f f e r e n t as compared f r o m t h o s e i n t h e m i c r o s o m a l p r e p a r a t i o n s . When o u a b a i n i s added t o t h e i n c u b a t i o n medium a f t e r i n -c r e a s i n g p e r i o d s o f a n a e r o b i o s i s , t h e n i t has p r o g r e s s i v e l y l e s s e f f e c t s on t h e r a t e o f a n a e r o b i c g l y c o l y s i s . T h i s may be due t o a f a l l i n t h e ATP c o n c e n t r a t i o n s s o t h a t when o u a b a i n i s added l a t e r i t has no more s t i m u l a t i n g e f f e c t . A d o s e r e s p o n s e c u r v e f o r t h e e f f e c t s o f d i f f e r e n t c o n -c e n t r a t i o n s o f o u a b a i n on t h e a n a e r o b i c g l y c o l y s i s show t h a t t h e g u i n e a p i g c e r e b r a l c o r t e x s l i c e s a r e more s e n s i t i v e t h a n t h e r a t c e r e b r a l c o r t e x s l i c e s t o o u a b a i n . T h i s i s p r o b a b l y b e c a u s e t h e s e n s i t i v i t i e s o f N a + , K + - A T P a s e t o o u a b a i n d i f f e r s w i t h t h e 163 s p e c i e s o f t h e a n i m a l u s e d . B o n t i n g , C r a v a g g i o and Hawkins + + h a v e i n f a c t shown t h a t o u a b a i n i n h i b i t i o n c u r v e s o f Na , K -ATP-a s e d i f f e r w i t h t h e s p e c i e s o f t h e a n i m a l , and t i s s u e u s e d . R e s u l t s o f e x p e r i m e n t s c a r r i e d o u t w i t h d e v e l o p i n g r a t b r a i n c o r t e x - s l i c e s show t h a t t h e r e s p o n s e o f i n f a n t r a t a n a e r -- 249 -o b i c g l y c o l y s i s t o o u a b a i n i n c r e a s e s w i t h age and p o s s i b l y + + t h i s i s r e l a t e d t o t h e r i s e i n Na , K -ATPase and g e n e r a l e n e r g y m e t a b o l i s m d u r i n g d e v e l o p m e n t . O t h e r e x p e r i m e n t s show t h a t t h e e f f e c t o f o u a b a i n i s a membrane phenomenon s i n c e a s t i m u l a t i o n o f a n a e r o b i c g l y c o l y s i s o f a c e t o n e powder e x t r a c t s o f b r a i n i n t h e p r e s e n c e o f o u a b a i n , i s n o t o b s e r v e d . The o u a b a i n s t i m u l a t e d g l y c o l y s i s d i f f e r s f r o m t h e TTX s t i m u l a t e d g l y c o l y s i s by t h e f a c t t h a t t h e f o r m e r i s n o t a t a l l a f f e c t e d by t h e p r e s e n c e o f NH^ + i n t h e i n c u b a t i o n medium. T h i s i s i n a c c o r d w i t h t h e c o n c l u s i o n t h a t K + and N a + movements p l a y a m i n o r r o l e w i t h o u a b a i n s t i m u l a t e d g l y c o l y s i s . However, a p a r t i a l i n h i b i t i o n was o b s e r v e d i n t h e p r e s e n c e o f L - g l u t a m -a t e and t h i s i s y e t t o be r e s o l v e d . L i k e TTX s t i m u l a t e d g l y c o -l y s i s , o u a b a i n s t i m u l a t e d g l y c o l y s i s i s i n h i b i t e d by c i t r a t e , due p r o b a b l y t o i n h i b i t i o n o f p h o s p h o f r u c t o k i n a s e a c t i v i t y ( T a b l e 28). When t h e N a + c o n c e n t r a t i o n o f t h e i n c u b a t i o n medium i s r e d u c e d , t h e r a t e o f a n a e r o b i c g l y c o l y s i s i s m a r k e d l y s t i m u -l a t e d by o u a b a i n . . When o u a b a i n and TTX b o t h a r e p r e s e n t t o -g e t h e r i n t h e i n c u b a t i o n medium, t h e n t h e r a t e o f a n a e r o b i c g l y c o l y s i s i s f u r t h e r i n c r e a s e d when compared w i t h t h e r a t e f o u n d when e i t h e r o f t h e s e d r u g s i s p r e s e n t a l o n e ( F i g u r e 3 4 ) . Under t h e s e c o n d i t i o n s , an i n c r e a s e i n K + / N a + r a t i o may s t i l l be o b s e r v e d ( F i g u r e 3 5 ) . T h e s e r e s u l t s d e m o n s t r a t e t h a t TTX a f f e c t s t h e p a s s i v e d o w n h i l l movements o f Na and K i n d e p e n d -e n t l y o f t h e o p e r a t i o n o f t h e s o d i u m pump ( N a + , K + - A T P a s e ) . I n t h e p r e s e n c e o f b o t h o u a b a i n and TTX, a c o m b i n a t i o n o f d e -+ + c r e a s e d ATP u t i l i z a t i o n and i n c r e a s e d K /Na r a t i o must be - 250 _ r e s p o n s i b l e f o r t h e v e r y h i g h r a t e s o f a n a e r o b i c g l y c o l y s i s o b t a i n e d . F u r t h e r m o r e , t h e e f f l u x o f amino a c i d s , w h i c h t a k e s p l a c e on t h e o n s e t o f a n a e r o b i o s i s , e i t h e r i n t h e p r e s e n c e o r a b s e n c e o f o u a b a i n , i s p a r t i a l l y b l o c k e d by TTX ( T a b l e 3 0 ) . T h e s e r e s u l t s f u r t h e r p o i n t o u t t h a t t h e a c t i o n o f TTX on t h e movement o f s u b s t a n c e s a c r o s s t h e b r a i n c e l l membrane i s i n d e p e n d e n t f r o m t h e a c t i o n o f o u a b a i n on N a + , K + - A T P a s e . No s i g n i f i c a n t e f f e c t o f 10 uM o u a b a i n was o b s e r v e d on a e r o b i c g l y c o l y s i s and t h i s i s i n a g r e e m e n t w i t h t h e f i n d i n g s 52 o f R o l l e s t o n and Newsholme 8.7 EFFECTS OF LOCAL ANESTHETICS ON THE ANAEROBIC GLYCOLYSIS The modes o f a c t i o n o f l o c a l a n e s t h e t i c s and TTX on t h e n e r v o u s t i s s u e seem t o be i d e n t i c a l . R e s u l t s o b t a i n e d w i t h l o c a l a n e s t h e t i c s on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r -t e x s l i c e s shows f u r t h e r s i m i l a r i t i e s b etween t h e s e d r u g s . T h u s , l i k e TTX, l o c a l a n e s t h e t i c s i n c r e a s e t h e r a t e o f a n a e r o -b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s ( T a b l e 3 1 ) . When l o c a l a n e s t h e t i c s a r e added t o t h e i n c u b a t i o n medium a f t e r t h e o n s e t o f a n o x i a , t h e y a r e i n e f f e c t i v e i n i n c r e a s i n g t h e r a t e o f a n a e r o b i c g l y c o l y s i s . As o b s e r v e d w i t h TTX, t h e K + / N a + r a t i o o f t h e c e r e b r a l c o r t e x s l i c e s i s a l s o i n c r e a s e d i n t h e p r e s e n c e o f l i d o c a i n e and p r e s u m a b l y t h i s i s r e s p o n s i b l e f o r t h e o b s e r v e d i n c r e a s e d r a t e s o f a n a e r o b i c g l y c o l y s i s . I n t h e p r e s e n c e o f 0.5 mM l i d o c a i n e , an i n c r e a s e i n t h e K + / N a + r a t i o was a l s o o b s e r v e d w i t h i n f a n t r a t c e r e b r a l c o r t e x s l i c e s ( T a b l e - 251 -32); however, no stimulation of anaerobic g l y c o l y s i s was ob-served with i n f a n t r a t cerebral cortex s l i c e s (Figure 36). Perhaps the infant (2-day old) r a t cerebral cortex g l y c o l y s i s i s not as se n s i t i v e as that of the adult to changes i n the K + and Na + concentrations. Further work i s necessary to c l a r i f y t h i s point. 8.8. EFFECTS OF OTHER NEUROTROPIC DRUGS ON THE ANAEROBIC GLY-LYSIS Results of experiments c a r r i e d out with pyrrole show that possibly i t s action on the anaerobic g l y c o l y s i s of guinea pig cerebral cortex s l i c e s i s mediated through an increase i n K +/ Na + r a t i o r e s u l t i n g i n stimulation of pyruvate kinase. The cerebral cortex s l i c e s from rats were found, however, to be more r e s i s t a n t to the organic bases tested than those of guinea pig s l i c e s and the reason for t h i s d i f f e r e n c e between these two animals i s not c l e a r . Preliminary r e s u l t s obtained with amytal and reserpine i n t h e i r e f f e c t s on the anaerobic g l y c o l y s i s of cerebral cortex s l i c e s , are of some i n t e r e s t . Thus i t was discovered that the anaerobic g l y c o l y s i s of adult r a t brain s l i c e s , and es p e c i a l l y those from the infant guinea pig brain s l i c e s i s enhanced by amytal. At anesthetic doses (0.25 mM), however, the anaerobic g l y c o l y s i s of r a t brain s l i c e s i s not much affected by amytal (Figure 39). The acc e l e r a t i v e e f f e c t s of amytal on anaerobic glyco-l y s i s of brain does not seem to be an a r t i f a c t due to i t s well known action i n suppressing NADH oxidation. Thus, as has been - 252 -p o i n t e d o u t e a r l i e r , t h e p r e s e n c e o f o x y g e n as an i m p u r i t y i n t h e N 2:C02 m i x t u r e c o u l d g i v e l o w e r r a t e s o f g l y c o l y s i s due t o P a s t e u r e f f e c t , and a m y t a l by p r e v e n t i n g t h e o x i d a t i o n o f NADH u n d e r t h e s e c o n d i t i o n s , w o u l d i n c r e a s e t h e r a t e o f a n a e r o b i c g l y c o l y s i s ; however, t h i s i s u n l i k e l y s i n c e a z i d e , u n d e r t h e same c o n d i t i o n s , h a s no e f f e c t on t h e a n a e r o b i c g l y c o l y s i s . (See C h a p t e r 7 ) . B a r b i t u r a t e s h a v e b e e n s t a t e d t o i n h i b i t t h e t r a n s -m i s s i o n o f i m p u l s e s , i n t h e s y m p a t h e t i c g a n g l i a . T h ey have b e e n f o u n d t o r e d u c e t h e N a + and K + c o n d u c t a n c e s i n l o b s t e r axons d u r i n g e x c i t a t i o n . I t has b e e n s u g g e s t e d t h a t b a r b i t u r a t e s i n t h e i r a n i o n i c f o r m s d i s s o l v e i n t h e membrane l i p i d s and t h e r e b y a f f e c t t h e b i n d i n g o f C a + + and t h u s a f f e c t i o n i c p e r m e a b i l i t i e s ^ An i n c r e a s e i n t h e K + c o n c e n t r a t i o n was o b s e r v e d w i t h i n f a n t g u i n e a p i g c e r e b r a l c o r t e x s l i c e s i n t h e p r e s e n c e o f 0.25 mM a m y t a l . A t t h i s c o n c e n t r a t i o n , t h e a n a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s i s n o t much a f f e c t e d by t h e d r u g . P e r h a p s t h e i n f a n t g u i n e a p i g b r a i n i s more s e n s i t i v e t o a m y t a l when compared w i t h a d u l t r a t b r a i n and a h i g h e r c o n c e n t r a t i o n o f a m y t a l c o u l d h a v e a g r e a t e r e f f e c t on t h e c a t i o n i c c o n t e n t s o f a d u l t r a t b r a i n s l i c e s . F u r t h e r s t u d y i s n e c e s s a r y t o s e t -t l e t h e mode o f a c t i o n o f b a r b i t u r a t e s on t h e a n a e r o b i c g l y c o -l y s i s o f c e r e b r a l c o r t e x s l i c e s . R e s e r p i n e was a l s o f o u n d t o i n c r e a s e t h e r a t e o f a n a e r -o b i c g l y c o l y s i s o f b r a i n s l i c e s , and i n i t s p r e s e n c e , t o o , an i n c r e a s e i n t h e K + / N a + r a t i o was o b s e r v e d . The e f f e c t o f r e -s e r p i n e d o e s n o t a p p e a r t o be due t o i t s known e f f e c t on t h e - 25 3 -r e l e a s e o f amines f r o m t h e i r s t o r a g e s i t e s a s amines a l o n e h a v e v e r y l i t t l e e f f e c t on t h e r a t e s o f a n a e r o b i c g l y c o l y s i s . The e f f e c t s o f r e s e r p i n e on t h e N a + and K + c o n t e n t s a r e unex-p e c t e d s i n c e i t i s n o t known t o b l o c k t h e g e n e r a t i o n o f a c t i o n p o t e n t i a l and t h e r e b y t h e movements o f c a t i o n s a c r o s s t h e neu-r o n a l membrane. F u r t h e r work i s i n p r o g r e s s t o c l a r i f y t h e mode o f a c t i o n o f r e s e r p i n e on t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . C h l o r .promazine, amphetamine and n i a l a m i d e h a v e no s i g n i f i c a n t s t i m u l a t o r y e f f e c t s on t h e r a t e s o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . From t h e r e s u l t s d i s c u s s e d i n t h i s t h e s i s , i t c a n be c o n -c l u d e d t h a t a number o f n e u r o t r o p i c d r u g s c a n a f f e c t t h e r a t e s o f a n a e r o b i c g l y c o l y s i s o f t h e c e r e b r a l c o r t e x s l i c e s . W i t h some d r u g s s u c h as TTX and l o c a l a n a e s t h e t i c s , t h e e x p e r i m e n t a l r e s u l t s c a n be e x p l a i n e d on t h e b a s i s t h a t i n t h e p r e s e n c e o f t h e s e d r u g s t h e r e i s a s p e c i f i c i n c r e a s e i n t h e c e l l K + / N a + r a t i o . P r e l i m i n a r y r e s u l t s w i t h p y r r o l e , a m y t a l and r e s e r p i n e h a v e shown t h a t t h e s e d r u g s t o o may a f f e c t t h e i o n i c f l u x e s a c r o s s t h e b r a i n c e l l membrane, b u t f u r t h e r work i s n e c e s s a r y t o s e t t l e t h i s m a t t e r . . 8.9 GENERAL CONCLUSIONS 1. TTX, a t low c o n c e n t r a t i o n s , a b o l i s h e s t h e g e n e r a t i o n o f a c t i o n p o t e n t i a l s i n a v a r i e t y o f e x c i t a b l e t i s s u e s . A t s i m i l a r c o n c e n t r a t i o n s , s u c h as 2 yM, i t e n h a n c e s t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s two t o t h r e e -f o l d . S u c h an e f f e c t o f TTX i s much g r e a t e r t h a n t h a t ob-t a i n e d on t h e a e r o b i c m e t a b o l i s m o f c e r e b r a l c o r t e x s l i c e s . - 254 -2. The a n a e r o b i c g l y c o l y s i s o f k i d n e y m e d u l l a o r 2-day o l d r a t b r a i n s l i c e s a r e n o t a f f e c t e d by TTX. . T h i s shows t h a t t h e e f f e c t o f TTX i s s p e c i f i c f o r m a t u r e c e r e b r a l t i s s u e . F u r t h e r m o r e , s i n c e t h e a n a e r o b i c g l y c o l y s i s o f a c e t o n e powder e x t r a c t s f r o m b r a i n i s n o t a f f e c t e d by TTX, i t f o l l o w s t h a t i n t e g r i t y o f t h e c e l l i s r e q u i r e d f o r i t s a c t i o n on t h e c e r e b r a l m e t a b o l i s m . 3. TTX has no a c c e l e r a t i n g e f f e c t on t h e r a t e o f a n a e r -o b i c g l y c o l y s i s when h i g h K + i s , p r e s e n t i n t h e i n c u b a t i o n med-ium c o n t a i n i n g n o r m a l N a + c o n c e n t r a t i o n . I f t h e N a + i s r e d u c e d a t t h e same t i m e when K + i s i n c r e a s e d , t h e r a t e o f a n a e r o b i c g l y c o l y s i s i n c r e a s e s and TTX h a s a p r o g r e s s i v e l y s m a l l e r p e r -c e n t a g e a c c e l e r a t i o n . The l a c k o f e f f e c t o f TTX i n a medium c o n t a i n i n g n o r m a l N a + and h i g h K + may be due t o t h e i n c r e a s e d N a - i n f l u x t h a t o c c u r i n c e r e b r a l c o r t e x s l i c e s i n p r e s e n c e o f h i g h e x t e r n a l K + c o n c e n t r a t i o n . 4. When TTX i s a d d e d a f t e r 15.min o f a n o x i a , i t no l o n g e r a f f e c t s t h e a n o x i c c e r e b r a l m e t a b o l i s m . T h e s e e x p e r i -ments l e a d t o t h e c o n c l u s i o n t h a t t h e r e i s an i n f l u x o f N a + i n t o , and e f f l u x o f K + f r o m t h e i n c u b a t e d c e r e b r a l t i s s u e a t t h e o n s e t o f a n o x i a and i f TTX i s added a f t e r e s t a b l i s h m e n t o f t h e s t e a d y s t a t e , i t i s n o t e f f e c t i v e i n i n c r e a s i n g t h e r a t e o f a n a e r o b i c g l y c o l y s i s . The e f f e c t s o f TTX a r e due t o c h a n -ges i n t h e N a + and K + c o n t e n t s by d i m i n i s h i n g t h e c a t i o n i c f l u x e s t h a t o c c u r a t t h e o n s e t o f a n o x i a . T h e s e e f f e c t s o f TTX a r e due t o i t s a c t i o n a t t h e b r a i n c e l l membrane, p o s s i b l y i n v o l v i n g p h o s p h o l i p i d s , r e s u l t i n g i n c h a n g e s i n t h e perme-a b i l i t y t o c a t i o n s . T h u s , i n t h e p r e s e n c e o f TTX, t h e i n i t i a l h i g h r a t e o f g l y c o l y s i s t e n d s t o be m a i n t a i n e d due t o o n l y a + + + s l o w d e c l i n e i n t h e c e l l u l a r K /Na r a t i o . The e f f e c t s o f Na and K + on t h e a n a e r o b i c g l y c o l y s i s a r e c o n s i d e r e d t o be m e d i -a t e d by c h a n g e s i n p y r u v a t e k i n a s e a c t i v i t y w h i c h i s e n h a n c e d by K and d i m i n i s h e d by Na . 5. I n t h e p r e s e n c e o f TTX, u n d e r a n o x i a , t h e c e l l ATP c o n c e n t r a t i o n i s a l s o i n c r e a s e d b u t t h i s may be due t o m a i n t e n -a n c e o f o r i g i n a l h i g h r a t e o f g l y c o l y s i s r a t h e r t h a n due t o d i r e c t e f f e c t s o f TTX on t h e e n e r g y u t i l i z i n g p r o c e s s e s o f t h e c e l l . 6. TTX a f f e c t s t h e a e r o b i c and a n a e r o b i c m e t a b o l i s m o f b r a i n i n v i t r o i n t h e same way as i t e f f e c t s t h e a c t i o n p o t e n -t i a l s . T h i s shows t h a t a c t i o n p o t e n t i a l s a r e g e n e r a t e d i n t h e i n c u b a t e d c e r e b r a l t i s s u e a t t h e o n s e t o f a n o x i a . T h i s i s b l o c k e d by TTX and m a n i f e s t s i t s e l f i n t h e h i g h e r r a t e o f a n a e r -o b i c g l y c o l y s i s . 7. The ab o v e c o n c l u s i o n i s f u r t h e r s u p p o r t e d by t h e f a c t t h a t i n t h e p r e s e n c e o f a g e n t s s u c h as p r o t o v e r a t r i n e o r L - g l u -t a m a t e , w h i c h l e a d t o l a r g e r i n f l u x o f N a + i n i s o l a t e d i n c u -b a t e d b r a i n , TTX do e s n o t a f f e c t t h e a n o x i c m e t a b o l i s m . F u r -t h e r m o r e , i n t h e p r e s e n c e o f c h e l a t i n g a g e n t s s u c h as EDTA and EGTA, w h i c h a l s o r e s u l t s i n g r e a t l y i n c r e a s e d i n f l u x o f N a + , TTX i s i n e f f e c t i v e i n e n h a n c i n g t h e r a t e o f a n a e r o b i c g l y c o -l y s i s o f c e r e b r a l c o r t e x s l i c e s . 8. The e f f e c t o f TTX on t h e N a + and K + c o n t e n t s may be - 256 -• g r e a t e r i n t h e n e u r o n s t h a n i n g l i a l c e l l s , b e c a u s e t h e f o r m e r a r e r e g a r d e d as t h e s i t e o f a c t i o n o f TTX. T h u s , c h a n g e s i n t h e n e u r o n a l K + / N a + r a t i o b r o u g h t a b o u t by TTX may be much + + g r e a t e r t h a n t h e c h a n g e s i n t h e K /Na r a t i o f o u n d i n t h e t i s -s u e as w h o l e . + + 9. I n a d d i t i o n t o i t s e f f e c t s on t h e Na and K f l u x e s , TTX a l s o p r e v e n t s t h e e f f l u x o f amino a c i d s f r o m .the i n c u b a t e d c e r e b r a l c o r t e x s l i c e s a t t h e o n s e t o f a n o x i a . T h i s e f f e c t o f TTX i s i n d e p e n d e n t o f t h e o p e r a t i o n o f t h e amino a c i d t r a n s -p o r t s y s t e m . 10. The a c c e l e r a t i n g e f f e c t o f C a + + on t h e a n a e r o b i c g l y -c o l y s i s i s p r e s u m a b l y m e d i a t e d by c h a n g e s i n t h e K + and N a + c e l l c o n t e n t s , by a mechanism r e s e m b l i n g t h a t o f TTX. 11. L o c a l a n e s t h e t i c s a c t l i k e TTX on c e r e b r a l g l u c o s e b r e a k d o w n i n a n o x i a b u t a t much h i g h e r c o n c e n t r a t i o n s . T he e f f e c t s o f some o r g a n i c , b a s e s , s u c h as p y r r o l e , w h i c h a l s o a c c e l e r a t e a n a e r o b i c g l y c o l y s i s o f g u i n e a p i g c e r e b r a l c o r t e x s l i c e s may be e x p l a i n e d on s i m i l a r l i n e s . 12. The r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s i n a C a + + - f r e e medium i s i n c r e a s e d i n t h e p r e s e n c e o f 10 pM o u a b a i n . I n a C a + + - c o n t a i n i n g medium, t h e s t i m u l a t i o n o f a n a e r o b i c g l y c o l y s i s i s n o t o b s e r v e d t o t h e same e x t e n t as i n a C a + + - f r e e medium. 13. The e f f e c t o f o u a b a i n i s p r e s u m a b l y due t o i n h i b i -t i o n o f t h e N a + , K + - A T P a s e , w h i c h consumes much o f t h e ATP i n t h e b r a i n c e l l . U n d er a n o x i c c o n d i t i o n s t h e r e i s a f a l l i n - 257 -ATP c o n c e n t r a t i o n and i t may become r a t e l i m i t i n g f o r t h e p h o s p h o r y l a t i o n o f g l u c o s e and F-6-P. The i n h i b i t i o n o f N a + , K + - A T P a s e r e s u l t s i n an e n h a n c e d ATP l e v e l . T h i s i n t u r n r e -s u l t s i n g r e a t e r a v a i l a b i l i t y o f ATP f o r s u g a r p h o s p h o r y l a -t t i o n r e a c t i o n s . Under t h e s e c o n d i t i o n s , c a t i o n c h a n g e s may p l a y a l e s s i m p o r t a n t p a r t i n t h e r e g u l a t i o n o f a n a e r o b i c g l y -c o l y s i s . 14. A m y t a l and r e s e r p i n e a l s o may i n c r e a s e t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . P r e l i m i n a r y r e s u l t s i n d i c a t e t h a t t h e i r a c t i o n may be m e d i a t e d t h r o u g h c h a n g e s i n t h e c a t i o n c o n t e n t s . L a r g e r t h a n a n e s t h e t i c c o n -c e n t r a t i o n o f a m y t a l a r e n e e d e d t o p r o d u c e a s i g n i f i c a n t e f -f e c t . It. i s l e s s e f f e c t i v e t h a n TTX o r l o c a l a n e s t h e t i c s . F u r t h e r work i s n e c e s s a r y t o e s t a b l i s h t h e mode o f a c t i o n o f . a m y t a l o r o f r e s e r p i n e . 15. C h l o r p r o m a z i n e , amphetamines and n i a l a m i d e h a v e l i t t l e o r no s t i m u l a t o r y e f f e c t s on t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f b r a i n s l i c e s . - 258 -BIBLIOGRAPHY 1. E. S. Gurdjian, W. E. Stone and J . E. Webster, Arch. Neurol. Psychiat. 51, 472 (1944). 2. E. S. Gurdjian, J . E. Webster and W. E. Stone, Am. J . Phys i o l . 156, 149 (1949). 3. L. J i l e k , J . Fischer, L. Kruiich and S. Trojan, Progr. Brain Res. 9_, 113 (1964). 4. W. Thorn, H. Sc h o l l , G. P f l e i d e r e r and B. Mueldener, J . Neurochem. 2, 150 (1958). 5. R. N. Lo l l e y and F. E. Samson, J r . , Am. J . Phy s i o l . 202, 77 (1962). , 6. 0. H. Lowry, J . V. Passonneau, F. X. Hasselberger and D. W. Schulz, J . B i o l . Chem. 239, 18 (1964). 7. M. A. Stewart, J . V. Passonneau and 0. H. Lowry, J . Neurochem. 12, 719 (1965) . 8. P. D. Ga t e f i e l d , O. H. Lowry, D. W. Schulz and J . V. Passonneau, J . Neurochem. 13_, 185 (1966) . 9. M. M. Cohen, J . Neuropathol. 19, 139 (1960). 10. M. M. Cohen, J . Neurochem. 9_, 337 (1962). 11. H. G., Albaum, W. K. Noell and H. I. Chinn, Am. J . PhysioL 174, 408 (1953) . 12. P. D. Swanson, J . Neurochem. 16_, 35 (1969). 13. H. Mcllwain, Biochem. J . 63_, 257 (1956). 14. R. B. Jennings, J . P. Kaltenbach and H. M. Somers, Arch. Pathol. 84_, 15 (1967) . 15. H. Mcllwain, J . Thomas and J . L. B e l l , Biochem. J . 6_4, 332 (1956) . 16. J . Thomas, Biochem. J . 6_4, 335 (1956). 17. R. Balazs i n Handbook of Neurochemistry, Vol. I l l (Ed. A. Lajtha), p. a. Plenum, New York (1970). 18. H. S. Bachelard and H. Mcllwain i n Comprehensive Biochem-i s t r y , Vol. 17 (Eds. M. F l o r k i n and E. H. Stotz) , p. 191. E l s e v i e r , Amsterdam (1969). - 25 9 " 19. J . H. Quastel i n Structure and Function of the Nervous Tissue Vol. 3 (Ed. G. H. Bourne), P. 61. Academic Press, New York (1969). 20. H. S. Bachelard i n Handbook of Neurochemistry, Vol. IV (Ed. A. Lajtha), p. 1. Plenum, New York (1970). 21. W. Sachs, J . Appl. Physiol. 2_0, 117 (1965). 22. H. B. Burch, 0. H. Lowry, A. M. Kuhlman, J . Skerjance, E. J . Diamant, S. R. Lowry and P. V. Dippe, J . B i o l . Chem. 238, 2267 (1963). 23. M. F. U t t e r , Ann. N. Y. Acad. S c i . 72^ 451 (1959). 24. M. F. U t t e r , Iowa State J . Sc. 3_8, 97 (1963). 25. O. H. Lowry and J . V. Passonneau, J . B i o l . Chem. 236, 41 (1964). 26. R. K. Crane i n the Enzymes Vol. 6 (Eds. P. D. Boyer, H. Lardy and K. Myrback), p. 47. Academic Press, New York (1962). 27. A. Sols and R. K. Crane, J . B i o l . Chem. 206, 925 (1954). 28. R. K. Crane and A. Sols, J . B i o l . Chem. 210, 597 (1954). 29. H. J . Fromm and V. Zewe, J . B i o l . Chem. 237, 1661 (1962). 30. E. J . A. Newsholme, F. S. Rolleston and K. Taylor, Biochem. 106, 193 (1968). 31a. K. Uyeda and E. Racker, J . B i o l . Chem. 240, 4682 (1965). 31b. K. Uyeda and E. Racker, J . B i o l . Chem. 240, 4689 (1965). 32. F. Raggi and D. S. Kronfeld, Nature 209, 1353 (1966). 33. H. S. Bachelard, Nature 215, 959 (1967). 34. M. F. Thompson and H. S. Bachelard, Biochem. J . 118, 25 (1970). 35. 0. H. Lowry and J . V. Passonneau, J . B i o l . Chem. 241, 2268 (1966). 36. B. Axelrod i n Metabolic Pathways, Vol. I (Ed. D. M. Greenberg), p. 112. Academic Press, New York (1967). 37. M. F. Utter, J . B i o l . Chem. 185, 499 (1950). - 260 -38. M. F. Utter, Ann. N. Y. Acad. Sc. 72_, 387 (1959). 39. J . A. Muntz and J . Hurwitz, Arch. Biochem. 3_2, 124, 137 (1951). 40. J . A. Bain and G. H. Pollock, Proc. Soc. Exp. B i o l . Med. 71, 495 (1949). 41. V. Bonavita, F. Ponte and G. Anore, J . Neurochem. 11, 39 (1964). 42. D. DiPietro and S. Weinhouse, Arch. Biochem. Biophys. 80_, 268 (1959) . 43. R. Balazs and J . R. Lagnado, J . Neurochem. 5_, 1 (1959). 44. , M. K. Johnson, Biochem.'J. 77, 610 (1960). 45. R. Tanaka and L. G. Abood, J . Neurochem. 10_, 571 (1963). 46. H. S. Bachelard, Biochem. J . 104, 286 (1967). 47. H. S. Bachelard and P. S. G. Goldfarb, Biochem. J . 112, 579 (1969). 48. 0. H. Lowry, N. J . Rosebrough, A. L. Farr and R. J . Randall, J . B i o l . Chem. 193, 265 (1951). 49. L. G. Abood, E. Brunngraber and M. Taylor, J . B i o l . Chem. 234, 1307 (1959). 50. H. Mcllwain, Biochemistry and the Central Nervous System. L i t t l e , Brown and Co., Boston (1966). 51. F. S. Rolleston, D. P h i l . Thesis. Univ. of Oxford (1966). 52. F. S. Rolleston and E. A. Newsholme, Biochem. J . 104, 524 (1967). 53. H. A. Lardy and R. E. Parks i n Enzymes: Units of B i o l o g i c a l Structure and Function fed. D. H. Gaebler), p. 584. Academic Press, New York (1956). 53a. V. D. Wiebelhaus and H. A. Lardy, Arch. Biochem. 21, 321 (1949). 54. T. E. Mansour and J . M. Mansour, J . B i o l . Chem. 237, 629 (1962). 55. J . V. Passonneau and 0. H. Lowry, Biochem. Biophys. Res. Comm. 7, 10 (1962). - 261 -56. J . V. Passonneau and 0. H. Lowry, Biochem. Biophys. Res. Comm. L3, 372 (1963). 57. H. Tiedemann and J . Born, Z. N a t u r f o r s c h . 14 B, 477 (1959). 58. M. Dixon and E. C. Webb, Enzymes, 2nd E d i t i o n , Longmans, London (1964) . 59. G. Takagaki, J . Neurochem. 15, 903 (1968). 60. F. L. Bygrave, Biochem. J . 101, 488 (1966). 61. R. C. Hanig and M. H. Aprison, Anal. Biochem. 21, 169 (1967). ~ 62. C. A. Ashford and K. C. Dixon, Biochem. J . 29,, 157 (1935) . 63. F. Dickens and G. D. G r e v i l l e , Biochem. J . 29, 1468 (1935). 64. E. Racker and I . Krimsky, J . B i o l . Chem. 161, 453 (1945). 65. G. Takagaki and Y. Tsukada, J . Neurochem. 2_, 21 (1957) . 66. H. M. Pappius, M. Rosenfeld, D. M. Johnson and K. A. C. E l l i o t t , Can. J . Biochem. P h y s i o l . 36_, 217 (1958) . 67. H. A. Lardy and J . A. Zi e g l e r , J . B i o l . Chem. 159, 343 (1945). 68. H. Mcllwain, Biochem. J . 52, 289 (1952). 69. M. Findlay, W. L. Magee and R. J . Rossiter, Biochem. J . 58, 236 (.1954) . 70. J . H. Quastel and A. H. M. Wheatley, J . B i o l . Chem. 119, LXXX (1937). 71. D. H. Adams and J . H. Quastel, Proc. Roy. Soc. 145 B, 472 (1956). 72. A. M. Shanes, Pharmacol. Revs. 10, 59 (1958). 73. A. M. Shanes, Pharmacol. Revs. 10_, 165 (1958). 74. K. A. C. E l l i o t t , I . H. Page and J . H. Quastel, Eds. Neurochemistry, 2nd Ed., C. C. Thomas, S p r i n g f i e l d (1962). 75. R. H. deJong, Physiology and Pharmacology of Local Anesthesia. C. C. Thomas, S p r i n g f i e l d (1970). - 262 -76. A. L. Hodgkin, Proc. Roy. Soc. 148 B, 1 (1958). 77. D. B. Tower i n Properties of Membranes and Diseases of the Nervous System (Eds. D. B. Tower, S. A. Luse and H. Grundfest). Chapter 1. Springer, New York (1962). 78. T. Narahashi, N. C. Anderson and J . W. Moore, J . Gen. Physiol. 50, 1412 (1967). 79. J . W. Moore and T. Narahashi, Fed. Proc. 26_, 1655 (1967). 80. A... L. Hodgkin, The conduction of the Nerve Impulse Liverpool University Press, Liverpool (1964). 81. H. T. Gordon and J . H. Welsh, J . C e l l . Comp. Phys i o l . 31, 395 (1948). 82. D. E. Goldman, Biophys. J . 4_, 167 (1964). 83. D. E. Goldman and M. P. Blaustein, Ann. N. Y. Acad. Sc. 137, 967 (1966). 84. D. C. Tosteson, Fed. Proc. 2_2 , 19 (1963) . 85. T. Narahashi, N. C. Anderson and J . W. Moore, Abstr. 10th Ann. Meeting Biophys. Soc. 147 (1966) 86. A. M. Shanes, Ann. Rev. Pharmacol. 3_, 185 (1963). 87. H. Schonenberger, A. Petter, and W. Zwez, Zum Wirkungsmechanismus der Localanasthetica. Arch. Pharm (Weinheim) 3_8, 209 (1968) , c i t e d i n R. H. de Jong, Physiology and Pharmacology of Local Anesthesia. C. C. Thomas, S p r i n g f i e l d (1970). 88. I. Tasaki i n Handbook of Physiology. Vol. I Section 1 (Ed. J . F i e l d ) , p. 75. American P h y s i o l o g i c a l Society, Washington (1959). 89. J . M. Ritchie and P. Greengard, Ann. Rev. Pharmacol. 6, 405 (1966). 90. R. E . Taylor, Am. J . Physiol. 196, 1071 (1959). 91. A. M. Shanes, W. H. Freygang, H. Grundfest and E. Amatnick, J. Gen. Ph y s i o l . £2, 793 (1959). 92. M. P. Blaustein and D. E. Goldman, Fed. Proc. 24_, 584 (1965). - 263 -93. G. A. Condouris, J . Pharmacol. Exp. Ther. 131, 243 (196ll 94. G. A. Condouris, J . Pharmacol. Exp. Ther. 141, 253 (1963X 95. P. W. Nathan and T. A. Sears, J . Physiol. 154, 4IP (1960). 96. P. w. Nathan and T. A. Sears, J. Physiol. 164 , 375 (1962)i 97. A. Rothstein, Ann. Rev. Physiol. 30_, 15 (1968). 98. M. P. Blaustein and D. E. Goldman, Science 153, 429 (1966). 99. B. H i l l e , Nature 210, 1220 (1966). 100. N. B. Anderson and J . S. Gravenstein, J . Pharmacol. Exp. Ther. 147, 40 (1965). 101. N. B. Anderson, J . Pharmacol. Exp. Ther. 16 3, 393 (1968). 102. R. E. Taylor, Am. J . P h y s i o l . 196, 1071 (1959). 103. F. Brink, Pharmacol. Revs. 6_, 243 (1954). 104. S. Weidmann, J. Physiol. 129, 568 (1955). 105. F. A. Davis and W. D. Dettbarn, Biochim. Biophys. Acta. 6_3, 349 (1962) . 106. R. Straub, Arch. Intern. Pharmacodyn. 107, 414 (1956). 107. M. B. F e i s t e i n , J . Gen. P h y s i o l . 47, 151 (1963). 108. S. Niwa, J . Pharmacol. Exp. Ther. 12, 323 (1919). 109. M. A. F. Sherif, J. Pharmacol. Exp. Ther. 38, 11 (1930). 110. D. T. Watt, J . Pharmacol. Exp. Ther. 96_, 325 (1949). 111. I. C. Geddes and J . H. Quastel, Anesthesiology 17_, 666 (1956). 112. B. R. Fink, G. E. Kenny and W. E. Simpson, Anesthesiology 30, 150 (1969). 113. B. E. Ryman and E. O'F. Walsh. J . Pharm. Pharmacol. 7, 341 (1955). 114. H. S. Mosher, F. A. Fuhrman, H. D. Buchwald and H. G. Fischer, Science 144, 1100 (1964) . - 264 -115. C. Y. Kao, Pharmacol. Revs. 18, 997 (1966). 116. M. H. Evans, B r i t . Med. B u l l . 25_, 263 (1969). 117. T. Furukawa, T. Sasooka and Y. Hosoya, Jap. J. Physiol. 9_, 143 (1959). 118. T. Narahashi, T. Deguchi, N. Urakawa and Y. Ohkubo, Am. J . Phys i o l . 198, 934 (1960). 119. T. Narahashi, J . W. Moore and W. R. Scott, J . Gen. Physi o l . £7, 965 (1964). 120. C. Y. Kao and F. A. Fuhrman. J . Pharmacol. Exp. Ther. 140, 31 (1963). 121. M. T a k a t a , J . W. Moore, C. Y. Kao and F. A. Fuhrman. J . Gen. P h y s i o l . 4_9, 977 (1966) . 122. J . W. Moore, M. P. B l a u s t e i n , N. C. A n d e r s o n and T. N a r a h a s h i , J . Gen. P h y s i o l . 5_0, 1401 (1967). 123. M. H. E v a n s , B r i t . J . P h a r m a c o l . 36_, 418 (1969). 124. J. W. Moore and T. N a r a h a s h i , F e d . P r o c . 2_6, 1655 (1967). 125. E . R o j a s and I. A t w a t e r , P r o c . N a t l . A c a d . S c i . 57, 1350 (1967). 126. T. N. P u l l m a n , A. R. L a v e n d e r and I. Aho, P r o c . N a t l . A c a d . S c i . 6_0 , 822 (1968). 12 7. T. N a r a h a s h i , N. C. A n d e r s o n and J . W. Moo r e , S c i e n c e 153, 765 (1966). 128. Y. Ogura and Y. Mori, Europ. J . Pharmacol. 3, 58 (1968). 129. H. Mcllwain, Biochem. Pharmacol. 16_, 1389 (1967). 130. S. L. Chan and J . H. Quastel, Science 156, 1752 (1967). 131. P. D. Swanson, B i o c h e m . P h a r m a c o l . 17_, 129 (1968) . 132. H. M c l l w a i n , J . A. H a r v e y a n d G. R o d r i g u e z , J . Neurochem. 16, 363 (1969). 133. I. P u l l , H. M c l l w a i n and R. L. Ramsay, B i o c h e m . J . 116, 181 (1970). 134. R. L. Ramsay and H. M c l l w a i n , J. Neurochem. 17, 781 (1970). ,- 265 -135. K. Okamoto and J . H. Quastel, Biochem. J . 120, 37 (1970). 136. Y. Itokawa and J . R. Cooper, Biochem. Pharmacol. 19, 985 (1970). 137. J . H. Quastel, Neurosciences Res. 3_, 1 (1970). 138. J . C. Skou, Biochim. Biophys. Acta 23_, 394 (1957) . 139. J . C. Skou, Biochim. Biophys. Acta £2, 6 (1960). 140. J . C. Skou, Physiol. Revs. £5, 596 (1965). 141. R. W. Albers, Ann. Rev. Biochem. 3_6, 727 (1967). 142. E. Heinz, Ann. Rev. Physiol. 29_, 21 (1967). 143. I. M. Glynn, B r i t . Med. B u l l . 24_, 165 (1968). 144. A. Rothstein, Ann. Rev. Physiol., 30_, 15 (1968). 145. R. Whittam and K. P. Wheeler, Ann. Rev. Ph y s i o l . 32, 21 (1970). 146. J . H. Schatzman, Helv. Physiol. Acta. 11, 346 (1953). 147. I. M. Glynn, J . Phys i o l . 136, 148 (1957). 148. V. Kofoed-Johnsen, Acta Ph y s i o l . Scand. 4_2 Suppl. , 145:87 (1958). 149. R. Whittam, J . Phys i o l . 140, 479 (1958). 150. R. L. Post, A. K. Sen and A. S. Rosenthal, J . B i o l . Chem. 240, 1437 (1965). 151. S. Fahn, G. J . Koval and R. W. Albers, J . B i o l . Chem. 243, 1993 (1968). 152. R. Gibbs, P. M. Roddy and E. T i t u s , J. B i o l . Chem. 240, 2181 (1965). 153. R. Rodnight, D. A. Hems and B. E. Lavin, Biochem. J . 101, 502 (1966). 154. R. Whittam, K. P. Wheeler and A. Blake, Nature 203, 720 (1964). - 266 -155. K. Nagano, N. M i z u n o , M. F u j i t a , Y. T a s h i m a , T. Nakao and M. Nakao, B i o c h i m . B i o p h y s . A c t a 143, 239 (1967). 156. A. K. Sen, T. T o b i n and R. L. P o s t , J . B i o l . Chem. 244, 6596 (1969). 157. W. Schoner, R. Beuisch and R. Kramer, Europ. J . Biochem. 7, 102 (1968). 158. A. H. Neufeld and H. M. Levy, J . B i o l . Chem. 245, 4962 (1970). 159. P. J . Garrahan and I. M. Glynn, J . Physiol. 192, 217 (1967) . 160. J . R. Murphy, J . Lab. C l i n . Med. 61, 567 (1963). 161. A. K. Sen and R. L. Post, J . B i o l . Chem. 239, 345 (1964) . 162. R. Whittam and M. E. Ager, Biochem. J . 9_7 , 214 (1965). 163. S. L. Bonting, L. L. Caravaggio and N. M. Hawkins, Arch. Biochem. Biophys. 9_8, 413 (1962) . 164. H. Yoshida, T. Nukada and H. Fujisawa, Biochim. Biophys. Acta '48, 614 (1961) . 165. A. Schwartz, H. Matsui and' A. H. Laughter, Science 159, 323 (1968). 166. J . S. Charnock and H. A. Potter, Arch. Biochem. Biophys. 134, 42 (1969). 167. A. Yoda and L. E . H o k i n , B i o c h e m . B i o p h y s . Res. Comm. 40, 880 (1970). 169. R. W h i t t a m and D. M. B l o n d , B i o c h e m . J . 9_2, 147 (1964). 170. O. Gonda and J . H. Q u a s t e l , B i o c h e m . J . 8 4 , 394 (1962). 171. M. M. de P i r a s and J . A. Z a d u n a i s k y , J . Neurochem. 12, 657 (1965). 172. P. D. Swanson and H. M c l l w a i n , J . Neurochem. 12^ , 877 (1965). 173. P. D. Swanson, J . Neurochem. 15_, 57 (1968). 174. P. D. Swanson and K. U l l i s , J . P h a r m a c o l . E x p . T h e r . 153, 321 (1966). - 267 -175. P. D. Swanson and W. L. Stahl, Biochem. Pharmacol. 19. 2394 (1970). 176. D.B. Tower, Exp. Brain Res. 6_, 273 (1968). 177. W. L. Stahl and P.D. Swanspn, N. Neurochem. 16>, 1553 (1969) . 178. G.B. Frank, J . Neurophysiol. 21, 263 (1958). 179. E.B.Wright and T. Tomita, J . C e l l . Physiol. £7,181 (1966). 180. S.M. Kupchan and W.E. Flack i n Antihypertensive Agente (Ed. . E. S c h l i t t l e r ) , p.429. Academic Press, New York (1967). 181. A. Wollenberger, Fed. Proe. 9_, 326 (1950). 182. A. Wollenberger, Biochem. J . 6_1, 68 (1955) . 183. A. Wollenberger, Biochem. J . 6^, 77 (1955). 184. J.H. Quastel, Proc. 4th Intern. Cong. Biochem.3_, 90 (1959) . 185. K.A.C. E l l i o t t and L.S. Wolfe i n Neurochemistry (Eds. K.A.C. E l l i o t t , I. H. Page and J.H. Quastel), p. 177. C. C. Tomas, S p r i n g f i e l d (1962). 186. M. M. K i n i and J . H. Quastel, Science 131, 412 (1960). 187. B. B. B r o d i e , A.K. Cho, F . J . E . S t e f a n o and G.L. G e s s a , A d v a n c e s i n B i o c h e m i c a l P s y c h o p h a r m a c o l o g y , V o l . 1 (Ed s . E . C o s t a a n d P. G r e e n g a r d ) p. 219. Raven P r e s s , New Y o r k (1969). 188. P. J . G. Mann a n d J.H. Q u a s t e l , N a t u r e 144, 943 (1939). 189. P. J . G. Mann and J.H. Q u a s t e l , B i o c h e m . J . 34_, 414 (1940). 190. I n t e r n a t i o n a l Symposium on Amphetamines and R e l a t e d compounds. P r o c e e d i n g s o f t h e M a r i o N e g r i I n s t i t u t e f o r P h a r m a c o l o g i c a l R e s e a r c h , M i l a n , I t a l y . ( E d s . E . C o s t a and S. G a r a t t i n i ) Raven P r e s s , New Y o r k ( 1 9 7 0 ) . 191. J.H. B i e l i n I n t e r n a t i o n a l Symposium on Amphetamines and R e l a t e d compounds. ( E d s . E . C o s t a and S. G a r a t t i n i ) , p. 3. Raven P r e s s , New Y o r k (1970). 192. S. J . S t r a d a , E . S a n d e r s - B u s h and F. S u l s e r , B i o c h e m . P h a r m a c o l . 19, 2621 (1970). - 268 -193. J . S c h u b e r t , B. F y r o , H. Nyback and G. S e c V a l l , J . Pharm. P h a r m a c o l . 2_2, 860 (1970). 194. F. Th. v o n B r i i c k e , 0. H o r n y k i e w i c z and E . B. S i g g , The P h a r m a c o l o g y o f P s y c h o t h e r a p e u t i c D r u g s , S p r i n g e r V e r l a g , New Y o r k (196 9). 195. J . H. Q u a s t e l and A. H. M. W h e a t l e y , P r o c . Roy. S o c . 1 112,B, 60 (1932). 196. J . H. Q u a s t e l , B r i t . Med. . B u l l . 21, 49 (1965). 197. J . H. Q u a s t e l i n P r i n c i p l e s o f P s y c h o p h a r m a c o l o g y ( E d s . W. G. C l a r k and J . de G i u d i c e ) , p. 141. A c a d e m i c P r e s s , New Y o r k (1970). 198. S. L . Chan and J . H. Q u a s t e l , B i o c h e m . P h a r m a c o l . 19, 1071 (1970). 199.. J . L. Webb and K. A. C. E l l i o t t , J . P h a r m a c o l ; E x p . T h e r . 103, 24 (1951). 200. 0. L i n d a n , J . H. Q u a s t e l and S. S v e d , Can. J . B i o c h e m . P h y s i o l . 3_5 , 1135, 1145 (1957). 201. H. M c l l w a i n and 0. G r e e n g a r d , J . Neurochem. 1, 348 (1957) . 202. A. A n d r e j e w and A. J . R o s e n b e r g , C. R. S o c . B i o l . 150, 639 (1956). 203. A. A n d r e j e w and A. J . R o s e n b e r g , C. R. S o c . B i o l . 151, 237 (1957). 204. A. A n d r e j e w , G. D u c e t , J . Louw and A. J . R o s e n b e r g , C. R. S o c . B i o l . 150, 4484 (1956). 205. H f Low, B i o c h i m . B i o p h y s . A c t a 3_2, 11 (1959). 206. K. Y a g i , T. Ozawa, M. Ando and T. N a g a t s u , J . N e u r o -chem. 5, 304 (1960). 207. M. J . R. D a w k i n s , J . D. J u d a h and K. R. R e e s , B i o c h e m . J . 73_, 16 (1959) . 208. M. A. S p i r i t e s and P. S. G u t h , N a t u r e 190, 274 (1961). 209. A. R. Freeman and M. A. S p i r i t e s , B i o c h e m . P h a r m a c o l . 12, 47 (1963). 210. P. M. Seeman and H. S. B i a l y , B i o c h e m . P h a r m a c o l . 12, 1181 (1963). - 26 9 -211. D. R i c h t e r i n N e u r o p s y c h o p h a r m a c o l o g y V o l . I I (Ed. E . R o t h l i n ) . p. 422, E l s e v i e r , Amsterdam (1961). 212. A. D o b k i n , R, G. B. G i l b e r t and K. I . M e l v i l l e , c i t e d i n W. F. T. T a t l o w , C. M. F i s c h e r and A. B. D o b k i n , Can. Med. A s s . 11, 380 (1954). 213. R. P. M a i c k e l , I n t . J . N e u r o p h a r m a c o l . 7, 23 (1968). 214. W. L. Magee and R. J . R o s s i t e r , Can. J . B i o c h e m . P h y s i o l . 41, 1155 (1963). 215. S. J . M u l e , B i o c h e m . P h a r m a c o l . 1S_, 339 (1969). 216. J . C h r i s t e n s e n , Y. S. L. F e n g , E . P o l l e y and A. W. Wase, F e d . P r o c . 17_, 358 (1958) . 217. J . A. B u z a r d , E x p e r i e n t i a 16, 153 (1960). 218. P. W. D a v i s and T. M. B r o d y , B i o c h e m . P h a r m a c o l . 15, 703 (1966). 219. T. A k e r a and T. M. B r o d y , M o l . P h a r m a c o l . A, 600 (1968). 220. T. A k e r a and T. M. B r o d y , M o l . P h a r m a c o l . 6, 557 (1970). 221. J . M. M i i l l e r , E . S c h l i t t l e r and H. J . B e i n , E x p e r i e n t i a 8, 338 (1952). 222. A. G i a c h e t t i and P. A. S h o r e , B i o c h e m . P h a r m a c o l . 19, 1621 (1970). 223. A. C a r l s s o n , P h a r m a c o l . R e v s . 18_, 541 (1966). 224. F. E . B l o o m and N. J . G i a r m a n , Ann. Rev. P h a r m a c o l . 8_, 229 (1968). 225. 0. C a r r i e r , J r . , B. H. D o u g l a s , L. G a r e t t and P. J . W h i t t i n g t o n , J . P h a r m a c o l . E x p . T h e r . 158, 494 (1967). 226. U. S. v o n E u l e r , S. R o s e l l and B. U v n a s , E d s . , Me c h a n i s m o f R e l e a s e o f B i o g e n i c A m i n e s , Pergamon P r e s s , O x f o r d (1966). 227. H. M. L a b o r i t a n d A. S a n s e i g n e i n P r i n c i p l e s o f P s y c h o p h a r m a c o l o g y ( E d s . W. G. C l a r k and J . de G i u d i c e ) , p. 259, A c a d e m i c P r e s s , New Y o r k (1970). 228. W. A. H i m w i c h , E d . , D e v e l o p m e n t a l N e u r o b i o l o g y , C. C. Thomas, S p r i n g f i e l d (1970). - 270 -229. J . Dobbing i n Developmental Neurobiology (Ed. W. A. Himwich), p 241. C. C. Thomas, S p r i n g f i e l d (1970). '230. J . S. O'Brien i n Developmental Neurobiology (Ed. W. A. Himwich) , p 262. C. C. Thomas, S p r i n g f i e l d (1970). 231. A. N. Davison and A.Peters, Myelination. C. C. Thomas, S p r i n g f i e l d (1970). 2 32. V. M. Tennyson i n Developmental Neurobiology (Ed. W. A. Himwich) , p. 47. C. C. Thomas, S p r i n g f i e l d (1970). 233. K. F. Swaiman i n Developmental Neurobiology (Ed. W. A. Himwich), p. 311. C. C. Thomas, S p r i n g f i e l d (1970). 234. F. E. Samson and D. J . Quinn, J . Neurochem. 14, 421 (1967). 235. H. E. Himwich, Brain Metabolism and Cerebral Disorders, Williams and Wilkins, Baltimore (1951). 2 36. S. Kawashima and T. ueda, Nippon Seirigaku Zasshi 28, 267 (1966), c i t e d i n J . H. Quastel, Neurosciences Res. 3, 1 (1970). 237. S. G. Schultz and P. F. Curran, Physiol. Revs. 50, 637 (1970) . 238. E . R i k l i s and J . H. Quastel, Can. J . Biochem. Physiol. 36_, 347 (1958) . 239. P. F. Curran, Fed. Proc. 2_4 , 993 (1965). 240. R. K. Crane, Fed. Proc. 24_, 1000 (1965). 241. D. M. Kipnis and J . E. P a r r i s h , Fed. Proc. 24, 1051 (1965). 242. J . H. Quastel, Proc. Roy. Soc. 163 B, 169 (1965). 243. G. Guroff, W. King and S. Udenfriend, J . B i o l . Chem. 236, 1773 (1961). 244. S. M. Shaneberg and N. J . Giarman, Biochim. Biophys. Acta 41, 556 (1960). 245. P. N. Abadom and P. G. S c h o l e f i e l d , Can. J . Biochem. Phys i o l . 40, 1575, 1591, 1603 (1962). - 271 -246. G. Takagaki, S. Hirano and Y. Nagata, J . Neurochem. 4 , 124 (1959) . 247. S. L a h i r i and A. Lajtha, J . Neurochem. 11., 77 (1964). 248. ' H. S. Bachelard, J . Neurochem. 18, 213 (1971). 249. 0. H. Lowry i n Nerve as a Tissue (Eds. K. Rodahl and B. Issekutz), p. 163. Harpur and Row, New York (1966). 250. J . F. R. Konig and R. A. K l i p p e l . The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Williams and Wilkins, Baltimore (1963). 251. R. P. Harpur and J . H. Quastel,' Nature 164, 693 (1949). 252. R. P. Harpur and J . H. Quastel, Nature 164, 779 (1949). 253. M. Nyman and V. P. Whittaker, Biochem. J . 87, 248 (1963) . 254. V. P. Whi t t a k e r , P r o g r . Biophys. Mol. B i o l . 15_, 39 (1965). 255. L. E. Hokin and A. Yoda, Proc. N a t l . Acad. S c i . 52, 454 (1964) . 256. M. G. Stanton, A n a l . Biochem.'22, 27 (1968). 257. H. J . Hohorst i n Methods o f Enzymatic A n a l y s i s (Ed. H. U. Bergmeyer),p. 266. Academic Press,New York (1965). 258. D. B. E l l i s and P. G. S c h o l e f i e l d , Cancer Res. 22, 305 (1962). 259. R. K. Crane and F. Lipmann, J . B i o l . Chem. 201, 235 (1953). 260. G. R. B a r t l e t t , J . B i o l . Chem. 234, 466 (1959). 261. 0. H. Lowry and J . V. P a s s o n n e a u i n Methods i n Enzym-o l o g y V o l VI ( E d s . S. P. C o l o w i c k and N. O. K a p l a n ) , p. 792. A c a d e m i c P r e s s , New Y o r k (1963). 262. 0. H. L o w r y , J . V. P a s s o n n e a u , D. W. S c h u l z and M. K. Rock, J . B i o l . Chem. 236, 2746 (1961). 263. 0. H. Lowry, J . V. Passonneau and M. K. Rock, J . B i o l . Chem. 236 , 2756 (1961) . - 272 -264. H. Shimizu, J . W. Daly and C. R. Creveling, J . Neurochem. 16, 1609 (1969). 265. W. Lamprecht and I. Trautschold i n Methods of Enzym-a t i c Analysis (Ed. H. U. Bergmeyer), p 543. Academic Press, New York (1965). 266. P. Greengard i n Methods of Enzymatic Analysis (Ed. H. U. Bergmeyer), p. 551. Academic Press. New York (1965) . 267. J . A. Harvey and H. Mcllwain, Biochem. J . 108, 269 (1968). 268. L. G. Abood and A. Matsubara, Biochim. Biophys. Acta. 163, 539 (1968). 269. M. J . Weidemann, D. H. Hems and H. A. Krebs, Biochem. J. 115, 1 (1969). 270. Advances i n Biochemical Psychopharmacology Vol. 3 (Eds. E. Costa and P. Greengard), Raven Press, New York (1970). 2 71. S. Azhar and C. R. Krishnamurti, Biochem. Biophys. Res. Comm. 43_, 58 (1971). 272. E. W. Sutherland, I. 0ye, and R. W. Butcher Recent Progr. Horm.' Res. 2_1, 623 (1965) . 273. E. W. Sutherland and T. W. R a i l , Pharmacol. Revs. 12, 265 (1960). 2 74. H. Maeno, E. M. Johnson and P. Greengard, J . B i o l . Chem. 246134 (1971). f 275. J . Dittmann and H. D. Herrmann, Experientia 26, 133 (1970). 276. M. H. Alper and W. Flacke, Ann. Rev. Pharmacol. 9, 273 (1969). 277. H. A. Krebs, Biochem. J . 2_9, 1951 (1935). 278. H. Weil-Malherbe, Biochem J . 3_2 , 2257 (1938). 279. 0. Meyerhof and J . R. Wilson, Arch. Biochem. 17, 153 (1948). 280. P. J . G. Mann and J . H. Quastel, Biochem. J . 35, 502 (1941). - 27 3 -281. M. Michaelis and J . H. Quastel, Biochem. J. 35, 518 (1941). 282. L. Ernster, H. Low and 0. Lindberg, Acta. Chem. Scand. 9, 200 (1955). 283. U. J a i l i n g , O. Lindberg and L. Ernster, Acta. Chem. Scand. 9, 198 (1955). 284. J . H. Quastel and A. H. M. Wheatley, Biochem. J . 32, 936 (1938). 285. J . R. Stern, L. V. Eggleston, R. Hems and H. A. Krebs, Biochem.. J. 4_4, 410 (1949) . 286. K. Krnjevic, Nature 228, 119 (1970). 287. A. W. Cuthbert, Pharmacol. Revs. 19, 59 (1967). 288. E. Heilbronn, J . Neurochem. 16, 627 (1969). 289. 0. Rosenthal and A. L a s n i t z k i , Biochem. Z. 196, 340 (1928). 290. A. Bernelli-Zazzera, G. Gaja and G. Ragnotti, Biochem. J. 100 , 114 (1966) . 291. G. Gaja, G. Ragnotti, F. Cajone and A. Bernelli-Zazzera, Biochem. J . 105, 647 (1967). 292. G. Gaja, G. Ragnotti, F. Cajone and A. Bernelli-Zazzera, Biochem. J . 109, 867 (1968). 293. G. Gaja, F. Cajone, M. E. Ferrero and A. B e r n e l l i -Zazzera, J . Natl. Cancer Inst. £4 , 1269 (1970). 294. G. Gaja and M. E. Ferrero, FEBS L e t t e r s , 6_, 31 (1970). 295. J . Thomas, Biochem. J . 66_, 655 (1957). 296. T. E. Mansour and D. B. Stone, Biochem. Pharmacol. 19, 1137 (1970). 2 97.