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Evidence in favour of glutamate as a mediator of synaptic transmission Haldeman, Scott 1973

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EVIDENCE IN FAVOUR OF GLUTAMATE AS A MEDIATOR OF SYNAPTIC TRANSMISSION by SCOTT HALDEMAN D.C., Palmer College of C h i r o p r a c t i c , 1964 B.Sc, University of P r e t o r i a , 1968 M.Sc, University of P r e t o r i a , 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In the Department of PHYSIOLOGY We accept t h i s thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA January, 1973 In present ing t h i s thes is in p a r t i a l f u l f i l m e n t of the requirements fo r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L ib ra ry sha l l make i t f r e e l y a v a i l a b l e f o r reference and study. 1 fu r ther agree that permission f o r extensive copying of th is t h e s i s for s c h o l a r l y purposes may be granted by the Head of my Department or by h is representa t ives . It i s understood that copying or p u b l i c a t i o n of th is thes is f o r f i n a n c i a l gain sha l l not be allowed without my wr i t ten permiss ion . Department of Physiology  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date January 1 1 . 1973 EVIDENCE IN FAVOUR OF GLUTAMATE AS A MEDIATOR OF SYNAPTIC TRANSMISSION ABSTRACT A microiontophoretic i n v e s t i g a t i o n of neurones i n the spinal cord, cuneate nucleus, thalamus and cerebral cortex i n the anaesthetized cat indicated that glutamic acid d i e t h y l ester (GDEE), a-methyl-glutamate (aMG) and DL-methionine-DL-sulphoximine (MSO) could reversibiy. block, and glutamic aci d dimethyl ester (GDME) and para-chloromercuriphenylsulphonate (pCMS) could enhance the ex c i t a t i o n of neurones produced by glutamate and by orthodromic nerve stimulation. The action of GDEE was r e l a t i v e l y s p e c i f i c i n that i t was possible to block glutamate-induced excitations without appreciably reducing the s e n s i t i v i t y of neurones to L-aspartate (Asp), L-cysteate (Cys), DL-homocysteate (DLH) and acetylcholine (ACh), whereas aMG and MSO showed no s p e c i f i c i t y of action and antagonized the excitatory e f f e c t s of a l l the amino acids to the same extent. GDME and pCMS enhanced the action of glutamate and aspartate to a greater extent than that of DLH. The responses of neurones i n the spi n a l cord and thalamus to e l e c t r i c a l stimulation of branches of the s c i a t i c nerve and of cuneate neurones to dorsal column stimulation were blocked by GDEE and enhanced by GDME i n the same manner as glutamate responses. GDEE blocked the responses of c o r t i c a l neurones to glutamate and thalamic stimulation but the depressant e f f e c t was not as commonly observed as i n other areas. Studies on the uptake of l a b e l l e d glutamate into crude synaptosomal preparations of rat cerebral cortex confirmed the existence of a high and low a f f i n i t y uptake mechanism f o r glutamate and showed that GDME i n h i b i t s the high a f f i n i t y uptake system. Glutamate and GDEE, but not aMG, enhanced the release of l a b e l l e d glutamate from s l i c e s of rat cortex. No competition between glutamate and GDEE was observed i n t h i s system. aMG was found to block the responses of the abdominal stre t c h receptor organ of the cra y f i s h to applied glutamate. GDEE blocked the- responses of the closer muscle i n the claw to e l e c t r i c a l stimulation of the excitatory nerve to thi s muscle. i v ' TABLE OP CONTENTS Page L i s t of t a b l e s . . v i i L i s t of figures . v i i i L i s t of abbreviations x i ^ Acknowledgements . x i i -CHAPTER I: Introduction . 1 The Mammalian C.N.S 5 1. D i s t r i b u t i o n of L-glutamate 5 2. Replacement of the transmitter 9 3. Glutamate release during synaptic a c t i v i t y 12 4. Ina c t i v a t i o n of glutamate 15 5. Comparison of action of glutamate and the p h y s i o l o g i c a l transmitter 18 6. The action of pharmacological agents on glutamate and sy n a p t i c a l l y evoked excitations 25 The Invertebrate Neuro-Muscular Junction. 26 1. D i s t r i b u t i o n of L-glutamate 27 2. Replacement of the transmitter 28 3. Glutamate release during synaptic a c t i v i t y 29 4. Inactivation of glutamate 30 5. Comparison of action of glutamate and the p h y s i o l o g i c a l transmitter 32 6. The action of pharmacological agents on glutamate and syn a p t i c a l l y evoked excitations 3^ V Page The present study 35 CHAPTER I I : Materials and Methods - Cat Preparations 39 Electrode Placement 40 a) Recording from thalamic neurones 4l b) Recording from cuneate neurones... 42 c) Recording from spinal cord neurones 43 d) Recording from the cerebral cortex 44 Recording Electrodes 46 E l e c t r i c a l Equipment 49 CHAPTER I I I : Results - Cat Preparations 52 a) Neuronal f i r i n g patterns and evoked responses - 52 b) Glutamic acid d i e t h y l ester 58 c) Glutamic acid dimethyl ester 74 d) a-methyl glutamate 85 e) DL-Methionine-DL-Sulphoximine 88 f) Other miscellaneous drugs 91 CHAPTER IV: Materials and Methods - Rat Cortex Preparations 98 a) Release of glutamate from brain s l i c e s 98 b) Glutamate uptake into synaptosomes 101 CHAPTER V: Results - Rat Cortex Preparations 104 a) Release of glutamate from brain s l i c e s 104 b) Glutamate uptake into synaptosomes 106 CHAPTER VI: Materials and Methods - Crayfish Preparations I l l a) The abdominal stretch receptor 116 v i Page b) The closer muscle of the claw 118 CHAPTER VIII: Discussion 122 The Primary Sensory Pathway 130 The Secondary Sensory Pathway 13^ The Thalamocortical Pathway 136 The Anaesthetic and Muscle Relaxant 139 The Release of Glutamate from Brain S l i c e s 140 The Crayfish Neuro-Muscular Junction. l 4 l SUMMARY AND CONCLUSIONS 145 BIBLIOGRAPHY 148 v i i LIST OP TABLES Table Page I. Drug solutions used for iontophoresis.... 48 I I . The action of derivatives of glutamate on thalamic (VPL) neurones 57 I I I . The e f f e c t of GDEE on single c e l l excitations 59 IV. The blocking action of GDEE on the f i r i n g of thalamic neurones . 63 V. The e f f e c t of GDME on single c e l l excitations 76 VI. The e f f e c t of aMG on single c e l l excitations 86 VII. The e f f e c t of MSO on single c e l l excitations 89 V I I I . The e f f e c t of pCMS and Cyclobenzaprine on single c e l l e x c i t a t i o n s . 93 IX. Kinetics of glutamate uptake into homogenates of rat brain 110 X. The e f f e c t s of a number of glutamate analogues on the f i r i n g rate of the stretch receptor i n the c r a y f i s h 117 v i i i " LIST OP FIGURES Figure Page 1. The p r i n c i p l e enzymes and products involved i n glutamate metabolism 3 2. A block diagram of the e l e c t r i c a l equipment used i n the cat experiments.. 51 3 . The eff e c t s of an intravenous i n j e c t i o n of 10 mg. Na-pentobarbitol on t i d a l CO2 and blood pressure 53 4. The response of a neurone i n the cuneate nucleus to increasing iontophoretic current strengths of DLH, Glut, Asp and Cys 56 5 . The ef f e c t s of GDEE on DLH, Glut and ACh ex c i t a t i o n of a VPL neurone 60 6. The e f f e c t of GDEE on the DLH, Glut and Cys e x c i t a t i o n of a neurone of the cuneate nucleus 61 7 . The eff e c t of GDEE on the response of a VPL neurone to the excitant amino acids 64 8. Dose-response curves for Glut excitations on a VPL neurone 66 9 . The e f f e c t of an i n t r a - a r t e r i a l i n j e c t i o n of GDEE on the responses of a VPL neurone to the excitant amino acids 67 1 0 . The eff e c t s of GDEE on amino acid and synap-t i c a l l y evoked excitations i n VPL 69 1 1 . The effect of GDEE on amino acid and synap-t i c a l l y evoked excitations of a spi n a l interneurone 70 1 2 . The eff e c t of GDEE on the amino acid and synaptic a l l y induced excitations of a neurone i n the cuneate nucleus 71 Figure Page 13. The effect of GDEE on amino acid and synap-t i c a l l y evoked excitations of a neurone i n the cerebral cortex 72 14. The eff e c t s of an i n t r a - a r t e r i a l i n j e c t i o n of GDEE on amino acid and synapti c a l l y evoked excitations i n VPL 73 15. The eff e c t of d i f f e r e n t current strengths of GDEE on the amino acid and evoked responses of a spinal interneurone . . 75 16. The effects of GDME on amino acid e x c i t a t i o n of a VPL neurone 78 17. The ef f e c t of GDME on amino acid e x c i t a t i o n of a neurone i n the cerebral cortex 79 18. A comparison of the ef f e c t s of GDEE and GDME on the amino acid induced excitations of a neurone of the cuneate nucleus 81 19. The effects of GDME on amino acid and synaptic e x c i t a t i o n of a VPL neurone 82 20. The eff e c t of GDME on the amino acid and synaptic a l l y evoked excitations of spinal interneurones 83 21. The eff e c t of GDME on the amino acid and synaptic a l l y evoked e x c i t a t i o n of a neurone of the cuneate nucleus ... 84 22. The potentiating e f f e c t s of aMG on amino acid e x c i t a t i o n of a VPL neurone 87 23. The ef f e c t of MSO on the amino acid and synaptically evoked e x c i t a t i o n of a spin a l interneurone 90 24. A comparison of the eff e c t s of GDEE and MSO on the amino acid excitations of a neurone i n the cerebral cortex 92 25. The ef f e c t of cyclobenzaprine on the amino acid and syna p t i c a l l y evoked excitations of a VPL neurone 94 X Figure Page 2 6 . The ef f e c t of pCMS on amino acid excitations of a VPL c e l l 96 2 7 . The eff e c t s of pCMS on amino acid and synap-t i c a l l y evoked excitations of a VPL neurone 97 2 8 . A diagram of the transfer holder used to transfer brain s l i c e s from one test tube of medium to another 99 2 9 . Plots of the eff e c t s of Glut and GDEE on the rate c o e f f i c i e n t of Glut e f f l u x from s l i c e s of cerebral cortex 105 3 0 . Plots of the eff e c t s of Glut and aMG on the rate c o e f f i c i e n t of Glut e f f l u x from s l i c e s of cerebral cortex 107 3 1 . Double r e c i p r o c a l plots of the v e l o c i t y of the uptake of L-Glut by homogenates of rat cerebral cortex 108 3 2 . A block diagram of the equipment used to record from the abdominal receptor i n cr a y f i s h 113 3 3 . A block diagram of the equipment used to record closer muscle contractions i n the claw of the c r a y f i s h 115 3 4 . The ef f e c t of aMG on the Glut excitations of the abdominal stretch receptor i n the cr a y f i s h 119 3 5 . The ef f e c t of GDEE on syn a p t i c a l l y induced contractions of the closer muscle of the claw of the c r a y f i s h 120 LIST OF ABBREVIATIONS acetylcholine L-aspartate adenosine triphosphate centimetre ce n t r a l nervous system L-cysteate d i r e c t current d i i s o p r o p y l phosphorofluoridate DL-homocysteate excitatory j u n c t i o n a l potentials excitatory postsynaptic potentials g r a v i t i e s Y-aminobutyric acid glutamic acid d i e t h y l ester glutamic acid dimethyl ester L-glutamate gram l-hydroxy-3-aminopyrrolidone-2 5-hydroxy-tryptamine intravenous kilogram Michaelis a f f i n i t y constant l y s e r g i c acid diethylamide molar millimetre m i l l i m o l a r m i l l i c u r i e milligram minute m i l l i l i t r e m illisecond DL-methionine-DL-sulphoximine megohm _ q nanoampere (10 ampere) noradrenaline p r o b a b i l i t y para-chloromercuriphenylsulphonate concentration second stimulus i s o l a t i o n unit v e l o c i t y Volt nucleus v e n t r a l i s l a t e r a l i s nucleus v e n t r a l i s p o s t e r o l a t e r a l i s maximum reaction rate a-methyl-L-glutamate Y-methyl-L-glutamate micron (10-"metrg) microampere (10,- ampere) m i c r o l i t r e (10 l i t r e ) ACKNOWLEDGMENTS I wish to acknowledge the f i n a n c i a l support of the Canadian and South Af r i c a n Medical Research Councils and the guidance and advice i n the understanding of ph y s i o l o g i c a l p r i n c i p l e s provided by the facu l t y and s t a f f i n the Department of Physiology at the University of B r i t i s h Columbia. I am indebted to Mrs. Y. Heap and Mr. R. Walker for t h e i r assistance with the s u r g i c a l procedures and h i s t o l o g i c a l preparations; to Mr. K. Henze who completed the figures and did a l l the photography for t h i s t h e s i s ; to Dr. C O . Parkes f o r his advice i n the f i e l d s of biochemistry and k i n e t i c s ; and to Dr. D.H. Copp, Dr. J.A. Pearson, Dr. H.D. Sanders, Dr.P. Vaughan and Dr. A.W. Duggan who formed my committee and helped to guide my Ph.D. programme. Dr. Hugh McLennan i s responsible for the most valuable and l a s t i n g benefits received i n the preparation of t h i s t h e s i s , and I am deeply indebted to him for his sound advice and guidance on educational, research and professional matters. F i n a l l y , I wish to thank my wife Joan for her support and encouragement and for the many hours which she spent i n the reading and typing of thi s t h e s i s . CHAPTER I INTRODUCTION The o r i g i n a l suggestion that chemical agents might be involved i n the transmission of nerve impulses came from T.R. E l l i o t t i n 1904, when he reported the conspicuous p a r a l l e l i s m between stimulation of sympathetic nerves and the action of adrenalin upon the v i s c e r a , and postulated that adrenalin might be released by the terminals of nerve endings and be responsible for the sympathetic a c t i o n . This hypothesis has been widely accepted and extended to include the parasympathetic nerves and transmission across neuro-muscular junctions and synapses within the central nervous system. Since i t was found that adrenalin was not the transmitter at a l l of these synapses the l i s t of possible mediators of synaptic transmission has been expanded to include a wide variety of compounds. Latecomers to t h i s l i s t of putative transmitters have been the amino a c i d s , of which L-glutamate, a dicarboxylic a-amino acid with a f i v e carbon chain, has shown the most promise as a possible excitatory transmitter. The history of glutamic acids dates back to 1866 when Ritthausen i s o l a t e d an organic acid from wheat gluten hydrolysates. He observed that when wheat protein was hydrolyzed with sulphuric acid and the reagent removed with 2 calcium hydroxide, an amino acid s u f f i c i e n t l y strong to decompose calcium carbonate was obtained. He named thi s substance glutamic a c i d , and was l a t e r able to i s o l a t e and c r y s t a l l i z e i t . The chemical synthesis of glutamic acid was c a r r i e d out by Wolff i n 1 8 9 0 , by means of a complex series of re a c t i o n s , beginning with l e v u l i n i c a c i d . There then followed investigations into the b i o l o g i c a l properties of t h i s amino a c i d , beginning with the demonstration that glutamic acid was capable of reducing methylene blue i n frog muscle preparations (Thunberg, 1920) and that i t could be oxidised by minced peripheral nerve (Thunberg, 1 9 2 3 ) . Since then the metabolic role of glutamic a c i d , e s p e c i a l l y i n nervous tissue because of i t s high concentration there (Krebs, Eagleston and Hems, 1 9 4 9 ) , and i t s probable importance i n maintaining brain ammonium le v e l s (Weil-Malherbe, 1 9 5 0 ) , has been studied i n great d e t a i l . The p r i n c i p a l metabolic pathways and enzymes considered to be of importance i n the synthesis and breakdown of glutamic acid are shown i n figure 1 . The observation of Krebs and Eagleston i n 1949 that glutamate could reduce the loss of potassium ions from brain s l i c e s incubated i n saline was the f i r s t i n d i c a t i o n that glutamate might have an influence on i o n i c fluxes i n nervous t i s s u e . Later Hayashi ( 1954) noted that a p p l i c a t i o n of sodium glutamate to the motor cortex caused cl o n i c convulsions and suggested that glutamate accelerates the s h i f t of sodium into nerve c e l l s , causing e x c i t a t i o n . However, i t was not u n t i l Curtis et a l . , ( i 9 6 0 ) showed that iontophoretic SUCCINIC SEMIALDEHYDE • Krebs Cycle - <i-KETOGLUTARATE NH3 H 2 0 <L- KETOGLUTARIC ACID AMIDE NH, rf-amino acid «t-keto acid \ H 2 0 GABA Glutamic Acid Decarboxylase ~~7 CO, ^ NADH+H+ GLUTAMATE ^ NAD * ^ H , 0 NH3 , \ Glutominase / Glutamine Syntetase T T T T NH, ATP ADP H 2 0 • A-amino acid • A- keto acid GLUTAMINE Figure 1. The p r i n c i p a l enzymes and products involved i n glutamate metabolism. 4 application of glutamate and other acidic amino acids to the immediate vicinity of individual neurones that the question of a possible synaptic role for glutamate was seriously raised. Before glutamate can be considered as a mediator of synaptic transmission i t must satisfy a number of c r i t e r i a which have been established for a l l putative transmitters (McLennan, 1963; Werman, 1966) . With the increased knowledge of the general mechanisms of synaptic transmission, the manner in which a criterion may be satisfied has changed, new c r i t e r i a have been established while others have been subdivided. However, the six basic c r i t e r i a remain as follows: 1. The putative transmitter must be present in the nerve endings where i t is presumed to be active. 2. There must be a replacement mechanism for the transmitter which is released. 3. It must be demonstrated that the transmitter is released during synaptic a c t i v i t y . 4. An inactivation mechanism must be present to terminate the action of the transmitter. 5. The substance must mimic the action of the physiological transmitter in a l l i t s aspects. 6. The substance must react in a similar manner to the transmitter in the presence of pharmacological agents. The similar reaction of a pharmacological agent at a specific synapse and on the action of a given compound, is strong evidence to suggest that the transmitter at that synapse and the compound are identical. For this reason the last c r i t e r i o n i s gaining importance i n determining which trans-mitters may be active i n s p e c i f i c pathways i n the b r a i n . The role of glutamate i n the nervous system has been reviewed by a number of authors ( C u r t i s , 1965; 1969; Kravitz et a l . , 1970; McLennan, 1970a; Johnson, 1972). Despite the numerous objections which have been r a i s e d , none of these a r t i c l e s seriously refutes the p o s s i b i l i t y that glutamate may act as a mediator of synaptic transmission. The reason for the d i f f i c u l t y i n firmly e s t a b l i s h i n g whether glutamate s a t i s f i e s a l l of the c r i t e r i a at any one synaptic junction has been that experiments have not been confined to one anatomical area of the nervous system and have been ca r r i e d out on a wide variety of test animals. This discussion w i l l be l i m i t e d to the two general areas i n which most of the research has been done, i . e . the mammalian cen t r a l nervous system and the invertebrate neuromuscular j u n c t i o n . THE MAMMALIAN C.N.S. 1. DISTRIBUTION OF L-GLUTAMATE. The presence of L-glutamate i n a l l areas of the nervous system has been established (Krebs et a l . , 19^9; T a l l a n , 1962; B a t t i s t o n et a l . , 1969; Duggan and Johnston, 1970a,b). In vertebrates, the concentration of free glutamic acid i s higher i n nervous tissue than i n other tissues and i n nervous tissue i t i s found i n larger quantities than any other amino acid ( T a l l a n , 1962). The concentration of glutamate i s not 6 constant throughout the nervous system, but instead, has a c h a r a c t e r i s t i c d i s t r i b u t i o n (Johnson and Aprison, 1971). It i s at i t s lowest i n sympathetic ganglia, increasing through the dorsal root ganglia, s p i n a l cord grey matter to the thalamus and cerebral cortex to i t s highest concentration (10.9 - 12.4 umoles/gm.) i n the caudate nucleus, pyriform lobe and the s y l v i a n gyrus. As the glutamate content varies between higher and lower le v e l s of the C.N.S., so i t varies between areas at the same neural l e v e l . This l a t t e r v a r i a t i o n i s perhaps less l i k e l y to be linked to d i f f e r e n t metabolic needs of the tissues than the former. There i s a higher l e v e l of glutamate i n dorsal roots than i n the peripheral nerves or ventral roots (Duggan and Johnston, 1970a,b), and glutamate content i n lumbosacral grey matter i s greater than i n the white matter. Furthermore the sensory nuc l e i of the medulla have a s i g n i f i c a n t l y higher glutamate content than other medullary regions (Johnson and Aprison, 1970) . It appears that anatomical l o c a t i o n i s not the only factor which exerts an influence over the concentration of brain amino a c i d s , since glutamate varies i n the maturing mammal. Of a l l the tested amino a c i d s , glutamate exhibited the greatest increase i n the developing cortex of the k i t t e n during maturation (Berl and Purpura, 1963) • Since morpho-l o g i c a l studies show that the growth of c o r t i c a l tissue and the development of neuronal connections i n this animal follows the same time course as the Increases i n glutamate 7 concentration, (Noback and Purpura, 1961) i t seems l i k e l y that these two processes are r e l a t e d . Experiments i n young dogs (Dravid et a l . , 1965) showed that the changes i n glutamate content up to maturity d i f f e r e d i n the various regions of the brain and these variations may possibly be related to h i s t o l o g i c a l or functional d i f f e r e n c e s . Although studies on the anatomical l o c a l i z a t i o n of the t o t a l glutamate content of the brain are of i n t e r e s t , only a small portion of the t o t a l i s l i k e l y to be important i n synaptic transmission since the greatest part i s involved i n numerous metabolic processes . Presumably th i s p o r t i o n , l i k e other p o t e n t i a l transmitters (Whittaker, 1965) should be located i n the axonal terminals of neurones. Indirect calculations based on the amount of glutamate i n grey and white matter have led Tower ( i 9 6 0 ) to suggest that most of the C.N.S. glutamate i s associated with neurones rather than g l i a . However, sub c e l l u l a r f r a c t i o n a t i o n studies (Mangan and Whittaker, 1966) have f a i l e d to show a uniquely synapto-somal l o c a l i z a t i o n for any amino a c i d . A l l amino acids examined had the same su b c e l l u l a r d i s t r i b u t i o n as cytoplasmic markers, with only a small portion being retained i n the pa r t i c u l a t e f r a c t i o n . It i s possible that this small amount i s that portion of the t o t a l brain glutamate involved i n synaptic transmission, since most of the p a r t i c u l a t e glutamate has been found to be associated with the synaptosomal f r a c t i o n ( R y a l l , 1962). Further there appears to be s u f f i c i e n t glutamate i n extracts of t h i s f r a c t i o n to e l i c i t excitatory 8 effects when applied i o n t o p h o r e t i c a l l y to single c e l l s (Krnjevic and'Whittaker, 1965). In addition a synaptic role for glutamate i s indicated by the observation that exogenously applied H-glutamate tended to l o c a l i z e i n the synaptosomal f r a c t i o n to a greater extent than other amino acids. The p o s s i b i l i t y that synaptosomal glutamate serves as a precursor for GABA as i t does i n other brain fractions ( B e r l , Nicklas and Clarke, 1970) seems unlikely since the synaptosomal fractions which accumulate glutamate d i f f e r from the corresponding fractions which accumulate GABA (Wofsey et a l . , 197D • The suggestion has been made that functional compartmenta-ti o n of glutamic acid i n the nervous system (Berl and Purpura, 1966; B e r l , Clarke and N i c k l a s , 1970; Berl et a l . , 1962; O'Neal and Koeppe, 1966) may be of si g n i f i c a n c e i n the determination of which portion of the t o t a l brain glutamate i s important i n synaptic transmission. This compartmentation may be unique to the brain since i t i s not evident i n other tissues such as the l i v e r (Berl et a l . , 1962). There are two d i s t i n g u i s h -able compartments or glutamate pools (Berl and Clarke, 1969); a small p o o l , associated with the so-called "synthetic t r i -carboxylic acid cycle" which appears to be the primary precursor of glutamine and has a rapid turnover; and a larger p o o l , associated with the " t r i c a r b o x y l i c acid energy cycle" which has a large quantity of transaminases, and i s involved i n energy metabolism of amino aci d s . It i s i n t e r e s t i n g to note that i n the immature animal a smaller percentage of the 9 t o t a l brain glutamate i s involved i n energy production than i n the mature animal (van den Berg, 1970). Although there i s no evidence as yet to connect either of these pools with a transmitter function, the p r o b a b i l i t y of a dual system separating metabolic and transmitter pools of th i s amino acid has been suggested (Johnson, 1972) . R i z z o l i (1968) presented further evidence i n favour of glutamate having a transmitter function when he observed that glutamate concentrations i n s p e c i f i c areas of the s p i n a l cord decreased following s p i n a l t r a n s e c t i o n . This i s u n l i k e l y to be due to metabolic changes since other amino acids (except glycine) remained at normal l e v e l s . He suggested that the degeneration of a descending pathway which u t i l i z e s a t r a n s -mitter associated with glutamate caused th i s depletion. 2. REPLACEMENT OF THE TRANSMITTER. Glutamate released from nerve endings must be replaced. Unlike transmitters such as ACh, numerous sources of glutamate exist which could f u l f i l l t h i s f u n c t i o n , however the problem l i e s i n determining which of these sources i s drawn upon as the replacement mechanism. An obvious consideration i s that the "transmitter pool" of glutamate may have the a b i l i t y to draw upon metabolic sources of glutamate precursors. Isolated nerve endings have the a b i l i t y to metabolize glucose and form glutamate (Bradford and Thomas, 1969). Isolated synaptic v e s i c l e s from rat cortex have been shown to contain l a b e l l e d glutamate following 10 an Intraventricular i n j e c t i o n of JH-acetate (Farrow and O'Brien, 1971), thereby i n d i c a t i n g an a b i l i t y of the vesicle s to pick up glutamate from the metabolic pool or to synthesize i t d i r e c t l y from acetate. Dobkin (1970) found that there was a decrease i n the glutamine content of the cerebral cortex but not i n that of glutamate following afferent nerve stimu-l a t i o n and Johnson (1972) suggested that this might be due to a conversion of glutamine to glutamate i n order to maintain the transmitter pool of glutamate. The primary enzyme involved i n glutamate synthesis i s glutamate dehydrogenase and the question arises whether i t i s involved i n synaptic transmission. When the sp i n a l cord of the chick embryo was sectioned i n such a manner that i t developed i n the absence of e x t r i n s i c neuronal connections, the glutamate dehydrogenase l e v e l i n the spinal cord was found to be s i g n i f i c a n t l y lower than i n control chicks (Burt and Narayanan, 1972). I f the enzymatic d e f i c i t i s associated d i r e c t l y with the operative d e f i c i t , these data would suggest that a ce r t a i n amount of the o v e r - a l l glutamate dehydrogenase a c t i v i t y i s associated with the development of synaptic connections i n the spinal cord. Topical a p p l i c a t i o n of di e t h y l - a - f l u o r o g l u t a r a t e , an i n h i b i t o r of glutamate dehydro-genase i n the cerebral cortex, causes a decrease i n the amplitude of the surface negative component of the response to thalamic s t i m u l i (Cohen et a l . , 1972). However, depletion of numerous other metabolic intermediates and energy reserves also takes place. This l a t t e r observation makes i t d i f f i c u l t 11 to conclude whether glutamate depletion i s the sole cause of the reduction i n e l e c t r i c a l a c t i v i t y . It i s i n t e r e s t i n g to note that a number of drugs such as chlorpromazine and phenothiazine which have d e f i n i t e behavioural e f f e c t s are i n h i b i t o r s of glutamate dehydrogenase (Skemisa and Pahien, 1971)• Kine t i c correlations between the i n h i b i t o r y action on glutamate dehydrogenase and the antipsychotic a c t i v i t y of these drugs suggests that the two a c t i v i t i e s are r e l a t e d . A second possible source i s the axonal flow of glutamate from the c e l l body to the nerve endings. Such a flow has been observed for glutamate i n the peripheral nerves of frogs and rats (Kerkut et a l . , 1967). It seems l i k e l y that a s i m i l a r process for glutamate would exist i n the C.N.S. since axoplasmic transport of other amino acids has been noted there (Poulkes and Robinson, 1970). This process i s un l i k e l y to be of prime importance i n replacing transmitter glutamate, since the transport i s i n both directions (Kerkut et a l . , 1967). Further, the ease with which glutamate can be formed by synaptosomes from basic metabolites would make t h i s mechanism superfluous. It has been known for some time (Stern et a l . , 19^9) that the brain has the a b i l i t y to a c t i v e l y accumulate g l u t a -mate from i t s environment which i s a further possible source of t h i s amino a c i d . It appears that the blood-brain b a r r i e r i n the adult allows f a i r l y large amounts of glutamate to pass into the brain (Roberts et a l . , 1959). This i s presum-ably balanced by an e f f l u x of a s i m i l a r amount since i n contrast to l i v e r , muscle and kidney there i s no net increase i n the glutamate content of the brain following intravenous i n j e c t i o n of large amounts of glutamate (Schwerin et a l . , 1950). This i s not true i n immature animals where the blood-brain b a r r i e r i s not completely formed (Himwich et a l . , 1957). It i s estimated that the contribution of plasma glutamate to the t o t a l glutamate pool of the brain i s approximately 10% (Roberts et a l . , 1959)> the remaining 90% o r i g i n a t i n g primarily from glucose and other metabolites. Although glutamine can act as a precursor to glutamate, t h i s interconversion i s more l i k e l y to be of homeostatic s i g n i f i c a n c e than d i r e c t supply, since glutamine does not appear to cross the blood-brain b a r r i e r more rap i d l y than glutamate (O'Neal and Koeppe, 1966). F i n a l l y , synaptosomal glutamate may be replaced by active accumulation from the e x t r a c e l l u l a r spaces. With crude synaptosomal preparations from b r a i n , glutamate (and aspartate) can be shown to be taken up by dual mechanisms, one of high a f f i n i t y s p e c i f i c for the two and a second of lower a f f i n i t y which i s probably common to a l l amino acids (Logan and Snyder, 1972) . Labelled glutamate taken up by brain s l i c e s can be released as glutamate following e l e c t r i c a l stimulation (Arnfred and Hertz, 1971), suggesting that glutamate taken up by synaptosomes may be recycled i n the transmitter p o o l . 3. GLUTAMATE RELEASE DURING SYNAPTIC ACTIVITY. Although only a l i m i t e d amount of research has been done 13 there are indications that glutamate i s released under ce r t a i n conditions following nerve stimulation. Jasper et a l . , (1965) showed that glutamate was released from the p i a l surface of the cerebral cortex of cats at a much higher rate during arousal states than when the EEG showed the constant spindle patterns c h a r a c t e r i s t i c of sleep. GABA showed the converse pattern of rele a s e . These data indicate a possible modula-t i o n of arousal and sleep by the two amino aci d s . Further studies Indicated that a s i m i l a r release of glutamate could be obtained following stimulation of the medial r e t i c u l a r formation (Jasper and Koyama, 1968, 1969). The arousal following stimulation of t h i s area was accompanied by release of both glutamate and acetylcholine from the cortex. Follow-ing thalamic stimulation only acetylcholine could be c o l l e c t e d , implying that the glutamate release following r e t i c u l o c o r t i c a l stimulation has some degree of s p e c i f i c i t y . A s i m i l a r release of glutamate has been observed follow-ing e l e c t r i c a l stimulation of brain cortex s l i c e s (Arnfred and Hertz, 1971) which appears to be s p e c i f i c since the ef f l u x of leucine by s i m i l a r stimulation was not affected (Srinivasan et a l . , 1969). The observation that glutamate, lysin e and leucine effluxes i n the corpus striatum are a l l equally enhanced by e l e c t r i c a l stimulation (Katz et a l . , 1969) may r e f l e c t some regional differences i n function. It has been postulated that spreading depression which occurs i n the cortex and r e t i n a i s caused by a release of glutamate from neurones (van Harreveld, 1959). I f glutamate was released 14 on stimulation of the cortex or the r e t i n a , i t would cause a depolarization of the area with a further e f f l u x of glutamate and K+ which i n turn would depolarize adjacent neurones thus causing the spreading depression. A study of the e x c i t a b i l i t y of c o r t i c a l neurones during spreading depression ( P h i l l i s and Ochs, 197D has confirmed that glutamate could be responsible for the excitatory and early depressive phases. Although attempts to detect a release of glutamate i n the cortex during spreading depression have been largely unsuccessful (van Harreveld and Kooiman, 1965), s i m i l a r attempts i n the r e t i n a have had p o s i t i v e r e s u l t s (van Harreveld and F i f k o v a , 1970, 1972). Another l o c a t i o n where glutamate e f f l u x has been observed following e l e c t r i c a l stimulation i s the peripheral nerves i n frogs (Wheeler et a l . , 1966) . Once again the release of glutamate appears to be highly s p e c i f i c as no e f f l u x of aspartic a c i d , l e u c i n e , glycine or lys i n e occurred. It was considered to be energy dependent as i t could be blocked by sodium azide. From experiments on desheathed nerves (De Feudis, 1971) i t appears that only a portion of the t o t a l exchangeable glutamate can be released by e l e c t r i c a l s t imulation. The question whether a l l nerve f i b r e s show an e f f l u x of glutamate on stimulation i s r a i s e d , however, the experiments by van Harreveld and Kooiman (1965) and Jasper and Koyama (1969) i n the cortex, suggest that this might not be the case. 15 4. INACTIVATION OF GLUTAMATE. There is a higher concentration of the enzymes involved in glutamate catabolism in non-cholinergic nerves than in cholinergic nerves. A study of these enzymes suggests that they are located intracellularly and tend to localize in specific fractions (Salganicoff and De Robertis, 1965). Most of the aminotransferases and other general metabolic enzymes can be found within the mitochondrial fraction, whereas glutamine synthetase is primarily located in the vacuolar system of the mitochondria and has a very low con-centration in the synaptosomal fraction. On the other hand, glutamic acid decarboxylase is located primarily in the nerve endings and axoplasm of neurones, making i t a more likely candidate for the catabolism of excess glutamate in this fraction. However, when labelled glutamate is taken up by isolated synaptosomes i t is possible to extract labelled aspartate, glutamine, alanine and GABA (Bradford and Thomas, 1969) indicating that a l l of the enzymes necessary for glutamate catabolism are present to some extent in nerve endings. Because of i t s location i t is unlikely that glutamine synthetase has any importance in the inactivation process. The fact that methionine sulphoximine which inhibits gluta-mine synthetase causes a decrease, i f anything in the excit-a b i l i t y of a c e l l to glutamate (Curtis et a l . , 1972) supports this view. Glutamic acid decarboxylase on the other hand may be involved, since systemic administration of the inhibitor thiosemicarbazide i s followed by an increased neuronal e x c i t a b i l i t y i n s u b c o r t i c a l structures (Preston, 1955)» and the drug has also been shown to enhance the e f f e c t of ionto-p h o r e t i c a l l y applied glutamate to single c e l l s (Steiner and Ruf, 1966, 1967)• The l a t t e r phenomena may be explained i n the following manner. Since glutamic acid decarboxylase i s located i n t r a c e l l u l a r l y and re-uptake of released glutamate appears to be the primary i n a c t i v a t i o n process i t i s possible that a buildup of i n t r a c e l l u l a r glutamate caused by the i n a c t i v a t i o n of the decarboxylase, might cause an increase i n the concentration gradient, a decreased uptake, and there-fore an enhanced a c t i o n . The lack of e x t r a c e l l u l a r catabolic mechanisms increases the p r o b a b i l i t y that re-uptake i s the preliminary step i n i n a c t i v a t i o n . The a b i l i t y of i s o l a t e d synaptosomes to accumulate glutamate i s well documented. The demonstration i n the cerebral cortex of a population of synaptosomes which s e l e c -t i v e l y accumulates glutamic and aspartic acid has been taken to indicate that t h i s mechanism may not be uniformly d i s t r i -buted about a l l c e l l s (Wofsey et a l . , 1971)• The presence of a high plus a low a f f i n i t y uptake mechanism (Logan and Snyder, 1972) would make t h i s system that much more e f f i c i e n t . The uptake appears to take place primarily into the synap-tosomal f r a c t i o n , rather than into the general cytoplasmic pool where the major part of endogenous glutamate i s found (Kuhar and Snyder, 1970). The existence of several d i s t i n c t types of transport systems for the uptake of amino acids i n i s o l a t e d nerve endings has been postulated (Blasberg and Lajtha, 1966; Peterson et a l . , 1972). An amino acid may have an a f f i n i t y to more than one transport system (Blasberg and Lajtha, 1966), so that i n addition to a high a f f i n i t y s i t e s p e c i f i c for that amino acid i t may have a lower a f f i n i t y at another. This may explain the two-affinity system for glutamate and aspartate uptake described by Logan and Snyder (1972). Experiments by Peterson et a l . , (1972) on aspartate uptake, which, according to Blasberg and Lajtha (1966) u t i l i z e s the same mechanism as glutamate, suggest that the transport system i s Na+ dependent. It i s possible that the transport of these amino acids may derive energy d i r e c t l y from a transmembrane Na+ gradient and follow the downhill movement of Na+ into the c e l l and the fact that the swelling observed when brain s l i c e s are incubated with glutamate i s also Na+ dependent (Lund-Andersen and Hertz, 1970), tends to confirm t h i s p o s s i b i l i t y . Organic mercurials have been shown to i n h i b i t i n t e s t i n a l amino acid transport (Reiser and Christiansen, 1965) and the uptake of amino acids into brain s l i c e s ( C u r t i s , Duggan and Johnston, 1970). As expected, iontophoretic a p p l i c a t i o n of these mercurials enhances glutamate action on s p i n a l neurones, presumably by blocking the uptake and increasing the e x t r a c e l l u l a r concentration ( C u r t i s , Duggan and Johnston, 1970). Thiosemicarbazide, which enhances glutamate a c t i v i t y when applied systemically, has no e f f e c t when applied ionto-p h o r e t i c a l l y i n amounts unl i k e l y to a f f e c t i n t r a c e l l u l a r 1 8 enzymes. This would be expected i f uptake was the f i r s t step i n the i n a c t i v a t i o n process. 5. COMPARISON OF ACTION OF GLUTAMATE AND THE  PHYSIOLOGICAL TRANSMITTER. Iontophoretic a p p l i c a t i o n of glutamate causes a powerful depolarization and e x c i t a t i o n of C.N.S. neurones (Curtis et a l . , I960) which appears rapidly with onset of the applied current and i s terminated equally rapidly with i t s cessation. This i s the f i r s t c r i t e r i o n of an excitatory transmitter candidate and t h i s observation has led to most of the remain-ing experimentation and speculation. Glutamate i s able to excite neurones i n most regions of the C.N.S. Examples of areas i n which c e l l s have been tested include the neocortex (Krnjevic and P h i l l i s , 1963a); paleocortex (Legge et a l . , 1966); thalamus (Curtis and Davis, 1962); caudate nucleus (McLennan and York, 1966); ce r e b e l l a r cortex (Krnjevic and P h i l l i s , 1963a); amygdala (Straughan and Legge, 1965); corpus striatum (Fifkova and van Harreveld, 1970); hippocampus (Biscoe and Straughan, 1966); cuneate nucleus (Galindo et a l . , 1967); brain stem (Curtis and Koizumi, 1961); olfactory bulb (von Baumgarten et a l . , 1963) r e t i n a (Kishida and Naka, 1968); and spinal cord (Curtis et a l . , i960). Glutamate also exerts a depolarizing action upon nerve terminals (Curtis and R y a l l , 1966). One v a l i d argument against glutamate as a transmitter i s that i t appears to have a widespread non-specific e f f e c t on a l l neuronal membranes. However, not a l l c e l l s are equally s e n s i t i v e to glutamate or respond i n the same way, thus Krnjevic ( 1964) described some c o r t i c a l c e l l s which responded i n a steady, e a s i l y graded discharge, while others reacted i n short bursts i n an "all-or-none" manner. The very high threshold of Betz c e l l s to glutamate described by Krnjevic ( 1964) as well as the observation that other c e l l s such as motorneurones (Curtis et a l . , i 9 6 0 ) , and Mauthner c e l l s i n f i s h (Diamond, 1963) are only depolarized subliminally by glutamate and very seldom f i r e an action p o t e n t i a l , may be due to the large size of these neurones. However, the same argument cannot be used to explain the difference i n the s e n s i t i v i t y of neurones i n separate areas of the thalamus (McLennan et a l . , 1968) and between d i f f e r e n t neurones i n the s p i n a l cord (Duggan, 1 9 7 1 ) , to the excitatory amino a c i d s . A s i m i l a r difference i n neuronal e x c i t a b i l i t y to i o n t o p h o r e t i c a l l y applied glutamate has been described i n the olf a c t o r y bulb (von Baumgarten et a l . , 1 9 6 3 ) , however i t seems probable that the lack of e f f e c t of glutamate on c e r t a i n c e l l s i n this area i s due to the action of smaller adjacent i n h i b i t o r y neurones which block the e x c i t a t i o n of the larger c e l l s (McLennan, 1971)• The fact that glutamate has no apparent e f f e c t i n motor nerves or s k e l e t a l muscles (Takeuchi and Takeuchi, i 9 6 0 ) suggests that the glutamate "receptor" might not exist on a l l excitable membranes. The a b i l i t y of glutamate to excite neurones i s not s p e c i f i c for t h i s amino acid since any compound possessing 20 two a c i d i c groups, one amino group and an optimal separation of 2 to 3 carbon atoms between the a-amino and w-acidic groups, can excite the same neurones which are s e n s i t i v e to glutamate (Curtis and Watkins, I960, 1963). A number of naturally occurring amino acids such as aspartate and cysteate are as active as glutamate, while many non-natural compounds, esp e c i a l l y those which have undergone N-alkylation (eg. N-methylaspartic acid) are considerably more potent than t h e i r natural counterparts. Glutamate i s therefore neither the strongest nor the weakest of t h i s group of e x c i t a n t s . Once again, however, there appears to be a regional difference i n the s e n s i t i v i t y to these relate d compounds. Spinal neurones seem to be equally s e n s i t i v e to the L- and D-enantiomorphs of glutamate (Curtis and Watkins, i960), whereas c o r t i c a l neurones are much more sen s i t i v e to the L form (Krnjevic and P h i l l i s , 1961). S i m i l a r l y , s p i n a l Renshaw c e l l s appear to be r e l a t i v e l y more sen s i t i v e to L-aspartate than to L-glutamate when compared with a population of dorsal s p i n a l interneurones (Duggan, 1971). McLennan et a l . , (1968) found a difference i n s e n s i t i v i t y of c e l l s i n the v e n t r o l a t e r a l thalamus to DLH and glutamate. This led to the proposal that two types of receptor for the excitant amino acids may be present:-an unspecific receptor common to a l l c e l l s , and a s p e c i f i c receptor activated only by glutamate. The excitatory action of glutamate on the membrane "receptor" appears to be explicable i n terms of changes i n the i o n i c permeability. Glutamate has been shown to increase 21 + + the Na permeability f i v e f o l d r e l a t i v e to that of K i n s l i c e s of cerebral cortex (Bradford and Mcllwain, 1966) and these changes r e s u l t in-a net i n f l u x of Na+, which has been calculated to be s u f f i c i e n t to cause depolarization (Harvey and Mcllwain, 1968). These changes i n permeability are probably due to the e x c i t a t i o n properties of glutamate since s i m i l a r changes could be observed with other excitatory amino ac i d s , but not with analogues which showed no excitatory . a c t i o n . A concomitant loss of energy-rich phosphate from the brain s l i c e s and the appearance of inorganic phosphate may be due to the acceleration of a Na+, K+-dependent ATPase by the a d d i t i o n a l Na+ entering the t i s s u e . Although changes ++ ++ i n membrane Ca with an increased Ca i n f l u x do occur following a p p l i c a t i o n of glutamate to the incubation medium (Ramsey and Mcllwain, 1970), the chelating properties of glutamate do not appear to be responsible for i t s mechanism of action ( P u l l et a l . , 1970). A loss of K+ and a gain of Na+, s i m i l a r to that observed on incubating b r a i n s l i c e s i n solutions containing glutamate, has been shown following prolonged e l e c t r i c a l stimulation of s i m i l a r preparations (Keesey et a l . , 1965). Since then attempts have been made to determine whether these two means of stimulation have s i m i l a r mechanisms of a c t i o n . The e f f e c t of glutamate on C a+ + fluxes d i f f e r s from that of e l e c t r i c a l s timulation, i n that the former causes a net i n f l u x ++ ++ of Ca , whereas the l a t t e r causes an increased Ca turnover but no net flux (Ramsey and Mcllwain, 1970) . These results indicate a- difference i n mechanism, as do experiments with tetrodotoxin. Tetrodotoxin completely i n h i b i t s the e l e c t r i -c a l l y induced Na+ entry i n n e o c o r t i c a l tissue (Mcllwain et a l . , 1969), but only p a r t i a l l y i n h i b i t s the Na+ entry induced by glutamate ( P u l l et a l . , 1970). I f one d i f f e r e n t i a t e s between the mechanism of slow Na+ i n f l u x during subthreshold depolarization and the mechanism of rapid Na+ i n f l u x c h a r a c t e r i s t i c of the actual spike p o t e n t i a l i t appears that tetrodotoxin blocks the spike generation caused by either e l e c t r i c a l or glutamate stimul a t i o n , but has no e f f e c t s on the conductance or depolarization caused by glutamate (Bernardi et a l . , 1972; Curtis et a l . , 1972; P u l l and Zieglgansberger, 1972). A number of mechanisms have been proposed to explain the changes i n membrane permeability caused by glutamate. One theory i s that depolarization may be due to a r e d i s t r i -bution of the pre-existing glutamyl groups of peptides l i n k -ing a transmembrane pore and i n t h i s manner remove the obstruction to the flow of Na+ and K+. A second p o s s i b i l i t y i s that glutamate may penetrate the membrane pore to i t s inner mouth, di s p l a c i n g C a+ +, which acts as a counter ion to the a c i d i c groups and l i m i t s the access of Na+ ( P u l l et a l . , 1970). Thirdly depolarization may be due to a c a r r i e r -type of process which transports the amino acid and Na+ into the c e l l , ( P u i l and Zieglgansberger, 1972). None of these theories can be substantiated. However, the l a s t suggestion seems unlikely since blocking of glutamate uptake enhances 23 rather than blocks the excitatory action of glutamate (Curtis et a l . , 1970). It i s e s s e n t i a l that the mechanism of glutamate e x c i t a t i o n should be shown to be i d e n t i c a l to that of the s y n a p t i c a l l y evoked p o t e n t i a l changes before glutamate can be accepted as a transmitter. However, attempts to compare the equilibrium potentials of glutamate and the evoked EPSP have been largely unsuccessful. The only serious attempt ( C u r t i s , 1965) showed that the apparent equilibrium p o t e n t i a l for the action of glutamate was at a higher l e v e l of p o l a r i z a t i o n than that of the EPSP. Most reviewers of these r e s u l t s (McLennan, 1970; Curtis et a l . , 1972; Johnson, 1972) f e e l that t h i s does not seriously weaken the l i k e l i h o o d of glutamate being a t r a n s -m i t t e r , since i t i s probable that the applied glutamate and the p h y s i o l o g i c a l transmitter act at d i f f e r e n t regions of the c e l l . For technical reasons the glutamate i s applied near the recording electrode, which i s located i n the perikaryon of the nerve c e l l . Since most of the synapses involved i n the monosynaptic r e f l e x appear to be located i n the dendritic regions of the c e l l ( R a i l et a l . , 1967), any estimate of the equilibrium p o t e n t i a l of the EPSP based on an impalement of the c e l l body may be seriously i n e r r o r . The observation that low doses of glutamate can produce a slow membrane' depolarization without any obvious conductance change (Bernadi et a l . , 1972) also suggests a possible dendritic s i t e of action of glutamate. The fact that a l t e r a t i o n of i n t r a -c e l l u l a r chloride concentration i s without e f f e c t on the 24 depolarization caused by glutamate a p p l i c a t i o n or the e x c i t a -tory transmitter (Curtis et a l . , 1972), further suggests that these two processes are s i m i l a r . In 1957j Lucas and Newhouse reported an i n t e r e s t i n g f i n d i n g which may or may not have any bearing upon the mechanisms of glutamate e x c i t a t i o n . They showed s t r i k i n g degenerative changes i n the infant mouse r e t i n a following subcutaneous treatment with monosodium glutamate. These findings were confirmed, and i n addition discrete lesions of the arcuate nucleus have been found (Olney and Ho, 1970; Hanson, 1970; Olney, 1971). The lesions i n the r e t i n a and arcuate nucleus are s i m i l a r , being characterized by rapid swelling of neuronal dendrites and c e l l bodies, followed by nuclear pyknosis, and occurring within 6 to 8 hours a f t e r administration. The hypothalamic l e s i o n i s accompanied by obesity and neuro-endocrine disturbances (Olney, 1969; Redding et a l . , 1971)> while the r e t i n a l l e s i o n produces c h a r a c t e r i s t i c changes i n the electroretinogram (Buckser, 1969). The p o s s i b i l i t y that the neurotoxicity and the excitatory properties of glutamate may be related was raised by Olney et a l . , (1971) when they showed that i d e n t i c a l lesions could be caused by aspartate, cysteate and DLH, whereas other non-excitatory amino acids were i n e f f e c t i v e (Olney and Ho, 1970). Experiments by Perez and Olney (1972) showed that the arcuate nucleus but not the adjacent hypothalamic areas accumulate glutamate following subcutaneous i n j e c t i o n i n young mice. If glutamate i s a transmitter, then the high a f f i n i t y uptake 25 mechanism needed to ina c t i v a t e i t could perhaps accumulate s u f f i c i e n t glutamate to cause c e l l u l a r edema. The other p o s s i b i l i t y i s that glutamate induces d e p o l a r i z a t i o n , i n -creases the permeability of the neural membranes to Na+ and i n this manner causes swelling. This has been shown to occur i n b r a i n s l i c e s i n v i t r o (Harvey and Mcllwain, 1968). 6. THE ACTION OF PHARMACOLOGICAL AGENTS ON GLUTAMATE  AND SYNAPTICALLY EVOKED EXCITATIONS. Experiments i n which the action of pharmacological agents on glutamate responses and on synap t i c a l l y evoked responses have been compared are very few. One of the few observations which has been made i s that thiosemicarbazide and the organic mercurials can enhance the e f f e c t s of i o n t o p h o r e t i c a l l y applied glutamate (Steiner and Ruf, 1966, 1967; Curtis et a l . , 1970). However, these studies did not include an i n v e s t i g a -t i o n of the e f f e c t s of these substances on evoked p o t e n t i a l s . Apparently these drugs are of l i t t l e use i n the i n v e s t i g a t i o n of possible glutaminergic pathways since thiosemicarbazide i s without e f f e c t on glutamate responses when applied i o n t o -p h o r e t i c a l l y to s p i n a l neurones (Curtis et a l . , 1970), and there i s great technical d i f f i c u l t y i n using the organic mercurials. Another i n v e s t i g a t i o n by Boakes et a l . , (1970) claimed that LSD could block the response of i o n t o p h o r e t i c a l l y applied glutamate to brain stem neurones. However, thi s e f f e c t was only seen with c e l l s which were excited by 5-hydroxytryptamine. Again the action of LSD on evoked responses was not tested. C e l l s not excited by 5-HT, but s t i l l excited by glutamate, were reported to be unaffected by LSD. The lack of s p e c i f i -c i t y of LSD e f f e c t s , and lack of LSD ef f e c t on c e l l s not excited by 5-HT makes i t a poor to o l for i n v e s t i g a t i n g glutaminergic synapses. A more promising recent series of experiments i n the search for glutamate antagonists were those performed by Curtis et a l . , (1972) using methionine sulphoximine and methoxy-aporphine. They were able to p a r t i a l l y block the effe c t s of ionto p h o r e t i c a l l y applied DLH and glutamate with-out appreciably a f f e c t i n g the action of a c e t y l c h o l i n e . They were not, however, able to demonstrate blocking of synaptic f i r i n g of neurones and they concluded that these compounds were unsuitable for the i n v e s t i g a t i o n of possible glutaminergic pathways. A s i m i l a r depression of amino acid responses with-out appreciably a f f e c t i n g ACh e x c i t a t i o n has been observed by Davies and Watkins (1972) using the compound 1-Hydroxy-3-aminopyrrolidone-2. Once again, however, no discrimination between glutamate and the other amino acid excitations was observed. THE INVERTEBRATE NEURO-MUSCULAR JUNCTION As early as 1952, Lewis reported the presence of high concentrations of glutamate and aspartate i n leg nerves of several invertebrate species. With the observation of 27 Robbins (1959) that glutamate could enhance neurally evoked contractions i n crustacean muscle at r e l a t i v e l y low concen-t r a t i o n s , and could induce contractions i n the absence of nerve stimulation at higher concentrations, glutamate became a candidate for the transmitter substance at the neuro-muscular junction i n invertebrates. The same c r i t e r i a which must be met for transmitters i n the mammalian C.N.S. must be s a t i s f i e d i n determining the v a l i d i t y of glutamate as the transmitter at the invertebrate neuromuscular j u n c t i o n . The advantage here, however, i s the r e l a t i v e ease of access to t h i s synapse, and the fact that apart from aspartate no other r i v a l for the p o s i t i o n of transmitter has been found. 1. DISTRIBUTION OF L-GLUTAMATE. In 19^1, S i l b e r showed that leg nerves of lobsters had a very high concentration of free amino a c i d s . Later studies (Lewis, 1952; Marks et a l . , 1970) have shown that most of these compounds were present i n concentrations some 100-fold higher, on a wet weight basis i n invertebrate nerves than i n mammalian nerves. Although the concentration of glutamate was not found to be conspicuously higher than other amino ac i d s , i t was the only compound i s o l a t e d from crab and lobster nerve by Kravitz et a l . , (1963), which showed excitatory a c t i v i t y . Kravitz et a l . , (1970) f e l t that glutamate could account for about h a l f the excitatory a c t i v i t y present i n i s o l a t e d axons, however, when the axon extracts were treated 28 with the enzyme glutamic acid decarboxylase, e s s e n t i a l l y a l l of the excitatory a c t i v i t y was destroyed. 2. REPLACEMENT OF THE TRANSMITTER. This c r i t e r i o n i s perhaps the least d i f f i c u l t of a l l to f u l f i l l f or glutamate, since i t i s a normal component of general metabolism. Although no research has been done on peripheral nerves Bradford et a l . , (1969) c a r r i e d out experiments on ganglia of the s n a i l and locust and showed that glutamate, aspartate and a number of other amino acids could be formed from glucose by nervous tissue and that metabolic patterns were s i m i l a r to those found i n rat c o r t i c a l t i s s u e . The large quantity of free amino acids i n invertebrate nerves might be due to a breakdown of proteins and peptides i n the axon. Marks et a l . , (1970) found large quantities of p r o t e o l y t i c enzymes i n the peripheral nerves of crabs and lobsters and they suggest that these enzymes may contribute to a complex l i b e r a t i o n of amino a c i d s , from p r o t e i n , during axoplasmic flow. It seems l i k e l y that free glutamate can also flow down the axon since i n brain-nerve-muscle prep-arations of the s n a i l , glutamate, inj e c t e d into the b r a i n , moved along the nerve and could be found i n the muscle (Kerkut et a l . , 1 9 6 7 ) . Its movement was rapid and seemed to be s p e c i f i c , since s i m i l a r movement was not observed for xylose. Apart from anabolism i n the nerve and flow from the c e l l body a t h i r d possible mechanism for the accummulation of 29 glutamate i n the nerve terminal i s uptake from the surrounding environment. Glutamate i s present i n the hemolymph of the lobster i n a concentration of 7x10 M, which i s too low to cause depolarization of muscle f i b r e s (Kravitz et a l . , 1970). Presumably portions of t h i s glutamate, plus any transmitter glutamate which may be released i n the i n t e r s t i t i a l spaces, could be reabsorbed by an active process. A Na+-dependent system capable of moving glutamate against a concentration gradient into a nerve-muscle preparation, has been demon-strated by Iversen and Kravitz (1968). The uptake mechanism seems to be s p e c i f i c for glutamate since close s t r u c t u r a l analogues had l i t t l e or no e f f e c t on the process. 3. GLUTAMATE RELEASE DURING SYNAPTIC ACTIVITY. A number of researchers have demonstrated the release of glutamate into a f l u i d perfusing neuromuscular junctions on stimulation of excitatory nerves (Kerkut et a l . , 1965; Usherwood, Machili and Leaf, 1968; Kravitz et a l . , 1970). Usherwood, Machili and Leaf (1968) also recovered s i g n i f i c a n t amounts of alanine, glycine and aspartate during t h i s procedure. An increase i n the e x t r a c e l l u l a r C a+ + was found to increase glutamate and alanine release, but had no effect on the release of glycine or aspartate. Since C a+ + potentiates nerve-muscle transmission, presumably by increasing the amount of transmitter released from the motor nerve terminals (Usherwood, 1963), the above phenomena would be expected to occur i f glutamate (or alanine which has no excitatory a c t i v i t y ) was the transmitter. Also as expected there was a greater glutamate release with an increased rate of stimulation of excitatory nerve (Usherwood, Machili and Leaf, 1968), while no glutamate was released during stimulation of i n h i b i t o r y nerves (Kravitz et a l . , 1970). Evidence to suggest that at least some of the glutamate released had been transported down the axon, came from Kerkut et a l . , (1967). He reported that l a b e l l e d glutamate placed i n the medium bathing the i s o l a t e d brain of the s n a i l , was not only transported to the nerve-muscle j u n c t i o n , but was released therefrom, on stimulation of the b r a i n . Once again the amount of r a d i o -active glutamate recovered on nerve stimulation was found to be proportional to the number of s t i m u l i given to the b r a i n . It i s i n t e r e s t i n g to note that under s i m i l a r circumstances of excitatory nerve stimulation but i n the presence of the metabolic i n h i b i t o r 2,4-dinitrophenol, there i s a depletion of round synaptic v e s i c l e s from c r a y f i s h nerve terminals (Atwood et a l . , 1972). Since these synaptic v e s i c l e s are thought to contain the p h y s i o l o g i c a l transmitter (McLennan, 1970) i t i s not u n l i k e l y that the glutamate released on stimulation originated from these round v e s i c l e s . 4. INACTIVATION OF GLUTAMATE. There are a number of enzymes i n invertebrate nervous tissue capable of converting glutamate to i n a c t i v e compounds. It i s possible to recover GABA, other amino acids and c i t r i c a cid cycle metabolites following the administration of 31 l a b e l l e d glutamate to preparations of s n a i l and locust ganglia (Bradford et a l . , 1 9 6 9 ) . As f a r as i s known, however, these enzymes are a l l located i n t r a c e l l u l a r l y , require other substances and cofactors for t h e i r a c t i v i t y , and are probably not d i r e c t l y involved with the removal of e x t r a c e l l u l a r glutamate. It seems more l i k e l y that e x t r a c e l l u l a r l y released glutamate i s inactivated through a transport system which removes i t from the i n t e r s t i t i a l spaces. As mentioned e a r l i e r , a transport system which i s activated by an increase i n external Na+, and i n h i b i t e d by an increase i n external K+, has been described by Iversen and Kravitz (1968) i n the l o b s t e r . Baker and Potashner (1971) describe two possible uptake systems; one of which can be competitively i n h i b i t e d by other a c i d i c amino acids but not by neutral compounds and a second which i s Na+-insensitive and more s p e c i f i c for glutamate. These data suggest that the glutamate uptake mechanism serves some s p e c i a l function as do the experiments of Faeder and Salpeter (1970) who showed that the uptake of glutamate i n insect nerve-muscle preparations was enhanced by nerve stimulation whereas a s i m i l a r uptake of leucine showed no increase and that uptake was greater at neuro-muscular junctions than i n other regions of the t i s s u e . They are of the opinion that the sheath c e l l s which surround the nerve terminals are probably very important i n the i n a c t i -vation process and could possibly act as a b a r r i e r to blood glutamate and control the supply of glutamate to the axon. 32 5. COMPARISON OF ACTION OF GLUTAMATE AND THE  PHYSIOLOGICAL TRANSMITTER. Robbins (1959) and van Harreveld and Mendelson (1959), showed that d i r e c t a p p l i c a t i o n of low concentration of glutamate could cause contractions i n crustacean muscle, accompanied by a depolarization of the muscle. Iontophoretic a p p l i c a t i o n of glutamate to c r a y f i s h muscle by Takeuchi and Takeuchi (1964) demonstrated that the s e n s i t i v i t y of the membrane to glutamate was not uniform. L-glutamate se n s i t i v e spots on the muscle were very circumscribed; moving the t i p of the glutamate pipette by less than 10 u eliminated or greatly attenuated the response and the spots coincided with points of innervation characterized by e x t r a c e l l u l a r l y recorded excitatory j u n c t i o n a l p o t e n t i a l s . I n t r a c e l l u l a r i n j e c t i o n s of L-glutamate had no e f f e c t on the muscle p o t e n t i a l . In the continued presence of L-glutamate there was an i n i t i a l increase i n s e n s i t i v i t y to both glutamate and the transmitter followed by tachyphylaxis to applied g l u t a -mate and a decline i n the response to the excitatory synaptic transmitter (Robbins, 1959; Takeuchi and Takeuchi, 1964; Usherwood and M a c h i l i , 1968). This led Takeuchi and Takeuchi (1964) to conclude that the receptors that respond to L-glutamate were i d e n t i c a l to those which respond to the p h y s i o l o g i c a l transmitter. Usherwood, Cochrane and Rees (1968) reduced the l i k e l i h o o d that the action of g l u t a -mate was to cause the release of the p h y s i o l o g i c a l t r a n s -mitter from the nerve terminal, by carrying out s i m i l a r 33 experiments on locust muscles denervated for a number of days. In these preparations the discrete glutamate-sensitive spots remained i n the absence of motor innervation. That the receptor i s r e l a t i v e l y s p e c i f i c for L-glutamate can be seen from the i n a b i l i t y of other compounds to cause e x c i t a t i o n of invertebrate muscle. Of the four amino acids g l y c i n e , alanine, aspartate and glutamate released on nerve stimulation, only glutamate and aspartate showed any excitatory a c t i v i t y (Usherwood and M a c h i l i , 1968). Crustacean muscle i s very i n s e n s i t i v e to D-glutamate even when applied d i r e c t l y to L-glutamate se n s i t i v e spots (Takeuchi and Takeuchi, 1964). The s e n s i t i v i t y to glutamine i s 100 times less than to L-glutamate and glutamine i n turn i s 10 times more potent than L-aspartate. Further studies by McDonald and O'Brien (1972) showed that cysteic acid and DLH, as well as a number of l i k e compounds have s i m i l a r but generally a lesser e f f e c t than L-glutamate. In an attempt to explain the mechanism of glutamate e x c i t a t i o n Dudel and K u f f l e r ( I 9 6 0 ) proposed that a conduc-tance increase to Na+, coupled with a decreased conductance to K+ or C l ~ or both, could account for the excitatory j u n c t i o n a l potentials i n c r a y f i s h muscle. The replacement + + of Na with L i or T r i s r e v e r s i b l y abolishes the e.j.p. as well as the e f f e c t of io n t o p h o r e t i c a l l y applied glutamate suggesting that the p h y s i o l o g i c a l transmitter and glutamate use s i m i l a r mechanisms of d e p o l a r i z a t i o n . Ozeki et a l . , (1966) showed that both glutamate e x c i t a t i o n and the e.j.p. are immune to tetrodotoxin and saxitoxin and concluded that both these processes d i f f e r from the mechanism of spike electrogenesis. Barctnek and M i l l e r (1968) found i d e n t i c a l r e v e r s a l potentials for glutamate and the miniature excitatory p o t e n t i a l s , at the glutamate sensi t i v e areas on insect muscles. Using d i f f e r e n t techniques Taraskevich (1971) found a s i m i l a r equality i n reversal potentials at the neuromuscular junction of c r a y f i s h . These observations further suggest that at least at the l a t t e r two synapses, the permeability changes brought about by glutamate and the excitatory trans-mitter are s i m i l a r . 6. THE ACTION OF PHARMACOLOGICAL AGENTS ON GLUTAMATE AND SYNAPTICALLY EVOKED EXCITATIONS. To date th i s c r i t e r i o n has been impossible to f u l f i l l , due to the lack of pharmacological agents which s p e c i f i c a l l y a f f e c t glutamate e x c i t a t i o n of c e l l s . The only piece of evidence which may prove to be s p e c i f i c for glutamate, i s the observation that 5^-ribonucleotides potentiate both the excitatory j u n c t i o n a l potentials of c r a y f i s h muscles and the e f f e c t s of ion t o p h o r e t i c a l l y applied glutamate (Ozeki and Sato, 1970). These authors are of the opinion that the potentiating e f f e c t of the nucleotides i s due to a f a c i l i t a t i o n of the binding of the transmitter or glutamate with the receptor of the post-synaptic membrane. 35 THE PRESENT STUDY I t becomes o b v i o u s from t h e r e v i e w o f the l i t e r a t u r e t h a t the weakest c r i t e r i o n f o r d e t e r m i n i n g the i m p o r t a n c e o f g l u t a m a t e i n s y n a p t i c f u n c t i o n i s t h e comparison o f t h e a c t i o n o f p h a r m a c o l o g i c a l agents on e x c i t a t i o n s produced by g l u t a m a t e and the p h y s i o l o g i c a l t r a n s m i t t e r . P h a r m a c o l o g i c a l agents w h i c h s p e c i f i c a l l y i n t e r a c t w i t h t h e p o s t u l a t e d g l u t -a m i n e r g i c synapses would be o f extreme i m p o r t a n c e i n d e t e r m i n -i n g w h i c h pathways u t i l i z e g l u t a m a t e as a t r a n s m i t t e r . These agents would i n t e r a c t w i t h t h e p o s t s y n a p t i c " r e c e p t o r " and c o u l d be e x p e c t e d t o g i v e i n f o r m a t i o n r e g a r d i n g the form o f the a c t i v e c e n t r e s and the s p e c i f i c i t y o f the r e c e p t o r . The g l u t a m a t e r e c e p t o r a p p a r e n t l y r e a c t s w i t h t h e t h r e e i o n i z e d s i t e s on t h e g l u t a m a t e m o l e c u l e ( C u r t i s and W a t k i n s , I 9 6 0 ) . By u t i l i z i n g a n alogues w h i c h have a s i d e c h a i n a t t a c h e d t o one o r more o f t h e p r o b a b l e a c t i v e s i t e s o f t h e g l u t a m a t e m o l e c u l e , i t may be p o s s i b l e t o f i n d a n o n - a c t i v e compound wh i c h would combine c o m p e t i t i v e l y w i t h t h e r e c e p t o r and i n t h i s manner b l o c k or reduce g l u t a m a t e a c t i v i t y . A number o f t h e s e a n a l o g u e s have been used i n t h e p a s t as a n t i m e t a b o l i t e s and have been shown t o a n t a g o n i z e or i n h i b i t enzymes i n v o l v e d i n g l u t a m a t e m e t a b o l i s m . a - M e t h y l g l u t a m a t e and 3 - h y d r o x y g l u t a m i c a c i d f o r example, have been shown t o i n h i b i t c o m p e t i t i v e l y the u t i l i z a t i o n o f g l u t a m i c a c i d by L a c t o b a c i l l u s a r a b i n o s i s (Ayengar and R o b e r t s , 1 9 5 2 ) . a-Methyl glutamate Is a competitive i n h i b i t o r of glutamic acid decarboxylase i n acetone powders of rat brain (Roberts, 1952), and has the a b i l i t y to i n h i b i t p a r t i a l l y glutamine synthetase (Weil-Malherbe, 1969) i n guinea pig brain homo-genates. I n h i b i t i o n of growth and c e l l d i v i s i o n of Euglena  g r a c i l i s has been obtained with the d i e t h y l and dimethyl esters of glutamic acid (Owens and Blum, 1969), presumably due to a competitive i n h i b i t i o n of the glutamic acid acyl t-RNA synthetase (Owens and Blum, 1966). I f one assumes that the glutaminergic postsynaptic receptor i s a protein with active s i t e s not unlike those of the glutamate enzymes, i t seems l i k e l y that compounds which compete with glutamate for these enzymes might very well compete i n a s i m i l a r manner for the receptor. It i s i n t e r e s t i n g to note that c e r t a i n glutamate analogues, when inje c t e d into test animals, cause profound neurological and behavioural reactions (Roberts, 1952; Desi et a l . , 1967) . The observation by Marshall (1971) that a-methyl glutamate was able to block the e f f e c t s of iont o p h o r e t i c a l l y applied glutamate on thalamic c e l l s , gave further promise to the i n v e s t i g a t i o n of these compounds. Another compound which has i n h i b i t o r y e f f e c t s on g l u t -amate enzymes, and which has been investigated by Curtis et a l . , (1972) f o r possible glutamate blocking a c t i o n , i s methionine sulphoximine. MSO i s a powerful convulsant and has been shown to cause a non-competitive i n h i b i t i o n of the glutamine synthetase of brain ( S e l l i n g e r and Weiler, 1963; Lamar and S e l l i n g e r , 1965). The period of i n h i b i t i o n of glutamine synthetase however, does not correspond with the period of neurological symptoms (Lamar, 1968) and since none of the other enzymes tested has been shown to be i n h i b i t e d by MSO (De Robertis et a l . , 1967) i t i s possible that the neurological seizures are due to interference with a d i f f e r e n t mechanism. Two other compounds, cyclobenzaprine (N,N-Dimethyl-5H-dibenzocycloheptene-A5,y-propylamine) and hydrastinine-HCL (l-Hydroxy-6,7-methylenedioxy-2-methyl-l,2,3,4-tetra-hydroisoquinoline) were also tested for possible blocking a c t i o n . Cyclobenzaprine i s a t r i c y c l i c antidepressant relate d to imipramine (Grof and Vinar, 1965; K l e i n and Davis, 1969), while hydrastinine i s related to narcotine which has central depressant properties (Stanek and Manske, 1954). • One d i f f i c u l t y encountered before commencing the experiments was to determine which areas i n the C.N.S. one should use for the i n v e s t i g a t i o n . Glutamate i s present throughout the nervous system and appears to excite almost a l l neurones. I f there are non-specific as well as s p e c i f i c receptors for glutamate as suggested by McLennan et a l . , (1968), i t i s quite l i k e l y that s p e c i f i c and non-specific pharmacological agents could be found to in t e r a c t with them. The s p e c i f i c receptors are more l i k e l y to exist i n glutami-nergic pathways, and ce r t a i n areas of the C.N.S. appear to be l i k e l y candidates f o r such a pathway. The d i s t r i b u t i o n of glutamate i n anterior and posterior roots has led to the postulation that glutamate could be the excitatory transmitter at primary afferent terminals (Graham et a l . , 1967; Duggan and Johnston, 1970a,b). The thalamus seems to be the most l i k e l y candidate for the " s p e c i f i c " glutamate receptor since i t was i n thi s area that McLennan et a l . , (1968) determined the difference i n c e l l u l a r responses to i o n t o -p h o r e t i c a l l y applied glutamate. The release of glutamate from the cortex during EEG arousal (Jasper et a l . , 1965) and on r e t i c u l a r formation stimulation (Jasper and Koyama, 1968, 1969), suggested that glutamate may also be involved i n th i s area. It was therefore decided to concentrate the in v e s t i g a t i o n i n these regions and to include the neuro-muscular junction i n the c r a y f i s h . 39 CHAPTER II MATERIALS AND METHODS - CAT PREPARATIONS Cats of either sex, weighing for the most part between 2.5 and 3-5 kg. were used. The cats were fasted for a minimum of sixteen hours p r i o r to anaesthesia and were anaesthetised with sodium pentobarbitone (Nembutal, Abbott Lab.) 35 mg./kg. administered i n t r a p e r i t o n e a l l y . Anaes-thesia was maintained by periodic a d d i t i o n a l doses of 10 mg. applied intravenously. A tracheotomy was performed and a glass Y-tube inserted into the trachea and secured. End-t i d a l CO2 lev e l s were monitored by connecting one arm of the Y-tube to a Beckman Medical Gas Analyzer or a Godart Capnograph. When the end-tidal CO2 l e v e l s deviated s i g n i -f i c a n t l y from the normal value of k% the animals were paralyzed with gallamine t r i e t h i o d i d e ( F l a x e d i l , Poulenc Ltd.,), 20 mg. intravenously and 20 mg. intramuscularly and the second arm of the Y-tube was attached to a Palmer a r t i f i c i a l r e s p i r a t i o n pump. A s i m i l a r procedure, together with a b i l a t e r a l pneumothorax was ca r r i e d out when i t became necessary to reduce recording a r t i f a c t s caused by excessive breathing movements. A permanent femoral venous cannula was inserted for the administration of anaesthetic or other drugs. In cases of low blood pressure or following excessive s u r g i c a l blood HO l o s s , a bo t t l e containing 5% Dextrose i n saline was attached to the venous cannula and a continuous i n f u s i o n of 0.2 ml./min. i n i t i a t e d . A femoral a r t e r i a l cannula was attached to a pressure transducer and blood pressure monitored on one channel of a polygraph, while a second channel of the p o l y -graph was connected to the COg monitor. This enabled a permanent record of the blood pressure and r e s p i r a t i o n to be kept. In c e r t a i n thalamic experiments a cardiac catheter for the i n t r a - a r t e r i a l a p p l i c a t i o n of drugs to the thalamus was inserted v i a the b r a c h i a l a r t e r y . In t h i s manner, i t was possible to bring about higher concentrations of the drug i n the brain than could be obtained intravenously. The depth of the cannula was estimated and no confirmation as to the lo c a t i o n of the t i p was made. During the experiments on thalamic and cuneate neurones .the r e c t a l temperature of the animal was monitored by an EKEG temperature control u n i t . Body temperature was main-tained at 37-38°C by a 6 volt battery-supplied heating pad placed under the animal. Electrode placement. Electrode placement was s i m i l a r to that used by Marshall (1971). Recording from the thalamic, c o r t i c a l and cuneate neurones required s t a b i l i z i n g of the head of the animal i n a stereotaxic apparatus (Precision Cinematographique). This instrument uses the external auditory meatus and lower orbit for head f i x a t i o n and f o r reference p o i n t s . In the experi-ments on the cerebral cortex s t e e l stimulating electrodes were ca l i b r a t e d beforehand with reference to the i n t e r - a u r a l l i n e at the midline p o s i t i o n . The l o c a t i o n and co-ordinates of the stimulation and recording s i t e s were determined from the stereotaxic atlasses of Snider and Niemer (1961) and Reinoso-Suarez ( 1 9 6 1 ) . The positions of the recording electrodes were confirmed by monitoring the spontaneous e l e c t r i c a l a c t i v i t y and the responses to a c t i v a t i o n of the known neural pathways. a) Recording from thalamic neurones. Following placement of the a r t e r i a l , venous and tracheal cannulae the cat's head was mounted i n the stereotaxic frame. After a midline s a g i t t a l i n c i s i o n , the s k u l l was exposed by scraping back the skin and temporal musculature. Using a trephine and bone forceps, the bone overlying the l e f t side of the brain was removed, exposing the cortex from the i n t e r -aural l i n e anterior for about 20 mm., and from the midline l a t e r a l for about 15 mm. Bleeding from dura or brain was cont r o l l e d by applying small pledgets of Gelfoam (Upjohn) while bleeding from bone was stopped with bone wax. The dura mater covering the exposed brain was removed and the surface vessels coagulated with a Birtcher coagulator. The c o r t i c a l grey and white matter overlying the thalamus was removed by suction revealing the f l o o r of the l a t e r a l v e n t r i c l e . This procedure prevented breakage and tissue plugging of the electrode t i p s which often occurred i f the 42 electrode was inserted through the c o r t i c a l tissue overlying the thalamus. The r e s u l t i n g cavity was kept p a r t l y f i l l e d with warm Locke s o l u t i o n , except when the recording electrode p o s i t i o n was being changed to a new track. In order to reach the VPL nucleus of the thalamus the electrode t i p was placed 7 mm. l a t e r a l to the midline so that i t passed just medial to the choroid plexus approximately 8 mm. anterior to the i n t e r -aural l i n e . The electrode track was v e r t i c a l . The r i g h t rear hindlimb was clamped i n an extended p o s i t i o n . The skin i n c i s i o n was made along the groove formed between the semitendinosus and the semimembranosus muscles on the medial s i d e , and the biceps femoris on the l a t e r a l s i d e . These muscles were then separated and the f a s c i a l a t a and p o p l i t e a l fat pad removed a f t e r c a r e f u l l y tying o f f the blood supply to the adipose t i s s u e . This procedure uncovered the f l o o r of the p o p l i t e a l fossa and revealed the s c i a t i c nerve with i t s numerous branches. Bipolar s i l v e r electrodes were attached to the two main branches of the s c i a t i c nerve i . e . the anterior t i b i a l and peroneal nerves and these nerves were cut d i s t a l to the electrodes. A reser v o i r was formed by tying the skin surrounding the area to supporting rods and the entire fossa was f i l l e d with warm p a r a f f i n o i l , which covered the nerves and electrodes. b) Recording from cuneate neurones. Following placement of the cannulae and mounting of the cat's head i n the stereotaxic frame, a midline skin i n c i s i o n was made from the top of the s k u l l p o s t e r i o r l y to the area of the fourth c e r v i c a l vertebra. The suboccipital and upper c e r v i c a l musculature were separated from the occiput, post-e r i o r arch of atlas and the spinous process of a x i s . Where necessary the Birtcher coagulator-desiccator was used to cut the muscles and to prevent bleeding. The posterior aspect of the occiput, the posterior arch of atlas and the spinous and lamina of axis were removed using bone forceps. The dura mater was cut away and the caudal aspect of the cerebellum removed by suction. This allowed easy access to the cuneate nucleus and the posterior columns. The recording electrode was placed v i s u a l l y i n the cuneate tubercle and bipolar s i l v e r stimulating electrodes were placed so that they just touched the dorsal columns on the same si d e . A rese r v o i r was made around the area and f i l l e d with warm p a r a f f i n o i l . c) Recording from spinal cord neurones. Following placement of the cannula a midline skin i n c i s i o n was made from the f i r s t s acral to the second lumbar spinous processes. The erector spinae muscles were dissected from both sides of the spine and the spinous processes and laminae of the t h i r d to the seventh lumbar vertebrae were removed with bone forceps. The dura mater was then cut away making the spinal cord e a s i l y a c c e s s i b l e . Extreme care was taken not to touch the spinal cord and the area was kept moist with wads of cotton wool dipped i n warm Locke s o l u t i o n . The animal was suspended by means of clamps t i g h t l y secured to the L2 and SI spinous processes. The anterior root of the L7 spinal nerve was severed and suspended over b i p o l a r s i l v e r stimulating electrodes. The recording electrode track was either v e r t i c a l just medial t o , or at an angle of 1 8° just l a t e r a l to the entrance of the L7 dorsal root into the spinal cord. Again a r e s e r v o i r was made and the area covered with p a r a f f i n o i l . Stimulating electrodes were placed on branches of the s c i a t i c nerve i n a manner s i m i l a r to that used when recording from the thalamus. In t h i s case, however, electrodes were placed on the following nerves; t i b i a l , p l a n t a r i s , gastroc-nemius, sural and peroneal. d) Recording from the cerebral cortex. The cannulae were f i x e d i n p o s i t i o n , the head mounted i n the stereotaxic frame and the s k u l l exposed. In order to avoid touching the b r a i n , a hand d r i l l f i t t e d with a small round dental burr was used to cut a window the same size as that used i n the thalamic experiments, but extend-ing a n t e r i o r l y as f a r as the f r o n t a l pole. The dura was removed and a small tear made i n the p i a i n the area of the precruciate gyrus. This allowed easy penetration of the cortex by the recording electrode. A small s l o t was made i n the c o n t r a l a t e r a l side of the s k u l l i n an area opposite the thalamus. A p a r a l l e l p a i r of s t e e l electrodes with 2 mm. separation was then lowered to co-ordinates previously calculated as being at the l e v e l of the VPL nucleus of the i p s i l a t e r a l thalamus. These electrodes were constructed from 25 gauge needle tubing and insulated to within 0 . 5 - 1 mm. of the t i p with Insl-X. A trephine hole i n the s k u l l was made posterior to the window and a concentric b i p o l a r electrode lowered to the co-ordinates of the i p s i l a t e r a l pyramids. These electrodes were con-structed by threading f i n e teflon-coated s t a i n l e s s s t e e l wire through 23 gauge s t a i n l e s s s t e e l tubing u n t i l the wire extended ^ - 1 mm. beyond the t i p of the tubing. The other end of the wire was led through an opening i n the side of the tubing and the outside of the electrode was coated with Insl-X. The t e f l o n i n s u l a t i o n of the inner electrode and the Insl-X coating of the outer electrode were removed from the t i p . The p o s i t i o n of the thalamic electrodes was confirmed h i s t o l o g i c a l l y . The h i s t o l o g i c a l technique used was a modification of that described by Marshall (1971) and Green (1958). A p o s i t i v e current was passed (20 uA for 30 sec.) through the electrode while i t was i n p o s i t i o n , i n order to deposit i r o n from the uninsulated t i p . The electrodes were removed, the dor s o l a t e r a l surfaces of the brain exposed, and a 1 cm. section of brain was cut p a r a l l e l to the e l e c -trode tracks and fixed i n 10% formalin for 2 - 3 days. The blocks were then dehydrated, cleared and embedded i n p a r a f f i n wax. Sections were cut 25 u t h i c k , mounted on ge l a t i n i z e d s l i d e s , placed i n formalin vapour, dried and brought to water. The sections were immersed i n a 1% E^O^ solution to prevent reduction of the i r o n to the ferrous form by formaldehyde and then placed for 5 min. i n a 5 - 10% solution of potassium ferrocyanide. 10$ HCL (half the volume of the potassium ferrocyanide) was then added and the sections l e f t for 20 - 30 min. during which time the Prussian Blue reaction took place and a blue spot i n the area where the iro n was deposited became obvious. The s l i d e s were washed i n d i s t i l l e d water, counterstained i n 1% s a f r a n i n , dehydrated, cleared and mounted. Paired sections were stained with c r e s y l v i o l e t to f a c i l i t a t e i d e n t i f i c a t i o n of topographical structures i n the neighbourhood of the electrode t i p . Recording electrodes. The centre b a r r e l of a 7-barrelled glass electrode assembly was used f o r the e x t r a c e l l u l a r recording while the outer barrels were used f o r iontophoretic a p p l i c a t i o n of chemical compounds. The glass electrode blanks were supplied by Vancouver S c i e n t i f i c Glassblowing as an array of seven fused glass c a p i l l a r i e s , drawn to an o v e r a l l diameter of 2 . 5 - 3-5 mm. These blanks were heated and pulled i n a v e r t i c a l Canberra-type micro-electrode p u l l e r and the r e s u l t -ing t i p s were broken back, under microscopic observation, to a t o t a l diameter of 5 - 10 u. The electrodes were then f i l l e d by b o i l i n g i n d i s t i l l e d water for 20 min. After c o o l i n g , the d i s t i l l e d water was removed from the wide part of the shaft with a syringe and a 30 gauge needle, and replaced by an e l e c t r o l y t e . 4M NaCl was used for the recording b a r r e l while a l l the drugs used i n the outer barrels are l i s t e d i n table I. These drugs were dissolved i n d i s t i l l e d water or 0.15M NaCl, the pH was adjusted as i n d i -cated to bring i t well away from the i s o - e l e c t r i c point (Krnjevic and P h i l l i s , 1963) and the solutions were f i l t e r e d through a m i l l i p o r e f i l t e r . The solutions diffused to the t i p within 24 - 48 hours. A platinum wire extending into the NaCl of the centre b a r r e l was connected to the a m p l i f i e r probe while s i l v e r wires extending into the remaining barrels connected the solutions i n these barrels to the iontophoretic panel. Before using the glass electrodes, the DC resistance of each b a r r e l was measured. The usual ranges were 5 - 12 Mfl for the recording b a r r e l and 70 - 100 Mfi for drug containing b a r r e l s . In i n i t i a l experiments a large Narashige micromanipulator mounted on a bar attached to the stereotaxic frame was used for holding and moving the glass electrode; l a t e r an AB Trans-vertex micromanipulator with an e l e c t r o n i c a l l y c o n t r o l l e d stepping motor was used. To prevent the unwanted d i f f u s i o n of active i o n s , r e t a i n i n g currents were applied to each of the drug-contain-ing b a r r e l s . Currents used to r e t a i n or pass drugs ionto-p h o r e t i c a l l y were measured on a Cambridge galvanometer and were passed to the barrels of the electrode through 1000 Mft series r e s i s t o r s . Relatively small variations i n resistance at the electrode t i p s therefore did not appreciably a f f e c t TABLE I DRUG SOLUTIONS USED FOR IONTOPHORESIS Drug Cone. Solvent pH Active ion 1 Acetylcholine Bromide 1 .00M water 4. 0 cation 2 Na-L-Glutamate 0 .50M water 8. 0 anion 3 Na-DL-Homocysteate 0 . 20M water 8. 0 anion i] Na-D-Glutamate 0 .50M water 8. 0 anion 5 Na-DL-N-methylglutamate 0 .50M water 8. 0 anion 6 Na-p-aminobenzoyl-L-glutamate 0 .50M water 8. 0 anion 7 Na-p-nitrobenzoyl-L-glutamate 0 .50M water 8. 0 anion 8 Na-L-glutamate-a-methylester 0 .50M water 8. 0 anion 9 L-glutamic acid-dimethylester.HCl 0 . 50M water 4. 0 cation 10 L-glutamic acid-diethylester-HCl 0 .50M water 4. 0 cation 11 Na-4-fluoroglutamate 0 • 50M water 8. 0 anion 12 Na-L-aspartate 0 .50M water 8. 0 anion 13 Na-L-cysteate 0 .50M water 8. 0 anion 14 DL-methionine-DL-sulphoximine 0 .50M water 3. 5 cation 15 Hydrastinine HCl 0 .01M 0.15M 3. 0 cation NaCl 16 Cyclobenzaprine HCl 0 .50M water 4. 0 cation 17 p-chloro-mercuri-phenyl-sulphonic acid 0 .02M 0 .15M 8. 0 anion NaCl 18 Atropine sulphate 0 .0£M 0.15M 7. 0 cation NaCl 49 the current flow. Occasionally depressant e f f e c t s s i m i l a r to those observed by Curtis and Koizumi (1961) and Krnjevic and P h i l l i s (1963a) on a p p l i c a t i o n of cation-ejecting currents to the drug con-t a i n i n g barrels were seen. In order to separate the drug ef f e c t s from those due to the current flow the depressant e f f e c t s of the current was counteracted with a current of equal strength but opposite p o l a r i t y , passing from another b a r r e l containing an ina c t i v e ion (usually Na+) (Salmoiraghi and Steiner, 1963)- The current e f f e c t s were distinguished by t h e i r instantaneous onset and termination, followed by rebound excitation(Krnjevic and P h i l l i s , 1963a). E l e c t r i c a l equipment. The stimulating electrodes were connected through a se l e c t i o n panel and stimulus i s o l a t i o n units to a Grass S8 stimulator. The signal from the recording electrode was''fed through a preamplifier or impedance converter and displayed on a dual beam oscilloscope which was triggered from the stimulator. A second p a r a l l e l oscilloscope which was not triggered from the stimulator monitored the continuous e l e c t r i c a l a c t i v i t y . The amplified signal from the o s c i l l o -scope was fed to a loudspeaker and an EKEG ratemeter, the intergrated ratemeter output being recorded on an E s t e r l i n e -Angus R e c t i l i n e a r recorder. The t r i g g e r i n g l e v e l was determined by feeding the ratemeter output into the second channel of the oscilloscope and matching the e l e c t r i c a l potentials with the t r i g g e r i n g p o t e n t i a l s . In thi s way the a c t i v i t y of a single c e l l could be represented i n the rate-meter output s i g n a l . The output from the ratemeter was also fed into a PDP-8/L D i g i t a l computer programmed to display post stimulus time and latency histograms. The computer was triggered from the stimulator so that i t counted potentials following each stimulus and added those following successive s t i m u l i which had the same poststimulus latency. The number of sweeps to be summed as well as the bin width and t o t a l number of bins could be v a r i e d , depending on the type of response being recorded. The histogram was ei t h e r displayed on an oscilloscope and photographed or plotted on an X/Y recorder. Figure 2 represents a block diagram of the e l e c t r i c a l equipment and connections used. O s c i l l o s -o p e cr C h a n n e l 1 T r i g g e r Channel 2 R a t e -m e t e r O s c i l l o s -Q c o p e ) C h a n n e l 1 C h a n n e l 2 R e c o r d e r T e l e t y p e Digital Comp. P u l s e Input T r i g g e r I n s t r u c t i o n s O s c i l l o s c o p e O X - Y P l o t t e r Figure 2. Block diagram of the e l e c t r i c a l equipment used i n the cat experiments. 52 CHAPTER III RESULTS - CAT PREPARATIONS The cats under barbiturate anaesthesia breathed spontan-eously with an end t i d a l C02 of 3.5 - .4.5$. As the animal started recovering from the anaesthetic, the respiratory rate increased and the end-tidal C0 2 decreased. Maintenance doses of 10 mg. Na-pentobarbitone caused, i n many cases, a momentary h e s i t a t i o n i n the respiratory movements, followed by a slowing of r e s p i r a t i o n and an increase i n the end-tidal C02- With i n j e c t i o n of the anaesthesia, the blood pressure rose during the apnoeic period following which the blood pressure was depressed for a considerable time. Figure 3 shows an example of these changes. The cats on gallamine t r i e t h i o d i d e and respired a r t i f i c i a l l y , maintained an end-t i d a l C02 of H%. A l l experiments were ca r r i e d out a f t e r the end-tidal C0 2 and blood pressure lev e l s had s e t t l e d to constant values. a) Neuronal f i r i n g patterns and evoked responses. Neurones i n VPL showed spontaneous bursts of action potentials separated by periods of i n a c t i v i t y c h a r a c t e r i s t i c of t h i s area (Andersen and C u r t i s , 1964; Andersen et a l . , 1967; Marshall, 1971). Stimulation of branches of the s c i a t i c nerve evoked bursts of 1 - 4 spikes at latencies 53 Figure 3. The e f f e c t s of an intravenous i n j e c t i o n of 10 mg. Na-pentobarbitol i n 1 ml. saline on t i d a l COp and blood pressure. Injection at arrow. between 8 and 20 msec, to the f i r s t action p o t e n t i a l . Spinal interneurones showed no consistent pattern of spon-taneous a c t i v i t y and the evoked response obtained by stimu-l a t i n g branches of the s c i a t i c nerve consisted of bursts of 1 - 1 0 spikes with minimum latencies usually between 6 and 10 msec. The cuneate neurones showed the t y p i c a l spon-taneous a c t i v i t y described by Schwartz et a l . (1964) and Galindo et a l . (1968) , i . e . double spikes occuring at a frequency of 5 - 10 per min. The response of these c e l l s to dorsal column stimulation was 1 - 3 .spikes at latencies between 1.5 and 8 msec. Under pentobarbitone anaesthesia the c o r t i c a l neurones showed l i t t l e or no spontaneous a c t i v i t y and were often completely s i l e n t u n t i l excited by one of the amino a c i d s . Evoked potentials i n the cortex e l i c i t e d by stimulation of VPL consisted for the most part of a single spike with a latency between 1.5 and 4 msec, and on only two occasions were multiple evoked spikes seen. The frequency of f i r i n g and the number of action potentials i n the evoked responses i n a l l areas was dependent on the strength of the stimulus, which could be decreased to a point at which the c e l l f i r e d with one or two spikes follow-ing every second or t h i r d stimulus. The attempts at blocking the response were done at th i s threshold stimulus strength. C e l l s i n the four areas tested were re a d i l y excited by the a c i d i c amino a c i d s . The time-lag between commence-ment of the iontophoretic current and e x c i t a t i o n of the c e l l by the drug varied from c e l l to c e l l , and from one excitant to another. Often the ef f e c t was immediate (figure 9 ) s at other times there was a period of 1 min. duration or longer before the c e l l was excited (figure 12). This was probably due to the variable distance between the t i p of the electrode and the stimulated c e l l . The response always stopped immediately a f t e r cessation of the current. The ej e c t i n g current needed to produce e x c i t a t i o n with DLH was considerably lower than that needed for Glut. Glut was generally equal to, or a more e f f e c t i v e excitant than Asp, whereas Cys was the least active of the four amino acids tested. Dose-responses of a single c e l l f or the four amino acids were determined i n the cuneate nucleus, and indicated that the sulphur-containing amino acids were active at lower ej e c t i n g currents and were able to induce higher maximum f i r i n g rates than did the two dicarboxylic amino acids (figure 4). The order of potency of the amino acids to excite the c e l l s i n the spinal cord was not as regular as that observed i n other areas. DLH was inv a r i a b l y the greater excitant, however some c e l l s showed a f a r greater s e n s i t i v i t y to Glut than to Asp, while others showed the reverse. The effects of nine Glut analogues on the spontaneous a c t i v i t y and e x c i t a t i o n of thalamic neurones by Glut are l i s t e d i n table I I . Only two, aMG and GDEE showed any a b i l i t y to antagonize the e x c i t a t i o n of neurones induced by Glut, whereas GDME caused a d e f i n i t e potentiating e f f e c t . Na-4-fluoroglutamate had a greater excitatory action than 56 I80n 0 40 80 120 160 200 240 280 E j e c t i n g C u r r e n t ( n A ) Figure 4. The response of a neurone in the cuneate nucleus to increasing iontophoretic current strengths of DLH, Glut, Asp and Cys. TABLE II THE ACTION OF DERIVATIVES OF GLUTAMATE ON THALAMIC (VPL) NEURONES Analogue Upon spontaneous a c t i v i t y Upon L-glutamate-induced a c t i v i t y Na-D-glutamate Na-DL-a-methylglutamate Na-DL-N-methylglutamate Na-p-amlnobenzoyl-L-glutamate Na-p-nitrobenzoyl-L-glutamate Na-L-glutamate-Y-methylester L-glutamic acid-dimethylester-HCl L-glutamic acid-diethylester-HCl Na-4-fluoroglutamate E x c i t a t i o n None None None None None None None Ex c i t a t i o n None Blockade None None None None Enhancement Blockade (Not tested) 58 L-Glut and could therefore not be tested for possible blocking action. aMG, yKG, GDEE and GDME occassionally caused an increase i n the background f i r i n g rate of c e l l s , but only when very high e j e c t i o n currents were used for prolonged periods of time. b) Glutamic acid d i e t h y l 'ester. Table III gives the re s u l t s of the action of GDEE on the induced f i r i n g of neurones i n the s p i n a l cord, cuneate nucleus, VPL and cerebral cortex, tabulated i n each case as a r a t i o of the t o t a l number of c e l l s examined. It should be noted that t h i s table gives no i n d i c a t i o n of the degree of blockade produced. It i s evident that i n the spin a l cord, cuneate nucleus and VPL GDEE more frequently antagonized excitations induced by Glut than those induced by the other amino acids or ACh. Two examples of the re s u l t s from which thi s table was compiled are i l l u s t r a t e d i n figure 5 ( i n the thalamus using DLH, Glut and ACh) and figure 6 ( i n the cuneate using DLH, Glut and Cys). The time taken for f u l l recovery of the Glut response varied a f t e r cessation of the GDEE eje c t i o n current, but usually complete recovery occurred within 5 - 1 0 min., as can be seen from figure 5. When the action of DLH was reduced by GDEE i t was always less affected than the response produced by Glut, and f u l l recovery of the DLH-induced response was also more rapid. In the thalamus GDEE had no appreciable e f f e c t on neuronal e x c i t a -tions caused by ACh even when the response to DLH was TABLE III THE EFFECT OF GDEE ON SINGLE CELL EXCITATIONS. EACH SET OF FIGURES INDICATES THE RATIO OF THE NUMBER OF CELLS IN WHICH ANY REVERSIBLE ACTION ON INDUCED FIRING WAS OBSERVED, TO THE TOTAL NUMBER EXAMINED. VPL SPINAL CORD CUNEATE NUCLEUS CORTEX Deer. Incr. Deer. Incr. Deer. Incr. Deer. Incr. response response response response response response response response Glut m 1 12 0 16 0 11 1 I F 18" 19 19 17 17 DLH 20 5 5 1 8 3 11 0 TJO TJ0 I F 16* I F I F 19 19 Asp 5 2 5 1 10 l 9 1 11 11 I F I ? I F 18" 17 17 Cys 3 1 l T\ F F ACh 9 l 19 19 Evoked 15 0 11 0 7 l 6 0 I F IF 13 13 11 l i 9 9 60 501 Control ODEE 80 (4min.) 50 T o 1- I 1 r H I H o « Recovery (I min.) 5 o r i 1 i — Recovery (8 min.) 50T 0 30 sec F i g u r e 5. The e f f e c t s o f GDEE on DLH, Glut and ACh e x c i -t a t i o n o f a VPL neurone. Ratemeter r e c o r d s o f the e x c i t a t i o n s e l i c i t e d by homocysteate (DLH), 25 nA; glutamate ( G L ) , 80 nA; and a c e t y l c h o l i n e (ACh), 84 nA. A: b e f o r e , B: d u r i n g GDEE a p p l i -c a t i o n f o r 4 min. wit h a c u r r e n t o f 80 nA; C: 1 min. a f t e r c e s s a t i o n o f the GDEE c u r r e n t , D: 7 min. l a t e r . 61 C. R e c o v e r y I min. 30 sec. Figure 6. The e f f e c t of GDEE on the DLH, Glut and Cys ex c i t a t i o n of a neurone of the cuneate nucleus. Ratemeter records are of excitations caused by DLH (160 nA), Glut (200 nA) and Cys (240 nA). A: c o n t r o l , B: following a p p l i c a t i o n of GDEE (80 nA) f o r 1 min., C: 1 min. following cessa-t i o n of the GDEE a p p l i c a t i o n . 62 depressed. The greater effectiveness of GDEE as a Glut antagonist was confirmed when quantitative comparisons of the extent of blockade were examined. The sustained frequency of f i r i n g of neurones i n the presence and absence of GDEE was regulated by adjusting the ej e c t i o n currents for the amino acids so that the control f i r i n g frequencies were roughly s i m i l a r . In the thalamus 34 d i r e c t comparisons were con-ducted between Glut and DLH, 11 between Glut, DLH and Asp, and 4 with a l l four amino a c i d s . The re s u l t s are given i n table IV, and indicate that the e f f e c t of GDEE on e x c i t a t i o n e l i c i t e d by Glut was s i g n i f i c a n t l y greater (p<0.02 i n every case using the paired T-test) than f o r any of the other three amino aci d s . On the basis of these experiments there was no s i g n i f i c a n t difference between the action of GDEE on DLH, Asp or Cys, although Asp does appear to be s l i g h t l y more affected than the sulphonate compounds ( i n the l a s t 4 c e l l s i n table IV the difference between the ef f e c t s of GDEE on Glut and Asp was not s i g n i f i c a n t ) . The mean maximal sus-tained frequencies of f i r i n g were estimated by eye (figure 7 ) -Occassionally a f t e r prolonged a p p l i c a t i o n at high ionto-phoretic currents GDEE had a s l i g h t excitatory action and increased the background f i r i n g rate of the neurone under i n v e s t i g a t i o n . Although i t i s d i f f i c u l t to draw conclusions (Curtis et a l . , 1 9 7 D , the antagonism between Glut and i t s derivative appears to be competitive. When the responses to increasing TABLE IV BLOCKING ACTION OF GDEE ON THE FIRING OF THALAMIC NEURONES. THE FIGURES INDICATE THE AVERAGE RATIOS OF THE FIRING FREQUENCIES ATTAINED IN THE PRESENCE AND ABSENCE OF GDEE, ± S.E. Glut DLH Asp Cys n 0.41 ± 0.049 0.42 ± 0.102 0.31 ± 0.106 0.82 + 0.034 0.95 ± 0.045 1.00 ± 0.000 0.79 ± 0.094 0.59 ± 0.227 0.85 ± 0.069 34 11 4 64 A. Control DLH 6 0 GLUT 1 2 0 ASP 2 0 0 CYS 2 0 0 B. GDEE 160 4 min. C. Recovery 5 min. i i 3 0 sec . Figure 7- The e f f e c t of GDEE on the response of a VPL neurone to the excitant amino acid s . Ratemeter records of the e x c i t a t i o n of a thalamic neurone induced by the electrophoretic a p p l i c a t i o n during the periods indicated of DLH (60 nA), Glut (120 nA), Asp (200 nA) and Cys (200 nA). A: c o n t r o l , B: following the ap p l i c a t i o n of glutamate d i e t h y l e s t e r (GDEE) f o r 4 min. from another b a r r e l of the e l e c -trode assembly, C: 5 min. a f t e r cessation of the GDEE-ejecting current. The horiz o n t a l l i n e s indicate the manner i n which the mean maximal sustained f i r i n g frequencies were estimated by eye. i n t e n s i t i e s o f the G l u t - e j e c t i n g c u r r e n t , i n t h e absence and p r e s e n c e o f the a n t a g o n i s t s were compared, the dose-r e s p o n s e c u r v e was s h i f t e d p a r a l l e l t o the c o n t r o l , i n d i c a t -i n g t h a t a h i g h e r G l u t c u r r e n t was r e q u i r e d t o e l i c i t a s i m i l a r degree o f n e u r o n a l e x c i t a t i o n i n the p r e s e n c e o f t h e i n h i b i t o r . The maximum s u s t a i n e d f i r i n g r a t e was t h e same i n t he i n h i b i t e d as i n t h e c o n t r o l s i t u a t i o n ( f i g u r e 8 ) . I n c r e a s e d "doses" o f G l u t i n b o t h c i r c u m s t a n c e s l e d t o d i m i n i s h e d r a t e s o f f i r i n g t h r o u g h e x c e s s d e p o l a r i z a t i o n and i n a c t i v a t i o n o f t h e c e l l membrane. . I n t r a - a r t e r i a l a d m i n i s t r a t i o n o f GDEE t h r o u g h a c a r d i a c c a n n u l a i n s e r t e d v i a the b r a c h i a l a r t e r y caused a s i m i l a r d e c r e a s e i n G l u t r e s p o n s e i n t h e t h a l a m u s . A c o n t r o l i n j e c t i o n o f 0 . 5 m l . Locke s o l u t i o n had no e f f e c t on t h e e x c i t a t i o n o f a s i n g l e c e l l caused by t h e i o n t o p h o r e t i c a p p l i c a t i o n o f t h e amino a c i d s ( f i g u r e 9A,B). A s i n g l e i n j e c t i o n o f 30 mg. GDEE ( o . 2 5 m l . o f a 0 . 5 N s o l u t i o n ) f o l l o w e d by 0 . 2 5 m l . Locke s o l u t i o n t o c l e a r the c a n n u l a caused a s u b s t a n t i a l d e c r e a s e i n t h e G l u t e x c i t a t i o n , w i t h -out a p p r e c i a b l y a f f e c t i n g t h e e x c i t a t i o n s p r o d u c e d by DLH, Asp and Cys ( f i g u r e 9C,D). The e f f e c t c o u l d be seen w i t h -i n 2 - 3 min. a f t e r t h e i n j e c t i o n , and r e c o v e r y o f t h e resp o n s e was complete w i t h i n 8 - 1 2 min. L a r g e r amounts o f GDEE r e s u l t e d i n a r e d u c t i o n o f the e x c i t a t i o n s caused by a l l f o u r amino a c i d s , a l t h o u g h G l u t was always r e d u c e d t o the g r e a t e s t e x t e n t and t o o k l o n g e s t t o r e c o v e r . T a b l e I I I a l s o i n d i c a t e s t h a t i o n t o p h o r e t i c a p p l i c a t i o n 66 20 40 60 80 100 Glutamate (nA) Figure 8. Dose-response curves f o r Glut excitations on a VPL neurone. Ordinate: maximum sustained frequency of c e l l f i r i n g : abscissa: i n t e n s i t y of Glut-ejecting current. 0-—0 before; X X during the a p p l i -cation of GDEE (160 nA): t — - • recovery. 67 A. Control B. 0.5 ml L o c k e 5 min. 100 r 6 o » <o N «> « DLH Glut Asp Cys 120 160 160 160 100 r 0 «= co C 3 0 mg. G D E E 3 min. D. R e c o v e r y 8 min. IOO r IOO r i i 30 sec Figure 9. The e f f e c t of an i n t r a - a r t e r i a l i n j e c t i o n of GDEE on the responses of a VPL neurone to the excitant amino a c i d s . Ratemeter records of the excitations e l i c i t e d by DLH (120 nA), Glut (160 nA), Asp (160 nA), Cys (160 nA). A: c o n t r o l , B: 5 min. following the i n j e c t i o n of 0.5 ml. Locke s o l u t i o n , C: 3 min. following the i n j e c t i o n of 30 mg. GDEE, D: 8 min. following the i n j e c t i o n of GDEE. 68 o f GDEE caused a r e v e r s i b l e r e d u c t i o n i n the r e s p o n s e s evoked by e l e c t r i c a l s t i m u l a t i o n o f a f f e r e n t n e r v e s i n a s i g n i f i c a n t number o f c e l l s i n a l l a r e a s t e s t e d . I n t h o s e c e l l s w h i c h showed b l o c k a d e o f the evoked r e s p o n s e s , e x c i t a t i o n s by G l u t were a l s o b l o c k e d c o n c u r r e n t l y and r e c o v e r y o c c u r r e d p a r i p a s s u w i t h r e s t i t u t i o n o f t h e G l u t s e n s i t i v i t y ( f i g u r e s 10,11,12,13). T h i s b l o c k a d e o f s y n a p t i c a c t i v a t i o n was not ob s e r v e d i n e v e r y c e l l i n wh i c h the r e s p o n s e t o G l u t had been a b o l i s h e d ; and when an i n h i b i t i o n was o b s e r v e d , t h e r e d u c t i o n i n t h e evoked r e s p o n s e was v e r y o f t e n not as g r e a t as the r e d u c t i o n i n G l u t e x c i t a t i o n . I n no case however, was a r e d u c t i o n i n the evoked r e s p o n s e found w i t h o u t a con-commitant d e c r e a s e i n t h e e f f e c t o f a p p l i e d G l u t . I n t h e thalamus s i m i l a r r e d u c t i o n s i n t h e evoked r e s p o n s e c o u l d be o b t a i n e d w i t h the i n t r a - a r t e r i a l i n j e c t i o n o f GDEE ( f i g u r e 1 4 ) . There appeared t o be some v a r i a t i o n i n t h e e f f e c t s on GDEE i n t h e d i f f e r e n t a r e a s o f t h e C.N.S. The r e d u c t i o n i n the evoked r e s p o n s e s i n the cuneate were g e n e r a l l y l e s s t h a n t h o s e o b s e r v e d i n o t h e r a r e a s . I n t h e s p i n a l c o r d , t h o s e c e l l s w h i c h showed a g r e a t e r s e n s i t i v i t y t o Asp t h a n t o G l u t r e a c t e d t o t h e a p p l i c a t i o n o f GDEE w i t h a e q u a l d e p r e s s i o n o f b o t h Asp and G l u t r e s p o n s e s w h i l e t h e DLH r e s p o n s e remained unchanged. A l t h o u g h i t was p o s s i b l e t o reduce G l u t r e l a t i v e l y s p e c i f i c a l l y ( f i g u r e 1 3 ) , t h e r e s u l t s i n the c o r t e x were not as c o n s i s t e n t as t h o s e o b s e r v e d i n o t h e r a r e a s . GDEE had an e f f e c t on a s m a l l e r p e r c e n t a g e o f c e l l s i n the c o r t e x t h a n i n the o t h e r a r e a s , and i t was a l s o more d i f f i c u l t t o 6 9 A. Control 5 0 DLH 20 Glut 100 B. GDEE 160 8 min. 5 0 - 1 5 0 -i 5 0 t 5 min. 30 sec. 25 msec. Figure 10, The e f f e c t of GDEE on amino acid and synapti-c a l l y evoked excitations i n VPL. A neurone was excited by the electrophoretic a p p l i c a t i o n of DLH (20 nA) and Glut (100 nA) and by e l e c t r i c a l stimulation of the c o n t r a l a t e r a l t i b i a l nerve (0 .1 msec, 3V). The figure shows ratemeter. records of the amino acid induced f i r i n g s on the l e f t , and on the righ t computed histograms show-ing the summed evoked responses to 50 s t i m u l i delivered at a frequency of 1/4 s e c , which i n usually consisted of two spikes. A: during a p p l i c a t i o n of GDEE,(l60 nA) for : 5-6 min. a f t e r cessation of. the GDEE In t h i s figure and a l l subsequent thi s case before, B: 6 min., C;current. figures arrows. the stimulus a r t i f a c t s are indicated by 70 50 LL Asp 80 Glut 80 DLH 80 B. GDEE 80 I min. 4 min. 1 2 Q. CO Recovery 4 min. 3 min. 30 sec. 20 msec. Figure 11. The ef f e c t of GDEE on amino acid and synap-t i c a l l y evoked excitations of a spi n a l i n t e r -neurone. Ratemeter records are of excitations produced by DLH (80 nA), Glut ( 80 nA) and Asp (80 nA). The histograms show the summed evoked responses to 40 s t i m u l i (1/2 s e c , 0.1 msec., 7V) applied to the i p s i l a t e r a l s u r a l nerve. A: c o n t r o l , B: following a p p l i c a t i o n of GDEE (80 nA) f o r 1-4 min., C: 3-4 min. follow-ing cessation of the GDEE ejec t i n g current. 71 Figure 12. The ef f e c t of GDEE on the amino acid and synap-t i c a l l y induced excitations of a neurone i n the cuneate nucleus. Ratemeter records are of excitations produced by DLH (120 nA) and. Glut (160 nA). Histograms show the summed responses to 50 s t i m u l i (1/2 s e c , 0.1 msec, 3V) applied to the i p s i l a t e r a l dorsal column. A: c o n t r o l , B: following a p p l i c a t i o n of GDEE (160 nA) f o r 12-13 min., C: 3-4 min. following cessation of the GDEE a p p l i c a t i o n . 72 A. Control 5 0 DLH 50 Glut 140 Asp 120 B. GDEE 160 3 min. 2.5 min. 5 0 1 30 sec. 6 msec. Figure 13. The ef f e c t of GDEE on amino acid and. synapti-c a l l y evoked excitations of a neurone i n the cerebral cortex. Ratemeter records are of e x c i -tations produced by DLH (50 nA), Glut (140 nA) and Asp (120 nA). Histograms show the summed responses to 40 st i m u l i (1/2 s e c , 0,1 msec, 4.5V) applied to VPL. A: c o n t r o l , B: fol l o w -ing a p p l i c a t i o n of GDEE (160 nA) f o r 2-3 min., C: 13-16 min. following cessation of the GDEE ejecting current. 73 A . Cont ro l 100 r 2 5 D L H 160 Glut 120 B. 50mg . G D E E 3 min. 100 o « CO \ <0 o JC a. CO 2 min. 25 1 co C. R e c o v e r y 8 min. 7 min. 100 r 0 »• 25 3 0 s e c . 2 0 m s e c . Figure 14. The e f f e c t s of an I n t r a - a r t e r i a l i n j e c t i o n of GDEE on amino acid and s y n a p t i c a l l y evoked e x c i -tations i n VPL. A neurone was excited by the electrophoretic a p p l i c a t i o n of DLH (160 nA) and Glut (120 nA) and by e l e c t r i c a l stimulation of the c o n t r a l a t e r a l t i b i a l nerve (0.1 m s ec, 4V). Ratemeter records of the amino acid induced f i r i n g i s shown on the l e f t and on the right the computed histograms of the summed evoked r e -sponses to 50 s t i m u l i delivered at a frequency of 1/2 s e c The stimulus a r t i f a c t s are i n d i -cated by arrows. A: before, B: 2-3 min. follow-ing a 50 mg. i n j e c t i o n of GDEE, C: 7-8 min. following the i n j e c t i o n of GDEE. 74 demonstrate a s p e c i f i c action towards Glut. However, as can be seen from the two examples given (figures 13,24), Glut responses could be blocked to a greater extent than those produced by DLH and Asp. In the cortex the evoked potentials were occasionally blocked completely and there-a f t e r took a long time for recovery which was often incomplete. The blocking e f f e c t s of GDEE were dependent on the strength of the ejecting current. At high iontophoretic currents (above 160 nA.) GDEE very often caused a reduction i n the s e n s i t i v i t y of a c e l l to a l l the excitatory amino ac i d s , whereas at low currents there was no s i g n i f i c a n t blocking of the excitations produced by any of the amino ac i d s . As the strength of the GDEE ejec t i n g current required to block Glut responses s p e c i f i c a l l y varied from one c e l l to another, i t was often necessary to try a number of ejecting current strengths. In the example i n figure 15 there was no s i g n i f i c a n t blocking e f f e c t at 80 nA, while at 125 nA GDEE blocked the Glut and evoked responses without reducing the s e n s i t i v i t y of the c e l l to Asp. c) Glutamic acid dimethyl e s t e r . The action of GDME on amino acid induced and evoked e x c i t a t i o n of c e l l s i n VPL varied s l i g h t l y from that observed i n the spinal cord, cuneate nucleus and cerebral cortex (table V ) . The eff e c t s of GDME on VPL neurones depended to a large extent on the iontophoretic current used. At currents of 160 nA or l a r g e r , there was usually a blockade 75 Control 50 Glut 20 Asp 100 B. GDEE 80 co "s co cu a. C. GDEE 125 50 5 0 50 : n a CO CD J£ CL ( 0 50 Recovery 50 30 s e c . 20 msec. F i g u r e 15. The e f f e c t o f d i f f e r e n t c u r r e n t s t r e n g t h s o f GDEE on t h e amino a c i d and evoked r e s p o n s e s o f a s p i n a l i n t e r n e u r o n e . The r a t e m e t e r r e c o r d s a r e o f e x c i t a t i o n s p r o d u c e d by G l u t (20 nA) and ASP (100 n A ) . The h i s t o g r a m s show t h e summed evoked r e s p o n s e s t o 40 s t i m u l i (1/2 s e c , 0.1 m s e c , 12V) a p p l i e d t o the i p s i l a t e r a l s u r a l n e r v e . A: c o n t r o l , B: f o l l o w i n g a p p l i c a t i o n o f 80 nA GDEE, C: f o l l o w i n g a p p l i c a t i o n o f 125 nA GDEE, D: r e c o v e r y . TABLE V THE EFFECT OF GDME ON SINGLE CELL EXCITATIONS. EACH SET OF FIGURES INDICATES THE RATIO OF THE NUMBER OF CELLS IN WHICH ANY REVERSIBLE ACTION ON INDUCED FIRING WAS OBSERVED, TO THE TOTAL NUMBER EXAMINED. VPL SPINAL CORD CUNEATE NUCLEUS CORTEX Deer Incr. Deer. Incr. Deer. Incr. Deer. Incr. response response response response response response response response Glut 3 7 0 4 1 3 0 5 11 11 5 5 If 5 5 DLH 2 3 1 2 1 1 1 1 11 11 5 5 3 3 F F Asp 4 3 0 3 0 2 0 5 F F 5 5 T\ F F Cys l 1 0 0 3 3 1 1 Evoked 3 4 0 3 0 2 7 7 5 5 3 3 of the Glut response (figure 16). The blocking e f f e c t was not as s p e c i f i c as that obtained with GDEE since a decrease i n the responses to Asp and Cys were usually observed. On cessation of the iontophoretic current the responses of the c e l l to the excitatory amino acids i n v a r i a b l y increased to values higher than the co n t r o l s , before f i n a l l y returning to the i n i t i a l l e v e l s a f t e r 5 - 1 5 min. As can be seen i n figure 16 the potentiation of the Glut and Asp responses was greater than the DLH and Cys responses. At iontophoretic currents below 100 nA GDME caused an increase i n the Glut-induced responses of 7 out of 8 c e l l s tested i n the thalamus. In 4 of these c e l l s where only Glut and DLH were tested, Glut responses increased to an average of twice the control value, while DLH responses showed no s i g n i f i c a n t increase. The r e s u l t s i n the spi n a l cord, cuneate and cortex appeared to be more consistent, with GDME causing an increase i n the responses to Glut and Asp at a l l current strengths (figures 17,20,21). Glut-induced excitations i n a l l areas usually showed a greater potentiation than Asp responses, which were i n turn increased to a greater extent than the DLH responses (figure 17) and the onset of the ex c i t a t i o n induced by Glut and Asp was often more rapid i n the presence of GDME. The changes could usually be observed within 5 min. a f t e r commencement of the application of GDME, and the responses of the c e l l s i n -variably returned to control values within 10 min. af t e r A. Control B. GDME 160 I min. 100 100 DLH 160 Glut 120 Asp 200 Cys 200 Recovery I min Recovery 5 min. 100 30 sec Figure 16. The ef f e c t s of GDME on amino acid excitations of a VPL neurone. Ratemeter records of the excitations produced by DLH (160 nA), Glut (120 nA), Asp (200 nA) and Cys (200 nA) are shown. A: before, B: a f t e r a p p l i c a t i o n of GDME f o r 1 min., C: 1 min. following cessation of the GDME-ejecting current, D: 5 min. follow-ing cessation of the GDME-ejecting current. 79 D L H 4 0 Olut 8 0 A s p 8 0 B. G D M E 6 0 6 min. i » 3 0 s e c . Figure 17. The eff e c t of GDME on amino acid e x c i t a t i o n of a neurone i n the cerebral cortex. Ratemeter records are of excitations produced by DLH (MO nA), Glut (80 nA) and Asp (80 nA). A: c o n t r o l , B: following a p p l i c a t i o n of GDME (60 nA) for 6 min., C: 5 min, following cessation of the GDME ejecti n g current. cessation. The recovery of the amino acid responses i n the thalamus tended to be slower than in.the other areas and very often i t was not complete even a f t e r 15 min. The effects of GDEE and GDME could be demonstrated at the same neurone. Figure 18 i s an example of a c e l l i n the cuneate i n which GDEE blocked the ex c i t a t i o n of Glut and Asp and, a f t e r recovery, GDME caused a reversible enhancement of the responses of the c e l l to these amino ac i d s , while the response to DLH was largely unaffected by both es t e r s . The e f f e c t of GDME on the evoked responses i n the thalamus, cuneate and spinal cord was si m i l a r to the ef f e c t on Glut responses. In those c e l l s i n the thalamus i n which GDME reduced Glut e x c i t a t i o n , the evoked response showed a concomitant decrease which returned with the recovery of the Glut response, whereas those c e l l s i n which a potentiation of the amino acid responses was observed showed an increase i n the evoked response (figures 1 9 , 2 0 , 2 1 ) . This increase could only be observed i f the stimulus was set near threshold, such that the c e l l f i r e d with every second or t h i r d v o l l e y . Under these circumstances the c e l l would f i r e more consis-tently on application of GDME. The increase i n evoked response with GDME was never as great as the increase i n Glut response, however, recovery to control values followed the same time course as the Glut response. In the two cuneate c e l l s which showed an altered evoked response the number of spikes following each stimulus was increased rather than the pr o b a b i l i t y of f i r i n g at short l a t e n c i e s . The number 81 A. Control 0. GOME 200 2 min. _W DLH 60 GLUT 120 ASPI40 to 8. GDEE 200 2 min. E. Recovery 5 min. w in Vs/1 30 sse. Figure 18. A comparison of the eff e c t s of GDEE and GDME on, the amino acid induced excitations of a neurone of the cuneate nucleus. Ratemeter records are of the excitations produced by DLH (60 nA), Glut (120 nA) and Asp (1*10 nA) . A: c o n t r o l , B: following a p p l i c a t i o n of GDEE (200 nA) fo r 2 min., C: 5 min. following cessation of GDEE a p p l i c a -t i o n , D: following a p p l i c a t i o n of GDME (200 nA) for 2 min., E: 5 min. following cessation of GDME a p p l i c a t i o n . 82 A. Control 50 p DLH 40 Glut 120 Asp 120 B. GDME 50 6 min. s 0) a. W 25 i 5 min. C. so r Recovery 10 min. 16 min. 30 sec. 20 msec. Figure 19. The e f f e c t s of GDME on amino acid and synaptic e x c i t a t i o n of a VPL neurone. Stimuli were applied to the c o n t r a l a t e r a l t i b i a l nerve (2.8V, 0.1 msec.) and the histogram shows the summed evoked responses to 40 st i m u l i (1/2 s e c ) . The histogram and ratemeter records of excitations produced by DLH (40 nA), Glut (120 nA) and Asp (120 nA) are shown A: before, B: following a p p l i -cation of GDME (50 nA) for 5-6 min., C: 10-16 min. following cessation of the GDME ejecting current. 83 A. Control 5 0 2 5 1 DLH 100 Glut 120 Asp 120 B. GDME 80 3 min. 5 min. Recovery 5 min. 50 4 min. 2 5 ! A i . L . 30 sec. 20 msec. F i g u r e 20. The e f f e c t o f GDME on th e amino a c i d and synap-t i c a l l y evoked e x c i t a t i o n s o f s p i n a l i n t e r n e u -r o n e s . The r a t e m e t e r r e c o r d s a r e o f e x c i t a t i o n s p r o d u c e d by DLH (100 n A ) , G l u t (120 nA) and Asp (120 n A ) . The h i s t o g r a m s show t h e summed evoked r e s p o n s e s t o 40 s t i m u l i (1/2 s e c , 0,1 msec., 10V) a p p l i e d t o t h e i p s i l a t e r a l s u r a l n e r v e . A: c o n t r o l , B: f o l l o w i n g a p p l i c a t i o n o f GDME (80 nA) f o r 3 - 5 min., C: 4-5 min. f o l l o w i n g c e s s a t i o n o f t h e GDME e j e c t i n g c u r r e n t . 84 Figure 21. The eff e c t of GDME on the amino acid and synap-t i c a l l y evoked excitations of a neurone of the cuneate nucleus. Ratemeter records are of excitations produced by DLH ( 8 0 nA), Glut ( 1 6 0 NA) and Asp (140 nA). Histograms show the summed responses to 50 s t i m u l i (1/2 s e c , 0.1 msec, 2-5V) applied to the i p s i l a t e r a l dorsal columns. A: c o n t r o l , B: following a p p l i c a t i o n of GDME (200 nA) fo r 4-8 min., C: 1-5 min. following cessation of the GDME ejecting current. of tests conducted i n thi s area was small, since GDME i t s e l f usually caused a marked increase i n the "spontaneous" f i r i n g of the cuneate neurones. The action of GDME on evoked responses i n the cortex was not examined. d) Alpha-Methylglutamate. Prom table VI i t i s evident that aMG blocked Glut e x c i t a t i o n i n approximately 40$ of the c e l l s tested i n the thalamus, but had no eff e c t on the responses of cuneate c e l l s to any of the exc i t a n t s . I n h i b i t i o n occurred only when high iontophoretic currents (above 200 nA) were used and i n most of the c e l l s where Glut excitations were.blocked there was a s i m i l a r i n h i b i t i o n of the responses to the other excitatory amino acids and ACh; however, the reduction of the DLH response was always less than that of the Glut e x c i t a -t i o n s . In 4 out of 10 c e l l s tested i n the thalamus, aMG caused a reduction i n the evoked response; however i n a l L of these cases there was a concommitant decrease i n the amino acid and ACh responses. No reductions i n the evoked responses were observed i n the cuneate. aMG often caused an increase i n the background rate of f i r i n g of neurones, for the most part following prolonged a p p l i c a t i o n at high current strengths, and indeed i n certain c e l l s , e s p e c i a l l y i n the cuneate, aMG appeared to be as ef f e c t i v e an excitatory agent as Glut i t s e l f . In a number of c e l l s aMG caused a potentiation of the responses to the excitatory amino acids (figure 22). The ef f e c t was TABLE VI THE EFFECTS OF aMG ON SINGLE CELL EXCITA-TIONS. EACH SET OF FIGURES INDICATES THE RATIO OF THE NUMBER OF CELLS IN WHICH ANY REVERSIBLE ACTION ON INDUCED FIRING WAS OB-SERVED, TO THE- TOTAL NUMBER EXAMINED. VPL CUNEATE NUCLEUS Deer. Incr. Deer. Incr. response response response response Glut 9 4 0 0 2T 24" 5 5 DLH 3 0 1 • 1 13 13 5 5 Asp 1 1 2 0 ? 5 5 Evoked 4 1 0 0 10 10 2 2 87 Figure 22. The potentiating effects of aMG on amino acid e x c i t a t i o n of a VPL neurone. Ratemeter records are of the excitations e l i c i t e d by DLH (80 nA), Glut (160 nA) and Asp (120 nA). A: before, B: following a p p l i c a t i o n of aMG (240 nA) f o r 7 min., C:' 8 min. a f t e r cessation of the aMG a p p l i c a t i o n . 88 non-specific, a f f e c t i n g DLH, Glut and Asp i n the same manner. e) DL-Methionine-DL-Sulphoximine. As can be seen from table VII, MSO had no marked or consistent e f f e c t s on the responses of VPL c e l l s to Glut, Asp, DLH, ACh or on the evoked response. Iontophoretic currents of 40 - 160 nA were used to apply the drug for periods up to 15 min. An i n t r a - a r t e r i a l i n j e c t i o n of 18 mg. MSO did not change the responses of c e l l s i n the thalamus. On two occasions when currents of 160 nA were passed, MSO increased the background "spontaneous" rate of f i r i n g , which returned to control le v e l s a f t e r the MSO-eje c t i n g current was terminated. In these c e l l s also there was no obvious e f f e c t on the amino acid or ACh responses. The e f f e c t s of MSO on sp i n a l interneurones and c o r t i c a l neurones were more dramatic than i t s e f f e c t s on thalamic neurones. At iontophoretic current strengths of 60 - 80 nA, MSO caused a decrease i n the responses of the c e l l to a l l amino acids tested, although the DLH excitations were often reduced to a les s e r extent than were those e l i c i t e d by Glut and Asp. Figure 23 i l l u s t r a t e s the responses of the only c e l l i n the spinal cord i n which MSO had a reversible e f f e c t on evoked potentials and i n which Glut responses were decreased to the greatest extent. In the other spinal interneurones i n which a reduction was observed, the Glut responses were reduced by no more than 30$, whereas i n the cortex the amino acid responses were often reduced to 70% TABLE VII THE EFFECT OF MSO ON SINGLE CELL EXCITATIONS. EACH SET OF FIGURES INDICATES THE RATIO OF THE NUMBER OF CELLS IN WHICH ANY REVERSIBLE ACTION ON INDUCED FIRING WAS OBSERVED, TO THE TOTAL NUMBER EXAMINED. VPL SPINAL CORD CORTEX Deer. Incr. Deer. Incr. Deer. Incr. response response response response response response Glut 1 1 3 0 3 0 9 9 7 7 T\ DLH 2 2 3 0 2 0 9~ 9 5 5 ? Asp 1 2 2 0 2 0 5 5 5 5 3 3 ACh 0 1 2 2 Evoked 0 0 1 0 0 0 5 5 1 1 90 A. Control 5 0 DLH 80 Glut 80 B. MSO 80 I min. 5 0 to 0 C. Recovery 2 min. 5 0 r 30 sec. 6 min. 5 min. 1 2 20 msec. Figure 23. The ef f e c t of MSO on the amino acid and synap-t i c a l l y evoked e x c i t a t i o n of a sp i n a l i n t e r -neurone. Ratemeter records are of excitations produced by DLH (80 nA) and Glut (80 nA). The histograms show the summed evoked responses to 40 s t i m u l i (1/2 s e c , 0.1 msec, 7V) applied to the i p s i l a t e r a l s u r a l nerve. A: c o n t r o l , B: following a p p l i c a t i o n of MSO (80 nA) for 1-6 min., C: 2-5 min. following cessation of the MSO ejecting current. 91 of control values. Figure 24 compares the eff e c t s of equal ejecting currents of GDEE and MSO on the same c o r t i c a l neurone. Although both GDEE and MSO reduced a l l the amino acid responses, GDEE reduced Glut to a far greater extent than DLH and Asp responses, whereas MSO affected a l l three responses to the same extent. f) Other miscellaneous drugs. Cyclobenzaprine was tested i n the thalamus and spi n a l cord where i t caused an equal reduction i n the s e n s i t i v i t y of neurones to a p p l i c a t i o n of Glut, Asp and DLH i n an appreciable percentage of the c e l l s tested (table V I I I ) . Reduction of amino acid s e n s i t i v i t y was usually accompanied by a reduction i n the evoked responses. At iontophoretic current strengths of 80 - 100 nA the i n h i b i t i o n of the evoked responses was seldom more than 40$ of the control lev e l s and was always less than the reduction i n the amino acid responses (figure 25). An i n t r a - a r t e r i a l i n j e c t i o n of 40 mg. of CB caused an immediate cessation of r e s p i r a t i o n i n the two cats tested. This was not investigated f u r t h e r . Only three c e l l s i n the thalamus were tested with hydrastinine. In one of these there was a s i g n i f i c a n t increase i n the spontaneous f i r i n g rate with 80 nA of the drug, however, none of the c e l l s showed any change i n t h e i r responses to Glut or ACh, nor was there any change i n the evoked response. pCMS caused an enhancement of the s e n s i t i v i t y to the 92 A. Control 5 0 DLH 60 Glut 60 Asp 80 B. ODEE 60 I min. 5 0 D. MSO 60 I min. f 1 \ 1 i i 5 0 .1 IF Li1 C. Recovery I min. E. Recovery I min. A 5 0 1 30 sec. Figure 2k. A comparison of the ef f e c t s of GDEE and MSO on the amino acid excitations of a neurone In the cerebral cortex. Ratemeter records are of excitations produced by DLH (60 nA), Glut (60 nA) and Asp (80 nA). A: con t r o l , B: following a p p l i -cation of GDEE (60 nA) f o r 1 min., C: 1 min. following cessation of GDEE ap p l i c a t i o n , D: follow ing a p p l i c a t i o n of MSO (60 nA) for 1 min., E: 1 min. following cessation of MSO ap p l i c a t i o n . TABLE VIII THE EFFECT OF pCMS AND CYCLOBENZAPRINE ON SINGLE CELL EXCI-TATIONS. EACH SET OF FIGURES INDICATES THE RATIO OF THE NUMBER OF CELLS IN WHICH ANY REVERSIBLE ACTION ON INDUCED FIRING WAS OBSERVED, TO THE TOTAL NUMBER EXAMINED. CYCLOBENZAPRINE - pCMS VPL SPINAL CORD VPL Deer. Incr. Deer. Incr. Deer. Incr. response response response response response response Glut 3 0 4 10 0 10 1 6 9 9 DLH 3 "4" 0 TT 4 10 0 10 2 3 9 9 Asp 3 0 3 F 0 F 1 5 9 9 Evoked l 0 3 0 2 3 2 2 11 11 9 9 94 A. Control 5 0 DLH 40 Glut 80 Asp 120 B. CB 80 I min. 5 0 u « CO CO 5 0 4 min. 5 0 El C. Recovery 16 min. 5 0 15 min. 5 0 30 sec. 25 msec. Figure 25. The ef f e c t of cyclobenzaprine on the amino acid and s y n a p t i c a l l y evoked excitations of a VPL neurone. Ratemeter records are of excitations produced by DLH (40 nA), Glut (80 nA) and Asp (80 nA). The histograms show the summed evoked responses to 50 s t i m u l i (1/2 s e c , 0.1 msec, 3V) applied to the c o n t r a l a t e r a l t i b i a l nerve. A: before, B: following a p p l i c a t i o n of CB (80 nA) for 1-4 min., C: 15-16 min. following cessation of the CB ejec t i n g current. excitatory amino acids of a high percentage of the VPL neurones tested (table V I I I , figure 26). The excitatory a c t i v i t i e s of Glut and Asp were enhanced to the same extent, and the e f f e c t of pCMS on these excitations was much greater than on that produced by DLH. The potentiation of amino acid responses was often accompanied by an increase i n the response evoked by a perithreshold stimulus (figure 27). In some of these experiments Glut and Asp caused a burst of spikes at high frequencies followed by depolarization of the c e l l during pCMS eje c t i o n and for some time a f t e r i t s cessation. This e f f e c t would l a s t up to 20 min. following a 10 min. a p p l i c a t i o n of pCMS, a f t e r which time normal amino acid responses would return. During t h i s period the evoked response was usually depressed. No increase i n background "spontaneous" f i r i n g of the c e l l s comparable to that described i n the spinal cord by Curtis et a l . (1970) was observed when the pCMS eje c t i n g current was terminated. r 96 A. C o n t r o l 50 0 «• D L H 3 0 Glut 8 0 A s p 8 0 7 min. B. P C M S 5 0 Ni 50 r o « (0 N CO <D J£ Q. C O 0 «• R e c o v e r y II min. 50 3 0 s e c . Figure 26. The e f f e c t of pCMS on amino acid excitations of a VPL c e l l . Ratemeter records of the exc i t a t i o n e l i c i t e d by DLH (30 nA), Glut (80 nA) and Asp (80- nA). A: before, B: following the iontophoretic a p p l i c a t i o n of pCMS (50 nA) for 7 min., C: 11 min. a f t e r cessation of the pCMS eject i n g current. 97 A. Cont ro l 5 0 25 D L H 4 0 A s p 160 B. P C M S 8 0 10 min. off 9 min. 5 0 • o CD <0 \ CO CO J£ Q . CO 0 25 CO CL CO 8 min. C R e c o v e r y 5 0 off 19 min. 2 5 2 2 min. 3 0 s e c . 2 5 m s e c . Figure 27- The ef f e c t s of pCMS on amino acid and synap-t i c a l l y evoked excitations of a VPL neurone.. Ratemeter records of excitations produced by DLH (40 nA) and Asp (160 nA) are shown. The h i s t o -grams show the summed evoked responses to 50 st i m u l i (1/2 s e c , 0.1 msec, 8V) to the contra-l a t e r a l t i b i a l nerve. A: before, B: 8-9 min. af t e r the cessation of the pCMS ejec t i n g current which was applied for 10 min., C: 19-22 min. following cessation of the pCMS ejecting current, 98 CHAPTER IV MATERIALS AND METHODS - RAT CORTEX PREPARATIONS Adult male rats of the Long-Evans s t r a i n were used. The animals were decapitated and the entire brain removed and placed i n cold Locke s o l u t i o n . Four s l i c e s weighing between 25 and 75 mg. were cut from the dorsal and l a t e r a l aspect of the cerebral cortex with the aid of an instrument s i m i l a r to that used by Stadie and Riggs (1944). a) Release of glutamate from brain s l i c e s . A technique s i m i l a r to that described by Arnfred and Hertz (1971) was used to study the eff e c t s of glutamate and i t s analogues on Glut release from brain cortex s l i c e s . After weighing, the c o r t i c a l s l i c e s were placed i n transfer holders and incubated at 37°C i n test tubes containing 4 ml. of the basic medium. The holders (figure 28) were s i m i l a r to those used by Arnfred et a l . (1970). They consisted of a glass tube with a nylon mesh bottom, and a rubber stopper through which passed a short glass tube as well as a long glass tube which reached almost to the nylon mesh. Gas containing- 95% 0^ and 5% C0 2 was continously passed through the longer tube. When the transfer apparatus was placed i n the test tube containing the basic medium, bubbling produced by the a i r kept the brain s l i c e s mobile i n the A diagram of the transfer f e r brain s l i c e s from one to another. holder used to trans-test tube of medium s o l u t i o n . The holder made i t possible to transfer the brain s l i c e from one soluti o n to another quickly and e a s i l y . The basic medium contained the following s a l t s , KC1 5.0mM NaCl 120.OmM NaHC03 15.OmM MgCl 2 l.OmM CaCl 2 1.5mM Glucose l.OmM In the Glut'-containing medium lOmM NaCl was replaced by lOmM Glut. In the media containing GDEE and aMG, 30mM NaCl was replaced by 30mM of the respective Glut analogue. In the combined media, 40mM NaCl was replaced by lOmM Glut and 30mM of the analogue. Glut and the analogues were 14 neutralized with NaOH before addition to the media. (U- C)-L-glutamic aci d (206 mCi/mM) was obtained from New England 14 Nuclear Corporation. The C-Glut medium was mixed by 14 adding 100 u l . C-Glut to 20 ml. of basic medium. The brain s l i c e s were incubated i n the basic medium containing the lZ*C-Glut for a period of 40 min. After incubation the s l i c e s were washed for approximately 5 sec. i n unlabelled medium and then incubated for 10 min. periods i n each of a series of twelve test tubes with non-radioactive medium. The f i r s t four test tubes contained basic medium, the second four contained the Glut and/or i t s analogues and the t h i r d set of four test tubes contained basic medium. The s l i c e s were then drained, weighed and dissolved i n 5 ml. of lM-NaOH i n a b o i l i n g water bath and the soluti o n 14 was di l u t e d with d i s t i l l e d water to 25 ml. The C released from the s l i c e s into each test tube was determined by counting 1.0 ml. of the medium from each tube i n a l i q u i d s c i n t i l l a t i o n counter. Each sample was dissolved i n 12 ml. of a mixture of 588 ml. ethanol and 412 ml. toluene i n which was dissolved 4 gm. of Omnifluor (NEN cat #NEF-906A) and counted for a minimum of 10 min. ( i . e . an e f f i c i e n c y of 1.0 - 5-0$ compared to an i n t e r n a l standard). One ml. of the sol u t i o n containing the dissolved brain s l i c e was a c i d i f i e d with 0.1 ml. formic acid before d i s s o l v i n g i t i n the counting s o l u t i o n . The amount of r a d i o a c t i v i t y found i n the series of test tubes a f t e r the washout, was added to that which was l e f t i n the t i s s u e , giving the t o t a l amount i n the s l i c e at 14 the s t a r t of the washout. No correction was made for C 14 l o s t from the system as C0 2. The amount l e f t at the beginning of each 10 min. period of the washout was c a l c u -lated by subtracting the amount of r a d i o a c t i v i t y already washed out i n previous test tubes from the t o t a l . The percentage loss of tracer into each test tube (rate c o e f f i c i e n t , Shanes and Bian c h i , 1959) was calculated as (c.p.m. i n the test tube) x 100  (c.p.m. i n the s l i c e when transferred to th i s test tube.) b) Glutamate uptake into synaptosomes. A technique s i m i l a r to that used by' Logan and Snyder (1971) to measure Glut uptake Into crude synaptosomal preparations of the cerebral cortex of rats was employed. The c o r t i c a l s l i c e s were homogenized i n 20 volumes of cold 0.32M sucrose solution using a Potter-Elvehjem glass homogenizer f i t t e d with a "Teflon" p e s t l e . The homogenate was centrifuged f o r 10 min. at 1000 g. and 0.2 ml. aliquots of the supernatant were added to 3-8 ml. of Krebs-Henseleit so l u t i o n containing: NaCl 1 1 8 . OmM KC1 4 . 7mM CaCl 2 2. 5mM NaHC03 25. OmM KH2P0lj 1. 2mM MgSO^ 1. 2mM Glucose 1 1 . ImM A series of 8 test tubes containing various concen-14 trations of (U- C)-L-Glut between 0.1 and ImM were used i n each experiment. The mixtures were eq u i l i b r a t e d with 95% C0 2 and 5% 0 2 and incubated for 4 min. at 3 7°C. The solutions were then immediately centrifuged at 27,000 g. for 15 min. at 0°C and the supernatants were poured o f f . • The r a d i o a c t i v i t y i n 50 y l . aliquots was assayed i n a l i q u i d s c i n t i l l a t i o n spectrometer. The p e l l e t s were washed i n 1 ml. of cold 0.15M NaCl and recentrifuged at 27,000 g. for 10 min.; the r a d i o a c t i v i t y i n the p e l l e t was extracted into 0 .5 ml. of IM hyamine hydroxide i n methanol, dissolved i n 12 ml. toluene and Omnifluor and counted. I d e n t i c a l measurements were made following incubation i n media contain-ing 0.25 or ImM GDME or ImM GDEE i n addition to the l a b e l l e d Glut. Prom these measurements the amount of Glut taken up by the synaptosomal preparations could be calculated and plotted against concentration i n the incubating medium. 104 CHAPTER V RESULTS - RAT CORTEX PREPARATIONS a) Release of glutamate from brain s l i c e s . 14 14 The loss of C from brain s l i c e s loaded with C-Glut and washed out i n sol u t i o n was increased by incubating the s l i c e s i n a medium containing 55mM K+ or lOmM Glut. The increased e f f l u x of l a b e l when Glut was added to the i n c u -bation medium was greatest i n the f i r s t test tube and then gradually decreased u n t i l i t was just above control values by the fourth test tube. With further incubation of the brain s l i c e i n Glut-free s o l u t i o n the e f f l u x of l a b e l usually continued at the same rate as the c o n t r o l . Thes.-j r e s u l t s are s i m i l a r to those observed by Arnfred and Hertz (1971). Four brain s l i c e s were incubated simultaneously, each with a d i f f e r e n t combination of Glut and/or i t s analogues added to the s o l u t i o n . Figure 29 i l l u s t r a t e s one example of a series i n which i ) Glut and analogue-free s o l u t i o n , i i ) lOmM Glut, i i i ) 30mM GDEE, iv) 30mM GDEE and lOmM Glut was added to the solutio n of the 5th through the 8th test tubes. The GDEE caused an increased e f f l u x of Glut s i m i l a r but smaller than that brought about by Glut. The effects of Glut and GDEE were not a d d i t i v e , the net increase i n 105 24-2 0 -c | 6 -pj • o o 8 -4 -O \\ // // W \ \ • \ * J' \ \ s\ \ x—x Control Glut GDEE ? T Glut + GDEE "i—> —r~ 20 —I— 40 —I— 60 80 100 -1 120 Time from beginning of Washout (min.) Figure 29. Plots of the effects of Glut and GDEE on the rate c o e f f i c i e n t of Glut e f f l u x from s l i c e s of cerebral cortex. A l l points marked with open markers and with crosses were obtained with washout i n the basic medium. The points marked with s o l i d markers were obtained when a) lOmM Glut, b) 30mM GDEE, c) lOmM Glut and 30mM GDEE was added to the basic medium between 40 and 80 min. from the beginning of the washout. 106 e f f l u x with both of these compounds i n the medium was equal to that caused by Glut alone. A s i m i l a r series of experiments using aMG instead of GDEE was carried out (figure 3 0 ) . The addition of 30mM aMG to the incubation solu t i o n had very l i t t l e , i f any, eff e c t on the Glut e f f l u x and the addition of lOmM Glut and 30mM aMG did not change the ef f l u x induced by Glut alone. b) Glutamate uptake into synaptosomes. The rate of accumulation of Glut by the homogenates incubated i n solutions.containing d i f f e r e n t concentrations of Glut, i n the absence or presence of GDEE or GDME, were drawn on double r e c i p r o c a l plots as functions of the Glut concentration (figure 31)• The points are mean values obtained from 8 control experiments, 5 experiments with * ImM GDME, 4 with 0.25mM GDME, and 4 with ImM GDEE. The regression l i n e s were calculated by a least squares f i t . Regression l i n e s were also calculated for the Glut data with appropriate weighting to allow f o r the increasing variance of the - values as - increased, by the method of constrained v s regression. Since these procedures resulted i n n e g l i g i b l e changes, simple regression equations were used for a l l experimental data. It was impossible to describe the experimental points by a single l i n e for the control and GDEE situations ( c f . Logan and Snyder, 1971, 1972); a l l of the points however f e l l close to a single l i n e when GDME was present i n the incubation s o l u t i o n . 107 2 8 - i 2 4 -*- 2 0 " C 0> O 16-<D O O <u o or 12-X-• -A-Control Glut <^MG Glut + «CMG 4 -— i — 4 0 - 1 — 60 —I— 80 —1 120 20 100 T i m e f r o m b e g i n n i n g o f W a s h o u t ( m i n ) Figure 30. Plots of the eff e c t s of Glut and aMG on the rate c o e f f i c i e n t of Glut e f f l u x from s l i c e s of cerebral cortex. A l l points marked with open markers and with crosses were obtained with washout i n the basic medium. The points marked with s o l i d markers were obtained when a) lOmM Glut, b) 30mM aMG, c) lOmM Glut and 30mM aMG was added to the basic medium between 40 and 80 min. from the beginning of the washout. 108 > 10 Control 8 6 4 2 i i / i . i i i 10 - 0.25 mM ODME 8 -6 4 ^ — " 2 SET • i l . l l l !mM 00EE I mM 00ME 40 -20 0 +20 40 60 80 100 -20 0 +20 40 60 80 100 l / S Figure 31. Double r e c i p r o c a l plots of the v e l o c i t y (v: umoles/gm. fresh tissue/min.) of the up-take of L-Glut by homogenates of rat cerebral cortex, as a function of the concentration (s: mM) of Glut i n the incubation medium. In the three graphs where GDME and GDEE were added, the dashed li n e s indicate the control values. The regression l i n e s have been calculated as described i n the t e x t . From these p l o t s , values of the Mlchaelis a f f i n i t y constant (K ) for a high and a low a f f i n i t y uptake were calculated by determining the r e c i p r o c a l of the i n t e r s e c -t i o n of the i a x i s . The p r o b a b i l i t y that the values d i f f e r e d from the control was computed from the res i d u a l variances about the regression l i n e s (table IX). The data indicate that neither ester exerted a s i g n i f i c a n t e f f e c t upon the low a f f i n i t y uptake of Glut, but that 0.25mM GDME p a r t i a l l y and ImM GDME completely i n h i b i t e d the high a f f i n i t y system. GDEE also appeared to reduce the high a f f i n i t y uptake of Glut, but the difference from the control s i t u a t i o n was not s i g n i f i c a n t . TABLE IX KINETICS OP GLUTAMATE UPTAKE INTO HOMOGENATES OF RAT BRAIN. K values (uM) High Low a f f i n i t y P a f f i n i t y P Glutamate 22 98 +0.25mM GDME 64 M3.025 64 >0.1 +1.00mM GDME 98 0.005 98 >0 .1 +1.00mM GDEE 31 ^0.100 109 >0.1 CHAPTER VI MATERIALS AND METHODS - CRAYFISH PREPARATIONS Experiments were ca r r i e d out on adult Columbia River c r a y f i s h of the species Pacifastacus l e n i u s c u l u s . Van Harreveld's (1936) s a l t s o l u t i o n consisting o f , NaCl 200.50mM KC1 5.37mM MgCl 2 1.25mM NaHC03 2.70mM CaCl 2 lO.OOmM was used as the bathing medium. A l l test solutions were made up i n thi s medium by replacing equimolar amounts of NaCl with the sodium s a l t s of the drugs used and adjusting the pH to 7-8 using NaOH. a) The abdominal stretch receptor. Methods s i m i l a r to those used by Wiersma et a l . ( 1953) and Florey (1957) were employed for d i s s e c t i n g and recording from the abdominal stretch receptors. The te r g a l parts of the abdominal segments were dissected free from the rest of the abdomen i n the form of a single s t r i p containing a l l of the extensor musculature. The proximal end of the f i r s t t e r g i t e was placed i n a clamp with the ventral surface up. The ventral and s u p e r f i c i a l extensor musculature with the exception of the musculus s u p e r f i c i a l i s l a t e r a l i s was removed. This muscle serves to protect the receptors from being stretched by p u l l on the nerve. A thread was fixed on the la s t segment by means of a hook, making i t possible to f l e x the abdominal s t r i p . The dorsal h a l f - s h e l l of the " t a i l " of the c r a y f i s h i n thi s way served as a receptacle for the test s o l u t i o n s . Removal of the tergal s t r i p necessarily severs the nerve trunk which supplies the stretch receptors, but a s u f f i c i e n t length of nerve remains to allow for the placement of electrodes. Solutions to be tested were applied t o p i c a l l y to the stretch receptor organ, and a f t e r 10 sec. exposure, the excess f l u i d was sucked away, and the ef f e c t upon the discharge of the slowly adapting receptor noted. The action potentials were recorded from the axoni.by a s i l v e r wire electrode and the clamp holding the preparation served as the i n d i f f e r e n t electrode. The action potentials generated by the slowly adapting stretch receptor, which were the only ones occurring spontaneously i n thi s prepara-t i o n , were amplified and displayed on an o s c i l l o s c o p e . T^he frequency of f i r i n g was determined with a ratemeter and -recorded on paper chart. Figure 32 gives a block diagram of the preparation and equipment. b) The closer muscle of the claw. The cheliped of the cr a y f i s h was removed at the is c h i o p o d i t e . The medial h a l f of the exoskeleton of the 113 Figure 32. A block diagram of the equipment used to record from the abdominal receptor i n cray-f i s h . meropodite was removed and the nerves exposed. The point of the fixed finger of the propodite was cut and a cannula inserted. Fluid running through this cannula circulated through the claw and leaked out at the meropodite. The entire claw was pinned firmly to a wax plate. The nerve was dissected free and divided. Each division was stimu-lated using bipolar si l v e r electrodes until the excitatory nerve to the closer muscle was found. In order to record the contraction of the closer muscle, the point of the dactylopodite of the claw was connected to a force transducer which in turn was connected to one channel of a polygraph. Figure 33 illustrates the preparation. A mariotte bottle containing van Harreveld's salt solution was elevated above the claw and attached to the cannula in the propodite. The continuing circulation of f l u i d kept the muscles and nerves moist. The drugs were applied through the same cannula and washed out again with van Harreveld's solution. 115 Polygraph Stimulator Tension . Tran sducer S. .U. Cannu la%v L^ . Figure 33. A block diagram of the equipment used to record closer muscle contraction in the claw of the crayfish. 116 CHAPTER VII RESULTS - CRAYFISH PREPARATIONS a) The abdominal stretch receptor. By f l e x i n g the dorsum of the t a i l of the c r a y f i s h i t was possible to make the stretch receptor f i r e at a constant r a t e . This rate of f i r i n g could be varied from zero to approximately 25 spikes/sec., by increasing the tension on the l i n e f l e x i n g the abdominal segments. The stretch receptor was sen s i t i v e to temperature, increasing i t s rate of discharge when flushed with a cold s o l u t i o n , while a decrease was observed when warm soluti o n was applied. A number of Glut analogues were tested on t h i s preparation and the ratemeter recording of the pattern of f i r i n g was used to determine whether these analogues had any excitatory or i n h i b i t o r y action on the stretch receptor. Table X gives the l i s t of substances used i n order of excitatory potency. N-methyl-dl-glutamic acid had by far the greatest excitatory e f f e c t on the stretch receptor and at high concentrations (10 mg./ml.) i t would cause a burst of potentials which increased i n frequency and decreased i n amplitude u n t i l the receptor was depolarized and stopped f i r i n g . Glut was a good exci t a n t , although r e p e t i t i v e a p p l i c a -tions caused successively decreasing responses. As could TABLE X THE EFFECTS OF A NUMBER OF GLUTAMATE ANA-LOGUES ON THE FIRING RATE OF THE STRETCH RECEPTOR IN THE CRAYFISH. THE COMPOUNDS ARE LISTED IN ORDER OF THEIR EXCITATORY POTENCY. Glut analogue Order of potency N-Methyl-DL-Glutamic acid 4-Fluoroglutamic acid L-Glutamic acid L-Glutamic acid d i e t h y l ester para-Nitrobenzoyl-L-Glutamic acid DL-Homocysteic acid L-Glutamic acid y-Mebhyl ester L-Aspartic acid N-Methyl-DL-Aspartic acid a-Methyl-L-Glutamic acid D-Glutamic acid a-Amino-L-Adipic acid Acetyl-L-Glutamic acid para-Aminobenzoyl-L-Glutamic acid Very strong Strong Weak be e x p e c t e d t h e g r e a t e s t e f f e c t s o f G l u t on the r e c e p t o r were o b s e r v e d when the f i r i n g r a t e was i n i t i a l l y low, whereas a t h i g h f i r i n g r a t e s a much s m a l l e r r e s p o n s e was o b s e r v e d . aMG had l i t t l e o r no e x c i t a t o r y e f f e c t s on the f i r i n g r a t e o f t h e s t r e t c h r e c e p t o r . I n th o s e p r e p a r a t i o n s where h i g h c o n c e n t r a t i o n (10 mg./ml.) o f aMG d i d not a f f e c t t he f i r i n g r a t e , a p p l i c a t i o n o f aMG p r i o r t o G l u t would reduce o r e l i m i n a t e the re s p o n s e o f t h e r e c e p t o r t o G l u t ( f i g u r e 3*0. The s e n s i t i v i t y o f the r e c e p t o r t o G l u t r e t u r n e d f o l l o w i n g t h o r o u g h washing w i t h f r e s h van H a r r e v e l d ' s s o l u t i o n . The e f f e c t s o f aMG d i d not appear t o be s p e c i f i c f o r G l u t s i n c e a s i m i l a r d e p r e s s i o n o f the e f f e c t s o f low c o n c e n t r a t i o n s o f N - m e t h y l - G l u t c o u l d a l s o be o b t a i n e d . GDEE was as e f f e c t i v e an e x c i t a n t as G l u t and i t was t h e r e f o r e i m p o s s i b l e t o d e t e r m i n e any b l o c k i n g a c t i o n by t h i s compound. b) The c l o s e r muscle o f t h e c l a w . S t i m u l a t i o n o f t h e nerve i n the me r o p o d i t e w i t h r e p e t i t i v e s t i m u l i a t a f r e q u e n c y o f 50/sec. caused a con-t r a c t i o n o f t h e c l o s e r muscle and e x e r t e d t e n s i o n up t o 20 gm. on the t r a n s d u c e r . The o n l y d r u g t e s t e d i n t h i s p r e p a r a t i o n was GDEE ( f i g u r e 35). A f t e r a c o n s t a n t l e v e l o f c o n t r a c t i o n w i t h s u c c e s s i v e s t i m u l i was o b t a i n e d , 0.2 mg. of GDEE i n van H a r r e v e l d ' s s o l u t i o n was p e r f u s e d t h r o u g h the claw . S t i m u l i f o l l o w i n g a p p l i c a t i o n o f GDEE a t f i r s t caused e r r a t i c c o n t r a c t i o n s f o l l o w e d by s u c c e s s i v e r e d u c t i o n s i n the f o r c e o f the c o n t r a c t i o n . A f t e r t h e claw was p e r f u s e d 119 12 r 30 sec. Figure 34. The effect of aMG on the Glut excitations of the abdominal stretch receptor in the crayfish. Ratemeter records show the effect of Glut (10 mg./ml.) alone, Glut following application of aMG (10 mg./ml.) and fi n a l l y after rinsing with van Harreveld's solution. 120 gm. 0 I S t i m . r 0 . 7 5 m l GDEE A . Rinse A. 1 8 sec. Figure 35. The effect of GDEE on synaptically induced contractions of the closer muscle of the claw of the crayfish. Records show the tension produced by the closer muscle on application of a stimulus (50/sec, 0 .2 msec, 50V) to the excitatory nerve of this muscle. 0.75 ml. of a ImM solution of GDEE was applied through a cannula in the claw. Rinsing was accomplished with the use of van Harreveld's solution. with fresh van Harreveld's solution the response to s t i m u l i slowly recovered u n t i l sustained contractions were again maintained. CHAPTER VIII DISCUSSION The r e s u l t s obtained i n th i s i n v e s t i g a t i o n demonstrate that i t i s possible to block r e v e r s i b l y or to enhance the excitatory action of glutamate on single neurones of the central nervous system by the l o c a l a p p l i c a t i o n of suitable pharmacological agents. The action of these agents appeared to be r e l a t i v e l y s p e c i f i c for glutamate, i n that the a c t i v a t i o n of the neurones by the exogenous a p p l i c a t i o n of other excitatory agents was often not a f f e c t e d . The synaptic a c t i v a t i o n of neurones i n a number of sensory pathways was affected by the pharmacological agents i n a s i m i l a r manner to that of the glutamate e x c i t a t i o n , which suggests that glutamate may be the p h y s i o l o g i c a l trans- . mitter of e x c i t a t i o n at these synapses. Of the compounds tested GDEE showed the greatest blocking action on glutamate responses. Blockade was most noticeable i n the sp i n a l cord, cuneate and thalamus and was observed following either iontophoretic or systemic administration of GDEE. That the glutamate e x c i t a t i o n was not depressed i n every c e l l may be at t r i b u t e d to the li m i t a t i o n s of the iontophoretic technique which makes i t impossible to determine the exact concentration of the 123 compound i n the region of the receptor (Curtis and Crawford, 1969). GDEE showed a large degree of s p e c i f i c i t y i n as much as i t antagonized the glutamate-induced neuronal excitations to a greater extent than those produced by the other e x c i t a -tory amino acids and ACh. The degree of s p e c i f i c i t y , however, was dependent on the amount of GDEE applied, thus with large iontophoretic currents, GDEE would usually block the action of a l l of the amino acid e x c i t a t i o n s , but even under these circumstances neuronal e x c i t a t i o n by ACh could be spared. With suitable adjustment of the GDEE current i t was usually possible to block the glutamate e f f e c t s p e c i -f i c a l l y without a f f e c t i n g the response of the c e l l to aspartate, cysteate and DLH, and therefore i n the present i n v e s t i g a t i o n the s e n s i t i v i t y of the c e l l to DLH was used as a c o n t r o l . The fact that ACh responses were very l i t t l e affected may explain why Curtis et a l . (1972) were unable to observe the same degree of s p e c i f i c i t y towards the amino acids, since they used the excitations produced by ACh as c o n t r o l s . The question whether DLH i s an appropriate compound to compare with glutamate however, requires cautious con-s i d e r a t i o n . The fact that i n every case the DLH-ejecting current used was lower than the glutamate current required to produce an equivalent e x c i t a t i o n (McLennan, 1970b), indicates that the net effectiveness of DLH upon the c e l l membrane i s greater. The r e l a t i v e f a i l u r e to antagonize the action of DLH might therefore merely r e f l e c t a quan-t i t a t i v e rather than a q u a l i t a t i v e difference between the two amino acids; however, the same consideration would not apply to the s p e c i f i c e f f e c t of GDEE on glutamate r e l a t i v e to ACh, aspartate and cysteate (figures 5,7)- The s p e c i -f i c i t y of action also indicates that GDEE does not exert i t s e f f e c t by depressing the o v e r a l l e x c i t a b i l i t y of the c e l l . Another consideration i s that since glutamate and aspartate are known to be transported into nerve endings by a high a f f i n i t y system (Logan and Snyder, 1971; Wofsey et a l . , 1971), whereas other amino acids (including presumably DLH and cysteate) are not, the non-natural sulphonic acids ejected into the perineuronal e x t r a c e l l u l a r space might di f f u s e to af f e c t more distant receptors than could be influenced by glutamate or aspartate ( C u r t i s , Duggan and Johnston, 1970a,b; Curtis et a l . , 1972). Such distant receptors might therefore be too f a r removed from the blocking action of GDEE to be affected by the drug, and thus the antagonist would appear less e f f e c t i v e against DLH and cysteate. On the other hand, the fi n d i n g that aspartate was antagonized to a lesser degree than glutamate (figures7,9,11) would argue against t h i s view and i n favour of the existence of neuronal s i t e s s p e c i f i c a l l y or at least p r e f e r e n t i a l l y s e n s i t i v e to glutamate. This concept of the d i v i s i o n of receptors into a group which i s unspecific and reacts with a l l the excitatory amino acids and one which i s more s p e c i f i c for glutamate was f i r s t proposed by McLennan et a l . (1968) when they showed that the s e n s i t i v i t y of thalamic neurones to DLH and g l u t -amate did not invariably run p a r a l l e l . McLennan (1970b) also found that excitations of c o r t i c a l neurones induced by i o n t o p h o r e t i c a l l y applied glutamate were depressed f o r a longer time by s t i m u l i to neighbouring cortex than were comparable excitations produced by DLH. The experiments of Boakes et a l . (1970) indicated that i n c e r t a i n brain stem neurones i t was possible to block glutamate but not DLH responses with LSD, and they also suggested that g l u t -amate and DLH react with d i f f e r e n t receptors. The observation that d i f f e r e n t populations of s p i n a l neurones show a greater r e l a t i v e s e n s i t i v i t y to aspartate than to glutamate (Duggan, 197D indicates that i n addition to a non-specific receptor s i t e f o r the a c i d i c amino acids and one which i s more se n s i t i v e to glutamate there i s . also l i k e l y to be a receptor which has a greater s e n s i t i v i t y to aspartate than to the other e x c i t a n t s . The existence of neurones with d i f f e r e n t r e l a t i v e s e n s i t i v i t i e s to glutamate and aspartate i n the s p i n a l cord was confirmed i n the present i n v e s t i g a t i o n , and i t i s i n t e r e s t i n g to note that i n those neurones which tended to be more sen s i t i v e to aspartate, GDEE blocked the e f f e c t s of glutamate and aspartate to the same extent. The receptor which reacts with ACh can be r e a d i l y distinguished from the receptors s e n s i t i v e to the a c i d i c amino acid s . Drugs such as atropine or dihydro-3-erythroidine which block ACh e x c i t a t i o n have very l i t t l e i f any e f f e c t on amino acid e x c i t a t i o n (Krnjevic, 1964; McLennan, 1970a), while MSO (present i n v e s t i g a t i o n ; Curtis et a l . , 1972), 2-methoxy-aporphine (Curtis et a l . , 1972) and the compound l-hydroxy-3-aminopyrrolidone-2 (HA-966) (Davis and Watkins, 1972) on the other hand appear to block amino acid-induced excitations without appreciably a f f e c t i n g those produced by ACh. The a b i l i t y of MSO to block the amino acid responses i n the present i n v e s t i g a t i o n was not as great as that observed by Curtis et a l . (1972). This i s probably due to the fact that Curtis et a l . used the L-isomer of MSO, whereas i n the present i n v e s t i g a t i o n the DL-isomer was used, e s p e c i a l l y since the convulsant a c t i v i t y and the a b i l i t y to i n h i b i t glutamate synthetase depends on the isomeric configuration (Rowe and Meister, 1970). In neither case, however, was any s i g n i f i c a n t degree of s p e c i f i c i t y towards glutamate excitations shown. The difference i n the e f f e c t s of MSO and GDEE becomes obvious when they are compared on the same c e l l (figure 24). aMG was the only other compound tested which showed any blocking a c t i o n . In contrast to the r e s u l t s reported by Marshall (1971) i t s e f f e c t was found to be rather weak and inconsistent. More commonly, effects s i m i l a r to those described by Curtis et a l . (1972) were observed, where aMG had a tendency to potentiate the e f f e c t s of a l l other e x c i t a -tory compounds (figure 22). The l a t t e r e f f e c t could well be due to a subthreshold depolarization of the neurone, thus increasing i t s e x c i t a b i l i t y , since aMG tended to increase the spontaneous f i r i n g rate of a number of c e l l s when applied at high iontophoretic current strengths. A potentiating e f f e c t on the excitations produced by the a c i d i c amino acids (especially glutamate and aspartate) was observed following a p p l i c a t i o n of GDME i n a l l areas of the nervous system tested. These e f f e c t s do not appear to be due to a subthreshold depolarization since the eff e c t s of DLH were not increased to the same extent (figures 17, 19,20). However, GDME may cause some degree of d e p o l a r i -zation since an increase i n the background f i r i n g rate was occasionally observed. The occasional blocking action observed with GDME (figure 16) may be due to i t s s t r u c t u r a l s i m i l a r i t y to GDEE; however, even i n these cases there was a potentiating e f f e c t on cessation of the GDME-ejecting current. The fact that the potentiating e f f e c t of GDME and the blocking e f f e c t of GDEE can be demonstrated on the same c e l l (figure 18) where the pH concentration and ejecting currents of the two esters are the same, indicates that the "receptors" which are responsible for the action of these drugs have a high degree of s p e c i f i c i t y . GDME occasionally caused blockade of the responses of neurones to glutamate (figure 16) and GDEE showed some i n h i b i t i o n of glutamate uptake into synaptosomes (figure 31). This suggests that glutamate e x c i t a t i o n and glutamate uptake are in h i b i t e d by both e s t e r s , GDEE having i t s predominant e f f e c t on glutamate e x c i t a t i o n whereas GDME had the greatest e f f e c t on glutamate uptake. The potentiating e f f e c t s of GDME can be explained i n terms of i n h i b i t i o n of the uptake system for glutamate and aspartate. The experiments on glutamate uptake confirmed those of Logan and Snyder ( 1 9 7 1 , 1 9 7 2 ) , which indicated that high a f f i n i t y as well as low a f f i n i t y mechanisms exist f o r the uptake of glutamate into crude synaptosomal preparations. The i n h i b i t i o n produced by GDME i s l i m i t e d to the high a f f i n i t y system (figure 3 1 ) . The marked increase i n K , the Michaelis a f f i n i t y constant, suggests a competitive i n h i b i t i o n ; however, concomitantly there i s an apparent increase i n V and the data therefore do not f i t any of max the c l a s s i c a l models of i n h i b i t i o n f o r a single enzyme system. Preliminary experiments on leucine uptake suggest that the i n h i b i t o r y action of the GDME i s not s p e c i f i c for glutamate uptake. The value determined f o r Km f o r the high a f f i n i t y uptake of glutamate i s comparable to that reported by Logan and Snyder; however, that f o r the non-s p e c i f i c low a f f i n i t y system i s only about one-tenth of the minimum value reported by those authors. No evident explanation for the difference presents i t s e l f . The data support the suggestion that transport Into c e l l u l a r elements may well be the mechanism for the i n -a c t i v a t i o n of the excitatory amino acids ( C u r t i s , Duggan and Johnston, 1 9 7 0 ) . It may be argued (McLennan, 1970a) that a prolonged excitatory action on cessation of the glutamate-ejecting current as well as a potentiation of the ex c i t a t i o n could be expected on i n h i b i t i o n of the uptake mechanism. However, thi s would not be the case i f the i n h i b i t i o n was only p a r t i a l , or as i n th i s case, only one of the two uptake systems was af f e c t e d . Assuming that under normal circumstances the uptake i s not close to saturation the re s i d u a l uptake mechanism a f t e r i n h i b i t i o n by GDME could well be s u f f i c i e n t to inact i v a t e the glutamate on cessation of the eje c t i n g current with no noticeable increase i n the i n a c t i v a t i o n time; however, the i n h i b i t i o n of the high a f f i n i t y uptake mechanism would allow a greater concentration of glutamate to accumulate e x t r a c e l l u l a r l y and thus cause a greater e x c i t a t i o n . Similar potentiating ef f e c t s without prolonged e x c i t a t i o n observed following systemic administration of thiosemicarbazide (Steiner and RUP, 1966) could be explained i n terms of i n h i b i t i o n of glutamate decarboxylase which i s located i n t r a c e l l u l a r l y (Salganicoff and De Robertis, 1965) thus increasing the i n t r a c e l l u l a r content of glutamate. This would have the effe c t of increasing the concentration gradient across the c e l l membrane and thus i n h i b i t the uptake mechanism. Both pCMS (which also i n h i b i t s amino acid uptake into s l i c e s of cerebral cortex (Curtis et a l . , 1970)) and GDME have a potentiating e f f e c t on evoked responses of c e l l s i n the thalamus as well as enhancing the excitations produced by glutamate and aspartate (figures 19, 27) and GDME has a si m i l a r e f f e c t i n the spi n a l cord and cuneate nucleus 130 (figures 20,21). This would suggest that the transmitter released at these synapses i s inactivated by a mechanism which i s i n h i b i t e d by these agents. However, the p o s s i b i l i t y that this e f f e c t i s the re s u l t of a subthreshold d e p o l a r i -zation cannot be excluded. The primary sensory pathway. The simultaneous blocking of the glutamate responses and the synapt i c a l l y evoked potentials i n spi n a l i n t e r -neurones and i n cuneate neurones by GDEE (figures 11,12,15) i s strong evidence to suggest that glutamate may be the mediator of synaptic transmission at primary afferent terminals. The preliminary observation by Davies and Watkins (1972) that HA-966, which has a depressant e f f e c t on the glutamate excitations of cuneate c e l l s blocks the response of these c e l l s to cutaneous stimulation further supports t h i s p o s s i b i l i t y . The suggestion that glutamate may be the transmitter at the primary sensory terminals was made by Aprison et a l . i n 1965 when they demonstrated a higher concentration of glutamate i n dorsal roots and spinal cord grey matter than i n the ventral roots or spi n a l white matter. Further studies (Duggan and Johnston, 1970a,b; Johnson and Aprison, 1970, 1971) have shown that the c e n t r a l l y directed portion of nerve roots from spinal ganglia have a higher glutamate content than the peripher a l l y directed nerves and that concentrations i n the dorsal regions of the spi n a l cord are higher than those of the v e n t r a l . S i m i l a r l y glutamate concentrations have been shown to be present i n s i g n i f i c a n t l y higher concentrations i n the cuneate and g r a c i l e nuclei than i n more ventral regions of the brain stem. These data are consistent with the suggestion that glutamate i s a transmitter i n these regions i f one accepts the idea that glutamate i s compartmentalized into metabolic and tran s -mitter "pools" i n the C.N.S. Under these circumstances one would expect those areas which u t i l i z e glutamate as a transmitter to show a higher l e v e l of t h i s amino acid by an amount equal to the transmitter p o o l . The fact that none of the other amino acids showed s i m i l a r differences i n concentration further suggests that glutamate serves some s p e c i a l i z e d function i n these regions. A number of other compounds have been suggested as mediators of synaptic transmission at primary afferent neurones. Amongst these are ACh (Biilbring and Burn, 1941), ATP (Holton and Holton, 1954), substance P (Lembeck, 1953) and histamine (Holton and Perry, 1951). The evidence suggests that ACh i s not the transmitter at primary afferent terminals since dorsal roots and the medullary sensory nuclei contain very l i t t l e ACh or choline-acetyltransferase (Mcintosh, 1941; Peldberg and Vogt, 1948). Further evidence against ACh serving as a transmitter at primary afferent terminals i s the i n a b i l i t y (Curtis et a l . , 196l; Steiner and Meyer, 1966; Galindo et a l . , 1967) or incon-si s t e n t a b i l i t y (Weight and Salmoiraghi, 1966) of ACh to excite motorneurones, interneurones and cuneate neurones when applied i o n t o p h o r e t i c a l l y , and the lack of e f f e c t of ACh blocking agents on the response of spinal neurones to sensory stimulation (Curtis et a l . , 1961). Substance P, histamine and ATP have been proposed as possible mediators of primary afferent synapses on the grounds that they are present i n d i s t a l portions of cutaneous nerves and are able to cause va s o d i l a t i o n i n the rabbit ear (McLennan, 1970a). This reasoning stems from the Dale hypothesis which states that a neurone releases the same compound at a l l of i t s terminal branches and since antidromic stimulation of peripheral sensory nerves can cause vaso-d i l a t i o n i t i s l i k e l y that the transmitter i n these nerves i s responsible for t h i s a c t i o n . It seems unl i k e l y however, that either substance P or histamine are involved i n central transmission at primary afferent synapses since neither of them have any excitatory a b i l i t y when applied d i r e c t l y to cuneate c e l l s (Galindo et a l . , 1967). It i s possible that the presence of histamine i n d i s t a l portions of cutaneous neurones may be the re s u l t of trauma (McLennan, 1970a) and that substance P may i n some manner bind with the tr a n s -mitter and influence i t s a c t i v i t y (Umrath and G r a l l e r t , 1967)-ATP on the other hand, does show powerful excitatory action when applied to cuneate and g r a c i l e neurones (Galindo et a l . , 1967)• This action has been a t t r i b u t e d to the calcium chelating properties of ATP, since other chelating agents show s i m i l a r excitatory a c t i v i t y ( C u r t i s , Perrin and 133 Watkins, I 9 6 0 ; Galindo et a l . , 1 9 6 7 ) . However, the observation that s p i n a l (Curtis et a l . , 1961) and c o r t i c a l (Krnjevic and P h i l l i s , 1963a) neurones are unaffected by ATP i n f e r s that cuneate neurones may be p a r t i c u l a r l y s e n s i -t i v e , and t h i s together with the observation that l i b e r a t i o n of catecholamine from the adrenal medulla i s accompanied by ATP release (Douglas and. Poisner, 1966) has led to the suggestion that ATP l i b e r a t e d together with the transmitter may act to modify the postsynaptic response (McLennan, 1 9 7 0 a ) . The suggestion that aspartate may act as an excitatory transmitter of s p i n a l interneurones was made by Davidoff et a l . ( 1967) when they observed that there was good c o r r e l a t i o n between the loss of s p i n a l interneurones and the decrease i n aspartate concentration following s p i n a l cord ischaemia. This p o s s i b i l i t y i s further enhanced by the observation that Renshaw c e l l s appear to be r e l a t i v e l y more se n s i t i v e to aspartate than to glutamate, i n comparison with a population of dorsal s p i n a l interneurones (Duggan, 1 9 7 D and the current observation that those neurones which tended to be more sensi t i v e to aspartate did not show the same degree of s p e c i f i c i t y to the glutamate blocker, GDEE. It i s i n t e r e s t i n g to note that glutamate increases the e x c i t a -b i l i t y of primary afferent terminals i n the cuneate nucleus whereas aspartate has no such e f f e c t (Davidson and Southwick, 1 9 7 1 ) . 134 The secondary sensory pathway. Neurones In the VPL nucleus of the thalamus showed the same response as spinal interneurones to the a p p l i c a t i o n of GDEE and GDME (figures 10,14,19), suggesting that glutamate may be the mediator of synaptic transmission at the secondary sensory synapses as well as at the primary afferent terminals. The fact that ventrobasal neurones i n the thalamus are excited by the a c i d i c amino acids has been known for some time (Curtis and Watkins, 1963) and as mentioned i n the i n t r o d u c t i o n , these neurones are r e l a t i v e l y more sensit i v e to glutamate than are more s u p e r f i c i a l thalamic neurones (McLennan et a l . , 1968). The thalamus contains f a i r l y large amounts of free glutamic acid (Johnson and Aprison, 1971). On autopsy the concentration of glutamate i n the thalamus of humans has been shown to be higher than i n the substantia nigra and red nucleus, but not as high as i n the basal ganglia and cortex (Perry et a l . , 1971). This f a i r l y s p e c i f i c l o c a l i z a t i o n might well be due to changes which occur following death, however, the lack of c o r r e l a t i o n between glutamate concentrations and those of GABA, glutamine and aspartate suggest that i t may be involved i n s p e c i f i c functions unrelated to metabolism i n those regions where i t i s present i n high concentrations. Additional evidence i n favour of glutamate as a trans-mitter at sensory synapses i n the thalamus i s the fact that none of the other putative transmitters appear to be involved. There are only moderate amounts of a c e t y l c h o l i n e , a c e t y l -cholinesterase and choline acetyltransferase i n the mammalian thalamus i n comparison to basal ganglia and ventral roots (Mcintosh, 1941; Feldberg and Vogt, 1948; Oliver et a l . , 1970) which suggests that the major afferent pathways to the thalamus are not c h o l i n e r g i c . There do appear to be some regional differences i n cholinesterase staining within the thalamus (Oliver et a r . , 1970), and i t seems l i k e l y that inputs other than the primary sensory ones may u t i l i z e a cholinergic mechanism (McCance et a l . , 1968; Marshall, 1971)• Further evidence to suggest that any cholinergic input to ventrobasal neurones i s unrelated to the primary afferent pathway i s the fact that whereas most of these neurones are r e a d i l y excited by acetylcholine i t i s possible to block t h i s e x c i t a t i o n with atropine or dihydro-8-erythroidine without a f f e c t i n g the response to cutaneous nerve stimulation (Andersen and C u r t i s , 1964). The catecholamines and 5-HT are present i n the thalamus (McGeer et a l . , 1963), and NA and 5-HT have been shown to excite a few c e l l s there ( P h i l l i s and Tebecis, 1976; but see Frederickson et a l . , 1971 and Jordon et a l . , 1972). However," there i s no evidence to suggest that these compounds are responsible for any excitatory synaptic mechanisms. On the contrary, they have been proposed as i n h i b i t o r y t r a n s -mitters since t h e i r prime e f f e c t on neurones i s one of depression. The observation that 5-HT i n h i b i t s the response of l a t e r a l geniculate neurones to optic nerve stimulation, but does not a f f e c t the responses of ventrobasal thalamic neurones to impulses i n cutaneous nerves, has led to the suggestion that the spinothalamic and opticogeniculate pathways u t i l i z e d i f f e r e n t transmitter mechanisms ( C u r t i s , 1966). The thalamocortical pathway. GDEE blocked the response of c o r t i c a l neurones to thalamic stimulation and glutamate a p p l i c a t i o n (figure 13); however, the action of GDEE i n thi s region was much less consistent than i n the spinal cord, cuneate nucleus and thalamus. This inconsistency could be due to the extreme neuronal complexity of t h i s structure and the l i k e l i h o o d that a variety of pathways with d i f f e r e n t synaptic trans-mitters are involved i n Its functi o n . The observation that GDEE was able to block the excitatory e f f e c t s of glutamate f a i r l y s p e c i f i c a l l y at cer t a i n c e l l s , whereas at others GDEE and MSO had either no ef f e c t or a non-specific e f f e c t on the s e n s i t i v i t y of the neurones to the excitatory amino acids, might be due to differences i n s e n s i t i v i t y of d i f f e r e n t c e l l s to glutamate. Crawford (1970) was unable to show any difference i n the r a t i o of the s e n s i t i v i t y of any population of c o r t i c a l c e l l s to DLH, aspartate and D-glutamate when compared to that of L-glutamate; however, the wide v a r i a t i o n i n the responses of neurones to DLH and L-glutamate which he attr i b u t e d to l i m i t a t i o n s i n the technique of iontophoresis, might well have been due to r e a l differences i n the sensi-t i v i t y of the neurones to the two amino a c i d s . McLennan (1970b) on the other hand, found that excitations of c o r t i c a l neurones induced by ion t o p h o r e t i c a l l y applied L-glutamate were depressed f o r a longer time by s t i m u l i to neighbouring cortex than were equivalent excitations produced by DLH. The differences seen were not the same at a l l c o r t i c a l depths, but were more pronounced i n layers II and V, suggesting that the pyramidal c e l l s giving r i s e to c o r t i c a l efferents which l i e i n those zones may be somehow s p e c i a l i z e d i n this regard. Additional evidence i n favour of glutamate playing some synaptic role i n the cortex i s that glutamate i s present i n high concentration i n c o r t i c a l tissue (Johnson and Aprison, 1971; Perry et a l . , 1971) and a portion of i t i s located i n the synaptosomal f r a c t i o n (Kuher and Snyder, 1969). In-creased amounts of glutamate and ACh can be c o l l e c t e d from f l u i d s bathing the cerebral cortex during arousal states and following r e t i c u l o c o r t i c a l stimulation (Jasper and Koyama, 1968, 1969), whereas on thalamic stimulation only ACh could be c o l l e c t e d . This might be interpreted as suggesting that the thalamocortical pathway u t i l i z e s a cholinergic transmission mechanism, and there i s a f a i r amount of a d d i t i o n a l evidence to suggest that ACh i s involved i n a thalamocortical pathway. ACh and i t s synthetising enzymes are present i n high concentrations i n the cortex (Feldberg and Vogt, 1948) and are decreased i n chronically undercut c o r t i c a l slabs ( C o l l i e r and M i t c h e l l , 1967), and c e l l s i n the deeper c o r t i c a l layers are strongly excited by acetylcholine (Krnjevic and P h i l l i s , 1963b; Crawford, 1970). ACh does not appear to be the transmitter i n the main sensory pathway, since atropine has been found to abolish the cholinoceptive responses without any ef f e c t upon the discharge evoked by sensory stimulation (Krnjevic and P h i l l i s , 1963b). However, the responses of c e l l s i n the cortex to thalamic stimulation can sometimes be blocked by the iontophoretic a p p l i c a t i o n of atropine (Stone, 1972). In these experiments the l a t t e r part of the burst response e l i c i t e d i n the cortex by stimulation of VPL was blocked by • atropine while the f i r s t few spikes were unaffected, and this suggests that two mechanisms may be involved i n the thalamocortical pathway. Similar conclusions can be drawn from the observation that i n h i b i t i o n of cholinesterase i n the cortex by DFP increases the amplitude of the late response to stimulation of the forepaws i n rats without a f f e c t i n g the primary complex of the evoked potentials (Bhargava, 1972). The presence of a "cholin e r g i c " as well as a possible "glutaminergic" pathway between the thalamus and the cortex could well explain the present inconsistent r e s u l t s . It i s possible that those c e l l s which have a cholinergic input have only the non-specific type of receptor and respond to GDEE with a decrease i n a l l the amino acid responses, while those which have a glutaminergic input respond to GDEE with a s p e c i f i c decrease i n the glutamate and evoked responses. It i s possible that glutamate i s responsible for the dir e c t pathway from somatic afferents and ACh f o r a second pathway of unknown function. The anaesthetic and muscle relaxant. Barbiturate anaesthesia has been shown to depress the mean spontaneous f i r i n g rate of c o r t i c a l neurones (Curtis and Crawford, 1970) and i s responsible for the spontaneous spindle a c t i v i t y i n the thalamus (Andersen et a l . , 1967; Baker, 1971)• In the spinal cord i t reduces the number of spikes i n the response evoked by peripheral nerve stimulation (Wall, 1967), but does not a f f e c t the i n i t i a l part of the response. S i m i l a r l y , the short latency responses of ventrobasal thalamic nuclei to stimulation of cutaneous nerves (Baker, 1971) appear to be unaffected by barbiturates. The e f f e c t s of barbiturates on i o n t o p h o r e t i c a l l y applied excitants seems to vary, depending on the area tested, however, i n a l l cases i t has either l i t t l e e f f e c t on the s e n s i t i v i t y of neurones to glutamate and DLH (Krnjevic and P h i l l i s , 1963a; Bloom et a l . , 1965; McCance et a l . , 1968) or depresses the s e n s i t i v i t y of the neurone to a l l forms of e x c i t a t i o n (Crawford and C u r t i s , 1966). The p o s s i b i l i t y that gallamine t r i e t h i o d i d e , which was used to paralyze the animals, might have some e f f e c t on the r e s u l t s should also be considered. This compound applied systemically has been shown to augment the a f t e r discharge of neurones i n the i n t a c t and i s o l a t e d cortex of unanaesthe-t i s e d cats (Halpern and Black, 1967), to increase the mean spontaneous f i r i n g rate of neurones i n the f e l i n e cuneate nucleus (Galindo et a l . , 1968) and can modify transmission through th i s nucleus. These observations suggest that gallamine, which has an excitatory e f f e c t on neurones when applied i o n t o p h o r e t i c a l l y (Crawford and C u r t i s , 1966; Galindo et a l . , 1968), penetrates the blood-brain b a r r i e r . However, i t i s possible that the eff e c t s which follow systemic a p p l i c a t i o n may r e f l e c t a l t e r a t i o n s i n the a c t i v i t y of peripheral sensory receptors (Curtis and Crawford, 1969). The release of glutamate from brain s l i c e s . In the experiments on s l i c e s of rat cortex the release of l a b e l l e d glutamate obtained by adding glutamate or K+ to the incubation medium i s thought to be due to a depolarization of the c e l l s i n the s l i c e (Arnfred and Hertz, 1971). I f th i s i s true , then the present r e s u l t s suggest that GDEE has a s i m i l a r depolarizing e f f e c t on the c e l l s and aMG i s unable to block the depolarization caused by glutamate. The t o t a l amount of l a b e l released, i n excess of the basal release seen i n the control experiments (figure 29; Arnfred and Hertz, 1971), appeared to be constant since the presence of glutamate and GDEE i n the bathing medium did not increase the glutamate release over that released by each of these compounds by i t s e l f , and the increased release over control values was i n i t i a l l y very marked but returned to control levels by the end of 40 min. even though the concentration of glutamate and/or GDEE i n the bathing medium remained constant. This suggests that the extra l a b e l l e d glutamate released by these compounds, and that released continuously into the soluti o n might possibly originate from d i f f e r e n t "pools". Similar conclusions were drawn by De Peudis ( 1 9 7 D , who showed that only a portion of the t o t a l exchangeable glutamate absorbed into desheathed nerves could be released on e l e c t r i c a l s t imulation. The c r a y f i s h neuromuscular j u n c t i o n . As can be seen from table X the r e l a t i v e s e n s i t i v i t y of the abdominal stretch receptor i n the cr a y f i s h to the excitatory amino acids d i f f e r s from that observed i n the mammalian C.N.S. Por example, the stretch receptor Is more sen s i t i v e to glutamate than to DLH, whereas the opposite i s true i n the mammalian C.N.S. The stretch receptor i s also very s e n s i t i v e to N-methyl-DL-glutamic acid and i n -sen s i t i v e to the corresponding aspartate compounds, whereas i n the sp i n a l cord, neurones show the opposite r e l a t i v e s e n s i t i v i t y (Curtis and Watkins, i960, 1963) • It i s possible that these compounds act on the stretch receptor neurones as does ACh (McLennan and York, 1966) rather than on the muscle d i r e c t l y , since no excitatory innervation of the muscle has been found (Jansen.et a l . , 1971). However, the fact that nerve-muscle preparations i n the grasshopper are also more sen s i t i v e to glutamate than to DLH (McDonald and O'Brien, 1972) suggests that the receptors for glutamate i n invertebrates do d i f f e r from those i n the mammalian C.N.S The blocking of the response of the stretch receptor to glutamate by aMG (figure 34) and of the contraction of the closer muscle by GDEE (figure 35) require cautious i n t e r p r e t a t i o n . It i s possible that these compounds compet t i v e l y depress the s e n s i t i v i t y of the membrane both to glutamate and to the p h y s i o l o g i c a l transmitter. However, both of the compounds cause some degree of e x c i t a t i o n of the stretch receptor and i t i s thus possible that they block the responses by causing a depolarization of the muscle, although i f t h i s was the case one would expect an i n i t i a l contraction on a p p l i c a t i o n of the drug. The a b i l i t y of GDEE to block glutamate responses s p e c i f i c a l l y , without a f f e c t i n g the s e n s i t i v i t y of c e l l s to DLH, aspartate or cysteate, together with the other observations on differences i n the r e l a t i v e s e n s i t i v i t y of neurones to the a c i d i c amino a c i d s , suggest that the active s i t e s on membranes for glutamate d i f f e r from those for the other amino a c i d s . Curtis and Watkins ( i 9 6 0 ) have shown that the size of the amino acid and the number of ionized groups i t contains determine to a large extent i t s a c t i v i t y . In an attempt to make the ionized groups on three excitatory amino acids conform to the c l a s s i c a l "3-point" receptor s i t e proposed by Curtis and Watkins ( i 9 6 0 ) , von Gelder (1971) had to propose that the amino acids adopt a folded molecular arrangement. I f , however, one assumes that there are d i f f e r e n t receptor s i t e s on the membrane which react with amino acids with d i f f e r e n t numbers of carbon atoms between the a c i d i c groups and with d i f f e r e n t s t e r i c configurations, then such complex proposals become unnecessary. The hypothetical existence of a receptor s i t e for a f i v e carbon dicarboxylic amino acid leads to the speculation of the existence of a receptor s i t e which i s p r e f e r e n t i a l l y s e n s i t i v e to the four carbon atom equivalent. I f t h i s i s the case, i t should be possible to f i n d a compound which s e l e c t i v e l y depresses the s e n s i t i v i t y of c e l l s to aspartate. The series of experiments i n t h i s thesis was undertaken i n an attempt to s a t i s f y one of the c r i t e r a for glutamate as a transmitter i . e . that the p h y s i o l o g i c a l transmitter at s p e c i f i c synapses must react i n the same manner i n the presence of pharmacological agents as does glutamate. That the evoked response at primary and secondary sensory afferent terminals responded to the presence of a variety of agents i n the same manner as glutamate-induced e x c i t a t i o n s , decreasing when glutamate responses decreased and increasing when they increased, suggests that the attempt has been succe s s f u l . It Is hoped that these re s u l t s may be useful i n future experiments to s a t i s f y more completely other c r i t e r i a for amino acids as transmitter agents at s p e c i f i c pathways. The use of GDME to block the i n a c t i v a t i o n of glutamate should permit better recovery of th i s compound from the region of a nucleus i f i t i s released from terminals which end there. The a b i l i t y of GDEE to block glutamate excitations s p e c i f i c a l l y and MSO to block a l l of the amino acid excitations should prove valuable i n i n t r a c e l l u l a r investigations i n further pursuit of the c r i t e r i o n of " i d e n t i t y of a c t i o n " . The substantiation of the proposed mode of action of the various blocking and potentiating agents w i l l , depend to a large extent on an i n t r a c e l l u l a r i n v e s t i g a t i o n ; and the p o s s i b i l i t y of s p e c i f i c "receptors" for aspartate and that t h i s compound too may act as a transmitter w i l l require a great deal of further research. SUMMARY AND CONCLUSIONS Following an examination of the l i t e r a t u r e on Glut as a p o t e n t i a l mediator of synaptic transmission, i t became apparent that the c r i t e r i o n which states that the p h y s i o l o g i c a l transmitter must respond to the actions of pharmacological agents i n the same manner as Glut had not been f u l f i l l e d . The iontophoretic or intravenous a p p l i c a t i o n of GDEE was found to block the responses of neurones i n the thalamus to Glut without appreciably a f f e c t i n g the responses to DLH, Asp, Cys and ACh. This s p e c i f i c action suggested that receptors exist which are p r e f e r e n t i a l l y s e n s i t i v e to Glut. The action of Glut and GDEE appeared to be competitive since the dose-response curve of a neurone to Glut s h i f t e d to the right i n the presence of GDEE. The concomitant blocking of Glut-induced excitations and the responses i n the thalamus and spinal cord evoked by e l e c t r i c a l stimulation of peripheral nerves, and those i n the cuneate nucleus evoked by dorsal column stimulation, were interpreted as suggesting that Glut could well be the ph y s i o l o g i c a l transmitter at primary and secondary afferent terminals. The response of c o r t i c a l neurones to ap p l i c a t i o n of Glut and thalamic stimulation could be blocked by GDEE, but the depression was less consistent than that observed i n other areas. This could be explained by the presence of two pathways between the thalamus and cortex of which only one u t i l i z e d Glut as a transmitter aMG and MSO also showed some a b i l i t y to block the responses of c e l l s to Glut; however, the i n h i b i t i o n produced by these compounds was never as great as that produced by GDEE, and did not show the same s p e c i f i c i t y of a c t i o n . MSO appeared to be more active i n the cerebral cortex and spi n a l cord than i n the thalamus and aMG often increased the spontaneous rate of f i r i n g and enhanced the a c t i v i t y of the excitatory amino acids The a p p l i c a t i o n of GDME potentiated the responses of neurones to Glut and Asp without appreciably a f f e c t i n g the responses to DLH. The simultaneous potentiation of the evoked responses i n the spinal cord, cuneate nucleus and thalamus further increased the p o s s i b i l i t y that Glut may act as a transmitter i n these regions. The potentiating e f f e c t s of GDME appeared to be due to an i n h i b i t i o n of Glut uptake mechanisms, since the high a f f i n i t y uptake mechanism for Glut i n crude synaptosomal preparations could be i n h i b i t e d by add-ing GDME to the incubation medium. A much smaller i n h i b i t i o n was obtained using GDEE. pCMS also caused a potentiation of the responses of thalamic neurones produced by Glut and Asp or by stimulation of peripheral nerves. 9 . The addition of GDEE or Glut to the incubation medium of s l i c e s of rat cortex caused a s i g n i f i c a n t increase i n the release of l a b e l l e d Glut. The amount of Glut which could be released over control lev e l s from the brain s l i c e by these compounds appeared to be constant and may well have originated from a s p e c i f i c "pool" of Glut. 10. The a p p l i c a t i o n of various Glut analogues to the abdominal stretch receptor i n the c r a y f i s h indicated that t h i s preparation was more sen s i t i v e to Glut and N-methyl-glutamate than to DLH or N-methyl-aspartate. Since the opposite i s true i n mammalian neurones i t i s possible that the receptors for the amino acids d i f f e r i n these two t i s s u e s . 11. aMG blocked the response of the abdominal stretch receptor to Glut, and GDEE blocked the response of the closer muscle i n the c r a y f i s h to e l e c t r i c a l stimulation of the motor nerve, suggesting that these compounds may have s i m i l a r properties at the cr a y f i s h neuromuscular junction and i n the mammalian C.N.S. 148 BIBLIOGRAPHY Andersen, P.; S.A. Andersson and T. Ljzfrno (1967). Nature of thalamo-cortical spindles during spontaneous barbiturate spindle a c t i v i t y . J . Physiol'., (Lond.), 192, 283-307. Andersen, P. and D.R. Curtis (1964). The pharmacology of the synaptic and acetylcholine-induced e x c i t a t i o n of ventrobasal thalamic neurones. Acta P h y s i o l . Scand., 6jLs 100-120. Aprison, M.H.; L.T. Graham J r . ; D.R. Livengood and R. Werman (1965). D i s t r i b u t i o n of glutamic acid i n the cat spinal cord and r o o t s . Fed. P r o c , 2_4, 462. Arnfred, T. and L. Hertz (1971). E f f e c t s of potassium and glutamate on brain cortex s l i c e s : Uptake and release of glutamic and other amino aci d s . J . Neurochem., 18, 259-265. Arnfred, T.; L.Hertz; L. L o l l e and H. Lund-Andersen (1970). An improved holder for transfer of brain s l i c e s during i n v i t r o incubation. Exp. Brain Res., 11, 373-375. Atwood, H.L.; F. Lang and W.A. Morrin (1972). Synaptic v e s i c l e s : Selective depletion i n c r a y f i s h e x c i t a -tory and i n h i b i t o r y axons. Science, 176, 1353-1355. Ayengar, P. and E. Roberts (1952). I n h i b i t i o n of u t i l i z a t i o n of glutamic acid by l a c t o b a c i l l u s arabinosus. Proc. Soc. Exp. B i o l . Med., 79, 476-481. ~ ~ Baker, M.A. (1971). Spontaneous and evoked a c t i v i t y of neurones i n the somatosensory thalamus of the waking cat. J . P h y s i o l . (Lond.), 217, 359-379-Baker, P.F. and S.J. Potashner (1971). The dependency of glutamate uptake by crab nerve on external Na and K . Biochemica et Biophysica Acta. BBA report, 249, 616-622. B a t t i s t o n , L.; A. Grynbaum and A. Lajtha (1969). D i s t r i -bution and uptake of amino acids i n various regions of the cat brain i n v i t r o . J . Neurochem., 16, 1459-1468. 149 Beranek, R. and P.L. M i l l e r (1968). The action of i o n t o -p h o r e t i c a l l y applied glutamate on insect muscle f i b r e s . J . Exp. B i o l . , 49, 83-93. B e r l , S. and D.D. Clarke (1969). Compartmentation of amino acid metabolism. i n : "Handbook of Neurochemistry. V o l . I I . " Ed: A. Lajtha. Plenum Press, New York. B e r l , S.; D.D. Clarke and W.J. Nicklas (1970). Compart-mentation of c i t r i c acid cycle metabolism i n b_£ain: E f f e c t of aminooxiacetic a c i d , ouabain and Ca on the l a b e l l i n g of^glutamate, glutamine, aspartate, and-, GABA by (1- C) acetate, (U- C) glutamate and (U- C) aspartate. J . Neurochem., 17, 999-1007. B e r l , S. and T.L. F r i g y e s i (1969). The turnover of glutamate, glutamine, aspartate and GABA l a b e l l e d with (1- C) acetate i n caudate nucleus, thalamus and motor cortex ( c a t ) . Brain Res., 12, 444-455. B e r l , S.; W.J. Nicklas and D.D. Clarke (1970). Compart-mentation of c i t r i c acid cycle metabolism i n brain: l a b e l l i n g of glutamate, glutamine, aspartate and GABA by several radioactive metabolites. J . Neurochem., 1_7, 1009-1015. B e r l , S. and D.P.Purpura (1963). Postnatal changes In amino acid content of k i t t e n cerebral cortex. J . Neurochem., 10_, 237-240. B e r l , S. and D.P. Purpura (1966). Regional development of glutamic acid compartmentation i n immature b r a i n . J . Neurochem., 13, 293-304. B e r l , S.; G. Takagaki; D.D. Clarke and H. Waelsch (1962). Metabolic compartments i n v i t r o . Ammonia and glutamic acid metabolism i n brain and l i v e r . J . B i o l . Chem., 237, 2562-2569. Bernardi, G.; W. Zieglgansberger; A. Hertz and E.A. P u i l (1972). I n t r a c e l l u l a r studies on the action of L-glutamic acid on s p i n a l neurones of the cat. Brain Res., 39, 523-525. Bhargava, V. (1972). E f f e c t of Diisopropyl phosphoro-f l u o r i d a t e (DFP) on the somatosensory evoked potentials i n r a t s . Psychopharm., 25, 376-379. 150 Biscoe, T.J. and D.W. Straugham (1966). Microelectro-phoretic studies of neurones i n the cat hippo-campus. J . Physiol. (Lond.), 183, 341-359-Blasberg, R. and A. Lajtha (1966). Heterogeneity of the mediated transport systems of amino acid uptake i n b rain. Brain Res., 1_, 86-104. Bloom, P.E.; and E. Costa and G.C. Salmoiraghi (1965). Anesthesia and the responsiveness of i n d i v i d u a l neurones of the caudate nucleus of the cat to acetylcholine, norepinephrine and dopamine administered by micro-electrophoresis. J . Pharmacol. Exp. Ther., 150, 244-252. Boakes, R.J.; P.B. Bradley; I. Briggs and A. Dray ( 1 9 7 0 ) . Antagonism of 5-hydroxytryptamine by LSD 25 i n the cent r a l nervous system: A possible neuronal basis . for the action of LSD 25. B r i t . J . Pharmacol., 40, 202-218. Bradford, H.P.; E.B. Chain; H.T. Cory and S.P.R. Rose (1969). Glucose and amino acid metabolism i n some inverte-brate nervous systems. J . Neurochem., 16, 969-978. Bradford, H.P. and H. Mcllwain (1966). Ionic basis f o r the depolarisation of cerebral tissues by excitatory a c i d i c amino acids. J . Neurochem., !L3, 1163-1177-Bradford, H.P. and A.J. Thomas (1969). Metabolism of glucose and glutamate by synaptosomes from mammalian cerebral cortex. J . Neurochem., l6_, 1495-1504. Buckser, S. ( 1 9 6 9 ) - The electroretinogram of the sodium glutamate treated albino r a t : i t s c h a r a c t e r i s t i c s and comparison with those of the untreated r a t . Jap. J. Physiol., 19, 547-568. Bulbring, E. and J.H.Burn ( 1 9 4 1 ) . Observations bearing on synaptic transmission by acetylcholine i n the spin a l cord. J. Physiol. (Lond.), 100, 337-368. Burt, A.M. and CH. Narayanan (1972). Development of glucose-6-phosphate, malate and glutamate dehydro-genase a c t i v i t i e s i n the ventral h a l f of the chick spi n a l cord i n the absence of e x t r i n s i c neuronal connections. Exp. Neurol., 34, 342-353. 151 Cohen, H.P.; C. Vasconetto and G.F. Aya l a (1972). The e f f e c t of t o p i c a l a p p l i c a t i o n of d i e t h y l - a -f l u o r o g l u t a r a t e on the metabolism and e l e c t r i c a l a c t i v i t y i n v i v o o f the cat c e r e b r a l c o r t e x . J . Neurochem., 19, 525-534. C o l l i e r , B. and J . F . M i t c h e l l (1967). The c e n t r a l r e l e a s e of a c e t y l c h o l i n e d u r i n g consciousness and a f t e r b r a i n l e s i o n s J . P h y s i o l . '(Lond.), 188, 83-98. Crawford, J.M. and D.R. C u r t i s (1966). s t u d i e s on f e l i n e Betz c e l l s . 186, 121-138. P h a r m a c o l o g i c a l J . P h y s i o l . (Lond.), Crawford, J.M. (1970). The s e n s i t i v i t y o f c o r t i c a l neurones to a c i d i c amino a c i d s and a c e t y l c h o l i n e . B r a i n Res., 17, 287-296. C u r t i s , D.R. (1965). The a c t i o n s o f amino a c i d s upon mammalian neurones. pp. 34-42, i n "Studies i n Phy s i o l o g y p r e s e n t e d t o J.C. E c c l e s " . Eds: D.R. C u r t i s and A.K. M c l n t y r e . S p r i n g e r , H e i d l e b u r g . C u r t i s , D.R. (1966). P h a r m a c o l o g i c a l s t u d i e s of tha l a m i c neurones, pp. 183-191, i n : "The Thalamus" Eds: D.P. Purpura and M.D. Yahr, Columbia U n i v . P r e s s , New York. C u r t i s , D.R.(1969). C e n t r a l S y n a p t i c T r a n s m i t t e r s . pp. 105-129, i n : " B a s i c Mechanisms o f the E p i l e p s i e s " . Eds: H.H. J a s p e r ; A.A. Ward J r . and A. Pope. L i t t l e , Brown and Co ( I n c . ) . C u r t i s , D.R. and J.M. Crawford (1969). C e n t r a l s y n a p t i c t r a n s m i s s i o n - m i c r o e l e c t r o p h o r e t i c s t u d i e s . Ann. Rev. Pharmacol., 9_, 209-240. C u r t i s , D.R. and R. Davis (1962). P h a r m a c o l o g i c a l s t u d i e s upon neurones o f the l a t e r a l g e n i c u l a t e nucleus o f the c a t . B r i t . J . Pharmacol., 18, 217-246. C u r t i s , D.R.-; A.W. Duggan; D. F e l i x ; G.A.R. Johnston; A.K. Teb e c i s and J.C. Watkins (1972). E x c i t a t i o n of mammalian c e n t r a l neurones by a c i d i c amino a c i d s B r a i n Res. 4 l , 283-301. C u r t i s , D.R.; A.W. Duggan and G.A.R. Johnston (1970). The i n a c t i v a t i o n of e x t r a c e l l u l a r l y a d m i n i s t e r e d amino a c i d s i n the f e l i n e s p i n a l c o r d . Exp. B r a i n Res;, 10, 447-462. C u r t i s , D.R.; A.W. Duggan and G.A.R. Johnston (1971). The s p e c i f i c i t y of strychnine as a glycine antago-n i s t i n the mammalian spi n a l cord. Exp. Brain Res., 12, 547-565. C u r t i s , D.R. and K. Koizumi (1961). Chemical transmitter substances i n brain stem of cat. J . Neurophysiol, 24, 80-90. C u r t i s , D.R.; D.D. Perrin and J.C. Watkins (i960). The e x c i t a t i o n of spinal neurones by the iontophoretic a p p l i c a t i o n of agents which chelate calcium. J . Neurochem., 6_, 1-20. C u r t i s , D.R.; J.W. P h i l l i s and J.C. Watkins (i960). The chemical e x c i t a t i o n of sp i n a l neurones by c e r t a i n a c i d i c amino a c i d s . J . P h y s i o l . (Lond.), 150, 656-682. C u r t i s , D.R..; J.W. P h i l l i s and J.C. Watkins (1961). Cholinergic and non-cholinergic transmission i n the mammalian s p i n a l cord. J . P h y s i o l . (Lond.), 158, 296-323. C u r t i s , D.R. and R.W. Ryall (1966). Pharmacological studies upon sp i n a l presynaptic f i b r e s . Exp. Brain Res., 1, 195-204. C u r t i s , D.R. and J.C. Watkins (i960). The e x c i t a t i o n and depression of spinal neurones by s t r u c t u r a l l y r e l a t e d amino ac i d s . J . Neurochem., 6^ , 117-141. C u r t i s , D.R. and J.C. Watkins (1963). A c i d i c amino acids with strong excitatory actions on mammalian neurones. J . P h y s i o l . (Lond.), 166, 1-14. Davidoff, R.A.; L.T. Graham J r . ; R.P. Shank; R. Werman and M.H. Aprison (1967). Changes i n amino acid concentrations associated with loss of sp i n a l interneurones. J . Neurochem., l4_, 1025-1-31. Davidson, N. and CP.P. Southwick (1971). Amino acids and presynaptic i n h i b i t i o n i n the rat cuneate nucleus. J . P h y s i o l . (Lond.), 219, 689-708. Davies, J . and J . C Watkins (1972). Is l-hydroxy-3-aminopyrrolidone-2 (HA-966) a s e l e c t i v e e x c i t a -tory amino acid antagonist. Nature New B i o l . , 238, 61-63. De F e u d i s , F.V. (1971). E f f e c t s o f e l e c t r i c a l s t i m u l a t i o n on the e f f l u x o f L-glutamate from p e r i p h e r a l nerve i n v i t r o . Exp. Neu r o l . , 30., 291-296. De R o b e r t i s , E.; O.Z. S e l l i n g e r ; G. Rodriguez de Lores A r n a i z ; A.M. A l b e r i c i and L.M. Zi e h e r (1967). Nerve endings i n methionine s u l f o x i m i n e c o n v u l -sant r a t s , a neurochemical and u l t r a s t r u c t u r a l study. J . Neurochem., l4_, 8I-89. D e s i , I . ; I.D.I. Far k a s ; J . Sos and A. Balogh (1967) . N e u r o p h y s i o l o g i c a l e f f e c t s o f glutamic a c i d e t h y l e s t e r . A c t a P h y s i o l . Acad. S c i . Hung., 32, 323-335. Diamond, J . (1963) • V a r i a t i o n i n the s e n s i t i v i t y t o gamma-amino-butyric a c i d of d i f f e r e n t r e g i o n s of the Mauthner neurone. Nature, (Lond.), 199, 773-775. Dobkin, J . (1970). R e v e r s i b l e changes i n glutamine l e v e l s i n the cat c e r e b r a l c o r t e x evoked by a f f e r e n t e l e c t r i c a l s t i m u l a t i o n and by a d m i n i s t r a t i o n o f pen t a r a e t h y l e n e t e t r a z o l e ( P e n t y l e n e t e t r a z o l ) . J . Neurochem., 17, 237-246. Douglas, W.W. and A.M. P o i s n e r (1966). On the r e l a t i o n between ATP s p l i t t i n g and s e c r e t i o n i n the a d r e n a l c h r o m a f f i n c e l l : e x t r u s i o n of ATP (unhydrolysed) d u r i n g r e l e a s e of catec h o l a m i n e s . J . P h y s i o l . , 183, 249-256. D r a v i d , A.R.; W.A. Himwich and J.M. Davis (1965) . Some f r e e amino a c i d s i n dog b r a i n d u r i n g development. J . Neurochem., 12_, 901-906. Dudel, J . and S.W. K u f f l e r (i960). E x c i t a t i o n at the c r a y f i s h neuromusclular j u n c t i o n w i t h decreased membrane conductance. Nature (Lond.), 187., 246-247. Duggan, A.W. (1971). Amino a c i d s as t r a n s m i t t e r s . Ph. D, T h e s i s . A u s t r a l i a n N a t i o n a l U n i v e r s i t y . Quoted by C u r t i s , D.R.; A.W. Duggan; D. F e l i x ; G.A.R. Johnston and A.K. Teb e c i s (1972) - E x c i t a t i o n o f mammalian c e n t r a l neurones by a c i d , amino a c i d s . B r a i n Res. 4 l , 283-301. Duggan, A.W. and G.A.R. Johnston (1970a). Glutamate and related amino acids i n cat, dog and rat sp i n a l r o o t s . Comp. gen. Pharmacol., 1, 127-128 . Duggan, A.W. and G.A.R. Johnston (1970b). Glutamate and related amino acids i n cat spinal r o o t s , dorsal root ganglia and peripheral nerves. J . Neurochem. 17, 1205-1208. E l l i o t t , T.R. ( 1904) . On the action of adrenalin. J . Ph y s i o l . , 3_lj xx-xxi. Faeder, I.R. and M.M. Salpeter ( 1970) . Glutamate uptake by a stimulated insect nerve muscle preparation. J . C e l l . B i o l . , _46, 300-307. Farrow, J.T. and R.D. O'Brien (1971) . Metabolites of ( H)- acetate bound to synaptic v e s i c l e s i s o l a t e d from rat cerebral cortex. J . Neurochem., 18, 963-973. Feldberg, W. and M. Vogt ( 1948) . Acetylcholine synthesis i n d i f f e r e n t regions of the central nervous system. J . Physiol.(Lond.), 107, 372-381. F i f k o v a , E. and A. van Harreveld ( 1970) . E f f e c t of glutamate on the striatum of the chicken. Fed. P r o c , 29, 264. F l o r e y , E. (1957). Chemical transmission and adaptation. J . Gen. P h y s i o l . , 40, 533-545-Foulkes, J.A. and N. Robinson ( 1970) . L a b e l l i n g of synaptosomes and axoplasmic flow i n the brain stem of the r a t . J . Neurochem., 17, 1429-1431. Frederickson, R.C.A.; L.M. Jordan and J.W. P h i l l i s ( 1 9 7 1 ) . The action of noradrenaline on c o r t i c a l neurones: eff e c t s of pH. Brain Res., 35., 556-560. Galindo, A.; K. Krnjevic and S. Schwartz (1967) . Micro-iontophoretic studies on neurones i n the cuneate nucleus. J . P h y s i o l . (Lond.), 192,- 359-377. Galindo, A.; K. Krnjevic and S. Schwartz ( 1968) . Patterns of f i r i n g i n cuneate neurones and some eff e c t s of F l a x e d i l . Exp. Brain Res., 5 , 87-101. Graham, L.T. J r . ; R.P. Shank; R. Werman and M.H. Aprison (1967) . D i s t r i b u t i o n of some synaptic transmitter suspects i n cord glutamic a c i d , aspartic a c i d , y-aminobutyric a c i d , glycine and glutamine. J . Neurochem., L 4 , 465-472. 155 Green, J.D. (1958). A simple microelectrode for recording from the C.N.S. Nature, (Lond.), 182,, 962. Grof, P. and 0. Vinar (1965). Preliminary comparative t r i a l of proheptatriene and imipramine i n the treatment of depressions. A c t i v . Nerv. Sup., 7, 288-289. Halpern, L.M. and R.G. Black (1967). P l a x e d i l (gallamine: t r i e t h i o d i d e ) : evidence for a central a c t i o n . Science, 155, I685-I687. Hansson, H.-A. (1970). U l t r a s t r u c t u r a l studies on the long term e f f e c t s of sodium glutamate on the rat r e t i n a . Virchows Arch. Abt. B Z e l l p a t h . , 6_, 1-11. Harvey, J.A. and H. Mcllwain (1968). Excitatory a c i d i c amino acids and the cation content and sodium ion flux of i s o l a t e d tissues from the b r a i n . Biochem. J . , 108, 269-274. Hayashi, T. (1954). E f f e c t s of sodium glutamate on the nervous system, Keio J . Med., 3, 183-192. Himwich, W.A.; J.C. Petersen and M.L. A l l a n (1957). Hematoencephalic exchange as a function of age. Neurology, 7, 705-710. Holton, F.A. and P. Holton (1954). The c a p i l l a r y d i l a t o r substances i n dry powders of spinal roots; a possible role of adenosine triphosphate i n chemical transmission from nerve endings. J . P h y s i o l . (Lond.), 126, 124-140. Holton, P. and W.L.M. Perry (1951). On the transmitter responsible for antidromic vas o d i l a t a t i o n i n the i n the rabbits ear. J . P h y s i o l . (Lond'.), 114, 240-251. Iversen, L.L. and E.A. Kravitz (1968). The metabolism of Y-aminobutycic acid (GABA) i n the lobster nervous system - uptake of GABA i n nerve-muscle preparations. J . Neurochem., 15., 609-620. Jansen, J.K.S.; A. Nja; K. Ormstad and L. Walljzfe (1971). On the innervation of the slowly adapting stretch receptor of the c r a y f i s h abdomen. An e l e c t r o -p h y s i o l o g i c a l approach. Acta. P h y s i o l . Scand., 81, 273-285. 156 Jasper, H.H.; R.T. Kahn and K.A.C. E l l i o t t (1965). Amino acids released from the cerebral cortex i n r e l a t i o n to i t s state of a c t i v a t i o n . Science, l47> 1448-1449. Jasper, H.H. and I. Koyama (1968). Amino acids released from the c o r t i c a l surface i n cats following stimulation of the mesial thalamus and midbrain r e t i c u l a r formation. Electroenceph. c l i n . Neurophysiol., 24_, 292. Jasper, H.H. and I. Koyama (1969). Rate of release of amino acids from the cerebral cortex i n the cat as affected by b r a i n stem and thalamic s t i m u l a t i o n . Can. J . P h y s i o l . Pharmacol., 47, 889-905. Johnson, J.L. (1972). Glutamic acid as a synaptic t r a n s -mitter i n the nervous system. A Review. Brain Res., 37, 1-19. Johnson, J.L. and M.H. Aprison (1970). The d i s t r i b u t i o n of glutamic a c i d , a transmitter candidate, and other amino acids i n the dorsal sensory neuron of the c a t . Brain Res., 24_, 285-292. Johnson, J.L. and M.H. Aprison (1971). The d i s t r i b u t i o n of glutamate and t o t a l free amino acids i n t h i r t e e n s p e c i f i c regions of the cat c e n t r a l nervous system. Brain Res., 26, 141-148.^ Jordan, L.M.; R.C.A. Frederickson; J.W. P h i l l i s and N. Lake (1972). Microelectrophoresis of 5-hydroxytrypta-mine: a c l a r i f i c a t i o n of i t s action on cerebral c o r t i c a l neurones. Brain Res., 4j0, 552-558. Katz, R.I.; T.N. Chase and I . J . Kopin (1969). E f f e c t of ions on stimulus-induced release of amino acids . from mammaliam brain s l i c e s . J . Neurochem., 16, 961-967. Keesey, J . C ; H. Wallgren and H. Mcllwain (1965). The sodium, potassium and chloride of cerebral t i s s u e s : Maintenance, change on stimulation and subsequent recovery. Biochem. J.m 9_5, 289-300. Kerkut, G.A.; L.D. Leake; A. Shapiro; S. Cowan and R.J. Walker (1965). The presence of glutamate i n nerve-muscle perfusates of H e l i x , Carcinus and Periplaneta. Comp. Biochem. P h y s i o l . , 15_, 485-502. 157 Kerkut, G.A.; A. Shaping and R.J. Walker (1967).' The transport of C-labelled material from C.N.S. 5=* muscle along a nerve trunk. Comp. Biochem. P h y s i o l . , 2_3, 729-748. Kishida, K. and K.I. Naka (1968). Interaction of excitatory and depressant amino acids i n the frog r e t i n a . J . Neurochem., 15_, 833-841. K l e i n , D.F. and J.M. Davis (1969). "Diagnosis and drug treatment of p s y c h i a t r i c disorders", pp. 187-298. Williams and Wilkins Co. (Baltimore). K r a v i t z , E.A.; S.W. K u f f l e r ; D.D. Potter and N.M. von Gelder (1963). Gamma-aminobutyric acid and other blocking compounds i n C r u s t a c e a . J . Neurophysiol., 26, 729-738. K r a v i t z , E.A.; C.R. Sl a t e r ; K. Takahashi; M.D. Bownds and R.M. Grossfeld (1970). Excitatory transmission i n invertebrates - Glutamate as a p o t e n t i a l neuromuscular transmitter compound, pp. 85-94, i n : "Excitatory synaptic mechanisms". Eds: Anderson, P. and J.K.S. Jansen. Scandinavian University Books (Oslo). Krebs, H.A.; L.V. Eggleston and R. Hems (1949). D i s t r i b u t i o n of glutamine and glutamic acid i n animal t i s s u e s . Biochem. J . , 44_, 159-163. Krebs, H.A. and L.V. Eggleston (1949). The e f f e c t of L-glutamate on the loss of potassium ions by brain s l i c e s suspended i n a saline medium. Biochem. J . , 44, v i i . K r n j e v i c , K. (1964). Micro-iontophoretic studies on c o r t i c a l neurones, .pp. 41-98, i n : "International review of neurobiology, Vol 7." Eds: C.C. P f e i f f e r J.R. Smythies. Academic Press (New York). Kr n j e v i c , K. and J.W. P h i l l i s (1961). The actions of ce r t a i n amino acids on c o r t i c a l neurones. J . Ph y s i o l . (Lond.), 159, 62P-63P. Krn j e v i c , K. and J.W. P h i l l i s (1963a). Iontophoretic studies of neurones i n the mammalian cerebral cortex. J . P h y s i o l . (Lond.), 165_, 274-304. Krnj e v i c , K. and J.W. P h i l l i s (1963b). Pharmacological properties of a c e t y l c h o l i n e - s e n s i t i v e c e l l s i n the cerebral cortex. J . P h y s i o l . (Lond.), 166, 328-350. 158 Krn j e v i c , K. and V.P. Whittaker (1965). E x c i t a t i o n and depression of c o r t i c a l neurones by brain f r a c t i o n s released from micropipettes. J . P h y s i o l . (Lond.), 179, 298-322. Kuher, M.J. and S.H. Snyder (1969). -.Localization of 3H-glutamic acid (JH-Glut) and -%-glycine ( H-GLY) i n synaptosomes of rat cerebral cortex. Fed. P r o c , 28, 578. Kuher, M.J. and S.H. Snyder,.(1970) . The s u b c e l l u l a r d i s -t r i b u t i o n of free H-glutamic acid i n rat cerebral c o r t i c a l s l i c e s . J . Pharmacol. Exp. Ther., 171, 141-152. Lamar, C. J r . (1968). The duration of the i n h i b i t i o n of glutamine synthetase by methionine sulfoximine. Biochem. Pharmacol., 17/ 636-640. Lamar, C. J r . and O.Z. S e l l i n g e r (1965) . The i n h i b i t i o n i n vivo of cerebral glutamine synthetase and glutamine transferase by the convulsant methionine sulfoximine. Biochem. Pharmacol., 14, 489-502. Legge, K.F.; M. Randic and D.W. Straugham (1966). The pharmacology of neurones i n the pyriform cortex. B r i t . J . Pharmacol., 26, 87-107. Lembeck, F. (1953). Zur Frage der zentralen ilbertragung afferenter Impulse. I l l Das Vorkommen und die Bedeutung der Substanz P i n den dorsalen Wurzeln des Ruckenmarks. Arch, exper. Path. u. Pharmakol., 219, 197-213. Lewis, P.R. (1952). The free amino acids of invertebrate nerve. Biochem J . , 5_2, 330-338 . Logan W.J. and S.H. Snyder (1971). Unique high a f f i n i t y uptake systems for g l y c i n e , glutamic and aspartic acids i n central nervous tissue of the r a t . Nature, (Lond.), 234, 297-299. Logan, W.J. and S.H. Snyder (1972). High a f f i n i t y uptake systems f o r g l y c i n e , glutamic and aspartic acids i n synaptosomes of rat central nervous t i s s u e s . Brain Res., _42, 413-431. Lucas, D.R. and J.P. Newhouse (1957). The toxic e f f e c t of sodium-L-glutamate on the inner layers of the r e t i n a . AMA. Arch. Ophth., 58, 193-201. 159 Lund-Andersen, H. and L. Hertz (1970). E f f e c t s of potassium and of glutamate on swelling and on sodium and potassium content i n brain-cortex s l i c e s from adult r a t s . Exp. Brain Res., 11, 199-212. Mangan, J.L. and V.P. Whittaker (1966). The d i s t r i b u t i o n of free amino acids i n su b c e l l u l a r fra c t i o n s of guinea-pig b r a i n . Biochem. J . , 9_8, 128-137 -Marks, N.; R.K. Datta and A. Lajtha (1970). D i s t r i b u t i o n of amino acids and of exo- and endopeptidases along vertebrate and invertebrate neurones. J . Neurochem., 17, 53-63. Marshall, K.C. (197D- Ph y s i o l o g i c a l and pharmacological studies of the f e l i n e thalamus. Ph.D. t h e s i s , University of B r i t i s h Columbia. McCance, I.; J.W. P h i l l i s and R.A. Westerman (1968). Acetylcholine-sensitivy of thalamic neurones:its re l a t i o n s h i p to synaptic transmission. B r i t . J . Pharmacol., 32, 635-651-McDonald, T.J.-and R.D.O'Brien (1972). Relative potencies of L-glutamate analogs on excitatory neuromuscular synapses of the grasshopper, Romelea Microptera. J . Neurobiol., 3, 277-290. McGeer, P.L.; E.G. McGeer and J.A. Wada (1963). Central aromatic amine level s and behaviour. II Serotonin and catecholamine l e v e l s i n various cat brain areas following administration of psycho-active drugs or amine precursors. Archs. Neurol.-(Chicago). 9, 81-89. Mcllwain, H.;. J.A. Harvey and G. Rodriguez (1969). Tetrodotoxin on the sodium and other ions of cerebral t i s s u e s , excited e l e c t r i c a l l y and with glutamate. J . Neurochem., IjS, 363-370 . Mcintosh, P.C. (1941). The d i s t r i b u t i o n of acetylcholine i n the peripheral and central nervous system. J . P h y s i o l . (Lond.), 99, 436-442. McLennan, H. (1963). "Synaptic transmission". W.B. Saunders Co. (P h i l a d e l p h i a ) . McLennan, H. (1970a). "Synaptic transmission, 2nd e d i t i o n . " W.B. Saunders Co. ( P h i l a d e l p h i a ) . 160 McLennan, H. (1970b). I n h i b i t i o n of long duration i n the cerebral cortex. A quantitave difference between excitatory amino a c i d s . Exp. Brain Res., 10, 417-426. McLennan, H. (1971). The pharmacology of i n h i b i t i o n of m i t r a l c e l l s i n the olfactory bulb. Brain Res., 29, 177-184. McLennan, H.; R.D. Huffman and K.C. Marshall (1968). Patterns of e x c i t a t i o n of thalamic neurones by amino acids and by a c e t y l c h o l i n e . Nature, (Lond.), 219, 387-388. McLennan, H. and D.H. York (1966). Cholinergic mechanisms inthe caudate nucleus. J . P h y s i o l . (Lond.), 187, 163-175. Noback, C.R. and D.P. Purpura (1961). Postnatal ontogenesis of neurons i n cat cortex. J . Comp. Neurol., 117, 291-301. O l i v i e r , A,; A. Parent and C.J. P o i r i e r (1970). I d e n t i f i -cation of the thalamic nuclei on the basis of t h e i r cholinesterase content i n the monkey. J . Anat., 106, 37-50. Olney, J.W. (1969). Brain l e s i o n s , obesity and other disturbances i n mice treated with monosodium glutamate. Science, 164, 719-721. Olney, J.W. (1971). Glutamate-induced neuronal necrosis i n the infant mouse hypothalamus: An electron microscopic study. J . Neuropath. Exp. Neurol., 30, 75-90. Olney, J.W. and O.L. Ho (1970). Brain damage i n infant mice following o r a l intake of glutamate, aspartate or cysteine. Nature, (Lond.), 227, 609-610. Olney, J.W.; O.L. Ho and V. Rhee (1971). Cytotoxic effects of a c i d i c and sulphur containing amino acids on the infant mouse central nervous system. Exp. Brain Res., 14, 61-76. O'Neal, R.M. and R.E. Koeppe (1966). Precursors i n vivo of glutamate, aspartate, and t h e i r derivatives i n rat b r a i n . J . Neurochem., 13, 835-847. 161 Owens, I. and J . J . Blum (1966). Amino acid esters as i n h i b i t o r s of aminoacyl-transfer ribonucleic acid synthetases i n Euglena and As t a s i a . J . B i o l . Chem., 242, 2893-2902. Owens, I.S. and J . J . Blum (1969). Induction of abnormal c e l l d i v i s i o n i n Euglena g r a c i l i s by glutamic d i e t h y l e s t e r . J . Protozoology., l6_, 211-215. Ozeki, M.; A.R. Freeman and H. Grundfest (1966). The membrane components of crustacean neuromuscular systems. I. Immunity of d i f f e r e n t electrogenic components to tetrodotoxin and s a x i t o x i n . J . Gen. Ph y s i o l . , 49, 1319-1334. Ozeki, M. and M. Sato (1970). Potentiation of excitatory j u n c t i o n a l potentials and glutamate-induced responses i n c r a y f i s h muscle by 5-ribonucleotides. Comp. Biochem. P h y s i o l . , 32, 203-218.. Perez, V.J. and J.W. Olney (1972). Accumulation of glutamic acid i n the arcuate nucleus of the hypothalamus of the infant mouse following sub-cutaneous administration of monosodium glutamate. J . Neurochem., 19, 1777-1782. Perry, T.L.; K. Berry; S. Hansen; S. Diamond and C. Mok (197D. Regional d i s t r i b u t i o n of amino acids i n human brain obtained at autopsy. J . Neurochem., 18, 513-519. Peterson, N.A.; CM. McKean and E. Raghupathy (1972). E f f e c t s of phenothiazines on amino acid transport and protein synthesis i n i s o l a t e d nerve endings. Biochem. Pharmacol., 21, 1275-1287-P h i l l i s , J.W. and S. Ochs (1971). E x c i t a t i o n and depression of c o r t i c a l neurones during spreading depression. Exp. Brain Res., 12, 132-149. P h i l l i s , J.W. and A.K. Tebecis (1967) . The response of thalamic neurones to ion t o p h o r e t i c a l l y applied monoamines. J . P h y s i o l . (Lond.), 192, 715-745-Preston, J.B. (1955). The influence of thiosemicarbazide on e l e c t r i c a l a c t i v i t y recorded i n the anterior brain stem of the c a t . J . Pharmacol. Exp. Ther., 115, 39-45. 162 P u l l , E.A. and W. Zieglgansberger (1972). E f f e c t of iont o p h o r e t i c a l l y applied tetrodotoxin on the action of L-glutamic acid i n the central nervous system. Proc. Can. Fed. B i o l . S o c , 15, number 107. P u l l , I.; H. Mcllwain and R.L. Ramsey (1970). Glutamate, calcium ion-chelating agents and the sodium and potassium ion contents of tissues from the b r a i n . Biochem. J . , 116, 181-187. R a i l , W.; R.E. Burke; T.G. Smith; P.G. Nelson and K. Frank (1967). Dendritic l o c a t i o n of synapses and possible mechanisms f o r the monosynaptic EPSP i n motoneurons. J . Neurophysiol., 30., 1169-1193. Ramsey, R.L.and H. Mcllwain (1970). Calcium content and exchange i n neoc o r t i c a l tissues during the cation movements induced by glutamates. J . Neurochem., 17, 781-787. Redding, T.W.; A.V. Schally; A. Arimura and I. Wakabayashi (1971). E f f e c t of monosodium glutamate on some endocrine functions. Neuroendocrinology, 8_, 245-255. Reisoso-Suarez, F. (1961). Topographischer Hirnatlas der Katze. E. Merck (Durmstadt). Reiser, S. and P.A. Christiansen (1965). I n t e s t i n a l transport of amino acids studied with L- v a l i n e . Amer. J . P h y s i o l . , 208, 914-921. Ritthausen, H. (1866). Ueber die Glutaminsaure. J . Prakt. Chem., 99_, 454-462. R i z z o l i , A.A. (1968). D i s t r i b u t i o n of glutamic a c i d , aspartic a c i d , y-a-minobutyric acid and glycine i n s i x areas of cat spinal cord before and a f t e r t r a n s e c t i o n . Brain Res., 1]., 11-18. Robbins, J . (1959). The e x c i t a t i o n and i n h i b i t i o n of crustacean muscle by amino a c i d s . J . P h y s i o l . (Lond.), 148, 39-50. Roberts, E. (1952). I n h i b i t i o n of b a c t e r i a l and brain glutamic acid decarboxylases. Fed. P r o c , 11, 275. 163 Roberts, R.B.; J.B. Plexner and L.B. Flexner (1959). Biochemical and ph y s i o l o g i c a l d i f f e r e n t i a t i o n during morphogenesis - XXIII. J . Neurochem., 4, 78-90. Rowe, W.B. and A. Meister (1970). I d e n t i f i c a t i o n of L-methionine-S-sulfoximine as the convulsant isomer of methionine sulfoximine. Proc. Nat. Acad. Sc., 66, 500-506. R y a l l , R.W. (1962). Subcellular d i s t r i b u t i o n of pharma-c o l o g i c a l l y active substances i n guinea pig brain. Nature, (Lond.), 196, 680-681. Salganicoff, L. and E. De Robertis (1965). Subcellular d i s t r i b u t i o n of the enzymes of the glutamic acid, glutamine and y-aminobutyric acid cycles i n rat brai n . J . Neurochem., 12, 287-309. Salmoiraghi, G.C. and P.A. Steiner (1963). Acetylcholine s e n s i t i v i t y of cat's medullary neurons. J . Neurophysiol., 26_, 581-597. Schwartz, S.; D. G i b l i n and V.E. Amassian (1964). Resting a c t i v i t y of cuneate neurons. Fed. Proc., 23, 466. ~~ Schwerin, P.; S.P. Bessman and H. Waelsch (1950). The uptake of glutamic acid and glutamine by brain and other tissues of the rat and mouse. J . B i o l . Chem., 184, 37-44. . S e l l i n g e r , O.Z. and P.Weiler (1963). The nature of the i n h i b i t i o n i n v i t r o of cerebral glutamine synthe-tase by the convulsant, methionine sulphoximine. Biochem. Pharmacol., 12, 989-1000. Shanes, A.M. and C P . Bianchi (1959). The d i s t r i b u t i o n and k i n e t i c s of release of radiocalcium i n tendon and s k e l e t a l muscle. J. Gen. Physiol., 42, 1123-1137. Shemisa, O.A. and L.H. Fahien (1971). Modifications of glutamate dehydrogenase by various drugs which a f f e c t behaviour. Molec. Pharmacol., 8-25. S i l b e r , R.H. (1941). The free amino acids of lobster nerve. J . C e l l . Comp. Physiol., 18, 21-30. 164 Stadie, W.C. and B.C. Riggs (1944). Microtome for the preparation of tissue s l i c e s f o r metabolic studies of surviving tissues i n v i t r o . J . B i o l . Chem., 154, 687-690. Stanek, J . and R.H.P. Manske (1954). Phthalideisoquinoline a l k a l o i d s , pp. l67-198,.in: "The a l k a l o i d s , chemistry and physiology, Vol IV." Eds: R.H.P. Manske and H.L. Holmes. Academic Press Inc,(N.Y.). Snider, R.S. and W.T. Neimer (1961). "A stereotaxic atlas of the cat brain." The University of Chicago Press Srinivasan, V; M.J. Neal and J.P. M i t c h e l l (1969). The eff e c t of e l e c t r i c a l stimulation and high potassium concentrations on the e f f l u x of ( H) Y_ a min o-b utyric acid from brain s l i c e s . J . Neurochem., 16, 1235-1244. Steiner, P.A. and M. Meyer (1966). Actions of L-glutamate, acetylcholine and dopamine on single neurones i n the nucleus cuneatus and g r a c i l i s of the c a t . Exp e r i e n t i a , 22_, 58-59. Steiner, P.A. and K. Ruf (1966). Excitatory effects of L-glutamic acid upon single unit a c t i v i t y i n rat brain and t h e i r modification by thiosemicarbazide and pyridoxal-5-phosphate. Helv. P h y s i o l . Acta., 24, 181-192. Steiner, P.A. and K. Ruf (1967). Interactions of L-glutamic a c i d , gamma-aminobutyric acid and pyrisoxal-5-phosphate at the neuronal l e v e l . Schweiz. Arch. Neurol. Neuroch. Psych., 100, 310-320. Stern, J.R.; L.V. Eggleston; R. Herns and H.A. Krebs (1949). Accumulation of glutamic acid i n i s o l a t e d b r a i n t i s s u e . Biochem. J . 44_, 410-418. Stone, T.W. (1972). Cholinergic mechanisms i n the rat somatosensory cerebral cortex. J . P h y s i o l . (Lond.), 225, 485-499. Straughan, D.W. and K.P. Legge (1965). The pharmacology of amygdaloid neurones. J . Pharm. Pharmacol., 17, 675-677. Takeuchi, A. and N. Takeuchi (i960). On the permeability of end-plate membrane during the action of trans -m i t t e r . J . P h y s i o l . (Lond.), 154, 52-67. T a k e u c h i , A. and N. T a k e u c h i (1964). The e f f e c t on c r a y f i s h muscle on i o n t o p h o r e t i c a l l y a p p l i e d g l u t a m a t e . J . P h y s i o l ( L o n d . ) , 170, 296-317. T a r a s k e v i c h , P.S. (1971). R e v e r s a l p o t e n t i a l s o f L-gl u t a m a t e and t h e e x c i t a t o r y t r a n s m i t t e r a t the neuromusc u l a r j u n c t i o n o f t h e c r a y f i s h . B i o c h i m . B i o p h y s . A c t a . , 241, 700-703-T a l l a n , H.H. (1962). A s u r v e y o f amino a c i d s and r e l a t e d compounds i n nervous t i s s u e . pp. 471-485, i n : "Amino a c i d p o o l s . " Ed: J.T. Hol d e n . E l s e v i e r P u b l . Co. (Amsterdam). Thunberg, T. (1920). Zur K e n n t n i s des i n t e r m e d i a r e n S t o f f w e c h s e l s und d e r d a b e i wirksamen Enzyme. Skand. A r c h . P h y s i o l . , 40_, 1-91. Thunberg, T. (1923). Zur K e n n t n i s d e r S t o f f w e c h s e l e n z y m e d e r N e r v e n f a s e r . Skand. A r c h . P h y s i o l . , 43, 275-286. Tower, D.B. (I960). The n e u r o c h e m i s t r y o f a s p a r a g i n e and g l u t a m i n e . pp. 173-204, i n : "The n e u r o c h e m i s t r y o f n u c l e o t i d e s and amino a c i d s " Eds: R.O. Brady and D.B. Tower. John W i l e y and Sons I n c . (N.Y.). Umrath, K. and M. G r a l l e r t (1967). Uber n e r v o s e Hemmungs-s u b s t a n z e n d e r W i r b e l t i e r e und i i b e r W i r kungs-mechanismen von Psychopharmaka. Z. B i o l . , 115, 322-364. Usherwood, P.N.R. (1963). Spontaneous m i n i a t u r e p o t e n t i a l s from I n s e c t muscle f i b r e s . J . P h y s i o l . ( L o n d . ) , 169, 149-160. Usherwood, P.N.R.; D.G. Cochrane and D. Rees (1968). Changes i n s t r u c t u r a l , p h y s i o l o g i c a l and pharma-c o l o g i c a l p r o p e r t i e s o f i n s e c t e x c i t a t o r y n e r v e -muscle synapses a f t e r motor nerve s e c t i o n . N a ture ( L o n d . ) , 218, 589-591.. Usherwood, P.N.R. and P. M a c h i l i (1968)'. P h a r m a c o l o g i c a l p r o p e r t i e s o f e x c i t a t o r y n e u r o m u s c u l a r synapses i n the l o c u s t . J . Exp. B i o l . , 49., 341-361. Usherwood, P.N.R.; P. M a c h i l i and G. L e a f (1968). L - g l u t a m a t e a t i n s e c t e x c i t a t o r y n e r v e - m u s c l e s y n a p s e s . Nature ( L o n d . ) , 219, 1169-1172. 166 van den Berg, G.J. (1970). Compartmentation of glutamate metabolism i n the developing brain: experiment with l a b e l l e d glucose, acetate, phenyalanine, tyrosine and p r o l i n e . J . Neurochem., 17_, 973-983. van Gelder, N.M. (1971). Molecular arrangement for ph y s i o l o g i c a l action of glutamic acid and Y- a min o -butyric a c i d . Can. J . P h y s i o l . Pharmacol., 49, 513-519. van Harreveld, A. (1936). A ph y s i o l o g i c a l s a l t s o l u t i o n f o r fresh-water crustaceans. Proc. Soc. Exp. B i o l . (N.Y.) 3_4, 428-432. van Harreveld, A. (1959). Compounds i n brain extracts causing spreading depression of cerebral c o r t i c a l a c t i v i t y and contraction of crustacean muscle. J . Neurochem., 3., 300-315. van Harreveld, A. and E. Fifkova (1970). Glutamate release from the r e t i n a during spreading depression. J . Neurobiol., 2, 13-29. van Harreveld, A. and E. Fifkova (1972). E f f e c t s of metabolic i n h i b i t o r s on the release of glutamate from the r e t i n a . J . Neurochem., 19, 1439-1450. van Harreveld, A. and M. Kooiman (1965). Amino acid release from the cerebral cortex during spreading depression and asphyxiation. J . Neurochem., 12, 431-439. van Harreveld, A. and M. Mendelson (1959). Glutamate-induced contractions i n crustacean muscle. J . C e l l . Comp. P h y s i o l . , 5_4, 85-94 . van Baumgarten, R.; F.E. Bloom; A.P. Oliver and G.C. Salmoiraghi (1963). Response of i n d i v i d u a l o l f a c t o r y nerve c e l l s to microelectrophoretically administered chemical substances. Pflugers Arch, ges. P h y s i o l . , 277, 125-140. Wall, P.D. (1967). The mechanisms of general anesthesia. Anesthesiology, 28, 46-52. Weight, F.F. and G.C. Salmoiraghi (1966). Responses of spi n a l cord interneurons to a c e t y l c h o l i n e , norepinephrine and serotonin administered by microelectrophoresis. J . Pharmacol. Exp. Ther., 153,420-427. Weil-Malherbe, H. (1950). Significance of glutamic acid for the metabolism of nervous t i s s u e . P h y s i o l . Rev., 30, 549-568. 167 Weil-Malherbe, H. (1969). Activators and i n h i b i t o r s of brain glutaminase. J . Neurochem., lj>, 855-864. Werman, R. (1966). A review - c r i t e r i a f or i d e n t i f i c a t i o n of a central nervous system transmitter. Comp. Biochem. P h y s i o l . , 18, 745-766. Wheeler, D.D.; L.L. Boyarsky and W.H. Brooks (1966). The release of amino acids from nerve during stimulation. J . C e l l . P h y s i o l . , 67, 141-148. Whittaker, V.P. (1965). The ap p l i c a t i o n of su b c e l l u l a r f r a c t i o n a t i o n techniques to the study of brain f u n c t i o n . Prog. Biophys. Moi. B i o l . , 15, 41-96. Wiersma, G.A.G.j E. Furshpan.. and E. Florey (1953). Phys i o l o g i c a l and pharmacological observations on muscle receptor organs of the c r a y f i s h , Cambarus C l a r k i i G i r a r d . J . Exp. B i o l . , 30, 136-150. Wofsey, A.R.; M.J. Kuhar and S.H. Snyder (1971). A unique synaptosomal f r a c t i o n , which accumulates glutamic and aspartic a c i d s , i n brain t i s s u e . Proc. Nat. Acad. S c i . (Wash.), 68, 1102-1106. Wolff, L. (1890). Ueber Glyoxylpropionsaure und einige Abkommlinge derselben. Liebigs Ann. Chem., 260, 79-136. 

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