UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies in the neural control of avian locomotion Sholomenko, Gerald Norman Weinstein 1989

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

Item Metadata

Download

Media
831-UBC_1989_A1 S56.pdf [ 14.63MB ]
Metadata
JSON: 831-1.0098328.json
JSON-LD: 831-1.0098328-ld.json
RDF/XML (Pretty): 831-1.0098328-rdf.xml
RDF/JSON: 831-1.0098328-rdf.json
Turtle: 831-1.0098328-turtle.txt
N-Triples: 831-1.0098328-rdf-ntriples.txt
Original Record: 831-1.0098328-source.json
Full Text
831-1.0098328-fulltext.txt
Citation
831-1.0098328.ris

Full Text

STUDIES IN THE NEURAL CONTROL OF AVIAN LOCOMOTION By GERALD NORMAN WEINSTEIN SHOLOMENKO B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1974 M.Sc, The U n i v e r s i t y of B r i t i s h Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (NEUROSCIENCE) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA March 1989 © G e r a l d Norman Weinstein Sholomenko, 198 9 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of A)&tAA>o S&C6~*C? The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT T h i s study examines aspects o f the n e u r a l s u b s t r a t e f o r locomotion i n b i r d s . E l e c t r i c a l s t i m u l a t i o n o f mid- and h i n d b r a i n r e v e a l e d t h r e e p r e v i o u s l y undefined brainstem r e g i o n s from which locomotion was e l i c i t e d i n the decerebrate animal. The s i t e s l i e w i t h i n the medial l o n g i t u d i n a l f a s c i c u l u s (MLF), i n t e r c o l l i c u l a r nucleus (ICo) and medial mesencephalic r e t i c u l a r f ormation (mMRF). These and p r e v i o u s l y d e f i n e d a v i a n locomotor r e g i o n s were f u r t h e r examined u t i l i z i n g the m i c r o i n j e c t i o n o f ag o n i s t s and a n t a g o n i s t s t o a c e t y l c h o l i n e , GABA, the e x c i t a t o r y amino a c i d s and Substance P t o determine the locomotor e f f e c t s o f p o t e n t i a l n e u r o t r a n s m i t t e r s at these s i t e s . C h o l i n e r g i c a g o n i s t s were e f f e c t i v e at e l i c i t i n g locomotion when i n j e c t e d i n t o the MLF, pontobulbar locomotor s t r i p (PLS) and medullary r e t i c u l a r f o rmation. GABAergic a n t a g o n i s t s evoked locomotion when i n f u s e d i n t o the PLS, ICo and pontine and medullary r e t i c u l a r formation. NMDA i n j e c t i o n i n t o the PLS, MLF, mMRF and medullary r e t i c u l a r formation e l i c i t e d locomotion or reduced the e l e c t r i c a l s t i m u l a t i o n t h r e s h o l d f o r locomotion, while Substance P i n j e c t i o n evoked locomotion when i n j e c t e d i n t o the pontine r e t i c u l a r formation. P h a s i c p e r i p h e r a l a f f e r e n t input was found not t o be e s s e n t i a l f o r the p r o d u c t i o n o f an a r r a y of av i a n locomotor p a t t e r n s when examined i n the spontaneous, e l e c t r i c a l l y s t i m u l a t e d and neurochemically s t i m u l a t e d p a r a l y z e d p r e p a r a t i o n s . However, a f f e r e n t feedback may have a r o l e i n s e t t i n g the a c t i v a t i o n l e v e l r e q u i r e d t o i n i t i a t e and set the frequency o f locomotor p a t t e r n s . The p r e s e r v a t i o n o f caudal d i e n c e p h a l i c n e u r a l s t r u c t u r e s allowed spontaneous locomotion i n i i the h i g h decerebrate b i r d , i m p l i c a t i n g the nucleus of the ansa l e n t i c u l a r i s , subthalamic nucleus and l a t e r a l hypothalamic area as p o s s i b l y modulating more caudal locomotor r e g i o n s . U t i l i z i n g an i n t e g r a t e d approach with the l i t e r a t u r e data c o l l e c t e d from a v a r i e t y of v e r t e b r a t e s , my r e s u l t s i n b i r d s suggest t h a t l o c o m o t o r - r e l a t e d n e u r a l pathways are h i g h l y conserved across a broad p h y l o g e n e t i c range. i i i TABLE OF CONTENTS Page A b s t r a c t i i Table of Contents i v L i s t o f Tables v i i i L i s t o f F i g u r e s i x Table of A b b r e v i a t i o n s x i Acknowledgement x v i Chapter 1 - Review of the L i t e r a t u r e and General 1 I n t r o d u c t i o n Review of the L i t e r a t u r e 2 S p i n a l Cord 5 Cortex 9 L a t e r a l V e s t i b u l a r Nucleus and Tectum 11 Red Nucleus 14 R e t i c u l a r Formation 15 Locomotor Regions and Locomotion-Related S t r u c t u r e s 18 Cerebellum 18 Pontobulbar Locomotor S t r i p 20 Mesencephalic Locomotor Region 23 Subthalamic Nucleus and Subthalamic Locomotor Region 29 B a s a l G a n g l i a 31 Limbic S t r u c t u r e s 32 Concl u s i o n s and Purpose of S t u d i e s i n the T h e s i s 34 The Decerebrate P r e p a r a t i o n 41 Nomenclature 4 6 Chapter 2 - E l e c t r i c a l S t i m u l a t i o n of Mesencephalic and 48 Pontine Regions E l i c i t s Locomotion i n Decerebrate B i r d s I n t r o d u c t i o n 49 M a t e r i a l s and Methods 52 F i g u r e 3 54 R e s u l t s 58 F i g u r e 4 59 F i g u r e 5 61 F i g u r e 6 64 F i g u r e 7 67 F i g u r e 8 71 D i s c u s s i o n 73 Concl u s i o n s 79 i v Chapter 3 - C h a r a c t e r i z a t i o n o f Avian Mid- and Hi n d b r a i n 81 S i t e s t h a t Produce Locomotion with L o c a l I n t r a c e r e b r a l I n f u s i o n of N e u r o t r a n s m i t t e r A g o n i s t s and A n t a g o n i s t s ( I ) : A c e t y l c h o l i n e . I n t r o d u c t i o n 82 M a t e r i a l s and Methods 85 R e s u l t s 88 Table 1 8 9 F i g u r e 9 90 F i g u r e 10 92 F i g u r e 11 96 F i g u r e 12 99 F i g u r e 13 101 F i g u r e 14 105 D i s c u s s i o n 108 Chapter 4 - C h a r a c t e r i z a t i o n o f Avian Mid- and Hi n d b r a i n 133 S i t e s t h a t Produce Locomotion with L o c a l I n t r a c e r e b r a l I n f u s i o n o f N e u r o t r a n s m i t t e r A g o n i s t s and A n t a g o n i s t s (II) : y-Aminobutyric A c i d (GABA). I n t r o d u c t i o n 134 M a t e r i a l s and Methods 137 R e s u l t s 138 Table 2 • 139 F i g u r e 15 140 F i g u r e 16 143 F i g u r e 17 145 . F i g u r e 18 148 F i g u r e 19 151 F i g u r e 20 154 D i s c u s s i o n 157 Chapter 5 - C h a r a c t e r i z a t i o n o f Avian Mid- and H i n b r a i n 166 S i t e s t h a t Produce Locomotion w i t h L o c a l I n t r a c e r e b r a l I n f u s i o n o f N e u r o t r a n s m i t t e r A g o n i s t s and A n t a g o n i s t s ( I I I ) : E x c i t a t o r y Amino A c i d s and Substance P. I n t r o d u c t i o n 167 M a t e r i a l s and Methods 168 R e s u l t s 169 Table 3 170 F i g u r e 21 171 F i g u r e 22 174 F i g u r e 23 176 F i g u r e 24 181 F i g u r e 25 187 F i g u r e 26 191 D i s c u s s i o n 195 v TABLE OF CONTENTS (CONTINUED) Page Chapter 6 - Avian Locomotion i n the Absence of Phasic 208 Af f e r e n t Input - The ^ F i c t i v e ' P r e p a r a t i o n I n t r o d u c t i o n 209 M a t e r i a l s and Methods 211 Resul t s 213 Figure 27 214 Figure 28 216 Figure 29 219 Figure 30 222 Figure 31 224 Figure 32 226 Figure 33 229 Dis c u s s i o n 231 Chapter 7 - Transection Level Determines Spontaneous Motor 236 A c t i v i t y i n the Decerebrate Avian P r e p a r a t i o n I n t r o d u c t i o n 237 Table 4 238 Figure 34 239 M a t e r i a l s and Methods 241 Results 243 Figure 35 244 Figure 36 24 6 Figure 37 248 Disc u s s i o n 253 Chapter 8 - Summary Di s c u s s i o n 258 Chapter 9 - L i s t of References 273 Appendix I 2 91 Appendix I I 2 94 v i LIST OF TABLES Table 1 - Time course, l a t e n c y and c o n c e n t r a t i o n s of a c e t y l c h o l i n e a g o n i s t s and a n t a g o n i s t s Table 2 - Time course, l a t e n c y and c o n c e n t r a t i o n s of GABA, i t s a g o n i s t s and a n t a g o n i s t s Table 3 - Time course, l a t e n c y and c o n c e n t r a t i o n s of glutamate, i t s a g o n i s t s and a n t a g o n i s t s and Substance P Page 89 139 170 Table 4 - Cat d e c e r e b r a t i o n l e v e l s . 238 v i i LIST OF FIGURES Chapter 1 F i g u r e 1. F i g u r e 2. Chapter 2 F i g u r e 3. F i g u r e 4. F i g u r e 5 F i g u r e 6. F i g u r e 7. F i g u r e 8. Chapter 3 F i g u r e 9. F i g u r e 10 F i g u r e 11 F i g u r e 12 F i g u r e 13 F i g u r e 14 Chapter 4 F i g u r e 15 F i g u r e 16 F i g u r e 17 Page Diagram of cat d e c e r e b r a t i o n l e v e l s . 6 Electromyographic a c t i v i t y , b l o o d p r e s s u r e 44 and heart r a t e r e s u l t i n g from e l e c t r i c a l s t i m u l a t i o n i n TTD Diagram of avian experimental apparatus. 54 Composite diagram of e l e c t r i c a l s t i m u l a t i o n 59 s i t e s i n pons and mesencephalon. S t i m u l a t i o n s i t e s on c o r o n a l s e c t i o n s i n the 61 avian b r a i n . EMG records of locomotion e l i c i t e d by MLF 64 s t i m u l a t i o n . EMG records of locomotion e l i c i t e d by mMRF 67 s t i m u l a t i o n and a f f e c t s of s t i m u l a t i o n frequency. EMG records of locomotion e l i c i t e d by ICo 71 s t i m u l a t i o n . Composite diagram of c h o l i n e r g i c a g o n i s t & 90 a n t a g o n i s t neurochemical i n j e c t i o n s i t e s . S t i m u l a t i o n and i n j e c t i o n s i t e s on c o r o n a l 92 s e c t i o n s i n the a v i a n b r a i n EMG r e c o r d s of locomotor a c t i v i t y e l i c i t e d by 96 e l e c t r i c a l s t i m u l a t i o n and c a r b a c h o l i n j e c t i o n i n t o the PLS. EMG records of locomotor a c t i v i t y e l i c i t e d by 99 e l e c t r i c a l s t i m u l a t i o n and c a r b a c h o l i n j e c t i o n i n t o the Cnd. EMG records of locomotor a c t i v i t y e l i c i t e d by 101 e l e c t r i c a l s t i m u l a t i o n and c a r b a c h o l i n j e c t i o n i n t o the Cnv. EMG records of locomotor a c t i v i t y e l i c i t e d by 105 e l e c t r i c a l s t i m u l a t i o n and c a r b a c h o l i n j e c t i o n i n t o the MLF. Composite diagram of GABAergic a g o n i s t and 14 0 a n t a g o n i s t neurochemical i n j e c t i o n s i t e s . EMG records of locomotor a c t i v i t y e l i c i t e d 143 by e l e c t r i c a l s t i m u l a t i o n and p i c r o t o x i n i n j e c t i o n i n t o the PLS. EMG records of locomotor a c t i v i t y e l i c i t e d by 145 p i c r o t o x i n and b i c u c u l l i n e i n j e c t i o n i n t o the PLS.. v i i i LIST OF FIGURES (CONTINUED) Page F i g u r e 18.- EMG records of locomotor a c t i v i t y e l i c i t e d by 148 e l e c t r i c a l s t i m u l a t i o n of the PLS bl o c k e d by GABA i n f u s i o n . F i g u r e 19.- EMG records showing GABA-reversible locomotor 151 a c t i v i t y e l i c i t e d by p i c r o t o x i n i n j e c t i o n i n t o Cnv. F i g u r e 20.- EMG records showing GABA-reversible locomotor 154 a c t i v i t y e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n and p i c r o t o x i n i n f u s i o n i n t o RP. Chapter 5 F i g u r e 21.- Composite diagram of EAA. & SubP a g o n i s t and 171 ant a g o n i s t neurochemical i n j e c t i o n s i t e s . F i g u r e 22.- EMG records showing locomotor a c t i v i t y e l i c i t e d 174 by e l e c t r i c a l s t i m u l a t i o n and NMDA i n j e c t i o n i n t o the PLS. Fi g u r e 23.- EMG records showing GDEE-reversible motor 17 6 e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n and NMDA i n j e c t i o n i n t o the Cnd. Fi g u r e 24.- EMG records showing GDEE-reversible locomotor 181 a c t i v i t y e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n and NMDA i n j e c t i o n i n t o the Cnv. Fi g u r e 25.- EMG records showing locomotor a c t i v i t y 187 e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n , Substance P and NMDA i n j e c t i o n i n t o the RP. Fi g u r e 26.- EMG and ENG records showing locomotor a c t i v i t y 191 e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n and NMDA i n f u s i o n i n t o the MLF. Chapter 6 F i g u r e 27.- B i l a t e r a l a l t e r n a t i n g walking a c t i v i t y i n a 214 spontaneously locomoting b i r d b e f o r e and a f t e r p a r a l y z a t i o n . F i g u r e 28.- Histogram o f e l e c t r i c a l s t i m u l a t i o n - i n d u c e d 216 and spontaneous step frequency d u r i n g p r e - p a r a l y z e d and p a r a l y z e d ^ f i c t i v e ' s t e p p i n g . F i g u r e 29.- B i l a t e r a l a l t e r n a t i n g walking a c t i v i t y evoked 219 by f o c a l e l e c t r i c a l s t i m u l a t i o n of the h i n d b r a i n b e f o r e and a f t e r p a r a l y z a t i o n . F i g u r e 30.- C o - a c t i v a t i o n o f l e g and wing a c t i v i t y evoked 222 by f o c a l e l e c t r i c a l s t i m u l a t i o n of the midbrain b e f o r e and a f t e r p a r a l y z a t i o n . F i g u r e 31.- B i l a t e r a l synchronous f l y i n g a c t i v i t y evoked 224 by f o c a l e l e c t r i c a l s t i m u l a t i o n of the h i n d b r a i n b e f o r e and a f t e r p a r a l y z a t i o n . F i g u r e 32.- Histogram o f e l e c t r i c a l s t i m u l a t i o n - i n d u c e d 226 wingbeat frequency d u r i n g p r e - p a r a l y z e d and p a r a l y z e d * f i c t i v e ' f l a p p i n g . i x LIST OF FIGURES (CONTINUED) Page Figure 33.- B i l a t e r a l * f i c t i v e ' hindlimb a c t i v i t y e l i c i t e d 229 by m i c r o i n j e c t i o n of carbachol and NMDA i n t o the pons and medulla. Chapter 7 Figure 34.- Diagram of a s a g g i t a l s e c t i o n through' the cat 239 brainstem showing neuraxis t r a n s e c t i o n l e v e l s and locomotor s i t e s important t o motor c o n t r o l . Figure 35.- Diagram of t r a n s e c t i o n l e v e l s of the avian 244 b r a i n which permit or e l i m i n a t e spontaneous locomotion i n the decerebrate b i r d . F igure 36.- B i l a t e r a l a l t e r n a t i n g walking a c t i v i t y i n a 246 spontaneously locomoting b i r d before and a f t e r p a r a l y z a t i o n . Figure 37.- EMG records showing spontaneous stepping and 24 8 f l y i n g a c t i v i t y i n a high decerebrate b i r d . x TABLE OF ABBREVIATIONS ACh a c e t y l c h o l i n e AChE a c e t y l c h o l i n e s t e r a s e AChM a c e t y l c h o l i n e m u s c a r i n i c r e c e p t o r AChN a c e t y l c h o l i n e n i c o t i n i c r e c e p t o r AHP area hypothalami p o s t e r i o r i s AL ansa l e n t i c u l a r i s AM nucleus a n t e r i o r m e d i a l i s hypothalami A n l nucleus a n n u l a r i s ANOVA a n a l y s i s of v a r i a n c e APH area parahippocampalis AQ c e r e b r a l aqueduct ATRO a t r o p i n e AVT area v e n t r a l i s o f T s a i BC brachium conjunctivum BICUC b i c u c u l l i n e BO o l f a c t o r y b ulb b.p. b l o o d p r e s s u r e CA a n t e r i o r commissure CARB car b a c h o l (carbamyl c h o l i n e ) Cb c erebellum CC . c e n t r a l c a n a l CCK c h o l e c y s t o k i n i n CCU constant c u r r e n t u n i t CCV v e n t r a l c e r e b e l l a r commissure CF cuneiform nucleus ChAT c h o l i n e a c e t y l t r a n s f e r a s e CHCS c o r t i c o h a b e n u l a r and c o r t i c o s e p t a l t r a c t CM mammillary body CNS c e n t r a l nervous system Cnd d o r s a l p a r t , c e n t r a l nucleus o f medulla Cnv v e n t r a l , p a r t , c e n t r a l nucleus of medulla CO o p t i c chiasm CoS s e p t a l commissural nucleus CP p o s t e r i o r commissure CPG c e n t r a l p a t t e r n generator (rhythmic o s c i l l a t o r ) CS c e n t r a l s u p e r i o r nucleus (Betcherew) CT t e c t a l commissure CI c e r v i c a l s p i n a l c o r d segment 1 . C2 c e r v i c a l s p i n a l c o r d segment 2 C3 c e r v i c a l s p i n a l c o r d segment 3 C4 c e r v i c a l s p i n a l c o r d segment 4 DBC d e c u s s a t i o n o f the brachium conjunctivum DLF d o r s o l a t e r a l f u n i c u l u s s p i n a l c o r d DM d e l t o i d e u s major muscle DMA dorsomedial a n t e r i o r t h a l a m i c nucleus DMP dorsomedial p o s t e r i o r t h a l a m i c nucleus DSCT d o r s a l s p i n o c e r e b e l l a r t r a c t x i DSD DSV s u p r a o p t i c d o r s a l d e c u s s a t i o n s u p r a o p t i c v e n t r a l d e c u s s a t i o n EAA e x c i t a t o r y amino a c i d s (glutamate, aspartate) EM ectomammillary nucleus EMG electromyogram E N G electroneurogram EW Edinger-Westphal nucleus F C L f l e x o r c r u r i s l a t e r a l i s muscle F D d o r s a l f u n i c u l u s F L M medial l o n g i t u d i n a l f a s c i c u l u s F R A f l e x o r r e f l e x a f f e r e n t F R L l a t e r a l mesencephalic r e t i c u l a r formation F R M medial mesencephalic r e t i c u l a r formation F T L l a t e r a l tegmental f i e l d FV v e n t r a l f u n i c u l u s G A B A y-aminobutyric a c i d GABAA GABAA r e c e p t o r subtype GC cuneate and g r a c i l e n u c l e i GCt c e n t r a l gray G D E E g l u t a m i c a c i d d i e t h y l e s t e r H A h y p e r s t r i a t u m accessorium Hb habenular nucleus Hp hippocampus HRP h o r s e r a d i s h peroxidase HV h y p e r s t r i a t u m v e n t r a l e Hz h e r t z (cycles/second) IC i n f e r i o r c o l l i c u l u s ICo nucleus i n t e r c o l l i c u l a r i s INC i n t e r s t i t i a l nucleus of C a j a l 10 i n f e r i o r o l i v a r y neucleus I P i n t e r p e d u n c u l a r nucleus Ipc nucleus i s t h m i , pars p a r v o c e l l u l a r i s I T C i l i o t i b i a l i s c r a n i a l i s muscle ( s a r t o r i u s muscle) i . v. intravenous KC1 potassium c h l o r i d e LC l o c u s c e r u l e u s L F M supreme f r o n t a l lamina L H h y p e r s t r i a t a l lamina L H A l a t e r a l hypothalamic area L I T C l e f t i l i o t i b i a l i s muscle LMD d o r s a l medullary lamina 1MLR l a t e r a l mesencephalic locomotor r e g i o n (see CF) LOCO locomotion L P A l a t e r a l p r e o p t i c area L P E C T l e f t p e c t o r a l i s muscle L P G locomotor p a t t e r n generator L P O p a r o l f a c t o r y lobe x i i LVN l a t e r a l v e s t i b u l a r nucleus ( D i e t e r s ' Nucleus) LRF p a r v o c e l l u l a r ( l a t e r a l ) r e t i c u l a r formation M molar MesV mesencephalic t r i g e m i n a l nucleus (see MNV) MLd l a t e r a l mesencephalic nucleus, d o r s a l p a r t MLF medial l o n g i t u d i n a l f a s c i c u l u s MLR mesencephalic locomotor r e g i o n mM m i l l i m o l a r mMLR medial mesencephalic locomotor r e g i o n (see PPN) mMRF medial mesencephalic r e t i c u l a r formation MNV mesencephalic t r i g e m i n a l nerve nucleus (see MesV) MPv mesencephalic nucleus, pars profundus MRF mesencephalic r e t i c u l a r formation MUSC muscimol MV motor t r i g e m i n a l nucleus MW molecular weight N ne o s t r i a t u m NA nucleus accumbens nAL subthalamic nucleus (named nucleus of the ansa l e n t i c u l a r i s i n b i r d s ) NC caudal n e o s t r i a t u m neurochemical a n e u r o t r a n s m i t t e r , i t s a g o n i s t or ant a g o n i s t NICO n i c o t i n e NR no response n i p r i d e sodium n i t r o f e r r i c y a n i d e NMDA N-methyl-D-aspartate NIII occulomotor nerve NV t r i g e m i n a l nerve NVI abducens nerve NX vagus nerve NXII h y p o g l o s s a l nerve 01 i n f e r i o r o l i v a r y nucleus (see 10) OMd d o r s a l p a r t , occulomotor nucleus OMv v e n t r a l p a r t , occulomotor nucleus OT o p t i c tectum Ov o v o i d nucleus P p i n e a l (Figure 32 only) P pons PaM paramedian nucleus PDA c i s - 2 , 3 - p i p e r i d i n e c a r b o x y l a t e PECT p e c t o r a l i s muscle PH plexus o f Ho r s l e y PICRO p i c r o t o x i n PILO p i l o c a r p i n e PLS pontobulbar locomotor s t r i p PMH p o s t e r i o r p a r t , p o s t e r i o r hypothalamic nucleus PMI paramedian i n t e r n a l t h a l a m i c nucleus POA a n t e r i o r p r e o p t i c nucleus POM medial p r e o p t i c nucleus PPN pedunculopontine nucleus (see mMLR) x i i i PRESTIM p r e v i o u s t o s t i m u l a t i o n PRF pontine r e t i c u l a r formation PrV p r i n c i p l e t r i g e m i n a l sensory nucleus PT p r e t e c t a l nucleus PVM p e r i v e n t r i c u l a r m a g n o c e l l u l a r nucleus R raphe nucleus R re d nucleus (Figures 1 & 31 only) Rgc g i g a n t o c e l l u l a r r e t i c u l a r formation RGC medullary g i g a n t o c e l l u l a r r e t i c u l a r nucleus RITC r i g h t i l i o t i b i a l i s c r a n i a l i s muscle RL l a t e r a l r e t i c u l a r nucleus RP caudal pontine r e t i c u l a r formation nucleus RPECT r i g h t p e c t o r a l i s muscle RPgc g i g a n t o c e l l u l a r p a r t , p ontine r e t i c u l a r nucleus Rpc pontine nucleus, p a r v o c e l l u l a r p a r t RPC caudal p o n t i n e r e t i c u l a r nucleus RPO pontine r e t i c u l a r nucleus, o r a l p a r t Ru red nucleus S s o l i t a r y nucleus SC s u p e r i o r c o l l i c u l u s SCE e x t e r n a l c e l l u l a r stratum SCI i n t e r n a l c e l l u l a r stratum SCOP scopolamine SG s u b s t a n t i a g e l a t i n o s a SGC c e n t r a l gray stratum SI s u b s t a n t i a innominata SL l a t e r a l s e p t a l nucleus SLR subthalamic locomotor r e g i o n SM medial s e p t a l nucleus SN s u b s t a n t i a n i g r a SNc s u b s t a n t i a n i g r a , pars compacta SP s u b p r e t e c t a l nucleus SpL l a t e r a l s p i r i f o r m nucleus SRCT s p i n o r e t i c u l o c e r e b e l l a r t r a c t SSP s u p r a s p i n a l nucleus ST s u b t r i g e m i n a l nucleus STIM s t i m u l a t i o n SV t r i g e m i n a l sensory nucleus T t r a p e z o i d body TFS t r i g e m i n a l f i e l d s t i m u l a t i o n Th thalamus TO o l f a c t o r y t u b e r c l e TPc s u b s t a n t i a n i g r a , pars compacta (named nucleus tegmentipedunculopontinus i n b i r d s ) TSM septomesencephalic t r a c t TTD descending t r i g e m i n a l t r a c t and nucleus TU t u b e r a l nucleus UDV u n i d i r e c t i o n a l v e n t i l a t i o n x i v V v e n t r i c l e VeD descending v e s t i b u l a r nucleus VeL l a t e r a l v e s t i b u l a r nucleus (see LVN) VeM medial v e s t i b u l a r nucleus VIP v a s o a c t i v e i n t e s t i n a l p o l y p e p t i d e vm ventromedial i n t e r n a l c e r e b e l l a r nucleus VS t r i g e m i n a l sensory nucleus (see PrV) VSCT v e n t r a l s p i n o c e r e b e l l a r t r a c t VTA v e n t r a l tegmental area of T s a i WGA-HRP wheat germ a g g l u t i n i n h o r s e r a d i s h peroxidase ZI zona i n c e r t a I I I occulomotor nerve and nucleus IV t r o c h l e a r nucleus VI abducens nucleus VII f a c i a l nucleus X d o r s a l motor nucleus vagus ATH i n c r e a s e d e l e c t r i c a l t h r e s h o l d f o r locomotion J'TH decreased e l e c t r i c a l t h r e s h o l d f o r locomotion uA microamp ul m i c r o l i t e r uM micromolar xv ACKNOWLEDGEMENT & ENDORSEMENT There are many people who took p a r t i n h e l p i n g me make i t through the t r i a l s and t r i b u l a t i o n s of doing the experiments, w r i t i n g the t h e s i s , doing the comprehensives and simply pushing forward t o the completion o f a Ph.D. S e v e r a l people were i n d i s p e n s i b l e . Greg Funk helped with the experiments, gave moral support, caught b i r d s , kept h i s good natured way even when I d i d n ' t and read t h i s t h e s i s with a f i n e t o o t h comb. Greg, I cannot thank you enough. Stephanie, you d i d n ' t c a t c h b i r d s , but you d i d l i v e w i t h me through t h i s d i f f i c u l t time, g i v i n g moral support, r e a d i n g the t h e s i s and keeping me on a s t r a i g h t and g e n e r a l l y even l i n e . I hope t o thank you f o r a long time i n t o the f u t u r e . P o l l y P, you have been unquestionably i n d i s p e n s i b l e f o r your u n f a i l i n g c onfidence i n my a b i l i t y t o f i n i s h . Thank you very much! Others a l s o helped i n the completion of t h i s t h e s i s . I would l i k e t o thank Dr. Steve V i n c e n t and Dr. Tony P h i l l i p s f o r r e a d i n g the t h e s i s and t h e i r very h e l p f u l comments. S p e c i a l thanks go t o Dr. John Steeves, who taught me t h a t independence i s e s s e n t i a l f o r success. Thanks a l s o t o D i e r d r e Webster, f o r a l l the c o f f e e , the neuroanatomy and j u s t the g e n e r a l good natured a t t i t u d e which always makes me f e e l good. Many other people deserve t o be here a l s o . They i n c l u d e Frank Smith, Ignacio V a l e n z u e l a , Marianne Morgan and B i l l Milsom. Thank you a l l ! ENDORSEMENT I t i s my wish t h a t no agency should ever d e r i v e m i l i t a r y b e n e f i t from the p u b l i c a t i o n o f t h i s t h e s i s . Authors who c i t e t h i s work i n support o f t h e i r own are requested t o q u a l i f y s i m i l a r l y the a v a i l a b i l t i y o f t h e i r r e s u l t s . x v i CHAPTER 1 REVIEW OF THE LITERATURE AND GENERAL INTRODUCTION 1 REVIEW OF THE LITERATURE Over the l a s t approximately 100 years a v a r i e t y of techniques have been brought t o bear on how the c e n t r a l and p e r i p h e r a l nervous systems exert c o n t r o l over the s k e l e t a l muscles which are the e f f e c t o r s of movement. O r i g i n a l l y , techniques as simple as the i s o l a t i o n of v a r i o u s p a r t s of the nervous system p r o v i d e d i n v e s t i g a t o r s with a good d e a l of i n f o r m a t i o n about what was being c o n t r o l l e d by those r e g i o n s . L e s i o n or a b l a t i o n experiments are s t i l l used to e l i m i n a t e c e r t a i n i n p u t s or outputs i n an e f f o r t t o d e l i n e a t e r e g i o n a l f u n c t i o n f o l l o w i n g s e l e c t i v e removal. N e v e r t h e l e s s , a v a r i e t y of new techniques have generated a v a s t amount of i n f o r m a t i o n which may both e l u c i d a t e and obfuscate, but w i l l undoubtedly complicate, the understanding of the mechanisms of motor c o n t r o l subserved by the nervous system. Powerful techniques such as l o c a l i z a t i o n of n e u r o t r a n s m i t t e r s with immunocytochemistry and t h e i r r e c e p t o r s w i t h r e c e p t o r autoradiography g i v e us i n f o r m a t i o n r e g a r d i n g the n e u r o t r a n s m i t t e r s used by the neurons and t h e i r p o s t s y n a p t i c t a r g e t s . F o c a l e l e c t r i c a l s t i m u l a t i o n has e l u c i d a t e d s e v e r a l b r a i n r e g ions which, when s t i m u l a t e d , evoke locomotor p a t t e r n s i n a v a r i e t y of v e r t e b r a t e s p e c i e s . E x t r a - and i n t r a c e l l u l a r r e c o r d i n g from s e l e c t e d neuronal p o p u l a t i o n s p r o v i d e s i n f o r m a t i o n about the f i r i n g p a t t e r n s and phase r e l a t i o n s h i p s of those c e l l s i n r e l a t i o n t o other neuronal p o p u l a t i o n s i n a d d i t i o n t o e s t a b l i s h i n g h o d o l o g i c a l r e l a t i o n s h i p s , while r e t r o g r a d e and anterograde neuroanatomical t r a c i n g techniques 2 have i n c r e a s e d the number of d e s c r i b e d pathways and attendant n e u r a l c i r c u i t s t o s u r p r i s i n g numbers. As w i l l be made c l e a r from the summary of the l i t e r a t u r e below, a great d e a l remains t o be e l u c i d a t e d r e g a r d i n g the pathways and mechanisms by which the c e n t r a l nervous system c o n t r o l s locomotion. V e r t e b r a t e s r e q u i r e the o r g a n i z a t i o n and i n t e g r a t i o n of n e u r a l components at a l l l e v e l s of the n e u r a x i s to perform t h e i r normal range of locomotor behaviours. The o r g a n i z a t i o n i s e s s e n t i a l l y h i e r a r c h i c a l , b e i n g l e a s t complex at the l e v e l of the s p i n a l c o r d and most complex i n the f o r e b r a i n . The most rudimentary motor response occurs at the l e v e l of the s p i n a l r e f l e x where only two neurons, comprising the n e u r a l c i r c u i t r y of the monosynaptic r e f l e x a r c , can subserve a r e l a t i v e l y simple motor behaviour such as occurs i n response t o a simple mechanical st i m u l u s (e.g. s t r e t c h i n g of the i n t r a f u s a l muscle f i b r e s ) . More complex o r g a n i z a t i o n can a l s o occur at s p i n a l l e v e l s v i a networks of i n t r i n s i c s p i n a l neurons ( s p i n a l locomotor rhythmic o s c i l l a t o r or p a t t e r n generator) which i n t e r a c t t o produce rhythmic mov.ement of the limbs. The most complex l e v e l of motor o r g a n i z a t i o n i s found w i t h i n and between n u c l e i of the hind-, mid- and f o r e b r a i n which are r e s p o n s i b l e f o r the c o n t r o l of v o l i t i o n a l ( i . e . g o a l - d i r e c t e d ) locomotion i n response t o e x t e r n a l or i n t e r n a l s t i m u l i . Before d i s c u s s i n g the h i e r a r c h i c a l o r g a n i z a t i o n of c e n t r a l motor s t r u c t u r e s , i t i s f i r s t necessary t o examine s e v e r a l of the types of p r e p a r a t i o n used i n the study of motor c o n t r o l (see F i g u r e 1, page 6). H i s t o r i c a l l y , s t u d i e s attempting to e l u c i d a t e the motor f u n c t i o n of d i f f e r e n t l e v e l s of the n e u r a x i s i n i t i a l l y 3 u t i l i z e d s e l e c t i v e removal or p a r t i t i o n o f p o r t i o n s of the b r a i n t o examine any d e f i c i t s of motor c a p a b i l i t y which r e s u l t e d from the l o s s o f t h i s n e u r a l c i r c u i t r y ( f o r review see G r i l l n e r , 1975; Wetzel and S t u a r t , 1976) . A number of i n v e s t i g a t o r s found t h a t a b l a t i o n o f the t e l e n c e p h a l o n ( c e r e b r a l c o r t e x -d e c o r t i c a t e p r e p a r a t i o n ) i n s p e c i e s i n c l u d i n g f i s h ( B i c k e l , 1900 c f . G r i l l n e r , 1975), f r o g s ( B i c k e l , 1900, c f . G r i l l n e r , 1975) and, sub-primate mammals (Goltz, 1892 c f . G r i l l n e r , 1975) (see F i g u r e 1, l i n e A) d i d l i t t l e t o i n t e r r u p t spontaneous locomotor behaviours ( G r i l l n e r , 1975). Indeed, Hinsey et al. (1930), u s i n g c h r o n i c p r e p a r a t i o n s , r e p o r t e d t h a t even t h a l a m i c (Figure 1 -l i n e B) and hypothalamic (Figure 1 - l i n e C) c a t s and r a b b i t s walked f o l l o w i n g surgery. In t h e i r p r e p a r a t i o n , i f a t r a n s e c t i o n was made from the r o s t r a l s u p e r i o r c o l l i c u l u s t o the o p t i c chiasm (see F i g u r e 1 - l i n e B), l e a v i n g a r o s t r a l p o r t i o n o f thalamus i n t a c t ( p r e - c o l l i c u l a r p r e o p t i c p r e p a r a t i o n ) , the animals would walk spontaneously and d i s p l a y behaviours which resembled motivated a c t i v i t i e s . Brainstem t r a n s e c t i o n l e a v i n g only the caudal t h i r d o f the thalamus i n t a c t (thalamic p r e p a r a t i o n ) (Figure 1, l i n e C), however, produced animals which were more r i g i d and locomoted only with s t r o n g e x t e r o c e p t i v e s t i m u l a t i o n (Laughton, 1924). L a t e r s t u d i e s i n c h r o n i c c a t s (Bard and Macht, 1958) demonstrated t h a t animals with even more caudal t r a n s e c t i o n s c o u l d locomote spontaneously. However, t r a n s e c t i o n o f the neuraxis from the r o s t r a l border o f the s u p e r i o r c o l l i c u l u s t o the caudal border o f the mammillary bodies ( p r e - c o l l i c u l a r post-mammillary or mesencephalic p r e p a r a t i o n ) (Figure 1 - l i n e D) (Hinsey et a l . , 1930; Orlovsky 4 and Shik, 1976) e l i m i n a t e d any spontaneous locomotion. The above p r e p a r a t i o n s , which w i l l be d i s c u s s e d more f u l l y below, have enabled i n v e s t i g a t o r s to examine locomotion under more e a s i l y c o n t r o l l a b l e c o n d i t i o n s . The v a r i o u s manipulations performed i n c l u d e , f o r example, e l i c i t i n g locomotion by 1 2 . e l e c t r i c a l or neurochemical s t i m u l a t i o n of s p e c i f i e d r e g i o n s of the b r a i n , examining spontaneous locomotor p a t t e r n s i n p a r a l y z e d animals and examining motor r e l a t e d s t r u c t u r e s at l e v e l s of the n e u r a x i s below the t r a n s e c t i o n d u r i n g c o n t r o l l e d locomotor behaviours. Such s t u d i e s have p r o v i d e d a great d e a l of i n f o r m a t i o n r e g a r d i n g the c o n t r o l of locomotion e x e r t e d by the c e n t r a l nervous system. S p i n a l Cord H i s t o r i c a l l y , Freusberg (1874), Freusberg and G o l t z (1874) and S h e r r i n g t o n (1910) were the f i r s t t o study motor performance by i s o l a t i n g components of the motor system. A f t e r h i g h s p i n a l c o r d t r a n s e c t i o n or d e c a p i t a t i o n , they found t h a t c a t s and dogs 1 S e l e c t i v e e l e c t r i c a l s t i m u l a t i o n presumably a f f e c t s voltage s e n s i t i v e channels located on any p o r t i o n of a neuron (e.g. terminal, axon, c e l l body) (for review see H i l l e , 1984), and therefore may e f f e c t a host of pre- and postsynaptic changes. 2 I n j e c t i o n of neurotransmitters, t h e i r agonists and antagonists (neurochemicals), presumably e f f e c t changes i n the s p e c i f i c neurotransmitter receptors f o r which they are e f f i c a c i o u s (for review, see Carpenter and Reese, 1981). Unlike e l e c t r i c a l s t i m u l a t i o n (see previous footnote), i t i s p o s s i b l e , using i n j e c t i o n of s e l e c t e d neurochemicals, to l o c a l i z e the receptor s i t e s at which the agonist or antagonist i s e f f e c t i v e . The s e l e c t i v i t y of neurochemical binding i s the basis f o r the autoradiographic l o c a l i z a t i o n of neurotransmitter receptors. Whether the neurochemical acts p r e s y n a p t i c a l l y , p o s t s y n a p t i c a l l y , or both, and the receptor changes which i t e l i c i t s are dependent on a v a r i e t y of f a c t o r s not the l e a s t of which i s receptor subtype. In vivo, the downstream e f f e c t s of neurochemical-induced receptor a c t i v a t i o n / i n a c t i v a t i o n are dependent upon the neural c i r c u i t r y . 5 F i g u r e 1. Diagram o f a s a g g i t a l s e c t i o n t h r o u g h t h e c a t b r a i n s t e m showing n e u r a x i s t r a n s e c t i o n l e v e l s and l o c o m o t o r s i t e s i m p o r t a n t f o r t h e s t u d y o f locomotor c o n t r o l . T r a n s e c t i o n l e v e l s a r e d e s i g n a t e d by l e t t e r s A-E. T r a n s e c t i o n l e v e l : A - t h a l a m i c , B -p r e c o l l i c u l a r premammillary ( h y p o t h a l a m i c of H i n s e y e t al., 1930), C - p r e c o l l i c u l a r p o s t m a m m i l l a r y , D - p r e c o l l i c u l a r p o s t - o c c u l o m o t o r , E - m i d c o l l i c u l a r p r e - o c c u l o m o t o r . Locomotor s i t e s i n c l u d e t h e s u b t h a l a m i c l o c o m o t o r r e g i o n (SLR) and m e s e n c e p h a l i c l o c o m o t o r r e g i o n (MLR). The h a t c h e d l i n e s s u r r o u n d i n g RPC and RGC r e p r e s e n t t h e p o n t i n e and m e d u l l a r y r e t i c u l a r f o r m a t i o n t h a t a r e thought t o be t h e major motor i n f o r m a t i o n p r o j e c t i o n systems t o the s p i n a l c o r d . A b b r e v i a t i o n s : CM - mammillary body, CO - o p t i c chiasm, IC - i n f e r i o r c o l l i c u l u s , MLR - m e s e n c e p h a l i c l o c o m o t o r r e g i o n , P - pons, R - r e d n u c l e u s , RGC - m e d u l l a r y g i g a n t o c e l l u l a r r e t i c u l a r n u c l e u s , RPC - c a u d a l p o n t i n e r e t i c u l a r n u c l e u s , SC - s u p e r i o r c o l l i c u l u s , SLR - s u b t h a l a m i c l o c o m o t o r r e g i o n , T - t r a p e z o i d body, Th - thalamus, I I I - o c c u l o m o t o r n e r v e . See t e x t f o r a d d i t i o n a l e x p l a n a t i o n . T h i s f i g u r e i s redrawn from: 1) S h i k e t a l . , 1968, 2) O r l o v s k y , 1970a, 3) G r i l l n e r and S h i k , 1973 and 4) W e t z e l and S t u a r t , 1976. 6 continued t o step i n a rhythmic a l t e r n a t i n g manner. T h i s " s p i n a l s t e p p i n g " p r o v i d e d the f i r s t d i r e c t evidence t h a t i n t a c t s u p r a s p i n a l i n f l u e n c e s were not e s s e n t i a l f o r the p r o d u c t i o n of the b a s i c p a t t e r n of locomotion. S h e r r i n g t o n (1910) suggested t h a t p o s t - t r a n s e c t i o n locomotion was dependent on r e f l e x a c t i o n s a c t i v a t e d by p e r i p h e r a l (sensory) i n p u t . However, Graham-Brown (1911) d i s p r o v e d t h i s s u g g e s t i o n by demonstrating t h a t p r e v i o u s l y d e a f f e r e n t e d animals a l s o performed s t e p p i n g movements f o l l o w i n g s p i n a l c o r d t r a n s e c t i o n . Graham-Brown l a t e r (1914) h y p o t h e s i z e d t h a t a n e u r a l c i r c u i t w i t h i n the s p i n a l c o r d a c t s as an i n t r i n s i c rhythm o s c i l l a t o r capable of producing the s p i n a l s t e p p i n g i n each limb ( h a l f - c e n t r e h y p o t h e s i s ) . The o s c i l l a t i n g c i r c u i t of each limb was p o s t u l a t e d t o i n t e r a c t w i t h the rhythmic o s c i l l a t o r s 'of other limbs to produce the rhythmic c o o r d i n a t e d movements of locomotor p r o g r e s s i o n ( G r i l l n e r , 1975). The rhythm was p o s t u l a t e d t o be an i n t r i n s i c p r o p e r t y of the i n t e r c o n n e c t i o n s of a n t a g o n i s t i c nerve c e l l s (Graham Brown, 1914) . More r e c e n t l y , G r i l l n e r and Zangger (1979) demonstrated t h a t a s p i n a l - t r a n s e c t e d p a r a l y z e d cat which i s d e v o i d of any rhythmic p e r i p h e r a l feedback would a l s o produce p a t t e r n s of a l t e r n a t i n g motor a c t i v i t y as recorded from p e r i p h e r a l nerve e f f e r e n t s . The electroneurograms (ENGs) ( f o r complete abbreviations l i s t , see pages xi-xv) recorded d u r i n g such f i c t i t i o u s or " f i c t i v e " (Perret et a l . , 1972) locomotion showed the same p a t t e r n as the ENGs recorded d u r i n g normal (unparalyzed) locomotion. Although the above f i n d i n g s have been g e n e r a l i z e d t o a v a r i e t y of v e r t e b r a t e s ( f o r review see 7 M c C l e l l a n , 1986), i t has yet t o be demonstrated t h a t x s p i n a l s t e p p i n g ' can occur i n the a d u l t of any primate s p e c i e s ( E i d e l b e r g , 1981) . S e v e r a l i n v e s t i g a t o r s have hypothesized t h a t the absence of x s p i n a l s t e p p i n g ' i n a d u l t primates r e s u l t s from an i n c r e a s e d dependency of s p i n a l s t e p p i n g c i r c u i t s on s u p r a s p i n a l i n f l u e n c e s ( E i d e l b e r g et al., 1981a/b; G r i l l n e r , 1975). E i d e l b e r g et al. (1981a) p o s t u l a t e d t h a t , i n primates, i n c r e a s e d descending t o n i c f a c i l i t a t o r y i n f l u e n c e s are necessary f o r the p a t t e r n generators t o t r i g g e r output from s p i n a l motorneurons. Des p i t e the i n a b i l i t y t o demonstrate ^ s p i n a l s t e p p i n g ' i n primates, s p i n a l c o r d p a t t e r n generators (rhythmic o s c i l l a t o r s ) t h a t are capable of producing the rhythmic a l t e r n a t i n g output necessary t o evoke locomotor a c t i v i t y have been found i n a l l lower v e r t e b r a t e s examined ( G r i l l n e r , 1975; M c C l e l l a n , 1986). S p i n a l l e s i o n s t u d i e s i n the lamprey and cat demonstrate t h a t the n e u r a l c i r c u i t s which comprise the generators can be l o c a l i z e d t o as few as 2-3 s p i n a l segments, but the c i r c u i t s themselves have yet t o be f u l l y c h a r a c t e r i z e d (Cohen and Wallen, 1980; G r i l l n e r and Zangger, 1979). The generators are undoubtedly modulated by p e r i p h e r a l feedback d u r i n g locomotion ( G r i l l n e r , 1975). Indeed, Jordan and co-workers (Shefchyk and Jordan, 1985; Jordan, 1986) have p o s t u l a t e d t h a t f l e x o r r e f l e x a f f e r e n t s (FRA) and pathways descending from s u p r a s p i n a l l e v e l s may impinge on a common in t e r n e u r o n f i r s t d e s c r i b e d by Jankowska et al. (1967) . However, a f f e r e n t s descending from s u p r a s p i n a l regions to the s p i n a l cord p a t t e r n generators appear to be the major c o n t r o l l i n g f a c t o r f o r 8 the i n i t i a t i o n and ongoing c o n t r o l of locomotion (Kuypers, 1982). The s u p r a s p i n a l n u c l e i thought to be i n v o l v e d i n motor c o n t r o l which p r o v i d e d i r e c t descending connections t o the s p i n a l c o r d i n c l u d e the c o r t e x , red nucleus, l a t e r a l v e s t i b u l a r nucleus, tectum and pontine & medullary r e t i c u l a r formation (for review see Kuypers, 1982; Holstege & Kuypers, 1988). Cortex T e l e n c e p h a l i c s t r u c t u r e s are known to be d i r e c t l y i n v o l v e d i n m o t i v a t i o n a l or v o l i t i o n a l commands which e l i c i t locomotor responses (Wetzel and S t u a r t , 1976). In mammals, the premotor and motor c o r t e x of the p r e c e n t r a l gyrus g i v e s r i s e t o descending c o r t i c o s p i n a l neurons of the pyramidal system (Kuypers, 1982). S t i m u l a t i o n of p a r t s of the motor c o r t e x e l i c i t d i s c h a r g e i n motoneurons i n n e r v a t i n g both proximal and d i s t a l limb muscles (Marsden et al., 1981; Kuypers, 1964). However, evidence suggests t h a t these d i r e c t c o r t i c o s p i n a l connections are c o n s i d e r a b l y more important to the h i g h l y f r a c t i o n a t e d muscle movements of the d i s t a l e x t r e m i t i e s than t o c o n t r a c t i o n s of the proximal limb muscles e s s e n t i a l t o b a s i c locomotor p a t t e r n s (e.g. walking) (Lawrence and Kuypers, 1968a,b; Kuypers, 1982) . High i n t e n s i t y e l e c t r i c a l s t i m u l a t i o n of pyramidal t r a c t neurons has been shown only to d i s r u p t locomotion (Orlpvsky, 1972a), while s t i m u l a t i o n at lower c u r r e n t s t r e n g t h s served to i n c r e a s e f l e x o r or extensor a c t i v i t y d u r i n g the a p p r o p r i a t e p o r t i o n of the step c y c l e (Shik et al., 1966; Orlovsky, 1972a; 9 f o r review see Armstrong, 198 6) . B i l a t e r a l pyramidotomy at the l e v e l of the caudal brainstem d i d not i n h i b i t v o l i t i o n a l locomotion i n the monkey, but s e v e r e l y reduced the animal's a b i l i t y t o perform p r e c i s e movements u s i n g the d i s t a l e x t r e m i t i e s (Lawrence and Kuypers, 1968a,b). In the c h r o n i c cat, l e s i o n of the l a t e r a l c o r t i c o s p i n a l t r a c t at m i d t h o r a c i c l e v e l s d i d not prevent recovery of hindlimb walking (Yu and E i d e l b e r g , 1981). A l s o , Steeves and Jordan (1980) u t i l i z e d a p r e c o l l i c u l a r - p o s t m a m m i l l a r y cat p r e p a r a t i o n t o demonstrate t h a t locomotion induced by s t i m u l a t i o n of a r e g i o n i n the midbrain (mesencephalic locomotor r e g i o n (MLR)) was not b l o c k e d by l e s i o n of the l a t e r a l c o r t i c o s p i n a l t r a c t s . In the p r e c o l l i c u l a r postmammillary c a t , the i n i t i a t i o n of locomotion by s t i m u l a t i o n of the MLR was u n a f f e c t e d by b i l a t e r a l medullary pyramidotomy. However, e l e c t r i c a l s t i m u l a t i o n o f pontine c o r t i c o f u g a l f i b r e s r o s t r a l t o the l e s i o n , which send c o l l a t e r a l s t o the r e t i c u l a r formation t h a t are a n t i d r o m i c a l l y a c t i v a t e d by the e l e c t r i c a l s t i m u l a t i o n (Shik et al., 1968; Orlovsky, 1972a), e l i c i t e d locomotion which c o u l d be bl o c k e d by complete d e s t r u c t i o n o f the MLR or m i d - c o l l i c u l a r t r a n s e c t i o n (Shik et a l . , 1968). The above f i n d i n g s i n d i c a t e t h a t the c o r t i c o f u g a l c o n t r i b u t i o n t o the i n i t i a t i o n and c o n t r o l of v o l u n t a r y locomotion, even i n primates, may be mediated v i a the p h y l o g e n e t i c a l l y o l d e r brainstem s t r u c t u r e s o f the ext r a p y r a m i d a l motor system. Neuroanatomical i n v e s t i g a t i o n s have demonstrated t h a t non-mammalian v e r t e b r a t e s i n c l u d i n g f i s h , amphibians, r e p t i l e s , and b i r d s l a c k d i r e c t t e l e n c e p h a l o - s p i n a l p r o j e c t i o n s (Cabot et al., 1982 ( b i r d ) ; Woodson and Kunzle, 1982 ( r e p t i l e s , t u r t l e ) ; 10 Leonard et al., 1979 ( f i s h ) . Indeed, these animals e x h i b i t few locomotor d e f i c i t s f o l l o w i n g d e c o r t i c a t i o n when compared to i n t a c t animals (Leonard et al., 1979; Gabbott and Jones, p e r s o n a l communication). F u r t h e r , sub-primate mammals, i n which the c o r t i c o s p i n a l connections are l e s s e x t e n s i v e than i n primates, e x h i b i t minimal locomotor impairment f o l l o w i n g c o r t i c a l a b l a t i o n ( f o r review see Wetzel and S t u a r t , 1976; G r i l l n e r , 1975). L a t e r a l V e s t i b u l a r Nucleus (LVN) ( D i e t e r s ' Nucleus) and Tectum The l a t e r a l v e s t i b u l a r nucleus (LVN) g i v e s r i s e t o descending axons which t r a v e l i n the s p i n a l c o r d v e n t r o l a t e r a l f u n i c u l u s and impinge on extensor motoneurons i n the s p i n a l c o r d (Pompeiano, 1984; Wilson and Yoshida, 1968). Orlovsky (1972b) found t h a t v e s t i b u l o s p i n a l neurons a r i s i n g predominantly from the LVN are r h y t h m i c a l l y modulated d u r i n g locomotion, e x e r t f a c i l i t a t o r y e f f e c t s on extensor motoneurons, and t h a t e l e c t r i c a l s t i m u l a t i o n of these c e l l s or t h e i r descending axons y i e l d s the same f a c i l i t a t o r y r e s u l t s d u r i n g both r e s t and locomotion (Orlovsky, 1972a) . Arshavsky et al. ( c f . Arshavsky and Orlovsky, 1986) found t h a t the r h y t h m i c a l a c t i v i t y of v e s t i b u l o s p i n a l c e l l s i s removed by c e r e b e l l a r a b l a t i o n while removal of a f f e r e n t p e r i p h e r a l i nput with p a r a l y z i n g agents ( ^ f i c t i v e ' p r e p a r a t i o n ) d i d not a f f e c t t h i s r h y t h m i c a l a c t i v i t y (Arshavsky and Orlovsky, 198 6). Wilson and Yoshida (1968) used e l e c t r o p h y s i o l o g i c a l techniques to show t h a t the LVN's s t r o n g e s t l i n k a g e , which was 11 monosynaptic, o c c u r r e d between v e s t i b u l o s p i n a l neurons and neck extensor motoneurons. They found t h a t v e s t i b u l o s p i n a l f i b r e s form synapses on some hindlimb extensor motoneurons (gastrocnemius-soleus) but c o u l d f i n d no monosynaptic l i n k s t o f o r e l i m b extensor neurons i n c a t s . They were, however, able t o demonstrate a s t r o n g p o l y s y n a p t i c connection between v e s t i b u l a r e f f e r e n t s and extensor motoneurons at a l l l e v e l s of the cord. T h e i r f i n d i n g s support the view t h a t LVN neurons r e c e i v e t o n i c a f f e r e n t l a b y r i n t h i n e input and i n t u r n exert l a b y r i n t h i n e r i g h t i n g r e f l e x e s on neck musculature. They p o s t u l a t e d t h a t "the monosynaptic l i n k t o neck motoneurons i s l e s s s u b j e c t t o descending and segmental c o n t r o l than i s the p o l y s y n a p t i c pathway", thus p l a c i n g the l a t e r a l v e s t i b u l a r nucleus i n an e x c e l l e n t p o s i t i o n t o modulate r i g h t i n g r e f l e x e s both at r e s t and d u r i n g locomotion. Pompeiano (1984) suggested t h a t p o s t u r a l adjustments of hindlimb muscles d u r i n g l a b y r i n t h r e f l e x e s are mediated by the v e s t i b u l o s p i n a l pathway and t h a t the "LVN e x e r t s an e x c i t a t o r y i n f l u e n c e on i p s i l a t e r a l extensor motoneurons". C o n s i d e r i n g the lumbosacral i n f l u e n c e o f v e s t i b u l o s p i n a l neurons, he p o s t u l a t e d t h a t the v e s t i b u l o s p i n a l and r e t i c u l o s p i n a l neurons have s y n e r g i s t i c i n f l u e n c e s t h a t r e s u l t from neck and macular v e s t i b u l a r input t o groups of motoneurons i n n e r v a t i n g hindlimb muscles (Pompeiano, 1984). The l a t e r a l v e s t i b u l o s p i n a l t r a c t t r a v e l s i n the v e n t r o l a t e r a l f u n i c u l u s of the s p i n a l c o r d . S e v e r a l i n v e s t i g a t o r s , i n c l u d i n g Steeves and Jordan (1980) i n the cat, E i d e l b e r g et al., (1981b) i n the monkey and Sholomenko and Steeves (1987) i n the b i r d , found t h a t l e s i o n s of the v e n t r o l a t e r a l f u n i c u l i a b o l i s h e d hindlimb locomotion i n these animals, i t appeared l i k e l y t h a t the v e s t i b u l o s p i n a l pathway was e s s e n t i a l f o r descending locomotor c o n t r o l . However, Yu and E i d e l b e r g (1981) found t h a t locomotor recovery c o u l d occur f o l l o w i n g b i l a t e r a l v e s t i b u l a r n u c l e i l e s i o n s i n c h r o n i c a l l y maintained c a t s , although t h e i r a b i l i t y t o walk at h i g h e r v e l o c i t i e s on a t r e a d m i l l was reduced compared t o u n l e s i o n e d or sham operated animals. F u r t h e r , they found only a t r a n s i t o r y r e d u c t i o n i n j o i n t extensor d r i v e , p a r t i c u l a r l y i n the h i n d l i m b s . They p o s t u l a t e d t h a t the v e s t i b u l o s p i n a l pathways a r i s i n g from LVN p l a y an a d j u n c t i v e r o l e i n the c o n t r o l of the s p i n a l c o r d p a t t e r n generators which c o n t r o l extensors d u r i n g the step c y c l e . J e l l et al. (1985) a l s o l e s i o n e d the LVN b i l a t e r a l l y and were s t i l l a b le to e l i c i t locomotion by MLR s t i m u l a t i o n i n the mesencephalic c a t . They found no major d e f i c i t s i n e i t h e r the amplitude or t i m i n g of f l e x o r and extensor EMGs of the h i n d l i m b s . Taken together, the above f i n d i n g s i m p l i c a t e the descending LVN-spinal pathway i n the c o n t r o l of both the r i g h t i n g r e f l e x e s v i a monosynaptic connections to neck motoneurons and the limb extensor p o s t u r a l muscles v i a a p o l y s y n a p t i c pathway. I t t h e r e f o r e appears l i k e l y t h a t the v e s t i b u l o s p i n a l p r o j e c t i o n p l a y s i t s most s i g n i f i c a n t r o l e i n the c o n t r o l of balance and posture d u r i n g locomotion, while having l i t t l e e f f e c t on the b a s i c locomotor rhythm. S i m i l a r t o the r o l e p l a y e d by the v e s t i b u l o s p i n a l pathways i n f i n e t u n i n g locomotion, the tectum appears t o i n f l u e n c e motor behaviours i n the cat (Huerta and H a r t i n g , 1982). The tectum has been demonstrated t o impinge monosynaptically on r e t i c u l a r formation neurons and, i n a d d i t i o n , g i v e s r i s e t o the t e c t o s p i n a l t r a c t . However, the t e c t o s p i n a l t r a c t has been demonstrated t o descend to only c e r v i c a l s p i n a l c o r d l e v e l s , thus presumably e l i m i n a t i n g a r o l e f o r t h i s pathway i n the c o n t r o l of hindlimb CPGs. The main r o l e of the t e c t o s p i n a l t r a c t i s p o s t u l a t e d t o be i n the " c o o r d i n a t i o n of head and eye movements" (Huerta and H a r t i n g , 1982), and t h e r e f o r e may be i n v o l v e d i n v i s u a l guidance d u r i n g locomotion, but the pathway has not, t o my knowledge, been p o s t u l a t e d to a f f e c t the b a s i c locomotor p a t t e r n . Red Nucleus (Ru) The red nucleus (Ru) g i v e s r i s e t o c r o s s e d r u b r o s p i n a l f i b r e s which f a c i l i t a t e f l e x o r motoneurons v i a p o l y s y n a p t i c pathways (Hongo et al., 1969a,b) d u r i n g the swing phase of locomotion (Orlovsky, 1972c). C e l l s i n the nucleus are r h y t h m i c a l l y a c t i v e d u r i n g swing (Orlovsky, 1972c) and e l e c t r i c a l s t i m u l a t i o n of the Ru enhances f l e x o r a c t i v i t y d u r i n g t h i s phase of locomotion (Orlovsky, 1972a). The r e d nucleus r e c e i v e s major a f f e r e n t i n p u t s from the cerebellum , c e r e b r a l c o r t e x (Orlovsky, 1972c), b a s a l g a n g l i a (Fanardzhyan and Sarkisyan, 1985) and d o r s a l column n u c l e i (Fanardzhyan and Sarkisyan, 1985). As i n the case of the LVN, c e r e b e l l a r a b l a t i o n e l i m i n a t e s the Ru rhythmic a c t i v i t y d u r i n g locomotion (Orlovsky, 1972c), while c o r t i c a l a b l a t i o n has l i t t l e e f f e c t on i t s c o i n c i d e n c e with the locomotor swing phase (Orlovsky, 1972c). However, b i l a t e r a l a b l a t i o n of the red n u c l e i or r u b r o s p i n a l t r a c t s (Steeves and Jordan,1980/ Yu and E i d e l b e r g , 1981/ Shik et al., 1968/ Ingram and Ranson, 1932/ J e l l et al., 1985/ Sholomenko and Steeves, 1987b/ Lawrence and Kuypers, 1968b) has l i t t l e e f f e c t on locomotion i n any c h r o n i c or acute animal s t u d i e d . While the red nucleus appears t o have l i t t l e importance f o r the b a s i c p a t t e r n s of locomotion, Kuypers (1982) p o s t u l a t e d t h a t the r u b r o s p i n a l t r a c t adds a second l e v e l of r e s o l u t i o n t o the s t r u c t u r e s which subserve b a s i c locomotor needs, p a r t i c u l a r l y w i t h r e s p e c t to the independent movement of d i s t a l p a r t s of i n d i v i d u a l limbs. S i m i l a r l y , Fanardzhyan and S a r k i s y a n (1985) envisage the red nucleus as an important motor/sensory i n t e g r a t i o n c e n t r e f o r the m o n i t o r i n g and c o r r e c t i o n of an i n i t i a t e d movement. I t appears, then, t h a t the r e d nucleus a c t s to " f i n e tune" motor c o n t r o l , but i s not e s s e n t i a l i n the c o n t r o l of the b a s i c motor rhythm. R e t i c u l a r Formation Mid- and h i n d b r a i n r e t i c u l a r formation neurons are the o r i g i n of the r e t i c u l o s p i n a l pathway which p l a y s an important r o l e i n the c o n t r o l of locomotion (Lawrence and Kuypers, 1968b/ E i d e l b e r g et a l . , 1981b/ E i d e l b e r g , 1981/ Steeves and Jordan, 1980/ A f e l t , 1974/ Steeves et al., 1987/ Sholomenko and Steeves, 1987/ Orlovsky, 1970a,b). L e s i o n s t u d i e s i n the monkey (Lawrence and Kuypers, 1968b/ E i d e l b e r g et al., 1981b), cat (Steeves and Jordan, 1980/ A f e l t , 1974/ E i d e l b e r g , 1981b), and b i r d (Sholomenko and Steeves, 1987b) demonstrate t h a t i n t e r r u p t i o n of the r e t i c u l o s p i n a l pathway or a b l a t i o n of i t s c e l l s of o r i g i n s e v e r e l y impairs locomotor a b i l i t y . Orlovsky (1970b), u s i n g i n t r a c e l l u l a r r e c o r d i n g i n the acute decerebrate cat, found t h a t a m a j o r i t y of r e t i c u l a r formation neurons were r h y t h m i c a l l y modulated d u r i n g : 1) spontaneous locomotion i n the t h a l a m i c p r e p a r a t i o n , 2) M L R - e l e c t r i c a l l y s t i m u l a t e d locomotion i n the mesencephalic p r e p a r a t i o n and 3) subthalamic locomotor r e g i o n ( S L R ) - s t i m u l a t i o n i n the t h a l a m i c p r e p a r a t i o n . He a l s o e s t a b l i s h e d the e x i s t e n c e of d i r e c t e x c i t a t o r y monosynaptic l i n k s between the regions s t i m u l a t e d (MLR or SLR) and r e t i c u l a r formation neurons. In a d d i t i o n , Orlovsky (1970a) found t h a t e l e c t r i c a l s t i m u l a t i o n of the pyramids r o s t r a l t o a pyramidal t r a n s e c t i o n e l i c i t e d locomotion i n the p r e c o l l i c u l a r postmammillary p r e p a r a t i o n t h a t was mediated m o n o s y n a p t i c a l l y v i a a n t i d r o m i c a c t i v a t i o n of r e t i c u l a r formation neurons. C o r t i c o f u g a l neurons, t h e r e f o r e , presumably send c o l l a t e r a l s t o r e t i c u l a r formation neurons and may exert some degree of motor c o n t r o l v i a t h i s pathway. Recent evidence shows t h a t e l e c t r i c a l s t i m u l a t i o n of the r e t i c u l a r formation e l i c i t s locomotion i n the cat (Mori et a l . , 1978; G a r c i a - R i l l and Skinner, 1987b; Jordan, 1986; Noga et al., 1988), r a t (Kinjo et al., 1988) and b i r d (Steeves et al., 1986). St u d i e s u s i n g the comparatively new approach of e l i c i t i n g or b l o c k i n g locomotor behaviour by i n j e c t i n g n e u r o t r a n s m i t t e r a g o n i s t s and a n t a g o n i s t s i n t o e l e c t r o p h y s i o l o g i c a l ! ^ i d e n t i f i e d locomotor regions has c o r r o b o r a t e d and extended p r e v i o u s f i n d i n g s . T h i s approach y i e l d s two types of i n f o r m a t i o n . F i r s t , n e u r o t r a n s m i t t e r a g o n i s t s and a n t a g o n i s t s are thought to act at r e c e p t o r s (Goodchild et al., 1982). Receptors are found on c e l l b odies, d e n d r i t e s and t e r m i n a l s but have not been l o c a l i z e d t o axons i n a p p r e c i a b l e numbers (Goodchild et al., 1982). Thus, a c t i v i t y evoked by d i r e c t i n t r a c e r e b r a l neurochemical i n f u s i o n would l i k e l y be due to the a c t i v a t i o n of these r e c e p t o r s and not due to the s t i m u l a t i o n of axons of passage which may t r a v e r s e an e l e c t r i c a l s t i m u l a t i o n s i t e . Second, the s e l e c t i v e i n j e c t i o n of these "locomotor" a f f e c t i n g neurochemicals leads to the c h a r a c t e r i z a t i o n of r e c e p t o r types, thereby a i d i n g i n the i d e n t i f i c a t i o n of neurons i n v o l v e d i n the locomotor p r o c e s s . Chemical s t i m u l a t i o n with v a r i o u s n e u r o a c t i v e agents i n j e c t e d i n t o the r e t i c u l a r formation has demonstrated t h a t locomotion can be a c t i v a t e d by c h o l i n e r g i c a g o n i s t s ( G a r c i a - R i l l et al., 1985; Sholomenko and Steeves, 1987a), GABAergic a n t a g o n i s t s ( G a r c i a - R i l l et al., 1985; Sholomenko and Steeves, 1987a; Noga et al., 1988), e x c i t a t o r y amino a c i d a g o n i s t s (glutamate, a s p a r t a t e , NMDA) ( G a r c i a - R i l l et al., 1985; Sholomenko and Steeves, 1987a; Noga et al., 1988) and Substance P (Noga et a l . , 1988; G a r c i a - R i l l et al., 1986). The combination of r e s u l t s from l e s i o n , s t i m u l a t i o n and neurochemical i n j e c t i o n s t u d i e s s t r o n g l y i m p l i c a t e r e t i c u l o s p i n a l neurons as b e i n g the o r i g i n of the major descending pathway c o n t r o l l i n g s p i n a l c o r d rhythmic o s c i l l a t o r s . However, the n e u r o t r a n s m i t t e r ( s ) u t i l i z e d by the r e t i c u l o s p i n a l pathway i t s e l f has yet to be determined (Jordan, 1986). 17 Locomotor Regions and Locomotion-Related Structures Higher order brainstem s t r u c t u r e s t h a t a p p a r e n t l y do not have d i r e c t descending connections to the s p i n a l c o r d locomotor generators or motoneurons have been i m p l i c a t e d as d r i v i n g and/or modulating the brainstem n u c l e i t h a t g i v e r i s e t o the d i r e c t s p i n a l pathways d e s c r i b e d above. Some of these s t r u c t u r e s do not have e a s i l y i d e n t i f i a b l e neuroanatomical s u b s t r a t e s and have been p h y s i o l o g i c a l l y i d e n t i f i e d as b eing i n v o l v e d i n locomotion by v i r t u e of the f i n d i n g t h a t e l e c t r i c a l s t i m u l a t i o n of these r e g i o n s can produce locomotor a l t e r a t i o n s . Thus, c o n t r o v e r s y s t i l l e x i s t s over the exact l o c a t i o n and h o d o l o g i c a l s u b s t r a t e s f o r the a c t i o n s observed with s t i m u l a t i o n of the pontobulbar locomotor s t r i p (PLS), the mesencephalic locomotor r e g i o n (MLR) and the subthalamic (SLR) locomotor r e g i o n . Other h i g h e r order s t r u c t u r e s such as the cerebellum, the l i m b i c system and b a s a l g a n g l i a are a l s o s t r o n g l y i m p l i c a t e d i n motor c o n t r o l . Cerebellum The cerebellum has the remarkable a t t r i b u t e of r e c e i v i n g d i r e c t and i n d i r e c t i n f o r m a t i o n from most p e r i p h e r a l r e c e p t o r s and from a l l of the CNS motor ce n t r e s (Arshavsky and Orlovsky, 1986). A f t e r p r o c e s s i n g the data, i t r e t u r n s the i n t e g r a t e d i n f o r m a t i o n back t o a l l of the motor c e n t r e s . Support f o r t h i s s u p p o s i t i o n comes from Arshavsky's group who have found t h a t the d o r s a l s p i n o c e r e b e l l a r t r a c t (DSCT), v e n t r a l s p i n o c e r e b e l l a r t r a c t (VSCT) and s p i n o r e t i c u l o c e r e b e l l a r t r a c t (SRCT) r e l a y 18 d i f f e r e n t types of i n f o r m a t i o n t o the cerebellum ( f o r review see Arshavsky and Orlovsky, 198 6). The DSCT r e l a y s i n f o r m a t i o n concerning a c t u a l movements from the p e r i p h e r a l motor system and i s s i l e n t i n the absence of muscle c o n t r a c t i o n s , w h ile both the VSCT and SRCT t r a n s m i t c e n t r a l l y generated n e u r a l i n f o r m a t i o n t o the cerebellum even i n the absence of rhythmic p e r i p h e r a l feedback such as i s found i n a f i c t i v e (paralyzed) p r e p a r a t i o n . F u r t h e r , Arshavsky's experiments support the hypothesis t h a t these pathways c a r r y i n f o r m a t i o n from c e n t r a l rhythmic o s c i l l a t o r elements r a t h e r than from output elements such as s p i n a l motoneurons (Arshavsky and Orlovsky, 1986). The cerebellum a l s o r e c e i v e s i n f o r m a t i o n concerning the v i s u a l system v i a the s u p e r i o r c o l l i c u l u s , the s t a t e of e q u i l i b r i u m v i a the v e s t i b u l a r system, the head and neck sensory system v i a the t r i g e m i n a l system and from the c o r t e x v i a the po n t i n e n u c l e i . I t s major h i n d b r a i n outputs i n c l u d e the r e t i c u l a r formation n u c l e i , r e d nucleus and LVN which g i v e r i s e t o the output pathways d i s c u s s e d above. The r e t i c u l a r formation, r e d nucleus and v e s t i b u l a r neurons a l l appear t o be r h y t h m i c a l l y modulated by output from the cerebellum, i n whose absence, the r h y t h m i c i t y and spontaneous a c t i v i t y of neurons was reduced (Orlovsky, 1970a,b; 1972a,c). In a d d i t i o n , the cerebellum a l s o i n f l u e n c e s the c o r t e x v i a the w e l l c h a r a c t e r i z e d t h a l a m i c loop. Arshavsky and Orlovsky (198 6) d e s c r i b e the cerebellum as an organ which can o r g a n i z e the i n t e r a c t i o n o f a v a r i e t y of locomotor 3 synergisms . Thus, i t can monitor i n f o r m a t i o n both from the 3 "Bernstein proposed the hypothesis of the m u l t i - l e v e l system of c o n t r o l of movements. According to h i s hypothesis higher sections of the nervous system determine the chains of motor a c t i v i t y , the lower l e v e l t i e s movements to s p a t i a l coordinates. S t i l l lower l e v e l s solve the motor problem as such by environment and the c u r r e n t s t a t e of each locomotor synergism, " s e l e c t e s s e n t i a l data" from t h i s i n f o r m a t i o n , and " r e g u l a t e the t r a n s m i s s i o n of s i g n a l s from one p a r t of the nervous system to another" (Arshavsky and Orlovsky, 1986). Although the cerebellum appears to p l a y a s t r o n g r o l e i n locomotor c o n t r o l , c e r e b e l l a r a b l a t i o n i n the cat does not prevent: 1) spontaneous locomotion i n the t h a l a m i c p r e p a r a t i o n , 2) locomotion i n i t i a t e d by e l e c t r i c a l s t i m u l a t i o n of the SLR i n the t h a l a m i c p r e p a r a t i o n or, 3) locomotion i n the mesencephalic p r e p a r a t i o n evoked by s t i m u l a t i o n of the MLR (Orlovsky, 1970a,b; 1972a,b,c). The above r e s u l t s i n d i c a t e t h a t while c e r e b e l l a r e f f e r e n t s can a f f e c t the q u a l i t y of locomotor output v i a a v a r i e t y of output pathways, the cerebellum i s not e s s e n t i a l f o r the genesis of the b a s i c locomotor rhythm. T h i s s u p p o s i t i o n does not, of course, negate the obvious importance of the cerebellum i n the c o n t r o l of ongoing motor a c t i v i t y i n the i n t a c t animal. Pontobulbar Locomotor S t r i p (PLS) The pontobulbar locomotor s t r i p (PLS) was p h y s i o l o g i c a l l y d e f i n e d by the f i n d i n g t h a t e l e c t r i c a l s t i m u l a t i o n of t h i s r e g i o n e l i c i t e d locomotion i n the p r e c o l l i c u l a r postmammillary cat (Shik and Yagodnitsyn, 1977; Mori et al., 1977). More recent s t u d i e s have g e n e r a l i z e d the e x i s t e n c e of the PLS to b i r d s (Steeves et al., 1987) and to a wide range of v e r t e b r a t e s p e c i e s ( f o r review, see M c C l e l l a n , 1986). The s t r i p ranges along the organizing the necessary i n t e r a c t i o n of elements (muscles, j o i n t s , limbs) and o p e r a t i v e l y c o n t r o l l i n g t h i s work." (cf. Shik e t al., 1966). At each l e v e l , "the neuronal mechanisms c o n t r o l l i n g a given motor act" (Arshavsky and Orlovsky, 1986) may be described as a locomotor synergism. 20 extent of the descending t r a c t and nucleus o f the t r i g e m i n a l nerve (Mori et al., 1977; Shik and Yagodnitsyn, 1977, 1978) r o s t r o c a u d a l l y from the l e v e l of the t r i g e m i n a l mesencephalic nucleus (MesV) t o the caudal medulla (Mori et al., 1977; Shik and Yagodnitsyn, 1977, 1978). Although not i n agreement with the r e g i o n proposed by G a r c i a - R i l l et al. (1986, 1987), Jordan (1986) p o s t u l a t e d t h a t the r o s t r a l head o f the PLS (MesV) i s synonymous with the medial mesencephalic locomotor r e g i o n (mMLR) d e f i n e d by G a r c i a - R i l l and Skinner (1986) and i s thought t o p r o j e c t i n t r a n u c l e a r i n t e r n e u r o n s t o more caudal PLS neurons (Jordan, 1986). A recent review, ( G a r c i a - R i l l et al., 1986), however, designates the pedunculopontine nucleus (PPN) as the medial MLR. Evidence t o supports t h i s s uggestion comes from neuroanatomical t r a c i n g s t u d i e s which demonstrate t h a t i n j e c t i o n of r e t r o g r a d e t r a c e r s i n t o the PLS l a b e l predominantly MesV neurons and only s m a l l numbers of PPN neurons, while anterograde neuroanatomical t r a c e r s i n j e c t e d i n t o the mMLR l a b e l mainly ventromedial r e t i c u l a r formation s t r u c t u r e s ( G a r c i a - R i l l et a l . , 1983b) . The name PLS may be a misnomer because the s t r i p a l s o appears t o extend c o n t i n u o u s l y caudalward i n t o the d o r s o l a t e r a l f u n i c u l u s (DLF) of the s p i n a l c o r d (Kazennikov et al., 1980, 1983a,b; Dubner and Bennett 1983; f o r review see M e C l e l l a n , 1986). I t has been p o s t u l a t e d t h a t the PLS may be a p o l y s y n a p t i c p r o p r i o s p i n a l pathway.(Mori et al., 1977; Shik and Yagodnitsyn, 1978; Kazennikov et a l . , 1983a,b) whose c e l l s of o r i g i n l i e ventromedial t o i t s descending f i b e r t r a c t ( Selionov and Shik, 1984). G a r c i a - R i l l (1983) o r i g i n a l l y d e s c r i b e d the PLS as being c o e x t e n s i v e w i t h Probst's t r a c t ( i . e . t r i g e m i n a l n u c l e a r f i b e r s c o u r s i n g j u s t v e n t r a l t o the nucleus from r o s t r a l medulla t o CI ( G a r c i a - R i l l and Skinner, 1986) ) which r e c e i v e s a f f e r e n t s from the mesencephalic t r i g e m i n a l nucleus ( G a r c i a - R i l l et al., 1983b). Jordan and co-workers (Jordan, 1986; Noga et al., 1988) argue s t r o n g l y t h a t c e l l bodies i n the l a t e r a l r e t i c u l a r formation and descending t r i g e m i n a l nucleus c o n s t i t u t e the PLS (Noga et al., 1988), and have a dual output both t o downstream t r i g e m i n a l ( p r o p r i o s p i n a l ) c e l l s (Matsushita et al., 1981,1982) and t o n u c l e i i n the medial r e t i c u l a r formation (Steeves and, Jordan, 1984). T h i s hypothesis i s supported by s e v e r a l o b s e r v a t i o n s i n c l u d i n g : 1) c o o l i n g o f the i p s i l a t e r a l medial r e t i c u l a r formation d u r i n g locomotion 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 the MesV/mMLR r e v e r s i b l y b l o c k s locomotion (Shefchyk et al., 1984). T h i s f i n d i n g suggests a blockade of the pathway from mMLR t o PLS to r e t i c u l a r formation t o s p i n a l c o r d between the PLS and r e t i c u l a r formation l i n k a g e , i n a d d i t i o n t o a b l o c k of the mMLR to r e t i c u l a r formation t o s p i n a l c o r d pathway between the mMLR and r e t i c u l a r formation. 2) c o o l i n g of the PLS a b o l i s h e s locomotion produced by mMLR s t i m u l a t i o n (Shefchyk et al., 1984). 3) PLS s t i m u l a t e d locomotion i s not a b o l i s h e d by t r a n s e c t i o n of the p r o p r i o s p i n a l PLS-DLF pathway at C2-C3 (Noga et a l . , 1988) and 4) e x t e r o c e p t i v e s t i m u l a t i o n of the t r i g e m i n a l r e c e p t i v e f i e l d and a v a r i e t y of other sensory a f f e r e n t s [e.g. pinna (Aoki and Mori, 1981) and f l e x o r r e f l e x a f f e r e n t (FRA) s t i m u l a t i o n , (Jankowska et a l . , 1967)] evokes locomotion ( f o r review see Jordan, 1986; Noga et al., 1988). Both Jordan and co-workers (Jordan, 1986; Noga et al., 1988) and G a r c i a - R i l l and Skinner (198 6) now suggest t h a t the locomotion e l i c i t e d by s t i m u l a t i o n o f the PLS-DLF system mimics t h a t which r e s u l t s from a f f e r e n t t r i g e m i n a l input t o t h i s locomotor r e g i o n . T h i s p l a c e s the descending t r i g e m i n a l n u c l e a r complex i n a p o s i t i o n very s i m i l a r t o t h a t of the l a t e r a l v e s t i b u l a r nucleus i n which a f f e r e n t i n put from the p e r i p h e r y , which may be modulated by comparator systems such as the cerebellum, i s able t o a l t e r motor output but i s not r e s p o n s i b l e f o r the primary locomotor rhythm. Recent s t u d i e s u t i l i z i n g neurochemical s t i m u l a t i o n t o e l i c i t locomotion suggest t h a t P L S / t r i g e m i n a l s t i m u l a t i o n - i n d u c e d locomotion i s under GABAergic i n h i b i t o r y c o n t r o l (Noga et a l . , 1988). Noga et al.(1988) found t h a t locomotion c o u l d be e l i c i t e d by i n j e c t i o n of the GABAergic a n t a g o n i s t p i c r o t o x i n i n t o the PLS. F u r t h e r , they found t h a t t r i g e m i n a l f i e l d s t i m u l a t i o n , which alone was i n e f f e c t i v e at e l i c i t i n g locomotion, would evoke locomotion f o l l o w i n g p i c r o t o x i n i n j e c t i o n i n t o the PLS. Other neurochemicals, i n c l u d i n g glutamate and Substance P, a l s o produced locomotor behaviour when i n j e c t e d i n t o the PLS (Noga et a l . , 1988). However, the anatomical s u b s t r a t e s through which these n e u r o t r a n s m i t t e r s a f f e c t locomotor c o n t r o l have yet to be determined. Mesencephalic Locomotor Region (MLR) The c l a s s i c a l MLR was c h a r a c t e r i z e d by Shik et a l . (1966) when they found t h a t f o c a l h i g h frequency e l e c t r i c a l s t i m u l a t i o n of a r e g i o n l a t e r i d e n t i f i e d as l y i n g i n c l o s e p r o x i m i t y to the cuneiform nucleus (Shik et al., 1967) evoked locomotion i n a mesencephalic cat p r e p a r a t i o n . They a l s o found t h a t v a r i a t i o n of the stimulus parameters c o u l d modulate the frequency of stepping, f o r c e of s t e p p i n g and g a i t , thereby making t h i s p r e p a r a t i o n i d e a l f o r the study of motor c o n t r o l mechanisms. L a t e r i n v e s t i g a t o r s have attempted to c l a r i f y the l o c a t i o n and input/output r e l a t i o n s of the MLR i n a v a r i e t y of animals i n c l u d i n g the monkey ( E i d e l b e r g et a l . , 1981), cat ( G a r c i a - R i l l et a l . , 1983a,b; G a r c i a - R i l l , 1983; Steeves and Jordan, 1984), r a t (Mogenson et al., 1985; Mogenson and Wu, 1986) and b i r d (Sholomenko and Steeves, i n p r e p a r a t i o n ; Webster and Steeves, i n p r e p a r a t i o n ) . As p r e v i o u s l y s t a t e d , Orlovsky (1970a) found t h a t the MLR had monosynaptic connections w i t h descending r e t i c u l o s p i n a l neurons. Steeves and Jordan (1984), u s i n g anterograde a u t o r a d i o g r a p h i c t r a c i n g techniques ( [ 3 H ] p r o l i n e & [ 3 H ] l e u c i n e ) , demonstrated descending p r o j e c t i o n s from the MLR t o the i p s i l a t e r a l (heavy p r o j e c t i o n ) and c o n t r a l a t e r a l ( l i g h t p r o j e c t i o n ) p ontine and medullary r e t i c u l a r formation. They a l s o found ascending p r o j e c t i o n s t o the more r o s t r a l cuneiform nucleus, i n f e r i o r c o l l i c u l u s , s u p e r i o r c o l l i c u l u s , c e n t r a l tegmental f i e l d , i p s i l a t e r a l p e r i a q u e d u c t a l gray, s u b s t a n t i a n i g r a (both pars compacta and r e t i c u l a t a ) , f i e l d s o f F o r e l and v e n t r a l tegmental area of T s a i (VTA). At d i e n c e p h a l i c l e v e l s , t e r m i n a l s were found i n n u c l e i which i n c l u d e d the t h a l a m i c i n t r a l a m i n a r n u c l e i , zona i n c e r t a , medial subthalamic nucleus and l a t e r a l hypothalamic nucleus. E l e c t r o p h y s i o l o g i c a l and neuroanatomical t r a c i n g techniques demonstrated a f f e r e n t p r o j e c t i o n s t o the f e l i n e MLR from the pars r e t i c u l a t a of the s u b s t a n t i a n i g r a , entopeduncular nucleus, c e n t r a l gray, nucleus of the ansa l e n t i c u l a r i s , medial and l a t e r a l hypothalamus and c e n t r a l nucleus of the amygdala ( G a r c i a - R i l l et al., 1983a,b; G a r c i a - R i l l , 1983). Anterograde t r a n s p o r t s t u d i e s u s i n g the t r i t i a t e d amino a c i d [ 3 H ] l e u c i n e i n j e c t e d i n t o the MLR gave s l i g h t l y d i f f e r e n t r e s u l t s than those of Steeves and Jordan (1984). While Steeves and Jordan (1984) found a predominance of i p s i l a t e r a l l a b e l l i n g i n the pontine and medullary r e t i c u l a r formation, G a r c i a - R i l l ' s group ( G a r c i a - R i l l et al., 1983b) found a h i g h e r p r o p o r t i o n of c o n t r a l a t e r a l r e t i c u l a r formation l a b e l l i n g a f t e r i n j e c t i o n . However, d i f f e r e n c e s i n p r o j e c t i o n l a t e r a l i t y c o u l d be a t t r i b u t e d t o a more medial placement of the i n j e c t i o n by G a r c i a - R i l l ' s group. In a d d i t i o n , G a r c i a - R i l l et al. (1983b) found a p r o j e c t i o n from the mMLR to Pr o b s t ' s t r a c t which was not r e p o r t e d by Steeves and Jordan (1984). More r e c e n t l y however, G a r c i a - R i l l and Skinner (1986, 1987b) r e i n t e r p r e t e d t h i s r e s u l t such t h a t the apparent MLR-Probst's t r a c t p r o j e c t i o n probably a r i s e s from the t r i g e m i n a l mesencephalic nucleus which i s known to send p r o j e c t i o n s t o the descending t r i g e m i n a l n u c l e a r complex ( G a r c i a - R i l l and Skinner, 1986). Combined with Orlovsky's f i n d i n g (1970a) t h a t MLR neurons have monosynaptic connections with s p i n a l p r o j e c t i n g r e t i c u l a r formation neurons, i t seems l i k e l y t h a t the MLR, whatever i t s anatomical s u b s t r a t e , e x e r t s c o n t r o l over motor f u n c t i o n v i a i t s monosynaptic connection w i t h the r e t i c u l a r - f o r m a t i o n . Recently, two d i v i s i o n s of the MLR have been p o s t u l a t e d . Thus, the c l a s s i c a l MLR d e s c r i b e d by Shik et al., (1966) [now 25 c a l l e d the l a t e r a l MLR (1MLR) Jordan, 1986; Noga et a l . , 1988] and a MLR (PPN/MLR) l o c a t e d w i t h i n the c o n f i n e s o f the pedunculopontine tegmental nucleus (PPN) ( G a r c i a - R i l l , 1986) have been d e s c r i b e d . Support f o r the e x i s t e n c e o f the PPN/MLR a l s o comes from s t u d i e s i n the r a t , where e l e c t r i c a l s t i m u l a t i o n of the PPN evoked locomotion i n the decerebrate animal ( G a r c i a - R i l l and Skinner, 1986) and i n c r e a s e d motor a c t i v i t y i n the i n t a c t f r e e l y moving animal (Mogenson et al., 1985; Brudzynski et al., 1988). As w i l l be d i s c u s s e d more f u l l y below, Brudzynski et a l . (1988) a l s o d e s c r i b e d more l a t e r a l s i t e s , p o s s i b l y e q u i v a l e n t t o the PPN/MLR, which evoke locomotion upon e l e c t r i c a l s t i m u l a t i o n but which show d i f f e r e n t c h a r a c t e r i s t i c s w i t h r e s p e c t t o chemical s t i m u l a t i o n . Mogenson et al. (1985), i n reviewing neuroanatomical connections t o the r a t PPN, l i s t e d the nucleus accumbens, the s u b p a l l i d a l area ( i n c l u d i n g the s u b s t a n t i a innominata and l a t e r a l p r e o p t i c a r e a ) , the zona i n c e r t a , the medial p r e o p t i c area, the subthalamic nucleus and the a n t e r i o r hypothalamus/preoptic area as a l l sending d i r e c t connections t o the PPN. They proposed t h a t the accumbens, s u b p a l l i d a l and zona i n c e r t a p r o j e c t i o n s t o PPN may p l a y a r o l e i n l i m b i c ("motivational") c o n t r o l over locomotor behaviour. In an attempt t o evoke or bl o c k locomotion, G a r c i a - R i l l et a l . (1985) i n j e c t e d a v a r i e t y of neurochemicals i n t o the PPN/MLR of the mesencephalic c a t . They found t h a t i n j e c t i o n of the GABAergic a n t a g o n i s t s p i c r o t o x i n and b i c u c u l l i n e i n t o the mMLR e l i c i t e d locomotion which c o u l d be bl o c k e d by the i n f u s i o n of GABA or muscimol at the same l o c a t i o n . F u r t h e r , they found t h a t glutamate (at h i g h c o n c e n t r a t i o n ) reduced the e l e c t r i c a l t h r e s h o l d and a c e t y l c h o l i n e was i n e f f e c t i v e i n e l i c i t i n g locomotion. A l s o , s t u d i e s from t h i s group ( G a r c i a - R i l l et al., 1985; G a r c i a - R i l l and Skinner, 1986) showed t h a t Substance P i n j e c t i o n i n t o the mMLR e l i c i t e d long l a s t i n g locomotion i n the c a t . In the i n t a c t f r e e l y moving r a t , i n j e c t i o n of p i c r o t o x i n and glutamate (Brudzynski et al., 1988) i n t o the PPN/MLR s i g n i f i c a n t l y i n c r e a s e d motor a c t i v i t y , w h i le i n j e c t i o n of the c h o l i n e r g i c a g o n i s t c a r b a c h o l reduced motor a c t i v i t y (Brudzynski et al., 1988). Why glutamate was more e f f e c t i v e i n the r a t than the cat remains to be e l u c i d a t e d , but s e v e r a l p o s s i b l e e x p l a n a t i o n s may be g i v e n . F i r s t , glutamate i s a n a t u r a l n e u r o t r a n s m i t t e r which i s r a p i d l y taken up or metabolized and u n l i k e e l e c t r i c a l s t i m u l a t i o n , where c u r r e n t spread and t h e r e f o r e r e c r u i t m e n t ( a c t i v a t i o n ) i s both i n t e n s i t y dependent and instantaneous w i t h the onset of s t i m u l a t i o n , the d i f f u s i o n o f glutamate through the t i s s u e t o a s u f f i c i e n t number of c e l l s t o evoke locomotion or change t h r e s h o l d would l i k e l y occur more r a p i d l y and w i t h l e s s d egradation i n the s m a l l e r more compact r a t PPN than i n the c a t . Second, G a r c i a - R i l l et al. (1985) used the mesencephalic p r e p a r a t i o n i n which a c t i v a t i o n l e v e l p l a y s a major r o l e i n the e l i c i t a t i o n o f locomotion, (see Mori et al., 1982), while Brudzynski et al. (1986, 1988) u t i l i z e d an i n t a c t f r e e l y moving animal i n which the a c t i v a t i o n l e v e l was a l r e a d y h i g h and i n some cases was made even hi g h e r by the i n t r o d u c t i o n of amphetamine i n t o the nucleus accumbens. I t i s l i k e l y t h a t i n the i n t a c t animal with h i g h b a s e l i n e a c t i v a t i o n , the recruitment of a small number of locomotor modulating neurons w i l l cause a s i g n i f i c a n t i n c r e a s e i n locomotor a c t i v i t y , w h i le i n the mesencephalic p r e p a r a t i o n , the a c t i v a t i o n l e v e l must f i r s t be i n c r e a s e d from a lower b a s e l i n e b e f o r e locomotion can be e l i c i t e d . Brudzynski et al. (1988) a l s o found t h a t i n j e c t i o n s of the c h o l i n e r g i c a g o n i s t c a r b a c h o l i n t o the PPN reduced locomotion, while more d o r s a l and l a t e r a l i n j e c t i o n s i n t o the r e g i o n bordered by the cuneiform nucleus, PPN and p e r i a q u e d u c t a l gray, p o s s i b l y coextensive with the 1MLR, i n c r e a s e d locomotion i n the f r e e l y moving animal. Both e f f e c t s c o u l d be r e v e r s e d by a t r o p i n e i n j e c t i o n i n t o the same s i t e . These d i f f e r e n t e f f e c t s may r e f l e c t the p r e v i o u s l y d i s c u s s e d d i f f e r e n t i a l d i s t r i b u t i o n o f r e c e p t o r s and/or p r o j e c t i n g f i b r e s from the two MLRs. U n l i k e Brudzynski et al. (1988), G a r c i a - R i l l et al. (1985) found no e f f e c t when a c e t y l c h o l i n e was i n j e c t e d i n t o the PPN/MLR of the mesencephalic c at p r e p a r a t i o n . Reasons f o r t h i s d i s c r e p a n c y remain t o be determined, however, the use of the s h o r t e r a c t i n g , q u i c k l y m etabolized a c e t y l c h o l i n e i n s t e a d of the longer a c t i n g c a r b a c h o l may be r e s p o n s i b l e f o r some of these experimental d i f f e r e n c e s (Taylor, 1 9 8 5 a ,b). P u t a t i v e n u c l e a r o r i g i n s of pathways impinging on the MLRs i n c l u d e t h e : 1) l a t e r o d o r s a l tegmental nucleus, which may send c h o l i n e r g i c (Brudzynski et al., 1988; F i b i g e r and Semba, 1988) and Substance P (Vincent et al., 1983) p r o j e c t i o n s t o the PPN, 2) s u b s t a n t i a innominata and l a t e r a l p r e o p t i c area, which may send c h o l i n e r g i c p r o j e c t i o n s t o the PPN (Brudzynski et al., 1 9 8 8 ) , 3) i n t r i n s i c c h o l i n e r g i c neurons present i n the PPN (Goldsmith and Van der Kooy, 1 9 8 8 ) , 4) S u b s t a n t i a n i g r a , pars r e t i c u l a t a , which appears t o send a GABAergic input t o PPN 28 ( G a r c i a - R i l l and Skinner, 1986), 5) entopeduncular nucleus, which sends input t o PPN ( G a r c i a - R i l l and Skinner, 198 6) and 6) Nucleus accumbens, which sends input to the PPN and cuneiform nucleus ( G a r c i a - R i l l and Skinner, 1986). Other p r o j e c t i o n s t o the MLRs have been d e s c r i b e d above, but the type of n e u r o t r a n s m i t t e r s employed i n these pathways remains to be e l u c i d a t e d . Subthalamic Nucleus and Subthalamic Locomotor Region The subthalamic nucleus and subthalamic r e g i o n ( i n c l u d i n g the p o s t e r i o r and l a t e r a l hypothalamus) a l s o appear t o be i n v o l v e d i n motor c o n t r o l . While b i l a t e r a l d e s t r u c t i o n of the subthalamus i n an otherwise i n t a c t cat does not prevent locomotion ( H a e r t i g and Masserman, 1940), u n i l a t e r a l d e s t r u c t i o n of the subthalamic nucleus u n d e r l i e s hemiballismus i n humans (Hammond et al., 1979). In a d d i t i o n , e l e c t r i c a l s t i m u l a t i o n of t h i s r e g i o n e l i c i t s a l t e r n a t i n g s t e p p i n g movements i n the i n t a c t ( S i e g e l and Flynn, 1968), l i g h t l y a n a e s t h e t i z e d (Waller, 1940; H a e r t i g and Masserman, 1940), t h a l a m i c (Orlovsky, 1969b), and hypothalamic cat ( E l d r i d g e et al., 1985), as w e l l as i n the decerebrate monkey ( E i d e l b e r g et al., 1981b). F u r t h e r evidence i m p l i c a t i n g the subthalamic r e g i o n as being important to locomotion comes from the o b s e r v a t i o n t h a t animals w i t h a t r a n s e c t i o n of the n e u r a x i s r o s t r a l t o the subthalamic nucleus (a p r e c o l l i c u l a r - p r e m a m m i l l a r y decerebration) w i l l spontaneously locomote, while a t r a n s e c t i o n removing t h i s nucleus (a p r e c o l l i c u l a r - p o s t m a m m i l l a r y decerebration) e l i m i n a t e s spontaneous a c t i v i t y (Shik et al., 1967; G a r c i a - R i l l and Skinner, 1986). Orlovsky (1969) found t h a t s t i m u l a t i o n of the subthalamic locomotor r e g i o n (SLR), a s i t e dorsomedial t o , but not w i t h i n , the subthalamic nucleus evokes t r e a d m i l l locomotion i n an acute t h a l a m i c c a t . F u r t h e r , he found t h a t b i l a t e r a l l e s i o n o f the MLR d i d not i n t e r r u p t SLR-stimulated locomotion but d i d reduce the bouts o f spontaneous locomotor a c t i v i t y a s s o c i a t e d w i t h the th a l a m i c p r e p a r a t i o n (Orlovsky, 1969). A l s o , decerebrate c a t s with b i l a t e r a l d e s t r u c t i o n of the SLR can be made to run and walk with e l e c t r i c a l s t i m u l a t i o n of MLR (Shik et al., 1966; S i r o t a and Shik, 1973). Thus, the SLR appears t o send e f f e r e n t s t o both the MLR and r e t i c u l a r formation (Orlovsky, 1970b). A n a t o m i c a l l y , the SLR appears t o be co e x t e n s i v e w i t h the zona i n c e r t a (ZI) and l a t e r a l hypothalamic area (LHA) which were l a b e l l e d with the anterograde t r a c e r PHA f o l l o w i n g i n j e c t i o n i n t o the r a t s u b s t a n t i a innominata (Mogenson et al.,1985). The ZI/LHA r e g i o n , l i k e the subthalamic nucleus, would be spared i n a spontaneously locomoting p r e c o l l i c u l a r - p r e m a m m i l l a r y t r a n s e c t i o n and may be damaged with the type of c e r e b r o v a s c u l a r a c c i d e n t a s s o c i a t e d with human hemiballismus. A l s o , the ZI r e c e i v e s a f f e r e n t p r o j e c t i o n s from the cortex, mesencephalic r e t i c u l a r formation and s u p e r i o r c o l l i c u l u s , w h i le sending e f f e r e n t s t o the PPN and b a s a l g a n g l i a (Mogenson and Wu, 1986). Mogenson and Wu d e s c r i b e the ZI r e g i o n as being s t r a t e g i c a l l y l o c a t e d i n a p o s i t i o n t o i n t e g r a t e i n f o r m a t i o n between the b a s a l g a n g l i a , l i m b i c system and MLR (Mogenson and Wu, 198 6). Recently, E l d r i d g e et al. (1985) and Waldrop et al, (1988) have demonstrated t h a t i n j e c t i o n of the GABAergic a n t a g o n i s t 30 p i c r o t o x i n i n t o the f e l i n e SLR evoked both a c t u a l and ^ f i c t i v e ' locomotion which c o u l d be b l o c k e d by muscimol (GABAA r e c e p t o r agonist) i n f u s i o n . T h i s data i m p l i c a t e s c e l l bodies or d e n d r i t e s c o n t a i n e d w i t h i n the SLR i n the a c t i v a t i o n of locomotion and c o n t r a d i c t s the suggestion by G a r c i a - R i l l and Skinner (1986) t h a t e l e c t r i c a l s t i m u l a t i o n of the SLR a c t i v a t e s b a s a l g a n g l i a - t h a l a m i c or entopedunculo-pedunculopontine n u c l e a r en  passant f i b r e s . As w i l l be d i s c u s s e d below, the r e g i o n of the SLR may act as a step i n the t r a n s d u c t i o n of motor i n f o r m a t i o n from l i m b i c systems (Mogenson et al., 1985). B a s a l G a n g l i a Damage to or degeneration of the b a s a l g a n g l i a n u c l e i has been shown to l e a d to movement d i s o r d e r s such as Parkinson's d i s e a s e ( S u b s t a n t i a n i g r a , pars compacta), and Huntington's chorea (caudate nucleus) ( c f . Carpenter, 1978). Although these d i s o r d e r s c o n s t i t u t e severe impairments t o motor performance, the major d e f i c i t s a s s o c i a t e d with these d i s e a s e s appear to be of a p o s t u r a l or i n t e n t i o n a l nature (Wetzel and S t u a r t , 1976; f o r review see G a r c i a - R i l l and Skinner, 1986). E l e c t r i c a l s t i m u l a t i o n of the globus p a l l i d u s produces i n c o n s i s t e n t locomotor responses i n the l i g h t l y a n a e s t h e t i z e d cat (Waller, 1940). Monkeys, dogs and c a t s w i t h b i l a t e r a l a b l a t i o n of v a r i o u s n u c l e i of the b a s a l g a n g l i a do not show i n c a p a c i t a t i n g impairment or l o s s of c o o r d i n a t i o n once locomotor p r o g r e s s i o n has begun (Waller, 1940; Denny-Brown, 1966; Hinsey et al., 1930). Therefore, the b a s a l g a n g l i a , while important to the i n i t i a t i o n of v o l u n t a r y locomotion, do not appear t o a f f e c t the s t e p p i n g mechanism i t s e l f (Martin, 1967) but probably modulate locomotor a c t i v i t y through connections w i t h e x t r a p y r a m i d a l motor c i r c u i t r y i n c l u d i n g the PPN/MLR ( G a r c i a - R i l l and Skinner, 1986). In accordance with t h i s i n f o r m a t i o n , the s u b s t a n t i a n i g r a , pars r e t i c u l a t a has been shown t o send a GABAergic p r o j e c t i o n t o the PPN/MLR (C h i l d s and Gale, 1983; McGeer et al., 1984; G a r c i a - R i l l et al., 1985), while entopeduncular nucleus and globus p a l l i d u s GABAergic e f f e r e n t s a l s o impinge on the PPN/MLR ( G a r c i a - R i l l and Skinner, 1986; McGeer et al., 1984). In a d d i t i o n , the b a s a l g a n g l i a - c o r t i c a l loop, v i a the centromedian, v e n t r a l a n t e r i o r and v e n t r a l l a t e r a l t h a l a m i c n u c l e i , may exert some i n f l u e n c e over the MLR, as c o r t i c a l e f f e r e n t s with an as yet u n s p e c i f i e d n e u r o t r a n s m i t t e r ( p o s s i b l y e x c i t a t o r y amino a c i d s (EAA) such as glutamate) appear t o connect w i t h the PPN/mMLR ( G a r c i a - R i l l and Skinner, 1986). Limbic S t r u c t u r e s Limbic s t r u c t u r e s have been i m p l i c a t e d i n m o t i v a t i o n a l aspects o f locomotor behaviours such as p r o c u r i n g food or escap i n g from p r e d a t o r s (Brudzynski and Mogenson, 1985). The ne u r a l c i r c u i t u n d e r l y i n g these behaviours i s b e l i e v e d t o be, i n p a r t , mediated v i a the PPN/MLR (Brudzynski and Mogenson, 1985). Two l i m b i c r e g i o n s , the hippocampal formation and amygdala, have been demonstrated t o p r o j e c t t o the s u b p a l l i d a l area [ s u b s t a n t i a innominata (SI) & l a t e r a l p r e o p t i c area (LPA)] and nucleus 32 accumbens (NA) i n the r a t (Mogenson et al., 1985; Mogenson and Wu, 1986, 1988). The amygdala appears t o send a d i r e c t p r o j e c t i o n to the MLR ( G a r c i a - R i l l et al., 1983b). The NA sends GABAergic e f f e r e n t s to the pedunculopontine and cuneiform n u c l e i ( G a r c i a - R i l l and Skinner, 1986; Jones and Mogenson, 1980) and a l s o to the s u b p a l l i d a l r e g i o n (Mogenson et a l . , 1985). The s u b p a l l i d a l r e g i o n , i n t u r n , sends a p r o j e c t i o n to PPN (Mogenson et al., 1985). The s u b p a l l i d a l region'' a l s o p r o j e c t s to the ZI/LHA (SLR) which, i n t u r n , sends e f f e r e n t s to PPN (Mogenson et al., 1985). Evidence to support the importance of t h i s l i m b i c motor c i r c u i t comes from a v a r i e t y of s t u d i e s u s i n g the f r e e l y moving r a t . These s t u d i e s have found t h a t : 1) the s u b s t a n t i a n i g r a , pars compacta sends a dopaminergic p r o j e c t i o n t o NA. Intra-accumbens i n j e c t i o n of dopamine or a g o n i s t s t h a t i n c r e a s e NA dopamine r e l e a s e (e.g. amphetamine) i n c r e a s e d locomotor a c t i v i t y (Brudzynski and Mogenson, 1985), 2) i n j e c t i n g p r o c a i n e , which b l o c k s s y n a p t i c and axonal t r a n s m i s s i o n ( f o r review, see Brudzynski and Mogenson, 1985) i n t o the PPN s i g n i f i c a n t l y reduced h y p e r a c t i v i t y e l i c i t e d by amphetamine i n j e c t i o n i n t o NA (Brudzynski and Mogenson, 1985), 3) I n j e c t i o n of GABA an t a g o n i s t s (e.g. p i c r o t o x i n ) i n t o the s u b p a l l i d a l r e g i o n (SI/LPA) i n c r e a s e d locomotor a c t i v i t y (Jones and Mogenson, 1980) and 4) i n j e c t i o n of p r o c a i n e i n t o the ZI/LHA a f t e r SI p i c r o t o x i n - i n d u c e d h y p e r a c t i v i t y reduces the a c t i v i t y (Mogenson et a l . , 1985). Combining the above f i n d i n g s with the neuroanatomical data showing t h a t these regions are connected t o the major l o c o m o t i o n - a s s o c i a t e d c e n t r e s , i t appears p o s s i b l e t h a t l i m b i c motor i n t e g r a t i o n may occur through t h i s n e u r a l c i r c u i t (Brudzynski and Mogenson, 1985/ Mogenson, 1984). Conclusions and Purpose of Studies i n the Thesis The survey of the l i t e r a t u r e p resented r e i t e r a t e s the p o i n t t h a t , w hile a great d e a l i s now known concerning pathways and mechanisms i n the CNS c o n t r o l of locomotion, much remains to be e l u c i d a t e d . For example, i t i s g e n e r a l l y accepted t h a t rhythmic o s c i l l a t o r s which are capable of producing the b a s i c locomotor p a t t e r n s e x i s t i n the s p i n a l cord. However, the n e u r a l c i r c u i t s which comprise the o s c i l l a t o r s remain unknown. The o r i g i n s of descending s u p r a s p i n a l pathways c o n t r o l l i n g the rhythmic o s c i l l a t o r s are perhaps the most c l e a r l y d e l i n e a t e d components of the c o n t r o l system i n non-primate v e r t e b r a t e s . In these animals, the r e t i c u l o s p i n a l pathway appears to p l a y an o b l i g a t o r y r o l e , as l e s i o n of the r e t i c u l a r formation or i t s descending t r a c t s a b o l i s h e s v o l u n t a r y locomotion. The v e s t i b u l o s p i n a l , r u b r o s p i n a l and c o r t i c o s p i n a l pathways are thought to add p o s t u r a l and " f i n e " c o n t r o l f e a t u r e s to the system. However, l i t t l e i s known about the exact connections and n e u r o t r a n s m i t t e r s the descending pathways u t i l i z e t o e x e r t c o n t r o l . Furthermore, while only l i m i t e d knowledge i s a v a i l a b l e c oncerning the n e u r a l elements impinging on these descending systems, more i s known about the hodology than about the n e u r o t r a n s m i t t e r s which act to c o n t r o l these pathways. Very l i t t l e i s known r e g a r d i n g the d i f f e r e n c e s between the locomotor c o n t r o l systems of primates versus lower v e r t e b r a t e s which c o u l d account f o r the occurrence of s p i n a l s t e p p i n g found i n a l l v e r t e b r a t e s except primates. Higher order s t r u c t u r e s i n c l u d i n g the locomotor regions, b a s a l g a n g l i a , l i m b i c system, sensory systems (e.g. t r i g e m i n a l , v e s t i b u l a r ) and comparator systems (e.g. cerebellum) appear t o ex e r t c o n t r o l over the descending s u p r a s p i n a l pathways, but comparatively l i t t l e i s known wit h c e r t a i n t y r e g a r d i n g the s p e c i f i c s of t h i s c o n t r o l . As d e s c r i b e d above, the m a j o r i t y of locomotor s t u d i e s have been c a r r i e d out i n complex mammalian systems (e.g. c a t , rat) t h a t employ quadrupedal locomotion (Dietz, 1987). The use of quadrupedal animals imposes c o m p l i c a t i o n s t o the study of motor systems (e.g. i n t r a g i r d l e c o o r d i n a t i o n , g a i t conversion) t h a t may, i n p a r t , be overcome by the study of a b i p e d a l system. The b i r d seemed t o me t o be i d e a l f o r the study of locomotor behaviour. L i k e humans, the b i r d i s a t r u e b i p e d d u r i n g overground locomotion. A l s o , l i k e humans, presumed i n t e r a c t i o n s between f o r e l i m b s and hindlimbs which occur i n quadrupedal animals are reduced or absent except d u r i n g the t r a n s i t i o n from walking t o f l y i n g . The two independent modes of a v i a n locomotion a l s o make the b i r d an i d e a l model f o r the study of locomotor behaviour, as i t may be p o s s i b l e t o more e a s i l y i s o l a t e the n e u r a l c i r c u i t r y i n v o l v e d i n these two d i f f e r e n t modes of locomotion. Perhaps most i m p o r t a n t l y , the b i r d does not possess the c o r t i c o s p i n a l t r a c t which complicates the study of motor c o n t r o l i n mammalian s p e c i e s and primates (Cabot et al., 1982; Webster and Steeves, 1988; Reiner and Karten, 1982). T h i s absence s t r o n g l y i m p l i c a t e s more caudal brainstem s t r u c t u r e s i n the c o n t r o l of locomotion and allows one to study a complex 35 motor system t h a t i s devoid of c o r t i c o s p i n a l i n f l u e n c e s , even i n the i n t a c t s t a t e . L a s t l y , b i r d s possess CNS motor c i r c u i t r y e q u i v a l e n t t o a l l mammalian v e r t e b r a t e s ( i n c l u d i n g primates) t o the l e v e l of the b a s a l g a n g l i a (Reiner et a l . , 1984), thus making p o s s i b l e the comparison of h i n d - and midbrain locomotor mechanisms across a broad p h y l o g e n e t i c range. The above a t t r i b u t e s make the b i r d an e x c e l l e n t model not only f o r the e l u c i d a t i o n of neuronal elements c o n t r o l l i n g v e r t e b r a t e motor systems, but a l s o f o r the study of other aspects of m o t o r i c i t y , i n c l u d i n g locomotor development and r e p a i r . P r i o r t o the b e g i n n i n g of t h i s work, a review of the l i t e r a t u r e i n d i c a t e d t h a t very l i t t l e was known about the p h y s i o l o g y and anatomy of s u p r a s p i n a l s t r u c t u r e s c o n t r o l l i n g locomotion i n b i r d s ( E i d e l b e r g , 1981), with the e x c e p t i o n t h a t b i r d s were capable of making s t e p p i n g movements i n the absence of s u p r a s p i n a l input ("spinal stepping") (Tarchanoff, 1895/ ten Cate, 1960). My s t u d i e s have examined aspects of the s u p r a s p i n a l mechanisms r e s p o n s i b l e f o r the i n i t i a t i o n and descending c o n t r o l of a v i a n locomotion (Weinstein et al., 1984; Sholomenko and Steeves, 1987a,b; Steeves et al., 1987). The Canada goose, Branta canadensis was chosen as the main experimental animal as i t i s an e x c e l l e n t walking b i r d as w e l l as a remarkable long d i s t a n c e f l i e r . F u r t h e r , i t i s a l a r g e animal, i s r e a d i l y a v a i l a b l e f o r study, i s easy to handle i n c a p t i v i t y and i s not an endangered s p e c i e s . The domesticated n o n - f l y i n g , Pekin duck, Anas platyrhynchos and the domesticated Brandies goose were a l s o u t i l i z e d i n some of the acute brainstem s t i m u l a t i o n experiments t o determine the a p p l i c a b i l i t y of the experimental f i n d i n g s i n the goose t o other avian s p e c i e s . My p r e v i o u s s t u d i e s have i n c l u d e d : 1) d e f i n i n g an index of normal locomotor muscle p a t t e r n s (Weinstein et al., 1984) u s i n g electromyographic techniques to determine which f o r e l i m b and hindlimb muscles best d e f i n e the f l i g h t and walking p a t t e r n s r e s p e c t i v e l y . The muscles which best d e f i n e the f l i g h t phase i n c l u d e the p e c t o r a l i s muscle (PECT) (major wing depressor) and d e l t o i d e u s major muscle (DM)(wing l e v a t o r ) . Muscles which d e f i n e the stance phase and swing phase of walking i n c l u d e the f l e x o r c r u r i s l a t e r a l i s (FCL) and i l i o t i b i a l i s c r a n i a l i s (ITC) (synonymous with the mammalian s a r t o r i u s muscle), r e s p e c t i v e l y , 2) u s i n g s e l e c t i v e l e s i o n s of the low t h o r a c i c s p i n a l c o r d to determine the descending s p i n a l c o r d pathways e s s e n t i a l to hindlimb locomotion i n both c h r o n i c a l l y maintained and i n acute decerebrate brainstem s t i m u l a t e d p r e p a r a t i o n . R e s u l t s from these s t u d i e s demonstrated t h a t motor i n f o r m a t i o n descending i n the v e n t r a l f u n i c u l i i s e s s e n t i a l f o r locomotion i n both p r e p a r a t i o n s . F u r t h e r , the pathway most s t r o n g l y i m p l i c a t e d as e s s e n t i a l t o locomotion was the r e t i c u l o s p i n a l pathway t r a v e l l i n g as a d i f f u s e p r o j e c t i o n i n the v e n t r a l s p i n a l c o r d (Sholomenko and Steeves, 1987b) and 3) d e f i n i n g r e g i o n s i n the mid- and h i n d b r a i n which, when e l e c t r i c a l l y s t i m u l a t e d , produce locomotor movements at low c u r r e n t i n t e n s i t y . Mapping of these s t i m u l a t i o n s i t e s has determined the l o c a t i o n of locomotor s i t e s i n the a v i a n brainstem which are e q u i v a l e n t t o those found i n mammals, thereby p r o v i d i n g a b e t t e r understanding of the s u p r a s p i n a l i n f l u e n c e s which i n i t i a t e , m aintain and modulate 37 locomotion i n b i r d s (Steeves et al., 1987). Locomotion evoking s t i m u l a t i o n s i t e s found to date i n c l u d e : 1) a s t r i p extending from the caudal pons t o caudal medulla subjacent to and w i t h i n the nucleus and t r a c t of the descending t r i g e m i n a l system. This r e g i o n appears to be e q u i v a l e n t to the mammalian pontomedullary locomotor s t r i p (PLS or P r o b s t ' s T r a c t of G a r c i a - R i l l et al., 1983b)/ 2) s i t e s i n the v e n t r a l g i g a n t o c e l l u l a r and m a g n o c e l l u l a r r e t i c u l a r formation of the h i n d b r a i n which l i e w i t h i n the d o r s a l and v e n t r a l c e n t r a l r e t i c u l a r n u c l e i and 3) a r e g i o n i n the midbrain which l i e s s l i g h t l y medial t o the l a t e r a l s p i r i f o r m n u cleus. The above f i n d i n g s have demonstrated t h a t s t r u c t u r e s r e s p o n s i b l e f o r a v i a n motor c o n t r o l are s i m i l a r t o those found i n other v e r t e b r a t e s . The purpose of the s t u d i e s found i n t h i s t h e s i s was t o f u r t h e r c h a r a c t e r i z e these and other r e g i o n s i n the a v i a n b r a i n which may c o n t r i b u t e to the c o n t r o l of locomotor behaviours. The c h a r a c t e r i z a t i o n of n e u r a l c i r c u i t r y which c o n t r o l s locomotion i s a necessary p r e r e q u i s i t e t o the f u n c t i o n a l r e p a i r of t h i s c i r c u i t r y f o l l o w i n g c e n t r a l nervous system (CNS) i n j u r y or degeneration. In Chapter 2, a s y s t e m a t i c survey u s i n g f o c a l e l e c t r i c a l s t i m u l a t i o n was performed i n an attempt to l o c a l i z e other avian locomotor r e g i o n s which may exert c o n t r o l over the regions d e s c r i b e d p r e v i o u s l y . Three p r e v i o u s l y u n c h a r a c t e r i z e d locomotion-evoking e l e c t r i c a l s t i m u l a t i o n r e g i o n s have been found. One r e g i o n l i e s i n c l o s e p r o x i m i t y t o the nucleus i n t e r c o l l i c u l a r i s (ICo) of the t e c t a l midbrain. The second can be l o c a l i z e d t o the medial midbrain r e t i c u l a r formation (MRF). A 38 t h i r d s i t e l i e s w i t h i n the c o n f i n e s of the pontine and r o s t r a l medullary medial l o n g i t u d i n a l f a s c i c u l u s (MLF). F o l l o w i n g the l o c a l i z a t i o n of the e l e c t r o p h y s i o l o g i c a l l y -d e f i n e d locomotor r e g i o n s , a s e r i e s of experiments were performed u s i n g m i c r o i n j e c t i o n of neurochemicals i n t o these r e g i o n s . While n e c e s s a r i l y incomplete, t h i s survey was undertaken f o r two reasons. F i r s t , neurochemical a c t i v a t i o n of locomotion from a locomotor r e g i o n should d i f f e r e n t i a t e between locomotion e l i c i t e d by a c t i v a t i o n of n e u r o t r a n s m i t t e r r e c e p t o r s and locomotion evoked by s t i m u l a t i o n of axons of passage. Second, s e l e c t i v e i n j e c t i o n of d i f f e r e n t locomotion-evoking neurochemicals may p r o v i d e i n f o r m a t i o n concerning both the n e u r o t r a n s m i t t e r s i n v o l v e d i n the locomotor c i r c u i t r y and the p o t e n t i a l n e u r a l pathways which c o n t a i n these n e u r o t r a n s m i t t e r s . Chapter 3 d e s c r i b e s and d i s c u s s e s the r e s u l t s of d i r e c t i n t r a c e r e b r a l i n j e c t i o n of c h o l i n e r g i c n e u r o t r a n s m i t t e r a g o n i s t s and a n t a g o n i s t s i n t o locomotion-evoking e l e c t r i c a l s t i m u l a t i o n s i t e s . C h o l i n e r g i c m u s c a r i n i c a g o n i s t s e l i c i t e d long l a s t i n g and r e v e r s i b l e (antagonist) locomotion when i n t r o d u c e d i n t o the pontobulbar locomotor s t r i p (PLS), d o r s a l p a r t of the medullary c e n t r a l nucleus (Cnd), v e n t r a l p a r t of the medullary c e n t r a l nucleus (Cnv), and MLF. I n j e c t i o n of a g o n i s t i n t o the medial mesencephalic r e t i c u l a r formation (MRF) reduced the t h r e s h o l d f o r e l e c t r i c a l l y s t i m u l a t e d locomotion. Chapter 4 d e s c r i b e s and d i s c u s s e s the r e s u l t s from the i n t r o d u c t i o n of y-aminobutyric a c i d (GABA) a n t a g o n i s t s i n t o a v a r i e t y of e l e c t r i c a l s t i m u l a t i o n - d e f i n e d s i t e s , i n c l u d i n g the PLS, Cnd, Cnv, pontine r e t i c u l a r formation (RP) and ICo. 39 A n t a g o n i s t i n j e c t i o n evoked long l a s t i n g locomotion' which was t r a n s i e n t l y r e v e r s e d by GABA. Chapter 5 d e s c r i b e s and d i s c u s s e s the r e s u l t s from m i c r o i n j e c t i o n o f e x c i t a t o r y amino a c i d and Substance P i n j e c t i o n i n t o a v a r i e t y o f locomotor r e g i o n s . I n j e c t i o n o f the g l u t a m a t e r g i c a g o n i s t N-methyl-D-aspartate (NMDA) i n t o s i t e s i n c l u d i n g the PLS, Cnd, Cnv, MLF and MRF e l i c i t e d , or reduced the e l e c t r i c a l t h r e s h o l d necessary t o induce, locomotion. These e f f e c t s were r e v e r s i b l e with the glutamate a n t a g o n i s t glutamic a c i d d i e t h y l e s t e r (GDEE). I n j e c t i o n of Substance P i n t o the pontine and medullary r e t i c u l a r formation e l i c i t e d walking or reduced the t h r e s h o l d f o r e l e c t r i c a l l y s t i m u l a t e d locomotion. P h a s i c p e r i p h e r a l a f f e r e n t input has been shown t o have a r o l e i n locomotor c o n t r o l i n a v a r i e t y o f v e r t e b r a t e s (see Chapter 6 ) . However, the extent t o which t h i s input may be important i n av i a n locomotion has not been determined. Therefore, s t u d i e s designed t o examine whether p h a s i c p e r i p h e r a l o a f f e r e n t input was e s s e n t i a l f o r avian locomotor p a t t e r n s were undertaken (Chapter 6 ) . My r e s u l t s demonstrate t h a t locomotor p a t t e r n s were evoked by both e l e c t r i c a l and chemical s t i m u l a t i o n of s e v e r a l mid- and h i n d b r a i n s i t e s i n the decerebrate p a r a l y z e d b i r d ( x f i c t i v e ' p r e p a r a t i o n ) . Furthermore, x f i c t i v e ' locomotion was found i n h i g h decerebrate spontaneously locomoting b i r d s . These r e s u l t s are d i s c u s s e d i n Chapter 6. The m u l t i l e v e l o r g a n i z a t i o n o f n e u r a l c i r c u i t r y s u b s e r v i n g locomotor c o n t r o l has been demonstrated by s e l e c t i v e t r a n s e c t i o n of the neu r a x i s i n a v a r i e t y o f decerebrate p r e p a r a t i o n s . As d e s c r i b e d i n the gen e r a l i n t r o d u c t i o n (see Locomotor Regions and L o c o m o t i o n - r e l a t e d S t r u c t u r e s ) , d e c e r e b r a t i o n to a l e v e l which p r e s e r v e s the mammalian subthalamic locomotor r e g i o n allows spontaneous locomotion i n the p r e p a r a t i o n . To determine whether a s i m i l a r o r g a n i z a t i o n e x i s t s i n b i r d s , and t o i d e n t i f y a r e g i o n which may exert c o n t r o l over locomotor behaviour, v a r y i n g l e v e l s of d e c e r e b r a t i o n were performed to examine t h e i r e f f e c t s on spontaneous versus non-spontaneous locomotion i n both p a r a l y z e d and unparalyzed decerebrate animals (Chapter 7). R e s u l t s from these s t u d i e s show t h a t i n b i r d s , as i n mammals, s t r u c t u r e s l y i n g near the r e g i o n of the subthalamic nucleus appear to subserve spontaneous locomotion and removal of these s t r u c t u r e s by more caudal t r a n s e c t i o n e l i m i n a t e s t h i s spontaneous a c t i v i t y . The Decerebrate Preparation The u n a n a e s t h e t i z e d decerebrate p r e p a r a t i o n has been u t i l i z e d i n a broad range of n e u r o b i o l o g i c a l s t u d i e s i n c l u d i n g the study of h i g h t h r e s h o l d t a c t i l e s t i m u l i (e.g. Besson and Le Bars, 1979/ Kajander and G i e s l e r , 1987), r e s p i r a t i o n r e s e a r c h (e.g. E l d r i d g e et a l . , 1985/ Funk et al., (submitted)) and locomotor c o n t r o l r e s e a r c h (e.g. Aoki and Mori, 1981/ Bard and Macht, 1958/ Budakova and Shik, 1970/ E i d e l b e r g et a l . , 1981b; G a r c i a - R i l l , 1983/ G a r c i a - R i l l et al., 1983a,b,c/ G a r c i a - R i l l and Skinner, 1987/ G r i l l n e r and Shik, 1973/ Hinsey et al., 1930/ J e l l et al., 1985/ Kazennikov et al., 1983a,b; Orlovsky, 1969, 1970a,b, 1972a,b; S e l i o n o v and Shik, 1984/ Shefchyk et al., 1984/ S h e r r i n g t o n , 1910, 1915/ Shik et al., 1966/ Sholomenko et al., 1987/ Steeves and Jordan, 1980/ Steeves et al., 1987; 41 V i l l a b l a n c a , 1962; Waller, 1940). Decerebrate animals are devoid of any concious perception of pain, a l l o w i n g experimental manipulations which are not acceptable i n i n t a c t , unanaesthetized animals which are unable to " i n d i c a t e or a r r e s t the onset of s u f f e r i n g " (e.g. paralyzed) (Wall, 1975; Wall and Sternbach, 1976). The f o l l o w i n g quotation, which supports the view that decerebrate animals are devoid of pain, (Adams, 1980) r e l a t e s t o human pain p e r c e p t i o n . "Perception of pain. Only upon the a r r i v a l of pain impulses at the thalamocortical level of the nervous system i s there concious awareness of the pain stimulus. C l i n i c a l study has not informed us of the exact l o c a l i z a t i o n of the nervous apparatus for t h i s mental process. It i s not e n t i r e l y abolished by a t o t a l hemispherectomy, including the thalamus on one side. It i s often said that impulses reaching the thalamus create awareness of the a t t r i b u t e s of sensation and that the p a r i e t a l cortex i s necessary for the appreciation of the i n t e n s i t y and l o c a l i z a t i o n of the sensation. This seems an o v e r s i m p l i f i c a t i o n . Probably a close and harmonious r e l a t i o n s h i p between thalamus and cortex must exist in order for a sensory experience to be complete. The t r a d i t i o n a l separation of sensation (in t h i s instance awareness of pain) and perception (awareness of the nature of the painful stimulus) has been abandoned in favor of the view that sensation, perception, and the various conscious and unconscious responses to a pain stimulus comprise an i n d i v i s i b l e process." T h i s view i s f u r t h e r supported by the f i n d i n g i n the f r e e l y moving r a t with v a r y i n g l e v e l s of d e c e r e b r a t i o n t h a t the medulla and brainstem support r e f l e x and s t a r t l e and f l i g h t r e a c t i o n s t o p a i n f u l s t i m u l i , while the rhinencephalon and c o r t e x support a f f e c t i v e and i n t e l l e c t u a l a l e r t n e s s , r e s p e c t i v e l y (Charpentier, 1968; f o r review of p a i n l e v e l s , see Loeser and Black, 1975). In r e g a r d t o p a i n r e s u l t i n g from the d e c e r e b r a t i o n procedure, s t r o n g evidence i s a v a i l a b l e from human s t u d i e s t h a t s u r g i c a l m a n i p u l a t i o n o f b r a i n t i s s u e does not e l i c i t p a i n responses. Many n e u r o s u r g i c a l procedures are o f t e n performed i n conscious p a t i e n t s with only the use of l o c a l a n a e s t h e t i c s at i n c i s i o n s i t e s ; such p a t i e n t s never r e p o r t the p e r c e p t i o n o f p a i n (Emmers, 1981). In t h i s t h e s i s , a l l s u r g i c a l procedures, i n c l u d i n g d e c e r e b r a t i o n procedures, were performed under h a l o t h a n e / n i t r o u s oxide i n h a l a t i o n a n a e s t h e s i a with d i r e c t i n f i l t r a t i o n o f l o c a l a n a e s t h e t i c at a l l i n c i s i o n s i t e s and pre s s u r e p o i n t s . F o l l o w i n g completion of the d e c e r e b r a t i o n procedure and subsequent removal of h a l o t h a n e / n i t r o u s oxide a n a e s t h e s i a , l o c a l a n a e s t h e t i c at p r e s s u r e p o i n t s and i n c i s i o n s i t e s was con t i n u e d u n t i l the t e r m i n a t i o n o f the experiment ( f o r d e t a i l s o f a l l s u r g i c a l procedures, see M a t e r i a l s and Methods, Chapters 2,3,6,7). No s i g n s of di s c o m f o r t (e.g. w r i t h i n g , v o c a l i z a t i o n , e l e v a t e d b l o o d p r e s s u r e , e l e v a t e d heart rate) were observed i n any decerebrate animal (see APPENDIX II) and, as shown i n F i g u r e 2, e l e c t r i c a l s t i m u l a t i o n caused no s i g n i f i c a n t i n c r e a s e i n heart r a t e or b l o o d p r e s s u r e , while the locomotion evoked by the s t i m u l a t i o n e l i c i t e d d i s t i n c t i v e c a r d i o v a s c u l a r changes. F i g u r e 2 . Electromyographic a c t i v i t y , b l o o d p r e s s u r e and heart r a t e changes r e s u l t i n g from ramped e l e c t r i c a l s t i m u l a t i o n i n the r e g i o n of the descending t r i g e m i n a l t r a c t and nucleus. The s t i m u l a t i o n t r a c e (STIM) demonstrates the ramped s t i m u l a t i o n i n t e n s i t y from OuA on the l e f t t o 80uA at the r i g h t . Blood p r e s s u r e (BP) remained r e l a t i v e l y constant from the b e g i n n i n g of s t i m u l a t i o n u n t i l t h r e s h o l d f o r locomotion was reached at approximately 60uA i n t e n s i t y . Upon the onset of locomotion, as demonstrated by electromyographic records from the l e f t (LPECT) and r i g h t (RPECT) p e c t o r a l i s muscles (major wing depressor muscles) and l e f t (LITC) and r i g h t (RITC) i l i o t i b i a l i s c r a n i a l i s muscles (major h i p f l e x o r synonymous with the mammalian s a r t o r i u s muscle), the b l o o d p r e s s u r e i n c r e a s e d (see c a l i b r a t i o n bar at r i g h t ) w i t h the locomotion and r e t u r n e d t o r e s t i n g l e v e l s f o l l o w i n g the t r i a l ( r e t u r n t o r e s t i n g BP not shown). The heart r a t e (HR) a l s o remained r e l a t i v e l y constant u n t i l the t h r e s h o l d f o r locomotion was reached and appeared to i n c r e a s e w i t h e x e r c i s e , r e t u r n i n g to r e s t i n g l e v e l s f o l l o w i n g the t r i a l (not shown) (nb. d u r i n g s t r o n g wing f l a p p i n g , movement a r t i f a c t makes i t d i f f i c u l t t o d i s t i n g u i s h the HR s i g n a l . However, the s i g n a l can be observed d u r i n g the i n t e r v a l between bout of s t r o n g f l a p p i n g ) . 44 eouA 80 uA OmmHg H R I 1MC I F u r t h e r i n d i r e c t evidence demonstrating t h a t decerebrate animals were not capable of p e r c e i v i n g a p a i n f u l stimulus come from s t u d i e s examining r e s p i r a t o r y changes as a consequence of evoking locomotion with brainstem s t i m u l a t i o n . In both cat [ e l e c t r i c a l and neurochemical s t i m u l a t i o n - subthalamic locomotor r e g i o n ( E l d r i d g e et a l . , 1985)] and b i r d [ e l e c t r i c a l s t i m u l a t i o n - t r i g e m i n a l nucleus and t r a c t (Funk et a l . , submitted) a l s o see Appendix II and F i g . 2], the v e n t i l a t o r y responses t o e l e c t r i c a l or chemical s t i m u l a t i o n - i n d u c e d locomotion were s i m i l a r t o those observed i n unoperated i n t a c t animals. Taken together, the above evidence supports the c o n t e n t i o n t h a t noxious s t i m u l a t i o n was not r e s p o n s i b l e f o r the r e s u l t s observed i n t h i s study. NOMENCLATURE To prevent c o n f u s i o n concerning s t r u c t u r e s which have homologous nomenclature but are not e q u i v a l e n t i n the a v i a n versus the mammalian l i t e r a t u r e , I have used the mammalian names where a d v i s a b l e . Thus, the a v i a n Nucleus tegmentipedunculopontinus, pars compacta (TPc) w i l l be- c a l l e d by the name of i t s mammalian e q u i v a l e n t , the s u b s t a n t i a n i g r a , pars compacta (SNc) (Brauth et a l . , 1978). A l s o , the a v i a n nucleus of the ansa l e n t i c u l a r i s (nAL) w i l l be c a l l e d the subthalamic nucleus, i t s mammalian e q u i v a l e n t (Brauth et a l . , 1978). Where homologies e x i s t between avian and mammalian s t r u c t u r e s w i t h d i f f e r e n t nomenclature, I have u t i l i z e d the avian nomenclature and p r o v i d e d the a p p r o p r i a t e mammalian counter p a r t (e.g. i l i o t i b i a l i s c r a n i a l i s (ITC) muscle i s e q u i v a l e n t t o the mammalian s a r t o r i u s muscle) (Weinstein et al., 1984). 47 CHAPTER 2 ELECTRICAL STIMULATION OF MESENCEPHALIC AND PONTINE REGIONS ELICITS LOCOMOTION IN DECEREBRATE BIRDS 48 INTRODUCTION The i n i t i a t i o n and c o n t r o l of s p i n a l locomotor mechanisms by s u p r a s p i n a l s t r u c t u r e s has been s t u d i e d i n many v e r t e b r a t e s p e c i e s (McClellan, 1986). U n t i l r e c e n t l y , however, very l i t t l e i n f o r m a t i o n was a v a i l a b l e concerning the r o l e p l a y e d by these s t r u c t u r e s i n the n e u r a l c o n t r o l of locomotion i n b i r d s ( E i d e l b e r g , 1981; Steeves et al., 1987). We are i n v e s t i g a t i n g a v i a n locomotion f o r s e v e r a l reasons. F i r s t , b i r d s d i s p l a y a s t r o n g s e p a r a t i o n between the f u n c t i o n a l a c t i v i t y of wing muscles i n v o l v e d d u r i n g f l i g h t from those used by the l e g s d u r i n g b i p e d a l walking. This suggests a f u n c t i o n a l uncoupling of f o r e l i m b and hindlimb s p i n a l locomotor p a t t e r n generators i n b i r d s (ten Cate, 1962), and may a l s o suggest t h a t the two p a t t e r n generators are c o n t r o l l e d by d i f f e r e n t descending s u p r a s p i n a l pathways (Jacobson and Hollyday, 1982). Second, b i r d s do not possess a t e l e n c e p h a l o - s p i n a l p r o j e c t i o n analogous to the mammalian c o r t i c o s p i n a l t r a c t (Cabot et al., 1982). F i n a l l y , u n l i k e most v e r t e b r a t e s , b i r d s are t r u e bipeds whose overground locomotor p a t t e r n s resemble human walking (Weinstein et al., 1984). E a r l i e r r e p o r t s from our l a b o r a t o r y (Steeves et al., 1986, 1987; Sholomenko and Steeves, 1987a,b) have demonstrated t h a t f o c a l e l e c t r i c a l s t i m u l a t i o n of d i s c r e t e h i n d b r a i n regions i n the low decerebrate b i r d p r e p a r a t i o n can e l i c i t the e n t i r e r e p e r t o i r e of a v i a n locomotor behaviours. The behaviours i n c l u d e b i p e d a l a l t e r n a t i n g stepping, b i p e d a l synchronous hopping, s t e p p i n g w i t h wing f l a p p i n g and f l y i n g alone. One locomotion-evoking r e g i o n was l o c a t e d w i t h i n the ventromedial pontomedullary r e t i c u l a r formation ( s p e c i f i c a l l y , the g i g a n t o c e l l u l a r r e t i c u l a r formation (Rgc)), while the second l a y d o r s o l a t e r a l l y w i t h i n the p a r v o c e l l u l a r r e t i c u l a r formation (LRF), subjacent to or w i t h i n the descending t r i g e m i n a l t r a c t and nucleus (TTD) (see Chapter 3, F i g s . 9 & 10). Retrograde t r a c i n g combined with e l e c t r i c a l s t i m u l a t i o n s t u d i e s demonstrated t h a t neuronal c e l l bodies i n Rgc, c o - l o c a l i z e d w i t h locomotion-evoking s t i m u l a t i o n s i t e s , p r o j e c t d i r e c t l y t o both c e r v i c a l and lumbar s p i n a l c o r d (Steeves et al., 1987a; Webster and Steeves, 1987). These r e s u l t s i n b i r d s , as i n a v a r i e t y of s p e c i e s i n c l u d i n g lamprey ( M c C l e l l a n and G r i l l n e r , 1984), s t i n g r a y (Leonard et al., 1979), t e l e p s t f i s h (Kashin et al, 1975), t u r t l e (Kazennikov et al., 1980), cat (Mori et al., 1978; Shik and Yagodnitsyn, 1977) and monkey ( E i d e l b e r g et al., 1981b) demonstrated t h a t r e t i c u l o s p i n a l neurons p l a y a major r o l e i n the descending c o n t r o l of s p i n a l c o r d rhythmic o s c i l l a t o r s which are e s s e n t i a l f o r the b a s i c motor p a t t e r n (Sholomenko and Steeves, 1987b; ten Gate, 1960, 1962; G r i l l n e r , 1985; Dubuc, 1988). The d o r s o l a t e r a l r e g i o n of the r e t i c u l a r formation appears to be the a v i a n homologue of the mammalian pontobulbar locomotor s t r i p (PLS) seen i n c a t s (Steeves et al., 1987; S e l i o n o v and Shik, 1984; f o r review see M c C l e l l a n , 1986) and s t i m u l a t i o n of t h i s r e g i o n a l s o e l i c i t e d locomotion i n b i r d s . The above evidence s t r o n g l y suggests a h i g h degree of c o n s e r v a t i o n of brainstem and s p i n a l c o r d motor neuronal c i r c u i t r y a cross a broad p h y l o g e n e t i c range of s p e c i e s . To f u r t h e r t e s t t h i s hypothesis and examine to what extent the c o n s e r v a t i o n of c i r c u i t r y observed i n the medulla and pons i s c a r r i e d t o more r o s t r a l b r a i n s t r u c t u r e s , we u t i l i z e d e l e c t r i c a l s t i m u l a t i o n i n the decerebrate b i r d to s y s t e m a t i c a l l y examine re g i o n s of the pons and mesencephalon to determine i f avian c o u n t e r p a r t s of more r o s t r a l mammalian locomotor r e g i o n s , i n c l u d i n g p o s s i b l e e q u i v a l e n t s of the mesencephalic locomotor r e g i o n (MLR), c o u l d be l o c a l i z e d . Our f i n d i n g s demonstrate t h a t locomotion c o u l d be evoked by f o c a l e l e c t r i c a l s t i m u l a t i o n of regions of the mesencephalon and pons. Two c e n t r e s were l o c a l i z e d to the a v i a n midbrain, one w i t h i n the medial mesencephalic r e t i c u l a r formation (mMRF) and a second i n c l o s e p r o x i m i t y to the i n t e r c o l l i c u l a r (Ico) and i s t h m a l (Ipc) n u c l e i of the tectum. The p ontine s i t e was l o c a l i z e d t o the d o r s a l m i d l i n e of the pons and r o s t r a l medulla w i t h i n the c o n f i n e s of the medial l o n g i t u d i n a l f a s c i c u l u s (MLF). 51 MATERIALS AND METHODS Surgery Each b i r d ( e i t h e r a Canada goose, Branta canadensis or a Pekin duck, Anas platyrhynchos) was anaesthetized throughout a l l s u r g i c a l procedures w i t h halothane (1-3%) and n i t r o u s oxide (20-30%) administered through an endotracheal tube (Figure 3). The a n t e r i o r a i r sac was cannulated to f a c i l i t a t e u n i d i r e c t i o n a l (flow through) v e n t i l a t i o n (UDV) of the lungs w i t h 0 2/C0 2 (95%/5%). The c a r o t i d a r t e r y and j u g u l a r v e i n were cannulated u n i l a t e r a l l y f o r monitoring blood pressure and f l u i d / c h e m i c a l i n f u s i o n r e s p e c t i v e l y . Body temperature was monitored w i t h a temperature probe i n s e r t e d i n t o the oesophagus and maintained (39-41°C) v i a a r e c t a l cooling/warming probe. The b i r d was then f i x e d i n a s t e r e o t a x i c head holder and the body supported i n a s l i n g mounted over a motorized t r e a d m i l l . F o l l o w i n g a craniotomy, a s u c t i o n decerebration was performed along a plane extending d o r s a l l y from the caudal margin of the habenular nucleus t o the ventrocaudal p o r t i o n of the o p t i c chiasm. To prevent blood l o s s during the decerebration procedure i n the geese, blood pressure was t r a n s i e n t l y depressed by intravenous ( i . v . ) i n f u s i o n of sodium n i t r o f e r r i c y a n i d e (nipride) (15mg/100ml) i n 10% dextrose s o l u t i o n . In ducks, no depression of blood pressure was necessary. Anaesthesia was d i s c o n t i n u e d f o l l o w i n g decerebration and the b i r d s were allowed a minimum of 30 minutes f o r a l l e f f e c t s of the anaesthetic t o wear o f f before any form of b r a i n s t i m u l a t i o n was i n i t i a t e d . All 52 pressure points (e.g. ear bars) and surgical s i t e s were r o u t i n e l y infused with xyolocaine hydrochloride (2%) after general anaesthetic removal. The l e v e l of t r a n s e c t i o n d e s c r i b e d above u s u a l l y e l i m i n a t e d spontaneous locomotion i n the decerebrate animal, however, r e f l e x responses t o deep pr e s s u r e and foot web p i n c h s t i m u l a t i o n appeared normal. B i p o l a r electromyographic (EMG) e l e c t r o d e s were implanted percutaneously i n the p e c t o r a l i s (PECT) and i l i o t i b i a l i s c r a n i a l i s (ITC) muscles t o monitor muscle a c t i v i t y (Weinstein et al., 1984). During f l i g h t , the PECT muscles act in-phase as the major depressors o f the wings. The ITC muscles o f the l e g s (synonymous t o the mammalian s a r t o r i o u s muscles) f u n c t i o n as the major h i p f l e x o r s and a l s o as weak knee extensors. A l l EMGs were recorded with the t r e a d m i l l on. EMG s i g n a l s were a m p l i f i e d (Grass P15/Framp) and f i l t e r e d (band pass 200-10K Hz) p r i o r t o moni t o r i n g on an o s c i l l o s c o p e and r e c o r d i n g on ch a r t r e c o r d e r (Gould ES100B) and tape ( A k a i ) ( F i g u r e 3). B r a i n S t i m u l a t i o n F o c a l e l e c t r i c a l s t i m u l a t i o n o f l o c a l i z e d r e g i o ns w i t h i n the avian brainstem was completed on 16 Canada geese and 11 Pekin ducks. Two types o f monopolar s t i m u l a t i n g e l e c t r o d e were u t i l i z e d . The f i r s t type was a commercially a v a i l a b l e s t a i n l e s s s t e e l e l e c t r o d e (Kopf model SNE 300, t i p diameter .= 0.1mm, impedance 60-70Kfi) while the second was c o n s t r u c t e d by i n s e r t i n g 0.0762mm s t a i n l e s s s t e e l wire (exposed t i p l e n g t h =s 0.1mm, impedance = 60-70kfi) through one b a r r e l of a p u l l e d three b a r r e l 53 - F Pi. f4 B r o i n S t i m u l a t i o n Exper imenta l Apparatus E.M.G movement potent iometer head holder inhatat ion anesthet ic i n te rna l carotids ligated vent i latory outf low treadmill D 55 m i c r o p i p e t t e ( t o t a l t i p diameter = 0.1mm). Constant c u r r e n t s t i m u l a t i o n t r i a l s , (Grass Model S88/ Grass Model CCU1A) were undertaken wi t h the f o l l o w i n g standard s t i m u l a t i o n parameters: square wave p u l s e d u r a t i o n = 1.0-2.0ms; p u l s e frequency = 6 0 H z ; c u r r e n t s t r e n g t h = 10-170/iA. S t i m u l a t i o n t r i a l s were undertaken by i n c r e m e n t a l l y lowering the e l e c t r o d e s t e r e o t a x i c a l l y (Karten and Hodos, 1967; Zweers, 1971) i n t o the brainstem while s t i m u l a t i n g with a c u r r e n t i n t e n s i t y of 50-100uA. When locomotion was observed, the c u r r e n t i n t e n s i t y was reduced to zero and then s l o w l y i n c r e a s e d u n t i l t h r e s h o l d was reached. The optimal e l e c t r o d e t i p p o s i t i o n f o r evoking locomotion was then e s t a b l i s h e d by s l o w l y lowering the e l e c t r o d e t o the p o i n t where c o o r d i n a t e d r e p r o d u c i b l e (stimulus l i n k e d ) locomotor movements were i n i t i a t e d with the lowest s t i m u l a t i o n c u r r e n t (Steeves et al., 1987). A f t e r r e c o r d i n g the s t i m u l a t i o n - e v o k e d electromyographic (EMG) a c t i v i t y , the s t i m u l a t i o n s i t e was marked f o r neuroanatomical i d e n t i f i c a t i o n with an e l e c t r o l y t i c l e s i o n made by p a s s i n g a d i r e c t c u r r e n t of 3 m i l l i a m p s f o r 5 seconds. At the end of each experiment, the animal was deeply a n a e s t h e t i z e d (5% halothane/70% n i t r o u s oxide) and s a c r i f i c e d with a bolus intravenous i n j e c t i o n of 2M KC1. The brainstem was removed and p l a c e d f o r at l e a s t 2 days i n 4% paraformaldehyde, 0.1M phosphate b u f f e r (pH = 7 . 4 ) . I t was then p l a c e d i n two changes of sucrose c y r o p r o t e c t a n t (25% sucrose/10% glycerine/0.1M phosphate b u f f e r (pH =7.4)) f o r at l e a s t 4 days. S e r i a l s e c t i o n s , at 50um t h i c k n e s s , were cut c o r o n a l l y on a f r e e z i n g microtome, mounted on g e l a t i n i z e d s l i d e s and s t a i n e d with E o s i n / C r e s y l V i o l e t dyes. The l o c a t i o n of e l e c t r o l y t i c l e s i o n s , i n d i c a t i n g e f f e c t i v e locomotor s t i m u l a t i o n s i t e s , were i d e n t i f i e d a c c o r d i n g t o the a t l a s e s of Karten and Hodos (1967) and Zweers (1971). 57 RESULTS Medial L o n g i t u d i n a l F a s c i c u l u s (MLF) E l e c t r i c a l s t i m u l a t i o n of the medial l o n g i t u d i n a l f a s c i c u l u s along a c o n s i d e r a b l e r o s t r o c a u d a l extent ( F i g s . 4 & 5) e l i c i t e d v a r i e d locomotor p a t t e r n s i n seven b i r d s (6 Canada geese, 1 Pekin duck) at t h r e s h o l d s t i m u l a t i o n i n t e n s i t i e s ranging from 30-160uA (mean = 92uA). The p a t t e r n s i n c l u d e d b i l a t e r a l s t e p p i n g i n 3 b i r d s ( F i g . 6A), combined s t e p p i n g and f l a p p i n g i n 3 animals ( F i g . 6B) and wing f l a p p i n g alone i n one p r e p a r a t i o n . These v a r i e d p a t t e r n s d i d not appear t o c o r r e l a t e with any r o s t r o c a u d a l or l a t e r a l o r i e n t a t i o n of the s t i m u l a t i o n s i t e s . Electromyographic records taken from 2 b i r d s showed a l t e r n a t i n g s t e p p i n g and s t e p p i n g with f l a p p i n g (Figure 6 ) . E l e c t r i c a l s t i m u l a t i o n of the l a t t e r animal (Figure 6B) e l i c i t e d a l t e r n a t i n g s t e p p i n g at low s t i m u l a t i o n i n t e n s i t y (not shown) which gave way to s t e p p i n g & wing f l a p p i n g as the c u r r e n t i n t e n s i t y was i n c r e a s e d . T h i s p a t t e r n , while found i n only one of the seven b i r d s s t i m u l a t e d , was r e m i n i s c e n t of t h a t seen d u r i n g e l e c t r i c a l s t i m u l a t i o n of the medullary r e t i c u l a r formation (Steeves et al., 1986). As a l s o seen d u r i n g MRF s t i m u l a t i o n , ramp i n c r e a s e s i n the stimulus i n t e n s i t y above t h r e s h o l d v a l u e s d u r i n g MLF s t i m u l a t i o n appeared to i n c r e a s e the f o r c e and frequency of the locomotor p a t t e r n (Steeves et al., 1986) . 58 F i g u r e 4. Composite diagram of c o r o n a l s e c t i o n s through the pons and mesencephalon i l l u s t r a t i n g s i t e s from which locomotion was evoked by e l e c t r i c a l s t i m u l a t i o n . The r o s t r o c a u d a l extent of the s e c t i o n s i s i n d i c a t e d by the numbers i n upper l e f t corner of each s e c t i o n [A=anterior, P=posterior ( i n m i l l i m e t e r s (mm))]. U n f i l l e d c i r c l e s r e p r e s e n t e f f e c t i v e s t i m u l a t i o n s i t e s which are d i s c u s s e d i n the t e x t . Rostrocaudal extent i s i n d i c a t e d by s t e r e o t a x i c l e v e l s i n the upper l e f t corner of each s e c t i o n . A b b r e v i a t i o n s : AL - ansa l e n t i c u l a r i s , AQ - c e r e b r a l aqueduct, BC - brachium conjunctivum, DBC - d e c u s s a t i o n of the brachium conjunctivum, EM - ectomammillary nucleus, EW - Edinger-Westphal nucleus, IP - i n t e r p e d u n c u l a r nucleus, IV - t r o c h l e a r nucleus, LC - l o c u s c e r u l e u s , MLd - l a t e r a l mesencephalic nucleus, d o r s a l p a r t , MLF - medial l o n g i t u d i n a l f a s c i c u l u s , MRF - mesencephalic r e t i c u l a r formation, MV - motor t r i g e m i n a l nucleus, NC - caudal neostriatum, NIII - occulomotor nerve, NV - t r i g e m i n a l nerve, OT - o p t i c tectum, PT - p r e t e c t a l nucleus, R - raphe nucleus, RP -pontine r e t i c u l a r formation, Rpc - pontine nucleus, p a r v o c e l l u l a r p a r t , RPO - o r a l p a r t , pontine r e t i c u l a r nucleus, Ru - red nucleus, SGC - c e n t r a l gray stratum, SP - s u b p r e t e c t a l nucleus, SV - t r i g e m i n a l sensory nucleus, TPc - s u b s t a n t i a n i g r a , pars compacta, V - v e n t r i c l e , VII - f a c i a l nucleus. (Redrawn from Karten and Hodos, 1967). 59 A 0.75 A 3.00 60 F i g u r e 5. C o r o n a l s e c t i o n s t h r o u g h v a r y i n g l e v e l s o f t h e n e u r a x i s i l l u s t r a t i n g s i t e s (L) f r o m w h i c h l o c o m o t i o n c o u l d be e l i c i t e d b y e l e c t r i c a l s t i m u l a t i o n . A : C o r o n a l s e c t i o n t h r o u g h t h e c a u d a l p o n s s h o w i n g a l o c o m o t i o n - e v o k i n g s t i m u l a t i o n s i t e (L) i n t h e M L F . B : C o r o n a l s e c t i o n t h r o u g h t h e m e s e n c e p h a l o n d e m o n s t r a t i n g a l o c o m o t i o n - e v o k i n g s t i m u l a t i o n s i t e (L) i n t h e m e d i a l m e s e n c e p h a l i c r e t i c u l a r f o r m a t i o n ( M R F ) . C : C o r o n a l s e c t i o n t h r o u g h t h e r o s t r a l m e s e n c e p h a l o n i l l u s t r a t i n g an e l e c t r i c a l l y s t i m u l a t e d l o c o m o t o r s i t e (L) i n t h e i s t h m a l p a r v o c e l l u l a r n u c l e u s ( I p c ) . D : C o r o n a l s e c t i o n t h r o u g h t h e m e s e n c e p h a l o n i l l u s t r a t i n g a l o c o m o t i o n - e v o k i n g s t i m u l a t i o n s i t e (L) i n t h e i n t e r c o l l i c u l a r n u c l e u s o f t h e t e c t u m . See R e s u l t s f o r d e t a i l s . A b b r e v i a t i o n s : F R L - l a t e r a l m e s e n c e p h a l i c r e t i c u l a r f o r m a t i o n , FRM - m e d i a l m e s e n c e p h a l i c r e t i c u l a r f o r m a t i o n , I C o - i n t e r c o l l i c u l a r n u c l e u s , I p c - i s t h m a l n u c l e u s , p a r v o c e l l u l a r p a r t , L - s t i m u l a t i o n s i t e m a r k e d b y an e l e c t r o l y t i c l e s i o n , MLd - d o r s a l p a r t , l a t e r a l m e s e n c e p h a l i c n u c l e u s , MLF - m e d i a l l o n g i t u d i n a l f a s c i c u l u s , MRF -m e d i a l m e s e n c e p h a l i c r e t i c u l a r f o r m a t i o n , NV -t r i g e m i n a l n e r v e , Omd - d o r s a l p a r t , o c c u l o m o t o r n u c l e u s , Omv -v e n t r a l p a r t , o c c u l o m o t o r n u c l e u s , P r V - p r i n c i p a l s e n s o r y t r i g e m i n a l n u c l e u s , R - r a p h e n u c l e u s , R P g c - g i g a n t o c e l l u l a r p a r t , p o n t i n e r e t i c u l a r f o r m a t i o n , Ru - r e d n u c l e u s , I I I - o c c u l o m o t o r n e r v e 61 62 63 F i g u r e 6. Electromyographic (EMG) records showing locomotor a c t i v i t y e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n of the medial l o n g i t u d i n a l f a s c i c u l u s (MLF). A: A l t e r n a t i n g s t e p p i n g r e p r e s e n t e d by EMG p a t t e r n s from the r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n of the MLF. ITC i s the major avian h i p f l e x o r and i s synonymous wi t h the mammalian s a r t o r i u s muscle. B: Stepping t o g e t h e r with wing f l a p p i n g EMGs evoked by e l e c t r i c a l s t i m u l a t i o n of the MLF. The top two t r a c e s are records from the r i g h t (RPECT) and l e f t (LPECT) p e c t o r a l i s muscles which are the major wing depressor muscles. The bottom 2 t r a c e s are records from the l e f t and r i g h t ITC f l e x o r muscles. 64 LPECT H H — * — T — r ~ 4 — ' RITC LITC 65 Medial Mesencephalic R e t i c u l a r Formation (mMRF) S t i m u l a t i o n of the medial mesencephalic r e t i c u l a r formation (Figure 4) at t h r e s h o l d c u r r e n t i n t e n s i t i e s ranging from 25-150uA (mean = 78jiA) e l i c i t e d locomotor p a t t e r n s i n 14 animals (4 Canada geese, 10 Pekin ducks) t h a t were s i m i l a r t o the p a t t e r n s seen i n response t o s t i m u l a t i o n o f the MLF. F i v e b i r d s d i s p l a y e d walking alone with no wing p a r t i c i p a t i o n even at s t i m u l a t i o n i n t e n s i t i e s up t o 170uA (maximum), while i n f i v e o t h ers, s t e p p i n g and f l a p p i n g began si m u l t a n e o u s l y (e.g. F i g u r e 7A) at the t h r e s h o l d i n t e n s i t y . F l y i n g behaviour alone was e l i c i t e d i n 4 other animals. The e f f e c t s o f changing the frequency o f s t i m u l a t i o n while m a i n t a i n i n g other s t i m u l a t i o n parameters constant were examined i n s e v e r a l animals. F i g u r e 7B i l l u s t r a t e s the hindlimb f l e x o r (ITC) and wing depressor (PECT) EMGs from one b i r d which demonstrated walking and f l y i n g t o g e t h e r at t h r e s h o l d s t i m u l a t i o n i n t e n s i t y (lOOuA). I n c r e a s i n g the stimulus frequency from 50Hz to 80Hz i n 10Hz increments decreased the frequency o f both f l a p p i n g (2.5-0.83Hz) and s t e p p i n g (2.3-1.3Hz) (only one l e g and wing are shown f o r each frequency). Nucleus I n t e r c o l l i c u l a r i s (ICo) and Nucleus Isthmi,  pars p a r v o c e l l u l a r i s (Ipc) Locomotion was e l i c i t e d i n nine Canada geese by f o c a l e l e c t r i c a l s t i m u l a t i o n ( t h r e s h o l d i n t e n s i t y range 25-100uA, mean = 71uA) of a r e g i o n i n c l o s e p r o x i m i t y t o the t e c t a l 6 6 Figure 7. EMG records showing locomotor a c t i v i t y produced by e l e c t r i c a l s t i m u l a t i o n of the medial mesencephalic r e t i c u l a r formation (mMRF). A: Stepping together w i t h wing f l a p p i n g EMGs e l i c i t e d at a s t i m u l a t i o n t h r e s h o l d of lOOuA. Records are from the l e f t (LITC) and r i g h t (RITC) ITC muscles together w i t h the l e f t (LPECT) and r i g h t (RPECT) muscles. B: EMG records from the same animal showing the e f f e c t s of changing s t i m u l a t i o n frequency on locomotion. The t r a c e s are p a i r e d t o i l l u s t r a t e the simultaneous stepping (LITC) and f l a p p i n g (LPECT) behaviour e l i c i t e d at a constant s t i m u l a t i o n i n t e n s i t y of lOOuA and square wave pulse d u r a t i o n (2.0ms). The frequency of s t i m u l a t i o n was v a r i e d from 50Hz i n the top p a i r to 80Hz i n the bottom p a i r i n 10Hz increments. The frequency of stepping decreased w i t h i n c r e a s i n g frequency of s t i m u l a t i o n (50Hz - 2.3 steps/sec, 60Hz - 1.7 steps/sec, 70Hz - 1.5 steps/sec, 80Hz - 1.3 s t e p s / s e c ) . The frequency of wing f l a p p i n g a l s o decreased w i t h i n c r e a s i n g s t i m u l a t i o n frequency (50Hz - 2.5 wingbeats/sec, 60Hz - 2.0 wingbeats/sec, 70Hz - 1.0 wingbeat/sec, 80Hz - 0.83 wingbeats/sec). 67 R P E C T L P E C T — • RITC i t 4+4- t r LITC 1 sec 68 B LPECT 50 Hz LITC LPECT 60 Hz LITC LPECT 70 Hz LITC LPECT 80 Hz LITC i n t e r c o l l i c u l a r and i s t h m a l n u c l e i (Figures 4 & 5 ). Threshold s t i m u l a t i o n produced s t e p p i n g t o g e t h e r with wing f l a p p i n g i n a l l animals. An example i l l u s t r a t i n g the c o a c t i v a t i o n of l e g and wing locomotor p a t t e r n s i s d i s p l a y e d i n F i g u r e 8. 70 F i g u r e 8. EMG records i l l u s t r a t i n g the locomotor a c t i v i t y e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n of the i n t e r c o l l i c u l a r nucleus of the mid-brain. The records were taken from the r i g h t (RPECT) and l e f t (LPECT) p e c t o r a l i s muscles s i m u l t a n e o u s l y with the r i g h t (RITC) and l e f t (LITC) ITC muscles. The c o a c t i v a t i o n of both limb g i r d l e s i n response to e l e c t r i c a l s t i m u l a t i o n was t y p i c a l f o r s i t e s i n t h i s r e g i o n . 71 R P E C T L P E C T R I T C L I T C i 1 1 s e c 7 2 DISCUSSION P r e v i o u s s t u d i e s i n our l a b o r a t o r y have demonstrated t h a t locomotion can be e l i c i t e d i n b i r d s by e l e c t r i c a l s t i m u l a t i o n of r e t i c u l a r formation n u c l e i t h a t g i v e r i s e t o the descending r e t i c u l o s p i n a l pathways e s s e n t i a l f o r the i n i t i a t i o n and ongoing c o n t r o l of locomotion (Steeves et al., 1986, 1987; Sholomenko and Steeves, 1987b; Webster and Steeves, 1988). Furthermore, a re g i o n corresponding t o the mammalian pontobulbar locomotor s t r i p was d e s c r i b e d (Steeves et al., 1986, 1987). The PLS appears t o p l a y a r o l e i n the sensorimotor a c t i v a t i o n of locomotion i n a v a r i e t y of s p e c i e s (McClellan, 1986; Jordan, 1986; Noga et al., 1988; see Chapter 1). To examine s t r u c t u r e s which may c o n t r o l or modulate the l o c o m o t o r - r e l a t e d r e t i c u l a r formation n u c l e i , we have u t i l i z e d e l e c t r i c a l b rainstem s t i m u l a t i o n t o examine s y s t e m a t i c a l l y more r o s t r a l p o r t i o n s o f the avian brainstem. E q u i v a l e n t s of the mammalian mesencephalic locomotor r e g i o n (MLR) and subthalamic locomotor r e g i o n (SLR) (Shik et al., 1966; Orlovsky, 1969) have not been p r e v i o u s l y i d e n t i f i e d i n b i r d s ( E i d e l b e r g , 1981). .Our p r e s e n t f i n d i n g s demonstrate one r e g i o n i n the pons and two i n the midbrain from which locomotion may be e l e c t r i c a l l y induced. These i n c l u d e the medial l o n g i t u d i n a l f a s c i c u l u s (MLF), the medial mesencephalic r e t i c u l a r formation (mMRF) and the i n t e r c o l l i c u l a r (ICo) and p a r v o c e l l u l a r i s t h m a l (Ipc) n u c l e i o f the t e c t a l midbrain. In b i r d s , as i n mammals (Karten and Hodos, 1967; Carpenter and S u t i n , 1983), the MLF d e s c r i b e s a f i b r e t r a c t which c a r r i e s 73 a v a r i e t y of p r o j e c t i o n s . The r o s t r o c a u d a l extent of the MLF i s from the l e v e l of the Edinger-Westphal nucleus t o the caudal medulla (Karten and Hodos, 1967). P r o j e c t i o n s which t r a v e r s e the MLF i n c l u d e : 1) descending axons t r a v e l l i n g from the pontine r e t i c u l a r formation (PRF) to the medulla and s p i n a l cord, 2)descending axons from the i n t e r s t i t i a l nucleus of C a j a l (INC) t o s p i n a l cord, P r o b s t ' s t r a c t , medial and descending v e s t i b u l a r n u c l e i and i n f e r i o r o l i v e (Carpenter and S u t i n , 1983; Skinner et al., 1984), 3) v e s t i b u l o s p i n a l f i b e r s which send c o l l a t e r a l s t o the medullary r e t i c u l a r formation (Carpenter and S u t i n , 1983) and 4) v i s u a l i n t e r n u c l e a r f i b r e s from c r a n i a l nerve n u c l e i VI, IV and I I I . In a d d i t i o n , some f i b r e s which impinge on the pedunculopontine tegmental nucleus (PPN) a l s o p r o j e c t v i a the MLF ( u n s p e c i f i e d o r i g i n ) (Rye et al., 1987). Se v e r a l of the above descending p r o j e c t i o n s t r a v e l l i n g i n the MLF have been i m p l i c a t e d i n locomotor c o n t r o l mechanisms which may subserve our r e s u l t s . Thus, e l e c t r i c a l s t i m u l a t i o n of these f i b r e s may a c t i v a t e locomotion i n a n o n - s p e c i f i c manner. F i b r e s a r i s i n g from the v e s t i b u l a r n u c l e i are c a r r i e d i n the MLF and p r o j e c t to the medullary r e t i c u l a r formation and s p i n a l c o r d and thus c o u l d be r e s p o n s i b l e f o r the locomotor responses a s s o c i a t e d w i t h MLF s t i m u l a t i o n . However, d i r e c t s t i m u l a t i o n of the l a t e r a l v e s t i b u l a r nucleus does not a c t i v a t e locomotion (Orlovsky, 1972a) but only f a c i l i t a t e s extensor tone i n the d ecerebrate c a t . S i m i l a r l y , although INC sends d i r e c t p r o j e c t i o n s v i a the MLF to the motor a s s o c i a t e d P r o b s t ' s T r a c t ( G a r c i a - R i l l et al, 1983a), e l e c t r i c a l s t i m u l a t i o n of the INC e l i c i t s only head or conjugate v e r t i c a l and r o t a t o r y eye movements (Skinner et al., 1984). A c t i v a t i o n of p ontine r e t i c u l a r formation f i b r e s have been i m p l i c a t e d i n the c o n t r o l . of p o s t u r a l tonus i n the cat (Mori et al., 1978, 1982). These f i b r e s have been demonstrated to impinge on the medullary r e t i c u l a r formation and s p i n a l c o r d s t r u c t u r e s i n b i r d s (Steeves et al., 1987, Webster and Steeves, 1988, i n p r e p a r a t i o n ) and c o u l d a l s o account .for the e f f e c t s of MLF s t i m u l a t i o n on locomotion. E l e c t r i c a l s t i m u l a t i o n of axons t e r m i n a t i n g on neurons of the PPN, a mesencephalic nucleus which appears to form a p o r t i o n of the mesencephalic locomotor r e g i o n (MLR) ( G a r c i a - R i l l et al., 1986) c o u l d a l s o be r e s p o n s i b l e f o r e l i c i t i n g locomotion through MLF s t i m u l a t i o n . However, while e l e c t r i c a l s t i m u l a t i o n of the axonal p r o j e c t i o n s a r i s i n g from these n u c l e i may have some r o l e i n locomotion evoked from the MLF r e g i o n , r e s u l t s from n e u r o t r a n s m i t t e r (or a g o n i s t s / a n t a g o n i s t s ) i n j e c t i o n s t u d i e s which w i l l be subsequently d i s c u s s e d (Chapters 3 & 5) i n d i c a t e t h a t i t i s u n l i k e l y t h a t the r e s u l t s of MLF s t i m u l a t i o n - i n d u c e d locomotion r e s u l t e d from the s t i m u l a t i o n of en passant f i b r e s alone. While the neuroanatomical connections of c e l l s i n t h i s r e g i o n which may be r e s p o n s i b l e f o r locomotion have yet to be determined i n b i r d s , s e r o t o n i n - c o n t a i n i n g neurons which s t a i n p o s i t i v e l y f o r a c e t y l c h o l i n e s t e r a s e have been i d e n t i f i e d i n c l o s e p r o x i m i t y to the locomotion-evoking s t i m u l a t i o n s i t e s (Dube and Parent, 1981; Taccogna et al., i n p r e p a r a t i o n ) . These s e r o t o n i n e r g i c c e l l s appear t o p r o j e c t to the nucleus p r e t e c t a l i s of the midbrain, a r e g i o n l y i n g i n c l o s e p r o x i m i t y t o e l e c t r i c a l l y i d e n t i f i e d locomotor s i t e s which w i l l be d i s c u s s e d below (Reiner et al., 1982). In the r a t , i n j e c t i o n of the GABAergic a g o n i s t muscimol i n t o e i t h e r the d o r s a l or median raphe n u c l e i i n c r e a s e s locomotor a c t i v i t y which i s b l o c k e d by pre-treatment w i t h b i c u c u l l i n e ( P a r i s and Lorens, 1987). A l s o , i b o t e n i c a c i d l e s i o n of c e l l bodies w i t h i n the median raphe produced h y p e r a c t i v i t y i n the r a t (Asin and F i b i g e r , 1983). These r e s u l t s , t h e r e f o r e , i m p l i c a t e t h i s r e g i o n i n locomotor processes i n the i n t a c t mammal. While the observed locomotor behaviour e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n o f the MLF i n b i r d s may r e s u l t from a c t i v a t i o n o f neurons i n t h i s r e g i o n , the mechanism and neuroanatomical pathways through which the locomotion i s evoked remains t o be determined. E l e c t r i c a l s t i m u l a t i o n of a second r e g i o n , the medial mesencephalic r e t i c u l a r formation (mMRF), a l s o e l i c i t e d locomotion i n decerebrate b i r d s . The r o s t r o c a u d a l extent of the e f f e c t i v e s t i m u l a t i o n s i t e s (from caudal r e d nucleus t o r o s t r a l pons) i n d i c a t e s the d i f f u s e nature of t h i s locomotor r e g i o n . In b i r d s , anterograde and r e t r o g r a d e t r a c i n g s t u d i e s have shown t h a t the mMRF has r e c i p r o c a l connections with deep l a y e r s ( l a y e r s 8-13) of the tectum (Hunt et al., 1977; Hunt and Brecha, 1984). In t u r n , deep t e c t a l l a y e r s r e c e i v e the bulk of t h e i r i n n e r v a t i o n from the l a t e r a l s p i r i f o r m nucleus (SpL), a r e l a y nucleus which i t s e l f r e c e i v e s the major outflow of the avian e q u i v a l e n t o f the mammalian b a s a l g a n g l i a (Reiner et al., 1984). B i l a t e r a l d e s t r u c t i o n of SpL r e s u l t s i n d e f i c i t s i n b i r d s t h a t are s i m i l a r , as i n monkeys wit h p a l l i d a l l e s i o n s and a l s o i n P a r k i n s o n i a n p a t i e n t s , t o those seen f o l l o w i n g d i s r u p t i o n of the outflow pathway from the mammalian b a s a l g a n g l i a (Bugbee, 197 9, c f Reiner et al., 1982). Thus, t h i s pathway may have a r o l e i n c o n t r o l l i n g the i n i t i a t i o n o f ongoing or impending movements which c o o r d i n a t e the b i r d ' s p o s i t i o n i n space (Bugbee, 1979, c f Reiner et al, 1982; Reiner et a l . , 1984). The mMRF p r o j e c t s i p s i l a t e r a l l y both t o the h i g h c e r v i c a l s p i n a l c o r d and t o the g i g a n t o c e l l u l a r r e g i o n of the medial medullary r e t i c u l a r formation (Webster and Steeves, i n p r e p a r a t i o n ) . T h i s c i r c u i t may, t h e r e f o r e , p r o v i d e a p o r t i o n of the outflow loop from the b a s a l g a n g l i a t o h i n d b r a i n s t r u c t u r e s which u n d e r l i e s some aspects of motor performance i n b i r d s (Reiner et al., 1984). W i t h i n the c o n s t r a i n t s o f the a v a i l a b l e data concerning a v i a n mMRF connections, those which have been i d e n t i f i e d above resemble the a f f e r e n t and e f f e r e n t pathways o f the mammalian mesencephalic locomotor regions ( G a r c i a - R i l l et al., 1983a; Steeves and Jordan, 1984; Rye et al., 1988). However, f u r t h e r comparison o f t h i s a vian r e g i o n t o the mesencephalic locomotor regions of mammals awaits i n c r e a s e d a v i a n and mammalian neuroanatomical, immunohistochemical and re c e p t o r subtype i n f o r m a t i o n . Locomotion was e l i c i t e d i n the low decerebrate b i r d p r e p a r a t i o n through e l e c t r i c a l s t i m u l a t i o n of a t h i r d s i t e , the deep t e c t a l r e g i o ns i n c l u d i n g the i n t e r c o l l i c u l a r nucleus (ICo) and p a r v o c e l l u l a r i s t h m a l nucleus ( I p c ) . The neuroanatomical connections through which these regions e l i c i t locomotor behaviour are p r e s e n t l y unknown. However, some pathways which may u n d e r l i e our r e s u l t s have been i d e n t i f i e d . ICo r e c e i v e s input from the s p i n a l c o r d (Hunt and Kunzle, 1976; Webster and Steeves, i n p r e p a r a t i o n ) , deep t e c t a l l a y e r s 77 (Hunt et al., 1977; Wild, 1987) and b o d y - r e l a t e d a f f e r e n t input from the cuneate and g r a c i l e n u c l e i (Wild, 1987). I t sends d i r e c t p r o j e c t i o n s t o the i p s i l a t e r a l r o s t r a l descending t r i g e m i n a l t r a c t and nucleus (TTD), i p s i - and c o n t r a l a t e r a l g i g a n t o c e l l u l a r r e t i c u l a r formation (Rgc), i p s i l a t e r a l c e n t r a l medullary nucleus, v e n t r a l p a r t (Cnv) (Reiner and Karten, 1982; Webster and Steeves, i n p r e p a r a t i o n ) and i p s i l a t e r a l h i g h c e r v i c a l s p i n a l c o r d (Reiner and Karten, 1982). Thus, ICo maintains connections t o l o c o m o t i o n - r e l a t e d n u c l e i and may be a r e l a y of the output c i r c u i t through which the a v i a n b a s a l g a n g l i a a f f e c t locomotor c o n t r o l (Reiner et al., 1984). These connections are r e m i n i s c e n t of those found f o r the mammalian mesencephalic locomotor r e g i o n s (MLR) which p r o j e c t d i r e c t l y t o r e t i c u l a r formation s t r u c t u r e s ( G a r c i a - R i l l et al., 1983a; Jordan, 1986; Noga et al., 1988). Indeed, Cabot et al. (1982) compare these connections t o those of the mammalian cuneiform nucleus, a p o r t i o n of which, i n mammals, i s b e l i e v e d to be the l a t e r a l MLR (Shik et al., 1966; Steeves and Jordan, 1984; Jordan, 1986; Noga et al., 1988). On the other hand, e l e c t r i c a l s t i m u l a t i o n of the more v e n t r a l l y l o c a t e d Ipc a l s o evoked locomotion i n our p r e p a r a t i o n . Ipc, a t e c t a l nucleus which s t a i n s p o s i t i v e l y f o r both c h o l i n e a c e t y l t r a n s f e r a s e (CHAT) (Taccogna et al., i n p r e p a r a t i o n ) and a c e t y l c h o l i n e s t e r a s e (AChE) (Hunt et al., 1977) r e c e i v e s c h o l i n e r g i c input from the s u p e r f i c i a l ( v i s u a l ) t e c t a l l a y e r s ( l a y e r s 2&3) (Hunt et al., 1977) and a l s o r e c e i v e s input from the deep t e c t a l l a y e r s ( l a y e r s 8-13) (Reiner et al,, 1982). In the t u r t l e and f r o g , C h A T - p o s i t i v e i s t h m a l nucleus neurons have 78 been demonstrated t o send a c h o l i n e r g i c p r o j e c t i o n t o the tectum (Desan et al., 1984), while i n the cat, C h A T - p o s i t i v e neurons have been l o c a l i z e d t o the parabigeminal nucleus [thought to be the mammalian e q u i v a l e n t of Ipc (Vincent and Reiner, 1987)]. In b i r d s , based on uptake of [ 3H]choline and HRP t r a n s p o r t s t u d i e s , a c h o l i n e r g i c p r o j e c t i o n has been p o s t u l a t e d from Ipc to l a y e r 2d of the l a t e r a l and caudal tectum (Hunt et al., 1982). S i m i l a r l y , r a d i o a c t i v e uptake s t u d i e s u s i n g [ 3 H ] g l y c i n e and [3H]GABA suggest t h a t Ipc neurons p r o j e c t g l y c i n e r g i c and GABAergic e f f e r e n t s t o the s u p e r f i c i a l t e c t a l l a y e r s (Hunt et al., 1977), i n d i c a t i n g t h a t Ipc, while h i s t o l o g i c a l l y homogeneous, may be heterogeneous with r e s p e c t t o n e u r o t r a n s m i t t e r type (Hunt et al., 1982). In r e t r o g r a d e l a b e l l i n g s t u d i e s , however, Ipc does not appear t o d i r e c t l y i n n e r v a t e m o t o r - r e l a t e d brainstem r e t i c u l a r formation (Rgc, Cnv) or sensorimotor (TTD) n u c l e i (Webster and Steeves, i n p r e p a r a t i o n ) . Therefore, while e l e c t r i c a l s t i m u l a t i o n of Ipc evokes locomotion i n decerebrate b i r d s , i t appears t h a t the behaviour i s evoked i n d i r e c t l y v i a an Ipc to t e c t a l neuronal c i r c u i t , the output of which remains to be determined. CONCLUSIONS The r e s u l t s of t h i s study demonstrate the presence of pontine and mesencephalic locomotor regions i n b i r d s . F u r t h e r i n v e s t i g a t i o n i s r e q u i r e d t o determine whether these regions are analogous t o the more r o s t r a l locomotor regions (MLR or SLR) of h i g h e r v e r t e b r a t e s p e c i e s ( f o r review, see Noga et al., 1988). One locomotor s i t e , l o c a t e d c l o s e t o or w i t h i n the i n t e r c o l l i c u l a r nucleus of the t e c t a l midbrain, appears t o possess s e v e r a l of the h o d o l o g i c a l c h a r a c t e r i s t i c s o f the mammalian MLR. However, c o n s i d e r a b l y more study i s r e q u i r e d t o determine whether t h i s s i t e i s an avian e q u i v a l e n t of the mammalian MLR. Stu d i e s which combine e l e c t r i c a l with neurochemical s t i m u l a t i o n o f these locomotor regions w i l l h e lp t o determine whether neuronal p o p u l a t i o n s or axons of passage are b e i n g s t i m u l a t e d . Some s t u d i e s u s i n g t h i s experimental paradigm have a l r e a d y been undertaken (see Chapters 2-4). Furthermore, neuroanatomical t r a c t t r a c i n g u s i n g anterograde and r e t r o g r a d e t r a c e r s combined wi t h r e c e p t o r a u t o r a d i o g r a p h i c b i n d i n g s t u d i e s of l o c o m o t o r - e f f e c t i n g r a d i o a c t i v e neurochemicals would h e l p t o determine the neuronal subpopulations from these r e g i o n s i n v o l v e d i n locomotor c o n t r o l . 80 CHAPTER 3 CHARACTERIZATION OF AVIAN MID- AND HINDBRAIN SITES THAT PRODUCE LOCOMOTION WITH LOCAL INTRACEREBRAL INFUSION OF NEUROTRANSMITTER AGONISTS AND ANTAGONISTS (I): ACETYLCHOLINE 81 INTRODUCTION The i n i t i a t i o n and ongoing c o n t r o l o f normal v e r t e b r a t e locomotion depend upon the p r o d u c t i o n , i n t e g r a t i o n and t r a n s f e r of both c e n t r a l l y and p e r i p h e r a l l y generated i n f o r m a t i o n i n t e r a c t i n g at many l e v e l s o f the c e n t r a l nervous system (CNS). In order t o understand the p o s s i b l e c o n t r i b u t i o n made by each l e v e l o f the complex network which c o n t r o l s locomotion, one approach has been t o examine the system by u t i l i z i n g only p o r t i o n s o f the CNS i n reduced p r e p a r a t i o n s ( G r i l l n e r and Zangger, 1979; G r i l l n e r and Kashin, 1976). One example which has y i e l d e d v a l u a b l e i n f o r m a t i o n i s the decerebrate e l e c t r i c a l l y s t i m u l a t e d cat p r e p a r a t i o n p i o n e e r e d by Shik and h i s co-workers (Shik et al., 1966). T h i s type of p r e p a r a t i o n has been used f o r the study of locomotion i n a v a r i e t y of v e r t e b r a t e s p e c i e s ( G a r c i a - R i l l , 1983, 1986; Jordan, 1986; f o r review see G r i l l n e r , 1975), i n c l u d i n g b i r d s (Sholomenko and Steeves, 1987a; Steeves et al., 1987). My s t u d i e s with decerebrate geese and ducks have shown t h a t e l e c t r i c a l s t i m u l a t i o n o f re g i o n s i n the mesencephalon, pons and medulla, i n c l u d i n g the pontobulbar locomotor s t r i p (PLS) mesencephalic r e t i c u l a r formation (MRF) and pontine and medullary r e t i c u l a r formation, e l i c i t a l l p a t t e r n s o f avian locomotion from walking t o f l y i n g . These f i n d i n g s , complementary t o those found i n a v a r i e t y of both h i g h e r and lower v e r t e b r a t e s ( f o r review see M c C l e l l a n , 1986), s t r o n g l y i m p l i c a t e n u c l e i of the mid- and h i n d b r a i n as p l a y i n g a major r o l e i n locomotor c o n t r o l . 82 Although locomotion can be e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n i n w e l l c i r c u m s c r i b e d r e g i o n s o f the brainstem ( f o r review see G r i l l n e r , 1975), t h i s type of s t i m u l a t i o n g i v e s only l i m i t e d i n f o r m a t i o n concerning the n e u r a l s t r u c t u r e s u n d e r l y i n g the behaviour. E l e c t r i c a l s t i m u l a t i o n a c t i v a t e s a l l neuronal elements i n c l u d i n g d i s t a n t l y o r i g i n a t i n g axons of passage (en  passant) which may t r a v e r s e the p o i n t o f s t i m u l a t i o n (Goodchild et al., 1982; G a r c i a - R i l l et a l . , 1985, 1987; Noga et a l . , 1988). To circumvent t h i s problem and to more c l o s e l y mimic the p h y s i o l o g i c a l a c t i v a t i o n of locomotion r e l a t e d r e g i o n s , we u t i l i z e d p h a r m a c o l o g i c a l s t i m u l a t i o n by i n j e c t i n g n e u r o t r a n s m i t t e r s , or n e u r o t r a n s m i t t e r a g o n i s t s and a n t a g o n i s t s (neurochemicals) i n t o s i t e s which produce locomotion when e l e c t r i c a l l y s t i m u l a t e d . These neurochemicals are thought t o a c t i v a t e n e u r o t r a n s m i t t e r r e c e p t o r s s i t u a t e d on d e n d r i t e s , c e l l bodies and t e r m i n a l s (Goodchild et al., 1982). Receptors have not, however, been l o c a l i z e d on axons (Goodchild et a l . , 1982). A c t i v a t i o n or blockade, then, o f locomotor behaviour by neurochemical i n j e c t i o n i n t o an e l e c t r i c a l l y d e f i n e d locomotor r e g i o n y i e l d s two types of i n f o r m a t i o n . F i r s t , i t d i f f e r e n t i a t e s between the a c t i v a t i o n o f r e c e p t o r s and en passant f i b e r s . Second, i t a i d s i n the d e f i n i t i o n o f r e c e p t o r types present on c e l l s b e l i e v e d t o g i v e r i s e t o pathways i n v o l v e d i n locomotor c o n t r o l . C o r r e l a t i o n o f t h i s type of f i n d i n g with neuroanatomical i n f o r m a t i o n makes neurochemical s t i m u l a t i o n a powerful paradigm f o r the study of locomotor c o n t r o l . The c h o i c e of neurochemicals used i n t h i s study was based on: p r e v i o u s i n v e s t i g a t i o n s u s i n g chemical s t i m u l a t i o n 83 ( G a r c i a - R i l l et al., 1985, 1987; Noga et al., 1988; E l d r i d g e et al., 1985 ; Brudzynski et al., 1986, 1988 (see Appendix I, pp. 291); neuroanatomical i n f o r m a t i o n ; and a u t o r a d i o g r a p h i c and immunocytochemical l o c a l i z a t i o n o f r e c e p t o r s types and t r a n s m i t t e r p r o f i l e s f o r n u c l e i i n the reg i o n s p r e v i o u s l y shown t o e l i c i t locomotion when e l e c t r i c a l l y s t i m u l a t e d . The neurochemicals u t i l i z e d i n t h i s chapter examine the locomotor e f f e c t s o f c h o l i n e r g i c a g o n i s t and an t a g o n i s t i n j e c t i o n i n t o p r e v i o u s l y d e f i n e d avian locomotor r e g i o n s . Because s i x regions have been found, and the number of n e u r o t r a n s m i t t e r s e x t e n s i v e , t h i s study was designed as a survey of these r e g i o n s , with more d e t a i l e d neurochemical c h a r a c t e r i z a t i o n of i n d i v i d u a l r e g i ons l e f t t o f u t u r e i n v e s t i g a t i o n s . Our r e s u l t s demonstrate t h a t n e u r o a c t i v e chemicals were e f f e c t i v e at s t i m u l a t i n g or b l o c k i n g avian locomotor p a t t e r n s i n s i t e s p r e v i o u s l y d e f i n e d u s i n g only e l e c t r i c a l s t i m u l a t i o n (Steeves et al., 1987). Locomotion was e l i c i t e d or the t h r e s h o l d f o r e l e c t r i c a l l y - i n d u c e d locomotion decreased i n a v a r i e t y of s i t e s f o l l o w i n g i n j e c t i o n o f c h o l i n e r g i c a g o n i s t s . The locomotion c o u l d be bl o c k e d or e l e c t r i c a l t h r e s h o l d i n c r e a s e d w i t h the corresp o n d i n g a n t a g o n i s t . The e f f e c t i v e a g o n i s t s / a n t a g o n i s t s appeared t o possess i n d i v i d u a l c h a r a c t e r i s t i c s f o r a c t i v a t i o n or blockade, with v a r y i n g times t o onset and l o n g e v i t y of a c t i o n (see Table 1). These r e s u l t s i n the b i r d w i l l be compared t o recent f i n d i n g s i n the cat and r a t , w i t h a view t o d e f i n i n g mid- and h i n d b r a i n pathways i n v o l v e d i n motor c o n t r o l . 84 MATERIALS AND METHODS Surgery The s u r g i c a l and d e c e r e b r a t i o n procedures and electromyographic r e c o r d i n g s from the l e g f l e x o r muscles (ITC) and wing depressor muscles (PECT) have been p r e v i o u s l y d e s c r i b e d i n Chapter 2. E l e c t r o n e u r o g r a p h i c records (ENGs) from v e n t i l a t e d animals p a r a l y z e d w i t h d-tubocurarine (0.025ml/kg) were recorded u s i n g b i p o l a r p l a t i n u m hook e l e c t r o d e s p l a c e d on p e r i p h e r a l nerves t o ITC and PECT wit h the same equipment used f o r EMGs but at highe r g a i n (10,000x) and with the t r e a d m i l l o f f . B r a i n S t i m u l a t i o n Both e l e c t r i c a l and chemical s t i m u l a t i o n of l o c a l i z e d r e g i o n s w i t h i n the brainstem were used t o e l i c i t locomotion from the p r e p a r a t i o n . A monopolar s t i m u l a t i n g e l e c t r o d e , c o n s t r u c t e d by i n s e r t i n g 0.0762mm s t a i n l e s s s t e e l wire (exposed t i p l e n g t h = 0.1mm, impedance = 60-70kQ) down one b a r r e l o f a p u l l e d three b a r r e l m i c r o p i p e t t e ( t o t a l t i p diameter = 0.1mm), was p o s i t i o n e d s t e r e o t a x i c a l l y i n t o s i t e s p r e v i o u s l y shown t o e l i c i t locomotion (Steeves et a l . , 1987; Sholomenko and Steeves, 1987a). The other two b a r r e l s of the m i c r o p i p e t t e were f i l l e d w ith n e u r o t r a n s m i t t e r a g o n i s t s , a n t a g o n i s t s or s a l i n e . These neurochemicals were d e l i v e r e d t o the m i c r o p i p e t t e from a mi c r o s y r i n g e (Hamilton) v i a f l e x i b l e v i n y l t u b i n g . Constant 85 c u r r e n t s t i m u l a t i o n t r i a l s (Grass Model S88/ Grass Model CCU1A) were undertaken with the f o l l o w i n g s t i m u l a t i o n parameters: square wave p u l s e d u r a t i o n - 1.0-2.Oms, p u l s e frequency - 60Hz, c u r r e n t s t r e n g t h - 25-170fiA. S t i m u l a t i o n t r i a l s were undertaken by i n c r e m e n t a l l y lowering the e l e c t r o d e i n t o the brainstem while s t i m u l a t i n g with a c u r r e n t i n t e n s i t y r anging from 50-100uA. When locomotion was observed, the s t i m u l a t i o n was turned o f f . The c u r r e n t i n t e n s i t y was then reduced t o zero. The s t i m u l a t o r was turned on and the c u r r e n t i n t e n s i t y was slow l y i n c r e a s e d u n t i l t h r e s h o l d was reached. The optimal e l e c t r o d e t i p p o s i t i o n f o r evoking locomotion was then e s t a b l i s h e d by slowly lowering the e l e c t r o d e to the p o i n t where c o o r d i n a t e d locomotor movements were i n i t i a t e d w i t h the lowest s t i m u l a t i o n c u r r e n t (Steeves et al., 1987). Once optimal e l e c t r o d e p o s i t i o n was e s t a b l i s h e d , chemical s t i m u l a t i o n was attempted by i n j e c t i o n of n e u r o t r a n s m i t t e r a g o n i s t s or a n t a g o n i s t s ( a g o n i s t / a n t a g o n i s t ) i n t o the s i t e (see Table 1). The i n j e c t i o n r a t e was 0.2ul/minute t o a maximum volume of 1. Oul f o r any one neurochemical s o l u t i o n (pH7.2-7.4) (unless otherwise stated, l u l volumes were injected). The c o n c e n t r a t i o n o f each s o l u t i o n v a r i e d , with i n i t i a l c o n c e n t r a t i o n s based on those used by other i n v e s t i g a t o r s (Noga et al., 1988; G a r c i a - R i l l et al., 1985; E l d r i d g e et al., 1985; Brudzynski et al., 1986, 1988). Over the course of many . experiments, an attempt was made t o s y s t e m a t i c a l l y t i t r a t e the c o n c e n t r a t i o n o f each a g o n i s t / a n t a g o n i s t t o f i n d the lowest s u p r a t h r e s h o l d c o n c e n t r a t i o n t h a t would s t i l l e l i c i t or blo c k locomotion (see Table 1). To examine the e f f i c a c y of each 86 neurochemical and i t s time course, the f o l l o w i n g manipulations were performed t o determine the e f f e c t s of a g o n i s t / a n t a g o n i s t i n j e c t i o n upon locomotion; 1) c o u n t e r a c t i n g a locomotion producing neurochemical by i n j e c t i o n of i t s corresponding a n t a g o n i s t and v i s e v e r s a 2) e l e c t r i c a l s t i m u l a t i o n f o l l o w i n g i n j e c t i o n o f a g o n i s t / a n t a g o n i s t t o examine the e f f e c t of i n j e c t i o n upon e l e c t r i c a l s t i m u l a t i o n t h r e s h o l d and 3) r e p l i c a t i o n o f each i n j e c t i o n e i t h e r i n the same s i t e and/or i n the homologous s i t e c o n t r a l a t e r a l l y . A f t e r r e c o r d i n g the locomotor a c t i v i t y (EMGs and/or ENGs) f o l l o w i n g e l e c t r i c a l s t i m u l a t i o n and neurochemical i n j e c t i o n , the s t i m u l a t i o n / i n j e c t i o n s i t e was marked f o r neuroanatomical i d e n t i f i c a t i o n with an e l e c t r o l y t i c l e s i o n made by p a s s i n g a d i r e c t c a t h o d a l c u r r e n t of 3 m i l l i a m p s f o r 5 seconds. H i s t o l o g i c a l procedures and s t i m u l a t i o n s i t e i d e n t i f i c a t i o n were the same as those d e s c r i b e d i n Chapter 2 . 87 RESULTS $> A c e t y l c h o l i n e A g o n i s t s and A n t a g o n i s t s A v a r i e t y of c h o l i n e r g i c m u s c a r i n i c (ACtm) and n i c o t i n i c (AChN) r e c e p t o r a g o n i s t s and a n t a g o n i s t s were i n f u s e d i n t o s i t e s i n the brainstem i n an attempt t o d e l i n e a t e the r e c e p t o r subtypes a c t i v a t e d . The neurochemicals used were: ca r b a c h o l , an AChM+N a g o n i s t , p i l o c a r p i n e , an AChM a g o n i s t ; n i c o t i n e , an AChN ag o n i s t ; a t r o p i n e s u l f a t e , an AChM an t a g o n i s t and; scopolamine, an AChM a n t a g o n i s t . Neurochemical i n j e c t i o n s i t e s , lowest e f f e c t i v e c o n c e n t r a t i o n s and time course of a c t i v i t y are l i s t e d i n Table 1 (also see Appendix I) and d e s c r i b e d i n the t e x t . The a g o n i s t s or a n t a g o n i s t s were i n j e c t e d i n t o s i x regions of the mid- and h i n d b r a i n from which locomotion was e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n ( F i g s . 9 & 10). These r e g i o n s i n c l u d e d the pontobulbar locomotor s t r i p (PLS) (a r e g i o n which l i e s w i t h i n or i n c l o s e ventromedial a p p o s i t i o n t o the descending t r i g e m i n a l t r a c t and nucleus (TTD)), the d o r s a l p a r t of the c e n t r a l nucleus (Cnd) i n the medullary r e t i c u l a r formation, the v e n t r a l p a r t o f the c e n t r a l nucleus (Cnv) i n the medullary r e t i c u l a r formation, the g i g a n t o c e l l u l a r p a r t of the pontine r e t i c u l a r nucleus (RPgc), the mesencephalic r e t i c u l a r formation (MRF) and the medial l o n g i t u d i n a l f a s c i c u l u s (MLF) of the pons. A composite diagram o f the e l e c t r i c a l s t i m u l a t i o n / n e u r o c h e i n i c a l i n j e c t i o n s i t e s i s shown i n F i g u r e 9 and examples o f l e s i o n s i n d i c a t i n g e f f e c t i v e s i t e s are shown i n F i g u r e 10. 88 TABLE 1 Acetylcholine Agonists and Antagonists Animal S i t * Chemical PH Concentrat ion! Injected Lowed Volume Effect ive Concentration Rate Time Cour t * (min) Latency Period decerebrate TTD Carbachol 7.2-7.4 25mM-100mM 2SmM I.Oul 0.2ul/min 2.2-12 7-45 bird Scopolamine '• 2SmM none •• — — Nicotine M 2SmM none •' — — Atropine '• 2SmM 25mM •• 6 25 Pilocarpine •• SOmM none " " 7 8 Cnd Carbachol " 11mM-100mM 11mM " 3.3-6 33 Scopolamine 25mM none " — — Atropine 30mM SOmM " <5 >26 Cnv Carbachol 7.2-7.4 27-100mM 27mM •• 2.2-6 7-46 Scopolamine " 25mM none " — — Nicotine 100mM none " — — Atropine 3-S0mM 20mM >7.6 21-40 Pilocarpine " 60mM none " — -RP Carbachol 64 mM none •• — — MRF Carbachol •• 100mM 100mM (RT) " •• — 45 MLF Carbachol •• 27-100mM 27mM •• 9-10 23-40 ABBREVIATIONS: Cnd — dorsal part, medullary central nucleus Cnv — ventral part, medullary oentral nucleus MLF — medial longitudinal faaoiculus MRF — mesencephalic reticular formation RP — pontine reticular nucleus RT — reduced threshold for electrically stimulated locomotion TTD — descending trigeminal tract and nucleus 89 F i g u r e 9. Composite diagram o f c h o l i n e r g i c a g o n i s t and ant a g o n i s t neurochemical i n j e c t i o n s i t e s . The diagram o f c o r o n a l s e c t i o n s through v a r i o u s l e v e l s o f the av i a n n e u r a x i s [numbers i n upper l e f t corner o f each l e v e l , A=anterior, P=posterior ( i n mm)] i l l u s t r a t e s the locomotor e f f e c t s of each neurochemical i n brainstem r e g i o n s from which locomotion was f i r s t e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n . Where a t r o p i n e i s shown as f i l l e d t r i a n g l e s , i t s e f f e c t was t o b l o c k locomotion. Key A b b r e v i a t i o n s : ATRO - a t r o p i n e ( t r i a n g l e ) , CARB - car b a c h o l (square), NICO - n i c o t i n e ( c i r c l e ) , PILO - p i l o c a r p i n e (hexagon), SCOP - scopolamine (diamond); LOCO - locomotion (except f o r a t r o p i n e ) , TH - decreased e l e c t r i c a l t h r e s h o l d i n t e n s i t y f o r locomotion, ^TH - i n c r e a s e d e l e c t r i c a l t h r e s h o l d i n t e n s i t y f o r locomotion, NR - no response. A b b r e v i a t i o n s : AL - ansa l e n t i c u l a r i s , AQ - aqueduct, CC -c e n t r a l c a n a l , Cnd - c e n t r a l nucleus medulla, d o r s a l p a r t , Cnv -c e n t r a l nucleus medulla, v e n t r a l p a r t , EM - ectomammillary nucleus, 10 - i n f e r i o r o l i v a r y nucleus, MLF - medial l o n g i t u d i n a l f a s c i c u l u s , MRF - mesencephalic r e t i c u l a r formation nucleus, MV - t r i g e m i n a l motor nucleus, N I I I - occulomotor nerve, N V - t r i g e m i n a l nerve, N XII - h y p o g l o s s a l nerve, OT -o p t i c tectum, R - raphe nucleus, RP - nucleus pontine r e t i c u l a r formation, Rpc - pontine p a r v o c e l l u l a r r e t i c u l a r nucleus, Ru -red nucleus, SSP - s u p r a s p i n a l nucleus, ST - s u b t r i g e m i n a l nucleus, SV - t r i g e m i n a l sensory nucleus, TTD - t r i g e m i n a l descending t r a c t and nucleus, VII - f a c i a l nucleus, X - d o r s a l motor nucleus vagus. 90 A 3.75 LOCO JTH 1TH \H PILO • © © O SCOP • • • o NICO • © © O CARS • B B • AM) A A A A Iff P3.75 P 4.50 91 F i g u r e 10 Coronal s e c t i o n s through the avian brainstem i l l u s t r a t i n g e l e c t r i c a l and neurochemical s t i m u l a t i o n s i t e s which e l i c i t e d locomotion i n the decerebrate b i r d . A: Coronal s e c t i o n through the caudal medulla showing a s t i m u l a t i o n / i n j e c t i o n s i t e (L) i n TTD. B: Coronal s e c t i o n through the caudal medulla showing a s t i m u l a t i o n / i n j e c t i o n s i t e (L) i n Cnd. C: Coronal s e c t i o n through the caudal medulla demonstrating a s t i m u l a t i o n / i n j e c t i o n s i t e (L) i n Cnv. D: Coronal s e c t i o n through the pons i l l u s t r a t i n g a s t i m u l a t i o n / i n j e c t i o n s i t e i n the pontine r e t i c u l a r formation (RP) near the raphe nucleus (R). For the e f f e c t s found i n each r e g i o n , see R e s u l t s . A b b r e v i a t i o n s : cc - c e n t r a l c a n a l , Cnd -d o r s a l p a r t , c e n t r a l medullary nucleus, Cnv - v e n t r a l p a r t , c e n t r a l medullary nucleus, IM - in t e r m e d i a t e nucleus, 10 -i n f e r i o r o l i v a r y nucleus, L - l e s i o n made at s t i m u l a t i o n / i n j e c t i o n s i t e , MLF - medial l o n g i t u d i n a l f a s c i c u l u s , NVIII - glossopharyngeal nerve, R - raphe nucleus, RP - pontine r e t i c u l a r formation, SSP - s u p r a s p i n a l nucleus, TTD - descending t r i g e m i n a l t r a c t and nucleus, X - d o r s a l motor nucleus o f the vagus. 92 93 94 Pontobulbar Locomotor S t r i p (PLS) E l e c t r i c a l s t i m u l a t i o n of the pontobulbar locomotor s t r i p (PLS) e l i c i t e d locomotor movements at low t h r e s h o l d i n t e n s i t i e s r a n ging from 30-80jiA i n nine animals. In four out of four b i r d s , f o l l o w i n g the establishment of a locomotion producing e l e c t r i c a l s t i m u l a t i o n s i t e (walking) (Figure 11A), i n j e c t i o n o f car b a c h o l (25-100mM) i n t o PLS e l i c i t e d a p a t t e r n o f a l t e r n a t i n g walking behaviour (no wing f l a p p i n g ) with a mean l a t e n c y t o the f i r s t d e t e c t a b l e movement f o l l o w i n g i n i t i a l i n j e c t i o n of 8.2 minutes (range 2.2-12 minutes). The s t e p p i n g (Figure 11B) was long l a s t i n g (mean 23 minutes: range 7-45 minutes), d u r i n g which time e l e c t r i c a l s t i m u l a t i o n enhanced the v i g o r of walking i n an i n t e n s i t y dependent manner. In the only animal t e s t e d , i n f u s i o n o f a t r o p i n e (25mM) i n t o the same s i t e d u r i n g c a r b a c h o l - i n d u c e d locomotion completely b l o c k e d not only the c h e m i c a l l y induced behaviour but a l s o a l l e l e c t r i c a l l y evoked s t e p p i n g up t o the maximal s t i m u l a t i o n i n t e n s i t y (170MA). I n t r o d u c t i o n o f p i l o c a r p i n e (50mM) i n t o PLS produced i n t e r m i t t e n t s h o r t b u r s t s of walking and f l y i n g behaviour i n one animal w i t h a time t o onset of 7 minutes. The b u r s t s were maintained f o r approximately 8 minutes. T h i s p e r i o d was f o l l o w e d by e x t e n s i o n o f both wings and l e g s , at which time e l e c t r i c a l s t i m u l a t i o n was i n e f f e c t i v e at e l i c i t i n g locomotor p a t t e r n s . N e i t h e r scopolamine (N=l) (25mM) nor n i c o t i n e (N=2) (25mM) was e f f e c t i v e at b l o c k i n g , e l i c i t i n g or changing the t h r e s h o l d of PLS e l e c t r i c a l l y s t i m u l a t e d locomotion. 95 F i g u r e 11. Electromyographic records (EMGs) showing locomotor a c t i v i t y e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n and car b a c h o l i n j e c t i o n i n t o the pontobulbar locomotor s t r i p (PLS). A: A l t e r n a t i n g s t e p p i n g r e p r e s e n t e d by EMG p a t t e r n s from the r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles (major h i p f l e x o r muscle) e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n of the PLS. B: A l t e r n a t i n g s t e p p i n g EMGs e l i c i t e d by i n j e c t i o n o f car b a c h o l (lOOmM) i n t o the same s i t e . 96 A 1 sec 97 C e n t r a l Nucleus, d o r s a l p a r t (Cnd) I n f u s i o n o f ca r b a c h o l (lOOmM) i n t o the d o r s a l p a r t of the medullary c e n t r a l nucleus (Cnd) produced long l a s t i n g s t e p p i n g i n only one of f i v e b i r d s t e s t e d ( F i g . 12A,B). In two animals, however, the t h r e s h o l d s f o r e l e c t r i c a l l y evoked locomotion were reduced (60/nA t o 30/iA and 80LLA t o 60/iA) a f t e r c a r b a c h o l (llmM & 50mM) i n f u s i o n . I n t r o d u c t i o n of a t r o p i n e (30mM) d u r i n g c a r b a c h o l s t i m u l a t e d locomotion b l o c k e d the ongoing locomotion, as w e l l as f u r t h e r attempts t o induce locomotion e l e c t r i c a l l y and ch e m i c a l l y ( c a r b a c h o l ) . S i m i l a r l y , i n t r o d u c t i o n o f a t r o p i n e i n t o one.of the animals t h a t showed decreased t h r e s h o l d (carbachol, 30mM) completely b l o c k e d any f u r t h e r e l e c t r i c a l l y evoked a c t i v i t y . Scopolamine (25mM) d i d not change the car b a c h o l - i n d u c e d decrease i n locomotor t h r e s h o l d of the other animal. C e n t r a l Nucleus, v e n t r a l p a r t (Cnv) In the v e n t r a l p a r t of the medullary c e n t r a l nucleus (Cnv), e l e c t r i c a l s t i m u l a t i o n , used t o e s t a b l i s h optimum e l e c t r o d e p o s i t i o n , e l i c i t e d b i l a t e r a l a l t e r n a t i n g hindlimb walking i n 9 out o f 10 b i r d s , and running and f l y i n g i n 1 out of 10. Subsequent i n j e c t i o n of ca r b a c h o l produced long l a s t i n g walking behaviour i n 6 of the 10 animals i n j e c t e d ( F i g . 13, ca r b a c h o l = 27mM). One b i r d showed both walking and f l y i n g behaviour i n response t o ca r b a c h o l i n j e c t i o n , while e l e c t r i c a l t h r e s h o l d f o r walking was reduced i n another. Two animals demonstrated no 98 F i g u r e 12. Electromyographic records (EMGs) showing locomotor a c t i v i t y e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n and ca r b a c h o l i n j e c t i o n i n t o the c e n t r a l medullary nucleus, d o r s a l p a r t (Cnd). A: A l t e r n a t i n g s t e p p i n g r e p r e s e n t e d by EMG p a t t e r n s from r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n of the Cnd. B: A l t e r n a t i n g s t e p p i n g EMGs e l i c i t e d by i n j e c t i o n of ca r b a c h o l (lOOmM/1.Oul) i n t o the same s i t e . 99 A 2 sec 100 F i g u r e 13. Electromyographic records (EMGs) showing locomotor a c t i v i t y e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n and ca r b a c h o l i n f u s i o n i n t o the c e n t r a l medullary nucleus, v e n t r a l p a r t (Cnv). PRESTIM: EMGs from r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles i l l u s t r a t i n g the l a c k of spontaneous locomotor a c t i v i t y i n the p r e - s t i m u l a t i o n decerebrate p r e p a r a t i o n . STIM: A l t e r n a t i n g s t e p p i n g r e p r e s e n t e d by EMG p a t t e r n s from the same muscles d u r i n g e l e c t r i c a l s t i m u l a t i o n (40LLA) of the s i t e (Cnv) shown by the squares i n the c o r o n a l s e c t i o n through the caudal medulla (bottom r i g h t ) . CARB: Carbachol i n j e c t i o n i n t o the same s i t e ( f i l l e d square) e l i c i t e d a l t e r n a t i n g s tepping, as demonstrated by the EMGs from RITC and LITC. ATRO: The car b a c h o l - i n d u c e d locomotor a c t i v i t y was b l o c k e d by i n j e c t i o n of a t r o p i n e ( u n f i l l e d square) i n t o the same s i t e . In a d d i t i o n , a f t e r a t r o p i n e i n j e c t i o n , as shown d u r i n g e l e c t r i c a l s t i m u l a t i o n of the s i t e , s timulus i n t e n s i t i e s up t o 170LLA d i d not evoke locomotor p a t t e r n s (some stimulus b l e e d through can be seen i n the EMG t r a c e s ) . 101 PRESTIM RITC LITC H i sec-* STIM Msec 4 C A R B m». im i 11 » — >! - f r . ,„fcRITC <•» • » -4 4* f « + H " | • I • LITC »-5sec-« e f f e c t s of c a r b a c h o l i n j e c t i o n . F i v e animals i n j e c t e d with a t r o p i n e (25mM) f o l l o w i n g c a r b a c h o l - i n d u c e d locomotion, i n c l u d i n g the w a l k i n g / f l y i n g b i r d , ceased a l l locomotor a c t i v i t y w i t h i n a mean time o f 7.5 minutes p o s t - i n j e c t i o n ( F i g . 13). In a l l cases where c a r b a c h o l - i n d u c e d locomotion was bl o c k e d by a t r o p i n e , locomotion d i d not r e t u r n i n the absence of e l e c t r i c a l s t i m u l a t i o n d u r i n g the experimental p e r i o d . However, the r e t u r n of e l e c t r i c a l l y s t i m u l a t e d locomotion f o l l o w i n g a t r o p i n e i n j e c t i o n appeared t o r e l a t e t o the c o n c e n t r a t i o n i n j e c t e d , with h i g h e r c o n c e n t r a t i o n s o f a t r o p i n e (50mM (range 25-50mM) b l o c k i n g f o r p e r i o d s o f up t o 40 minutes ( s t i m u l a t i o n maximum 170^A). I n j e c t i o n s of n i c o t i n e (N=2) (lOOmM) and p i l o c a r p i n e (N=2) (50mM) were i n e f f e c t i v e at producing c h e m i c a l l y s t i m u l a t e d locomotion when i n f u s e d i n t o Cnv and d i d not appear t o have any e f f e c t on e i t h e r the t h r e s h o l d or type o f locomotion e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n . Scopolamine (N=l) (25mM) a l s o had no e f f e c t when i n j e c t e d i n t o t h i s s i t e . Pontine (RP) and Mesencephalic (MRF) R e t i c u l a r Formation I n j e c t i o n o f c a r b a c h o l (54mM) i n t o the pontine r e t i c u l a r formation (RPgc) (N=l) had no e f f e c t on e l e c t r i c a l l y s t i m u l a t e d locomotor t h r e s h o l d or on locomotor p a t t e r n . In the MRF, however, c a r b a c h o l (lOOmM) i n j e c t i o n decreased the e l e c t r i c a l s t i m u l a t i o n t h r e s h o l d f o r locomotion (100—>60fiA) i n one animal, but was i n e f f e c t i v e at e l i c i t i n g locomotion or changing the e l e c t r i c a l s t i m u l a t i o n t h r e s h o l d i n t e n s i t y f o r locomotion i n a second b i r d . 103 Medial L o n g i t u d i n a l F a s c i c u l u s (MLF) E l e c t r i c a l s t i m u l a t i o n (70-80uA) of the MLF evoked hindlimb and f o r e l i m b locomotion i n f i v e b i r d s . Low stimulus i n t e n s i t i e s evoked walking alone ( F i g . 14A) which gave way t o f l y i n g and running at h i g h e r i n t e n s i t i e s . I n j e c t i o n of c a r b a c h o l (27-100mM) i n t o these s i t e s i n th r e e out of f i v e animals evoked e i t h e r walking (N=l) ( F i g . 14B) or running and f l y i n g (N=2). The animals were i n i t i a l l y e l e c t r i c a l l y s t i m u l a t e d , then c u r a r i z e d , and the s t i m u l a t i o n i n t e n s i t y necessary t o evoke locomotor p a t t e r n s was i n c r e a s e d over the p r e - p a r a l y z e d c o n d i t i o n t o a mean i n t e n s i t y of 170uA (see Chapter 6). However, the c o n c e n t r a t i o n s of c a r b a c h o l necessary t o evoke locomotor p a t t e r n s i n these p a r a l y z e d b i r d s ( ^ f i c t i v e ' locomotion) were not s i g n i f i c a n t l y d i f f e r e n t from those found i n unparalyzed p r e p a r a t i o n s . COMPARISON BETWEEN ELECTRICAL AND CHEMICAL STIMULATED LOCOMOTION The locomotion e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n t y p i c a l l y v a r i e d with d i f f e r e n t i n t e n s i t i e s of s t i m u l a t i o n and from r e g i o n to r e g i o n i n the b i r d (Sholomenko and Steeves, 1987). For example, e l e c t r i c a l s t i m u l a t i o n i n some s i t e s produced walking at low c u r r e n t i n t e n s i t y (e.g. 30uA), running and f l y i n g at hi g h e r i n t e n s i t y (e.g. 90fxA) and f l y i n g alone at s t i l l h i g h e r i n t e n s i t y (e.g. 120uA) (see F i g . 3 , Steeves et al., 1987). In other s i t e s , t h r e s h o l d i n t e n s i t y s t i m u l a t i o n would, evoke running and f l y i n g t o gether, while i n s t i l l other l o c a t i o n s , only 104 F i g u r e 14. Electromyographic records (EMGs) showing locomotor a c t i v i t y e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n and ca r b a c h o l i n j e c t i o n i n t o the medial l o n g i t u d i n a l f a s c i c u l u s (MLF). A: A l t e r n a t i n g s t e p p i n g as re p r e s e n t e d by EMGs from r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles d u r i n g e l e c t r i c a l s t i m u l a t i o n of the MLF. B: A l t e r n a t i n g s t e p p i n g EMGs e l i c i t e d by car b a c h o l (27mM) i n f u s i o n i n t o the same s i t e . 105 LITC M- 111,1 "•>*MI>» "4 ^ m *"* I 1 sec 106 walking c o u l d be e l i c i t e d at a l l s t i m u l a t i o n i n t e n s i t i e s . However, e l e c t r i c a l s t i m u l a t i o n always evoked the same p a t t e r n of behaviour i n a s t i m u l a t i o n dependent and s i t e s p e c i f i c manner. Thus, repeated t r i a l s i n the same s i t e always e l i c i t e d the same locomotor p a t t e r n . These p a t t e r n s , with few exceptions, were r e p l i c a t e d by the neurochemical i n j e c t i o n s which e l i c i t e d locomotion. 107 DISCUSSION Neurochemical s t i m u l a t i o n of s e l e c t e d r e g i o n s i n the avian mid- and h i n d b r a i n e l i c i t e d a spectrum of locomotor p a t t e r n s i n decerebrate b i r d s . My r e s u l t s demonstrated t h a t c h o l i n e r g i c a g o n i s t s evoke locomotion and mu s c a r i n i c a n t a g o n i s t s b l o c k locomotion when i n j e c t e d i n t o a v a r i e t y of s i t e s . These r e s u l t s w i l l be d i s c u s s e d r e g i o n by r e g i o n and c o n c l u s i o n s w i l l be drawn as t o the p o s s i b l e a v i a n neuroanatomical pathways i n v o l v e d . These data from b i r d s w i l l be compared with those found i n mammalian s p e c i e s f o r which s i m i l a r data e x i s t s . Pontobulbar Locomotor S t r i p E l e c t r i c a l s t i m u l a t i o n of the pontobulbar locomotor s t r i p (PLS) e l i c i t s locomotion i n b i r d s (Steeves et al., 1987; Funk et al., submitted) and i n a wide range of v e r t e b r a t e s p e c i e s from 0 lamprey t o cat (f o r review, see Chapter 1 & M c C l e l l a n , 1986). As w i l l be d i s c u s s e d i n g r e a t e r d e t a i l below, the neuroanatomical input/output r e l a t i o n s o f the av i a n TTD are s i m i l a r t o those found i n a v a r i e t y of v e r t e b r a t e s with the ex c e p t i o n t h a t the major t r i g e m i n o t h a l a m o c o r t i c a l r e l a y found i n mammals i s r e p l a c e d i n b i r d s by a d i r e c t t e l e n c e p h a i i c p r o j e c t i o n (Arends et al., 1984; Arends and Dubbeldam, 1984; Wi l d et al., 1984). My p r e v i o u s s t u d i e s i n b i r d s demonstrate t h a t e l e c t r i c a l s t i m u l a t i o n of the descending t r i g e m i n a l nucleus and the Plexus of H o r s l e y (PH) , i n a d d i t i o n t o adjacent regions of the 108 pontomedullary l a t e r a l r e t i c u l a r formation ( p a r v o c e l l u l a r r e t i c u l a r nucleus), which are v i r t u a l l y i n d i s t i n g u i s h a b l e from the nucleus i n t e r p o l a r i s of TTD (Arends and Dubbeldam, 1984), e l i c i t s locomotion i n the decerebrate animal, thus d e f i n i n g t h i s a region as the avian equivalent of the mammalian PLS (Steeves et al., 1987). In my experiments, locomotion was e l i c i t e d by i n t r o d u c t i o n of carbachol or p i l o c a r p i n e i n t o the PLS. Carbachol, a c h o l i n e r g i c agonist w i t h a s t r u c t u r e s i m i l a r to that of a c e t y l c h o l i n e (Taylor, 1985) i s an e f f e c t i v e cholinomimetic at both n i c o t i n i c and muscarinic a c e t y l c h o l i n e r g i c receptor subtypes (Burgen, 1983), i n c l u d i n g four c u r r e n t l y recognized muscarinic receptor subtypes which have been i d e n t i f i e d i n the CNS (Buckley et a l . , 1988). Receptor b i n d i n g s t u d i e s demonstrate that carbachol binds t o high and low a f f i n i t y receptors i n the CNS, although the b i n d i n g v a r i e s by a f a c t o r of 100 depending upon the l o c a t i o n (Burgen, 1983). The b i n d i n g s t u d i e s a l s o demonstrate t h a t at carbachol concentrations of lOOmM, a l l muscarinic (Mi & M2) receptor s i t e s should be bound (Potter et al., 1983). Thus, at the s i t e of carbachol i n j e c t i o n i n t o locomotor regions i n the avian b r a i n at high concentrations (lOOmM), a l l muscarinic receptors should be a c t i v a t e d . I t was not p o s s i b l e t o determine, however, the concentration gradient from the i n j e c t i o n p o i n t to the periphery of the a f f e c t e d volume. In t h i s study, i t was a l s o not p o s s i b l e t o d i s t i n g u i s h between the d i f f e r e n t receptor subtypes a c t i v a t e d , as no agonists or antagonists are yet a v a i l a b l e which are s p e c i f i c f o r a s i n g l e muscarinic receptor subtype (Buckley et al., 1988). Therefore, i t was d i f f i c u l t t o d i s t i n g u i s h between the car b a c h o l induced a c t i v a t i o n o f m u s c a r i n i c r e c e p t o r s which 1) i n c r e a s e potassium conductance i n some neurons, 2) decrease the potassium conductance i n some neurons, 3) i n c r e a s e the c a t i o n (most i m p o r t a n t l y sodium) conductance i n some neurons or 4) reduce c a l c i u m conductance i n some neurons (North, 1985). S i m i l a r d i f f i c u l t i e s come i n determining the s p e c i f i c r e c e p t o r subtype a c t i v a t e d by i n j e c t i o n o f the m u s c a r i n i c a g o n i s t p i l o c a r p i n e i n t o the PLS, as p i l o c a r p i n e r e c o g n i z e s a l l m u s c a r i n i c r e c e p t o r subtypes, although with d i f f e r i n g a f f i n i t i e s (Brown, 1983). However, the a c t i v a t i o n o f locomotion f o l l o w i n g p i l o c a r p i n e i n j e c t i o n lends credence t o the hypothesis t h a t c h o l i n e r g i c r e c e p t o r s are i n v o l v e d i n the a c t i v a t i o n , although t h i s a g o n i s t was employed i n a s i n g l e t r i a l o n ly. F u r t h e r evidence i m p l i c a t i n g m u s c a r i n i c r e c e p t o r s as being r e s p o n s i b l e f o r the i n d u c t i o n of locomotion was t h a t c a r b a c h o l induced locomotion c o u l d be bl o c k e d by i n j e c t i o n o f the mu s c a r i n i c a n t a g o n i s t a t r o p i n e s u l f a t e i n t o the same s i t e . A t r o p i n e has been demonstrated as a n o n - s e l e c t i v e a n t a g o n i s t which b l o c k s a l l m u s c a r i n i c r e c e p t o r s (Mitchelson, 1983), and which has no n i c o t i n i c a c t i o n (Mitchelson, 1983). However, as only a s i n g l e PLS s i t e was i n j e c t e d with a t r o p i n e , t h i s r e s u l t can only be c o n s i d e r e d s u g g e s t i v e o f a r o l e f o r m u s c a r i n i c r e c e p t o r s u n d e r l y i n g the a c t i v a t i o n of locomotion from t h i s r e g i o n . C o r r e l a t i v e support f o r t h i s s uggestion comes from the f i n d i n g t h a t n i c o t i n e , the c l a s s i c c h o l i n e r g i c n i c o t i n i c r e c e p t o r a g o n i s t , had no e f f e c t when i n j e c t e d i n t o t h i s r e g i o n . However, scopolamine, a potent m u s c a r i n i c a n t a g o n i s t ( K i l b i n g e r , 110 1983), r a t h e r unexpectedly a l s o had no e f f e c t on e l e c t r i c a l s t i m u l a t i o n - i n d u c e d locomotion when i n j e c t e d i n t o the PLS i n one animal. These above r e s u l t s i n the b i r d , while somewhat e q u i v o c a l , do suggest a r o l e f o r c h o l i n e r g i c m u s c a r i n i c r e c e p t o r s u n d e r l y i n g the observed r e s u l t s . The r e s u l t s are a l s o supported, i n p a r t , by neuroanatomical and r e c e p t o r l o c a l i z a t i o n s t u d i e s d i s c u s s e d below. N-[ 3H]methylscopolamine (AChM antagonist) b i n d i n g s t u d i e s i n pigeon b r a i n show heavy l a b e l l i n g of c h o l i n e r g i c r e c e p t o r s i n TTD and s u b s t a n t i a g e l a t i n o s a (SG) of the medulla and s p i n a l cord, ( D i e t l et a l . , 1988 see F i g u r e 1H,J,K,G,I). Thus, t h i s r e g i o n c o n t a i n s the type of r e c e p t o r which c o r r e l a t e s with our observed r e s u l t s . However, t o our knowledge, no c h o l i n e r g i c i n p u t s t o TTD or SG have been r e p o r t e d i n b i r d s . In an attempt t o i s o l a t e p o s s i b l e c h o l i n e r g i c i n p u t s t o these r e g i o n s , our l a b o r a t o r y has u t i l i z e d immunohistochemical techniques to l o c a l i z e c h o l i n e a c e t y l t r a n s f e r a s e (ChAT) c o n t a i n i n g c e l l bodies i n the avian b r a i n (Steeves and Taccogna, unpublished o b s e r v a t i o n s ) . T h i s study has been combined with r e t r o g r a d e t r a n s p o r t s t u d i e s u s i n g f l u o r e s c e i n - and rhodamine conjugated dextran amines (Glover et a l . , 1986) i n t o TTD (Webster and Steeves, unpublished observations) to l o c a t e p u t a t i v e c h o l i n e r g i c p r o j e c t i o n s i n t o t h i s r e g i o n . These f i n d i n g s i n d i c a t e t h a t p o s s i b l e sources of c h o l i n e r g i c input to TTD may a r i s e from ChAT p o s i t i v e neurons o r i g i n a t i n g i n : 1) the pontine and medullary r e t i c u l a r formation, i n c l u d i n g the l a t e r a l r e t i c u l a r nucleus (RL), g i g a n t o c e l l u l a r r e t i c u l a r nucleus (Rgc) and o r a l p a r t of the pontine r e t i c u l a r formation (RPO), 2) the n u c l e i o f VII, which send e f f e r e n t s t o TTD i n re g i o n s caudal t o the obex, 3) the descending v e s t i b u l a r nucleus (VeD), which p r o j e c t s t o r o s t r a l r e g i o n s of TTD, 4) the nucleus raphe p a l l i d u s , which impinges on r o s t r a l TTD, 5) the paramedian nucleus, a l s o p r o j e c t i n g t o r o s t r a l TTD and 6) the p a r a b r a c h i a l r e g i o n which l i e s v e n t r a l t o the v e n t r a l subceruleus nucleus and p r o j e c t s t o r o s t r a l TTD. ChAT c o n t a i n i n g neurons have a l s o been l o c a l i z e d t o TTD and the s u b t r i g e m i n a l n u c l e a r r e g i o n s , a l l o w i n g f o r the p o s s i b i l i t y t h a t i n t r i n s i c c h o l i n e r g i c i n t e r n e u r o n s may modulate TTD n e u r a l c i r c u i t s and subserve the ca r b a c h o l - i n d u c e d locomotion from t h i s r e g i o n . In mammals, a f f e r e n t s from the t r i g e m i n a l and glossopharyngeal nerves impinge on the descending t r i g e m i n a l nucleus and are thought t o c o n t a i n Substance P, c h o l e c y s t o k i n i n (CCK), v a s o a c t i v e i n t e s t i n a l p o l y p e p t i d e (VIP) and somatostatin (Dubner and Bennett, 1983) but are ap p a r e n t l y not c h o l i n e r g i c . In the cat the ChAT-containing c e l l s (Vincent and Reiner, 1987) of PPN/mMLR may send a smal l c h o l i n e r g i c i n n e r v a t i o n t o PLS neurons ( G a r c i a - R i l l , 1985) which, i n t u r n , p r o j e c t upon the brainstem r e t i c u l a r formation and p r o p r i o s p i n a l pathways o u t l i n e d above. A l s o i n mammals, MesV i s known t o send i n t e r n u c l e a r f i b r e s t o TTD ( G a r c i a - R i l l et al., 1983; Ikeda et al., 1984) but no ChAT a c t i v i t y has been found i n the MesV (Vincent and Reiner, 1987). Th i s p r e c l u d e s the p o s s i b i l i t y t h a t MesV p r o v i d e s c h o l i n e r g i c i nput t o TTD (Reiner and V i n c e n t , 1987). Other p o s s i b l e c h o l i n e r g i c i n p u t s t o TTD may a r i s e from the caudal cuneiform nucleus, where both ACh and AChE have been l o c a l i z e d i n the r a t ( P a l k o v i t s and Jacobowitz, 1974; 112 Ramon-Moliner and Dansereau, 1974) and cat (Kimura et al., 1981). However, some co n t r o v e r s y e x i s t s as t o whether the cuneiform nucleus c o n t a i n s ACh neurons, as Reiner and Vin c e n t (1987) found no evidence o f ChAT c o n t a i n i n g neurons i n t h i s nucleus ( f o r review see p. 521 of Rye et al., 1981). Pharmacological evidence o f a r o l e f o r ACh i n TTD s t i m u l a t i o n - i n d u c e d locomotion has a l s o been found i n the r a t where 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 ACh i n c r e a s e d f i r i n g r a t e s o f c e l l s i n the nucleus c a u d a l i s o f TTD ( S a l t and H i l l , 1981). While, t o our knowledge, no c h o l i n e r g i c a f f e r e n t s t o the r e g i o n we i n j e c t e d have been u n e q u i v o c a l l y e s t a b l i s h e d i n b i r d s , our s t u d i e s p o i n t t o s e v e r a l p o t e n t i a l c h o l i n e r g i c pathways which may p l a y some r o l e i n locomotor c o n t r o l v i a the PLS pathway. However, the p r e c i s e r o l e o f these p r o j e c t i o n s i n motor c o n t r o l remains t o be determined. In b i r d s , TTD e f f e r e n t s p r o j e c t t o a v a r i e t y of re g i o n s i n c l u d i n g cerebellum, s p i n a l c o r d d o r s a l horn (C4), Cnv, Cnd, ST, PH, RL, Rpc, d o r s a l column n u c l e i , PrV, and p a r a b r a c h i a l nucleus (Arends and Dubbeldam, 1982, 1984; Arends et al., 1984; Webster and Steeves, p e r s o n a l communication). The s p i n a l c o r d p r o j e c t i o n may be e q u i v a l e n t t o the t r i g e m i n a l p r o p r i o s p i n a l network found i n mammals, while the e f f e r e n t s t o PrV and the r e t i c u l a r n u c l e i have been i m p l i c a t e d i n the c o n t r o l of grasping, f e e d i n g and pecking i n b i r d s (Wild et al., 1984). Connections of TTD wit h the r e t i c u l a r formation n u c l e i w i l l be d i s c u s s e d below i n r e l a t i o n t o locomotion e l i c i t e d from these r e g i o n s . On the b a s i s of i t s h o d o l o g i c a l connections, combined w i t h i t s locomotor e l i c i t i n g p r o p e r t i e s f o l l o w i n g e l e c t r i c a l and chemical s t i m u l a t i o n , however, TTD appears t o p l a y a major r o l e i n the sensory c o n t r o l of a v a r i e t y of motor behaviours i n b i r d s . T h i s data, t h e r e f o r e , agrees with the p r o p o s a l by Jordan and co-workers (Jordan, 1986; Noga et a l . , 1988) f o r mammalian sp e c i e s t h a t the descending t r i g e m i n a l nucleus i s concerned with the sensorimotor c o n t r o l of locomotion. Our s t u d i e s suggest t h a t t h i s c o n t r o l i s mediated, i n p a r t , by c h o l i n e r g i c m u s c a r i n i c mechanisms. C e n t r a l Nucleus, d o r s a l p a r t (Cnd) Previo u s s t u d i e s have shown t h a t e l e c t r i c a l s t i m u l a t i o n of the d o r s a l p a r t o f the medullary c e n t r a l nucleus (Cnd) e l i c i t s locomotion i n the decerebrate b i r d (Steeves et al., 1986). Retrograde t r a n s p o r t s t u d i e s u s i n g True Blue (Steeves et al., 1987) and HRP (Cabot et al., 1982) demonstrated t h a t Cnd g i v e s r i s e , i n p a r t , t o the r e t i c u l o s p i n a l f i b e r s t r a v e l l i n g i n the s p i n a l c o r d v e n t r a l f u n i c u l u s , which have been shown through s e l e c t i v e low t h o r a c i c s p i n a l c o r d l e s i o n s , t o be e s s e n t i a l f o r v o l u n t a r y or e l e c t r i c a l l y induced walking both i n c h r o n i c and decerebrate b i r d s (Sholomenko and Steeves, 1987b), cat (Steeves and Jordan, 1980; E i d e l b e r g , 1981) and monkey ( E i d e l b e r g et al,, 1981). Thus, i t appears t h a t c e l l s o r i g i n a t i n g from t h i s nucleus g i v e r i s e t o a descending pathway important f o r the descending c o n t r o l o f locomotion. F u r t h e r evidence f o r a r o l e of Cnd i n locomotor c o n t r o l i n b i r d s comes from neuroanatomical s t u d i e s which show t h a t Cnd r e c e i v e s a f f e r e n t input from the s u b n u c l e i c a u d a l i s and i n t e r p o l a r i s of TTD (Arends et al., 1984; Arends and Dubbeldam, 1984), the nucleus i n t e r c o l l i c u l a r i s (ICo) (Webster, p e r s o n a l communication), the tectum (Hunt and Kunzle, 1976) and from the a r c h i s t r i a t u m intermedium v i a the o c c i p i t o m e s e n c e p h a l i c t r a c t (Wild, 1985, W i l d et a l . , 1984). The a f f e r e n t input from the a r c h i s t r i a t u m has been i m p l i c a t e d as b eing p a r t of a c i r c u i t t e r m i n a t i n g i n the pontomedullary r e t i c u l a r formation which i s e s s e n t i a l f o r the c o n t r o l of f e e d i n g , g r a s p i n g and pecking i n the pigeon ( W i l d et a l . , 1984). Input to Cnd from the deep t e c t a l l a y e r s (Hunt and Kunzle, 1976; Reiner and Karten, 1982) appears t o be p a r t of the outflow c i r c u i t i n v o l v i n g the avian b a s a l g a n g l i a ( T P c ) / l a t e r a l s p i r i f o r m nucleus/tectum, which i s p o s t u l a t e d t o be i n v o l v e d i n v i s u o - s p a t i a l motor c o n t r o l (Reiner et a l . , 1984). In t u r n , Cnd sends ascending p r o j e c t i o n s t o the motor n u c l e i of c r a n i a l nerves V & VII, g i g a n t o c e l l u l a r r e t i c u l a r nucleus (Rgc) and the p a r a b r a c h i a l nucleus (Arends and Dubbeldam, 1984), i n a d d i t i o n t o i t s descending r e t i c u l o s p i n a l component. The p r o j e c t i o n s from PLS/TTD t o brainstem r e t i c u l a r n u c l e i , i n c l u d i n g Cnd, appear to have d i r e c t sensorimotor i m p l i c a t i o n s as d i s c u s s e d from our r e s u l t s above. In b i r d s , ICo, and FRL are thought to c o n t a i n neuronal p o p u l a t i o n s homologous t o those found i n the the mammalian cuneiform nucleus (Cabot et a l . , 1982; Edwards, 1975) and w i l l be d i s c u s s e d subsequently. The e f f e r e n t connections of Cnd to the p a r a b r a c h i a l and c r a n i a l nerve V and VII motor n u c l e i i m p l i c a t e t h i s r e g i o n i n the c o n t r o l of f e e d i n g and d r i n k i n g behaviours (Arends and Dubbeldam, 1982). These h o d o l o g i c a l r e l a t i o n s h i p s , t h e r e f o r e , p l a c e Cnd i n an e x c e l l e n t p o s i t i o n t o act as an i n t e g r a t o r y and descending output region from which both sensory and higher b r a i n centers may i n f l u e n c e motor c o n t r o l . To f u r t h e r our understanding of Cnd's r o l e i n locomotor c o n t r o l , we have begun to examine i t s neuropharmacology. Carbachol i n f u s i o n i n t o Cnd e l i c i t e d (or reduced t h r e s h o l d for) walking behaviour which was blocked by the muscarinic antagonist a t r o p i n e , while n i c o t i n i c agonists were i n e f f e c t i v e at producing locomotion or changing the t h r e s h o l d f o r e l e c t r i c a l l y s t i m u l a t e d locomotion. Autoradiographic s t u d i e s of receptor b i n d i n g w i t h N-[ 3H]methylscopolamine i n d i c a t e the presence of muscarinic c h o l i n e r g i c receptors i n the Cnd of the pigeon ( D i e t l et a l . , 1988). Although no c h o l i n e r g i c a f f e r e n t s to Cnd have been unequivocally i d e n t i f i e d i n b i r d s , neuroanatomical (Webster and Steeves, unpublished'observations) and immunohistochemical st u d i e s from our la b o r a t o r y (Steeves and Taccogna, unpublished observations) demonstrate the presence of ChAT c o n t a i n i n g c e l l bodies i n the subtrigeminal nucleus, TTD, pontine r e t i c u l a r formation, l a t e r o d o r s a l tegmental nucleus and nucleus i s t h m i , pars p a r v o c e l l u l a r i s , a l l of which have been shown to p r o j e c t t o Cnd. Cnd i t s e l f a l s o contains ChAT p o s i t i v e neurons. S i m i l a r l y , i n mammals, the equivalent s t r u c t u r e , FTL, ( l a t e r a l tegmental f i e l d ) r e c e i v e s a f f e r e n t input from the region of the PLS/trigeminal area (Noga et a l . , 1988; G a r c i a - R i l l et al.,1983b) and al s o appears t o re c e i v e descending c h o l i n e r g i c input from the MesV region and PPN/mMLR ( G a r c i a - R i l l et a l . , 1983b). Further, i n cat, c e l l bodies c o n t a i n i n g ChAT have been l o c a l i z e d i n the r e t i c u l a r tegmental f i e l d (Vincent and Reiner, 1987), p o s s i b l y i m p l i c a t i n g i n t r i n s i c c h o l i n e r g i c neurons and m u s c a r i n i c r e c e p t o r s as producing some of the observed r e s u l t s . Taken t o g e t h e r w i t h those from mammalian s t u d i e s , our r e s u l t s p r o v i d e s t r o n g evidence t h a t Cnd neurons g i v e r i s e t o p a r t o f the descending r e t i c u l o s p i n a l pathway which i s e s s e n t i a l f o r locomotor c o n t r o l i n v e r t e b r a t e s . F u r t h e r , i t appears t o act as an i n t e g r a t i o n c e n t r e f o r sensory i n f o r m a t i o n from the descending t r i g e m i n a l nucleus and a l s o from h i g h e r b r a i n s t r u c t u r e s c o n t r o l l i n g locomotion i n b i r d s . Based on the f i n d i n g s t h a t c a r b a c h o l i n j e c t i o n e l i c i t e d a t r o p i n e - r e v e r s i b l e locomotor behaviour, the nucleus appears t o be i n p a r t under c h o l i n e r g i c m u s c a r i n i c c o n t r o l and p o s s i b l e c o n t r o l pathways have been d e s c r i b e d . However, as only a s i n g l e animal d i r e c t l y demonstrated locomotion f o l l o w i n g c a r b a c h o l i n f u s i o n , i t may be p o s s i b l e t h a t neurochemical spread t o Cnv or TTD was r e s p o n s i b l e f o r the locomotion. S t u d i e s u t i l i z i n g s m a l l e r i n j e c t i o n volumes' (presumably w i t h l e s s spread) may a l l e v i a t e t h i s problem. A l s o , why scopolamine d i d not bl o c k locomotion i n the one animal i n j e c t e d awaits f u r t h e r r e p l i c a t i o n . Future s t u d i e s are a l s o r e q u i r e d t o determine both the mechanisms and source(s) of c h o l i n e r g i c i n put (or i n t r i n s i c neurons) which may exert c o n t r o l over t h i s descending pathway. C e n t r a l Nucleus, v e n t r a l p a r t (Cnv) The Cnv a l s o appears t o p l a y and important r o l e i n locomotor c o n t r o l . E l e c t r i c a l s t i m u l a t i o n of the v e n t r a l p a r t of the medullary c e n t r a l nucleus (Cnv) produced locomotion i n b i r d s (Steeves et al., 1986; Sholomenko and Steeves, 1987b). Evidence from the b i r d (Sholomenko and Steeves, 1987), as i n the cat (Steeves and Jordan, 1980) and monkey ( E i d e l b e r g et al., 1984), demonstrated t h a t a l e s i o n of the descending pathways t r a v e l l i n g i n the s p i n a l c o r d v e n t r a l f u n i c u l u s b l o c k s v o l u n t a r y locomotion i n the i n t a c t animal and e l e c t r i c a l l y s t i m u l a t e d locomotion i n the decerebrate p r e p a r a t i o n . In the c a t , G a r c i a - R i l l ' s group ( G a r c i a - R i l l and Skinner, 1987b) u t i l i z e d r e t r o g r a d e t r a n s p o r t of bisbenzimide and n u c l e a r y e l l o w from e l e c t r i c a l l y / c h e m i c a l l y d e f i n e d locomotor s i t e s i n the VRN to demonstrate i t s a f f e r e n t i n p u t . They found i p s i l a t e r a l and c o n t r a l a t e r a l p r o j e c t i o n s t o VRN from the c e n t r a l gray, cuneiform nucleus, subcuneiform nucleus, PPN, midbrain r e t i c u l a r formation, a n t e r i o r l a t e r a l r e t i c u l a r nucleus, l a t e r a l r e t i c u l a r nucleus and s c a t t e r e d r e t i c u l a r formation c e l l s . Steeves and Jordan (1984) u t i l i z e d anterograde t r a n s p o r t of r a d i o a c t i v e amino a c i d s t o demonstrate d i r e c t p r o j e c t i o n s t o the pontine and medullary r e t i c u l a r formation from the c l a s s i c a l MLR, while Orlovsky, (1970a) used e l e c t r o p h y s i o l o g i c a l techniques t o show a monosynaptic l i n k a g e between the 1MLR and medullary r e t i c u l a r formation. In b i r d s , neuroanatomical r e t r o g r a d e t r a n s p o r t s t u d i e s demonstrate t h a t c e l l s i n Cnv g i v e r i s e t o r e t i c u l o s p i n a l neurons t r a v e l l i n g the l e n g t h of the s p i n a l c o r d i n the v e n t r a l f u n i c u l u s (Steeves et a l . , 1987; Webster and Steeves, 1988) and t h a t e l e c t r i c a l s t i m u l a t i o n i n and around these c e l l s e l i c i t s locomotion (Steeves et al., 1987). In b i r d s , r e t r o g r a d e (True 118 blue, HRP, dextran amines) and anterograde (WGA-HRP) neuroanatomical t r a c i n g techniques demonstrate t h a t a f f e r e n t s t o the medial medullary r e t i c u l a r formation (Cnv) i n c l u d e the nucleus i n t e r c o l l i c u l a r i s (ICo), cerebellum, Area v e n t r a l i s of T s a i (AVT), Red nucleus, nucleus raphe magnus, l a t e r a l and medial mesencephalic r e t i c u l a r formation (FRL, FRM), more r o s t r a l .pontine r e t i c u l a r formation, (RPc, RPgc) and TTD (Webster, p e r s o n a l communication). A l l of the above a f f e r e n t s t r u c t u r e s have been i m p l i c a t e d i n motor c o n t r o l (as d i s c u s s e d above; f o r review see Kuypers, 1981) and s i m i l a r t o Cnd, p l a c e Cnv i n an i d e a l p o s i t i o n t o i n t e g r a t e and output motor i n f o r m a t i o n t o the s p i n a l cord. H o d o l o g i c a l c o n s i d e r a t i o n s i m p l i c a t e Cnv as b e i n g homologous to the mammalian v e n t r a l r e t i c u l a r nucleus ( G a r c i a - R i l l and Skinner, 1987a,b) (magnocellular r e t i c u l a r formation of Noga et a l . , 1988). To examine the m o t o r - a s s o c i a t e d r o l e which a c e t y l c h o l i n e may have i n the a c t i v a t i o n of locomotion from t h i s r e g i o n , c h o l i n e r g i c a g o n i s t s and a n t a g o n i s t s were i n j e c t e d i n t o Cnv s i t e s from which locomotion c o u l d be e l e c t r i c a l l y induced. Carbachol i n j e c t i o n i n t o the v e n t r a l p a r t of the medullary c e n t r a l nucleus (Cnv) produced a t r o p i n e - r e v e r s i b l e locomotor behaviour i n b i r d s i n a manner s i m i l a r t o t h a t found f o l l o w i n g i n j e c t i o n of a c e t y l c h o l i n e , c a r b a c h o l or a c e t y l c h o l i n e s t e r a s e i n h i b i t o r s i n t o the cat medullary v e n t r a l r e t i c u l a r nucleus ( G a r c i a - R i l l and Skinner, 1987a). However, p i l o c a r p i n e i n j e c t i o n d i d not e l i c i t any locomotor behaviour. Although i t i s p r e s e n t l y unknown whether any avian a f f e r e n t p r o j e c t i o n s t o Cnv are d e f i n i t i v e l y c h o l i n e r g i c , s t u d i e s from our l a b o r a t o r y p o i n t t o 119 s e v e r a l p o t e n t i a l n u c l e i w h i c h p o s s e s s b o t h ChAT c o n t a i n i n g n e u r o n s a n d h a v e b e e n f o u n d t o p r o j e c t t o C n v . T h e s e i n c l u d e , f r o m c a u d a l t o r o s t r a l , T T D , N u c l e u s r e t i c u l a r i s g i g a n t o c e l l u l a r i s ( R g c ) , N u c l e u s r e t i c u l a r i s p a r v o c e l l u l a r i s (Rpc) a n d N u c l e u s m e s e n c e p h a l i c u s , p a r s p r o f u n d u s ( M P v ) . A l s o , a s i n t h e c a t ( J o n e s e t al., 1 9 8 6 ) , C n v i t s e l f e n c o m p a s s e s ChAT c o n t a i n i n g n e u r o n s . We c a n s p e c u l a t e t h a t T T D , R g c a n d R p c a r e t h e mos t l i k e l y c a n d i d a t e s t o h a v e t h i s c h o l i n e r g i c e f f e c t on C n v n e u r o n s , a s e l e c t r i c a l s t i m u l a t i o n o f t h e s e r e g i o n s e l i c i t s l o c o m o t i o n . H o w e v e r , t h e s e p a t h w a y s a r e s t i l l o n l y p o t e n t i a l l y c h o l i n e r g i c . F u r t h e r s t u d y i s n e e d e d t o r e s o l v e t h i s p r o b l e m . I n t h e r a t , PPN p r o j e c t i o n s t o VRN h a v e b e e n shown t o c o n t a i n a c e t y l c h o l i n e (Rye e t al., 1987 , 1 9 8 8 ) . Some c o n t r o v e r s y e x i s t s r e g a r d i n g t h e s e c o n n e c t i o n s , a s G o l d s m i t h a n d V a n D e r K o o y ( 1 9 8 8 ) , u s i n g N A D P H - d i a p h o r a s e h i s t o c h e m i s t r y , f o u n d no e v i d e n c e o f d e s c e n d i n g c h o l i n e r g i c p r o j e c t i o n s f r o m PPN i n t h e r a t . H o w e v e r , Rye e t al. (1988) h a v e r e c e n t l y r e p o r t e d a s i g n i f i c a n t c h o l i n e r g i c p r o j e c t i o n f r o m PPN t o t h e r e t i c u l a r f o r m a t i o n i n t h e r a t . J o n e s e t al. ( 1 9 8 6 ) , a l s o i n t h e r a t , u s e d r e t r o g r a d e t r a n s p o r t o f [ 3 H ] C h o l i n e t o f i n d e v i d e n c e f o r t h e p r e s e n c e o f c h o l i n e r g i c n e u r o n s d e s c e n d i n g f r o m t h e PPN t o h i g h c e r v i c a l l e v e l s . I n a d d i t i o n , t h e y d e m o n s t r a t e d r e t r o g r a d e l y l a b e l l e d c e l l s i n t h e m e d i a l g i g a n t o c e l l u l a r r e t i c u l a r f o r m a t i o n o f t h e m e d u l l a w h i c h d e s c e n d t o c e r v i c a l s p i n a l l e v e l s , p o s s i b l y i m p l i c a t i n g t h e s e d e s c e n d i n g c h o l i n e r g i c n e u r o n s i n t h e c o n t r o l o f l o c o m o t i o n . C e l l s a r i s i n g f r o m t h e VRN a r e known t o g i v e r i s e t o r e t i c u l o s p i n a l n e u r o n s i m p o r t a n t t o l o c o m o t o r c o n t r o l ( S t e e v e s e t a l . , 1 9 8 7 ) . A s i n t h e b i r d , ChAT i m m u n o r e a c t i v i t y 120 has been found i n the tegmental f i e l d o f the cat (medial to l a t e r a l tegmental f i e l d and d o r s a l t o i n f e r i o r o l i v e ) (Vincent and Reiner, 1987), s u p p o r t i n g the p o s s i b i l i t y t h a t neurons i n t r i n s i c t o t h i s r e g i o n may p r o v i d e i n t e r n e u r o n a l input (or be output neurons) t o descending . r e t i c u l o s p i n a l neurons. The above s t u d i e s i n d i c a t e t h a t i n mammals, as i n b i r d s , the d e f i n i t i v e locomotor r e l a t e d c h o l i n e r g i c i n p u t s to the medullary r e t i c u l a r formation r e s p o n s i b l e f o r the observed r e s u l t s remain to be e l u c i d a t e d . I t appears l i k e l y , however, t h a t r e t i c u l o s p i n a l neurons o r i g i n a t i n g from the avian Cnv, l i k e the mammalian VRN, are at l e a s t p a r t i a l l y under c h o l i n e r g i c m u s c a r i n i c c o n t r o l . Pontine R e t i c u l a r Formation (RP) E l e c t r i c a l s t i m u l a t i o n of the v e n t r a l p ontine r e t i c u l a r formation has been shown to e l i c i t locomotion i n b i r d s (Sholomenko and Steeves, 1987). Prev i o u s i n v e s t i g a t o r s have u t i l i z e d both chemical and e l e c t r i c a l s t i m u l a t i o n of the pontine r e t i c u l a r formation to evoke a v a r i e t y of motor responses from t h i s r e g i o n . I n t e r e s t i n g l y , Katayama et al. (1984) found t h a t i n j e c t i o n of c a r b a c h o l i n t o the d o r s a l aspects of the pontine r e t i c u l a r formation i n the i n t a c t cat produced p o s t u r a l a t o n i a s i m i l a r t o t h a t seen w i t h d o r s a l m i d l i n e pontine e l e c t r i c a l s t i m u l a t i o n i n the mesencephalic cat (Mori et a l . , 1978). Mori et al. (1978) d e s c r i b e d two regions i n the pontine tegmentum which gave very d i f f e r e n t r e s u l t s when e l e c t r i c a l l y s t i m u l a t e d . The f i r s t r e g i o n , which l a y d o r s a l l y w i t h i n the c e n t r a l s u p e r i o r p o n t i n e nucleus, produces a decrease i n hindlimb extensor muscle tone when s t i m u l a t e d . The second more v e n t r a l r e g i o n , l y i n g w i t h i n the boundaries of the nucleus raphe magnus, produces an i n c r e a s e i n extensor tone when s t i m u l a t e d . In b i r d s , r a p h e - s p i n a l connections are known t o impinge on p r e g a n g l i o n i c sympathetic neurons i n the s p i n a l c o r d and have not been i m p l i c a t e d i n motor c o n t r o l (Cabot et al., 1982). L i k e Mori's r e s u l t s i n c a t s , i n b i r d s , v e n t r a l pontine e l e c t r i c a l s t i m u l a t i o n r e s u l t e d i n locomotion. In b i r d s , the pontine r e t i c u l a r formation i s known to r e c e i v e a f f e r e n t input from the n u c l e i c a u d a l i s and i n t e r p o l a r i s o f TTD (Arends and Dubbeldam, 1982; Webster, p e r s o n a l communication) and from the tectum (Hunt and Kunzle, 197 6). I t p r o j e c t s e f f e r e n t s t o caudal TTD, motor n u c l e i V and VII, nucleus p a r a b r a c h i a l i s (Arends and Dubbeldam, 1982) and to lower brainstem r e t i c u l a r formation n u c l e i (Webster, p e r s o n a l communication). RP a l s o g i v e s r i s e t o sparse descending p r o j e c t i o n s t o both c e r v i c a l and lumbar s p i n a l c o r d which t r a v e l i n the v e n t r a l f u n i c u l u s of the s p i n a l c o r d (Cabot et al., 1982; Webster and Steeves, 1988), a r e g i o n which l e s i o n s t u d i e s show supports locomotion (Sholomenko and Steeves, 1987), Mammalian neuroanatomical s t u d i e s demonstrate t h a t RP r e c e i v e s p r o j e c t i o n s from the r e g i o n of the v e n t r a l tegmental area, caudal cuneiform (Steeves and Jordan, 1984) and PPN ( G a r c i a - R i l l et al., 1983) i n the cat and r a t ( G a r c i a - R i l l et al.,1986). I t a l s o r e c e i v e s a f f e r e n t input from the l a t e r o d o r s a l tegmental nucleus (TLD) i n the r a t (Brudzynski et al., 1988). I n j e c t i o n of c a r b a c h o l i n t o RP was i n e f f e c t i v e at e l i c i t i n g locomotion or a f f e c t i n g the stimulus t h r e s h o l d necessary to evoke locomotion i n the one p r e p a r a t i o n t e s t e d . F u r t h e r study i s r e q u i r e d t o determine whether c h o l i n e r g i c a g o n i s t s and a n t a g o n i s t s a f f e c t locomotor p a t t e r n s i n t h i s r e g i o n . However, i n j e c t i o n of other n e u r o t r a n s m i t t e r a g o n i s t s and a n t a g o n i s t s were e f f e c t i v e at producing locomotor changes i n t h i s r e g i o n and w i l l be d i s c u s s e d i n subsequent chapters (Chapters 4&5). Mesencephalic R e t i c u l a r Formation (MRF) E l e c t r i c a l s t i m u l a t i o n of the mesencephalic r e t i c u l a r formation, i n c l u d i n g s i t e s i n the nucleus i n t e r c o l l i c u l a r i s (ICo), l a t e r a l mesencephalic r e t i c u l a r formation (FRL) and medial mesencephalic r e t i c u l a r formation (FRM), e l i c i t e d locomotion i n the decerebrate b i r d (Sholomenko and Steeves, 1988). ICo r e c e i v e s a f f e r e n t input from the s p i n a l cord, d o r s a l column n u c l e i , and tectum (Hunt and Kunzle, 197 6; Webster, p e r s o n a l communication). I t sends e f f e r e n t p r o j e c t i o n s t o the pontine and medullary r e t i c u l a r formation, as w e l l as t o the h i g h c e r v i c a l s p i n a l c o r d (Reiner and Karten, 1982; Webster, p e r s o n a l communication). FRL and FRM appear to have s i m i l a r a f f e r e n t s and e f f e r e n t s t o those of ICo. Cabot et al. (1982) compared these connections to those of the mammalian cuneiform nucleus (see Chapter 2), a p o r t i o n of which, i n mammals, i s b e l i e v e d t o be the l a t e r a l MLR (Steeves and Jordan, 1984; Shik et al., 1966). In one of two b i r d s , i n j e c t i o n of c a r b a c h o l i n t o the MRF reduced the e l e c t r i c a l s t i m u l a t i o n t h r e s h o l d f o r locomotion. T h i s e q u i v o c a l r e s u l t suggests t h a t d e t a i l e d study i s r e q u i r e d t o determine any p o s s i b l e r o l e f o r a c e t y l c h o l i n e i n t h i s locomotor r e g i o n . As i s d i s c u s s e d below, such a study i n the b i r d may p r o v i d e important i n f o r m a t i o n concerning the c o n t r o l of locomotion by the c u r r e n t l y i d e n t i f i e d mammalian mesencephalic locomotor r e g i o n s . N- [ 3H]methylscopolamine b i n d i n g s t u d i e s i n the b i r d show the presence o f mu s c a r i n i c c h o l i n e r g i c r e c e p t o r b i n d i n g i n ICo but l i t t l e b i n d i n g i n e i t h e r FRL or FRM ( D i e t l et a l . , 1988). ChAT immunohistochemical s t u d i e s demonstrate sparse or no l a b e l l i n g o f p o t e n t i a l c h o l i n e r g i c c e l l s i n these r e g i o n s (Taccogna and Steeves, unpublished o b s e r v a t i o n s ) , thus negating any homology between these midbrain s t r u c t u r e s and the ACh-containing c e l l bodies o f the mammalian PPN/mMLR d e f i n e d by G a r c i a - R i l l and co-workers ( G a r c i a - R i l l et a l . , 1983a). I n t e r e s t i n g l y , G a r c i a - R i l l et a l . (1985) found t h a t i n j e c t i o n o f a c e t y l c h o l i n e i n t o the PPN/MLR was i n e f f e c t i v e at e l i c i t i n g locomotion i n the decerebrate cat and Brudzynski et al. (1988) found t h a t c a r b a c h o l i n j e c t i o n i n t o PPN decreased locomotion i n the f r e e l y moving i n t a c t r a t . However, ca r b a c h o l i n j e c t i o n s p l a c e d between the PPN, cuneiform nucleus and p e r i a q u e d u c t a l gray, p o s s i b l y w i t h i n the r e g i o n of the r a t e q u i v a l e n t of the cat 1MLR, i n c r e a s e d locomotion i n the r a t (Brudzynski et a l . , 1988). The above r e s u l t s i n cat and r a t appear t o l e n d credence t o the e x i s t e n c e o f two MLRs, each p o s s e s s i n g d i f f e r e n t c h a r a c t e r i s t i c s t h a t may be more f u l l y d e f i n e d by neurochemical i n j e c t i o n s t u d i e s . The pha r m a c o l o g i c a l p r o p e r t i e s o f neurons i n corresponding anatomical s t r u c t u r e s i n the midbrain o f b i r d s and mammals i n d i c a t e t h a t d i f f e r e n t p o p u l a t i o n s o f neurons are under somewhat d i f f e r e n t c o n t r o l mechanisms, but are both a p p a r e n t l y a c t i v e i n the c o n t r o l of locomotor behaviours. We suggest t h a t r e g i o n s i n the av i a n midbrain, s p e c i f i c a l l y i n the regions of the nucleus i n t e r c o l l i c u l a r i s and mMRF, are e q u i v a l e n t t o those proposed as the l a t e r a l and medial MLRs of mammals. F u r t h e r s t u d i e s are r e q u i r e d , however, b e f o r e any homology between the mammalian and avian MLRs can be completed. Medial L o n g i t u d i n a l F a s c i c u l u s (MLF) Locomotion was e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n o f the pontine and r o s t r a l medullary medial l o n g i t u d i n a l f a s c i c u l u s (MLF). Carbachol i n j e c t i o n i n t o the MLF e l i c i t e d s i m i l a r locomotor p a t t e r n s . The MLF i s a f i b r e t r a c t which c a r r i e s ascending p r o j e c t i o n s from a l l v e s t i b u l a r n u c l e i t o v i s u a l motor n u c l e i and p r o j e c t i o n s t o the i n t e r s t i t i a l nucleus of C a j a l (InC) (Carpenter and S u t i n , 1983). I t c a r r i e s f i b r e s from the 1) pontine r e t i c u l a r formation t o the s p i n a l c o r d (Carpenter and S u t i n , 1983), 2) InC, which forms the i n t e r s t i t i o - s p i n a l t r a c t (Carpenter and S u t i n , 1983), t o the Probst's t r a c t (Skinner et al., 1984), 3) r o s t r a l brainstem (e.g. InC) n u c l e i t o the i n f e r i o r o l i v a r y nucleus (Carpenter and S u t i n , 1983; Skinner et a l . , 1984)), 4) v e s t i b u l o s p i n a l t r a c t which sends c o l l a t e r a l s t o the medullary r e t i c u l a r formation (Carpenter and S u t i n , 1983) and 5) i n t e r n u c l e a r v i s u a l motor n u c l e i ( I I I , IV and VI) (Carpenter and S u t i n , 1983). In a d d i t i o n , the MLF c a r r i e s f i b r e s which impinge on the pedunculopontine nucleus i n the r a t (source u n s p e c i f i e d ) (Rye et al., 1987). While i t was not s u r p r i s i n g t h a t e l e c t r i c a l s t i m u l a t i o n o f t h i s t r a c t e l i c i t e d locomotor responses, as many of i t s f i b r e t r a c t s are r e l a t e d t o locomotor f u n c t i o n (e.g. pontine r e t i c u l a r formation n u c l e i , pedunculopontine n u c l e u s ) , i t was s u r p r i s i n g t h a t neurochemical s t i m u l a t i o n with the c h o l i n e r g i c a g o n i s t c a r b a c h o l e l i c i t e d long l a s t i n g locomotor behaviours. To our knowledge, no c h o l i n e r g i c r e c e p t o r s have been r e p o r t e d i n the MLF, although s e r o t o n i n - c o n t a i n i n g neurons which s t a i n p o s i t i v e l y f o r a c e t y l c h o l i n e s t e r a s e have been l o c a l i z e d i n t h i s r e g i o n i n the chi c k e n and duck (Dube and Parent, 1981; Taccogna et a l . , i n pr e p a r a t i o n ) (see Chapter 2 ) . The h o d o l o g i c a l f e a t u r e s of these neurons and t h e i r p o s s i b l e r e l a t i o n t o locomotor f u n c t i o n , however, remain t o be determined. PHARMACOLOGICAL CONSIDERATIONS Neurochemical i n j e c t i o n i n t o some of the above locomotor r e g i o n s demonstrated t h a t these r e g i o n s c o n t a i n n e u r o t r a n s m i t t e r r e c e p t o r s which can be a f f e c t e d by c h o l i n e r g i c a g o n i s t s and a n t a g o n i s t s . The mode of a c t i o n by which the neurochemical a f f e c t s the r e c e p t o r i s dependent upon the agent u t i l i z e d . A g o n i s t s are b e l i e v e d t o induce conformation changes i n r e c e p t o r p r o t e i n s . T h i s can be achieved through d i r e c t c o u p l i n g t o the ionophore or i n d i r e c t l y v i a a c t i v a t i o n of a second messenger system (muscarinic r e c e p t o r s - see Buckley et al., 1988). The ag o n i s t presumably produces an a l t e r a t i o n i n the i o n i c p e r m e a b i l i t y o f the membrane, l e a d i n g t o a neuronal response which e l i c i t s downstream responses (e.g. locomotion). A n t a g o n i s t s , on the other hand, are b e l i e v e d t o form an i n e r t complex wi t h the r e c e p t o r p r o t e i n , which, i n the case of a com p e t i t i v e a n t a g o n i s t , can be d i s p l a c e d by an a p p r o p r i a t e a g o n i s t at s p e c i f i c c o n c e n t r a t i o n s ( f o r review of co m p e t i t i v e b i n d i n g s t u d i e s , see Watson et al., 1984). In my study, the r e c e p t o r s , when a c t i v a t e d or i n a c t i v a t e d by the i n j e c t e d agent, presumably a f f e c t a s u f f i c i e n t number of neurons t o e l i c i t or bl o c k decerebrate a v i a n locomotion. The a c t i v a t i o n (or blockade) o f neurons necessary t o evoke (or block) locomotion i s dependent on the a b i l i t y o f the neurochemical to spread through the t i s s u e ( a c t i v a t i n g or i n a c t i v a t i n g r e c e p t o r s ) u n t i l enough neuronal r e c e p t o r s (and t h e r e f o r e neurons) have been r e c r u i t e d so t h a t the outward b e h a v i o u r a l s i g n s o f locomotion can be recorded with EMGs or ENGs. Thus, neurochemicals which are not r e a d i l y m e tabolized [e.g. a c e t y l c h o l i n e s t e r a s e which does not hydr o l y z e c a r b a c h o l (Taylor, 1985)] w i l l be more e f f e c t i v e s t i m u l a t o r s / i n h i b i t o r s than r e a d i l y m e tabolized neurochemicals such as a c e t y l c h o l i n e (Taylor, 1985). A l s o , the a b i l i t y of one neurochemical (e.g. atropine) t o counter a c t the e f f e c t s of another neurochemical (e.g. carbachol) i s dependent upon t h e i r r e l a t i v e b i n d i n g a f f i n i t i e s [atropine»>carbachol (Furchgott and Cherry, 1984)] and the c o n c e n t r a t i o n of each agent, i f the p a i r are a c t i n g at the same s i t e . In the case of the ca r b a c h o l versus a t r o p i n e mainly used i n t h i s study, a t r o p i n e has been shown t o d i s p l a c e r e c e p t o r bound 127 c a r b a c h o l (and p i l o c a r p i n e ) at mu s c a r i n i c r e c e p t o r s (Taylor, 1985). Recent f i n d i n g s ( C h r i s t i e and North, 1988) suggest t h a t the c o m p e t i t i v e antagonism o f ca r b a c h o l by a t r o p i n e at mu s c a r i n i c r e c e p t o r s i s not d i r e c t l y on the ionophore, but on mu s c a r i n i c r e c e p t o r s l o c a t e d d i s t a n t t o the ionophore ( C h r i s t i e and North, 1988). While i t i s not yet p o s s i b l e t o determine the exact nature o f the l o c o m o t i o n - a s s o c i a t e d c h o l i n e r g i c r e c e p t o r s b e i n g s t i m u l a t e d by the neurochemical i n j e c t i o n technique. I t appears l i k e l y t h a t m u s c a r i n i c r e c e p t o r s can be i m p l i c a t e d i n t h i s c o n t r o l . The p h a r m a c o l o g i c a l a c t i v a t i o n o f locomotion i s not, however, without l i m i t a t i o n s . F i r s t , i t i s d i f f i c u l t t o t i t r a t e the i n j e c t i o n dose t o produce t h r e s h o l d a c t i v a t i o n . Second, the locomotion e l i c i t e d by neurochemical s t i m u l a t i o n i s not as r e a d i l y m o d i f i e d as i s e l e c t r i c a l s t i m u l a t i o n - i n d u c e d locomotion. T h i r d , l i k e e l e c t r i c a l s t i m u l a t i o n , the l a c k of a response t o any giv e n neurochemical may r e f l e c t the e f f i c a c y of the p r e p a r a t i o n at the time of i n j e c t i o n . Fourth, the spread o f neurochemical, and t h e r e f o r e , e f f e c t i v e d i s t r i b u t i o n , i s d i f f i c u l t t o c o n t r o l (see below). F i f t h , the c o n c e n t r a t i o n s of i n t r a c e r e b r a l l y i n j e c t e d neurochemicals are probably not p h y s i o l o g i c a l , although i t may be argued t h a t i n order t o r e c r u i t a s u f f i c i e n t number of neurons t o e l i c i t locomotion, i t i s necessary t o use highe r than p h y s i o l o g i c a l c o n c e n t r a t i o n at the i n j e c t i o n p o i n t . S i x t h , the i n t r a c e r e b r a l i n f u s i o n technique p r o v i d e s no i n f o r m a t i o n concerning the pre - or p o s t - s y n a p t i c l o c a t i o n o f the r e c e p t o r s . L a s t l y , the technique cannot account f o r the p o s s i b i l i t y t h a t the neurochemical i s a c t i v a t i n g 128 r e c e p t o r s present on neurons which normally have no a f f e r e n t i n p u t s a s s o c i a t e d w i t h t h a t t r a n s m i t t e r and which may not normally be i n v o l v e d i n the locomotor process (e.g. a c h o l i n e r g i c r e c e p t o r may be present on a c e l l which has no c h o l i n e r g i c i n p u t , a l s o see Stone and Burton, 1988). The l i m i t a t i o n s of t h i s technique do not, however, p r e c l u d e i t s u s e f u l n e s s i n demonstrating t h a t the e l e c t r o p h y s i o l o g i c a l l y - d e f i n e d locomotor regions a l s o c o n t a i n neuronal r e c e p t o r s (presumably d e n d r i t i c , somal or on te r m i n a l s ) which may u n d e r l i e the p h y s i o l o g i c a l l y evoked locomotor responses. F u r t h e r , the technique p r o v i d e s i n f o r m a t i o n concerning p o s s i b l e n e u r o t r a n s m i t t e r c o n t r o l l i n g i n p u t s t o the locomotor r e g i o n s and has p r e d i c t i v e value i n determining the nature of n e u r a l pathways i n v o l v e d i n locomotor c o n t r o l . The i n t r a c e r e b r a l neurochemical i n f u s i o n technique a l s o has p r e d i c t i v e v a l u e f o r the use of other techniques. For example, p r o s p e c t i v e n e u r o t r a n s m i t t e r s i d e n t i f i e d with neurochemical i n f u s i o n c o u l d be examined u s i n g in-vivo m i c r o d i a l y s i s (Sabol and Freed, 1988; Becker et a l . , 1988; Ajima and Kato, 1988; P h i l l i p s et a l . , 1988). The d i a l y s i s technique may be used t o q u a n t i f y the r e l e a s e of n e u r o t r a n s m i t t e r s d u r i n g evoked locomotion and thereby e l u c i d a t e which n e u r o t r a n s m i t t e r s have a r o l e i n brainstem locomotor c o n t r o l . NEUROCHEMICAL SPREAD AND TIME COURSE OF ACTIVATION/INACTIVATION Although t h i s study and those i n the f o l l o w i n g chapters (Chapters 4 & 5) d i d not i n c l u d e the i n j e c t i o n of dye marker 129 chemicals i n t o the i n j e c t i o n s i t e s to mark the degree of neurochemical spread, i t i s l i k e l y t h a t the s i z e of our s t i m u l a t i o n s i t e s were smaller than those shown i n the cat ( G a r c i a - R i l l et al., 1985, G a r c i a - R i l l and Skinner, 1987). The slow i n j e c t i o n r a t e used i n the present study (0.2/il/min) , as compared to tha t u t i l i z e d by other groups(e.g. G a r c i a - R i l l et al., 1985 - 1/il/min; G a r c i a - R i l l and Skinner, 1987a - 1/jl/min; Noga et al., 1988 - l j i l / m i n ; L a i and S i e g e l , 1988 - 0.5nl/min) and the small volumes (maximum l.Ofil) as compared t o others (e.g. G a r c i a - R i l l et al., 1985 - 1.5-3.0jxl; Noga et a l . , 1988 -5/il (see Appendix I)) i n j e c t e d would have served to reduce the neurochemical spread through the t i s s u e . A l s o , a f f e c t e d areas were probably w i t h i n <0.5mm ra d i u s , f o r i t was noted i n s e v e r a l t r i a l s of the present study that i n f u s i o n of a neurochemical 0.5mm from an e f f e c t i v e s i t e d i d not e l i c i t locomotion. Studies u t i l i z i n g autoradiographic t r a c i n g of dispersed s i l v e r g rains f o l l o w i n g i n j e c t i o n of r a d i o a c t i v e agonists and antagonists i n t o i n j e c t i o n s i t e s might serve to confirm the degree of spread a f t e r i n t r a c e r e b r a l i n f u s i o n of neurochemicals. With regard t o the late n c y f o r a c t i v i t y of the various neurochemicals u t i l i z e d , there appears t o be a general t r e n d t h a t small molecular weight neurochemicals (e.g. carbachol (molecular weight (MW) = 182.6)(this chapter), GABA (MW = 103.1) (Chapter 4) and NMDA (MW = 147.1) (Chapter 5)) acted more q u i c k l y than those w i t h l a r g e molecular weight (e.g. atropine (MW = 676.8)(this chapter), p i c r o t o x i n (602.6)(Chapter 4 ) ) . Whether t h i s t r e n d r e s u l t s from a d i f f e r e n t i a l d i f f u s i o n rate through the t i s s u e s remains to be determined. A l s o , the time 130 course (see Table 1) over which the a g o n i s t s and a n t a g o n i s t s maintained t h e i r locomotor e f f e c t s appeared t o be s i m i l a r t o those r e p o r t e d by other i n v e s t i g a t o r s (see Appendix I ) . For example, a t r o p i n e i n j e c t i o n i n t o the cat v e n t r a l r e t i c u l a r nucleus (NRV - see Appendix I) bl o c k e d e l e c t r i c a l s t i m u l a t i o n - i n d u c e d locomotion f o r 1-2 hours ( G a r c i a - R i l l and Skinner, 1987a), while i n t h i s study, a t r o p i n e i n j e c t i o n i n t o an e q u i v a l e n t a v i a n brainstem r e g i o n , the v e n t r a l r e t i c u l a r formation (Cnv), b l o c k e d e l e c t r i c a l l y s t i m u l a t e d locomotion f o r at l e a s t the l e n g t h of the experimental p e r i o d (21-40min). Thus, a t r o p i n e appears t o produce long l a s t i n g e f f e c t s i n both b i r d and cat, although i t i s d i f f i c u l t t o compare these e f f e c t s due to d i f f e r e n c e s i n both c o n c e n t r a t i o n and i n j e c t i o n volume. COMPARATIVE CONSIDERATIONS The neuroanatomical and n e u r o p h y s i o l o g i c a l comparison of b i r d s w i t h mammals i s somewhat hi n d e r e d by the d i f f e r e n t neuroanatomical terminology used f o r the two groups. However, i t appears t h a t n e u r a l c i r c u i t r y i n the b i r d midbrain and h i n d b r a i n i s comparable t o t h a t of mammals (Reiner et a l . , 1984), only d i v e r g i n g at the l e v e l of the output of the b a s a l g a n g l i a (Reiner et a l . , 1984). A l s o , the h i n d b r a i n descending pathways i n the b i r d appear t o be fundamentally comparable t o those of mammals (Cabot et a l . , 1982; Webster and Steeves, 1988). My r e s u l t s , t h a t demonstrate both e l e c t r i c a l and neurochemical c h o l i n e r g i c s t i m u l a t i o n (carbachol, p i l o c a r p i n e ) of r e s t r i c t e d b rainstem r e g i o n s produce a v i a n locomotion, are s i m i l a r to those 131 brainstem r e g i o n s i n mammals. These s i m i l a r i t i e s t h e r e f o r e underscore the suggestion t h a t the neuroanatomical s u b s t r a t e u n d e r l y i n g the c o n t r o l of locomotion i s h i g h l y conserved f o r these groups of v e r t e b r a t e s . Furthermore, our r e s u l t s demonstrate t h a t s e v e r a l locomotion-promoting r e g i o n s i n the a v i a n h i n d b r a i n are under c h o l i n e r g i c m u s c a r i n i c c o n t r o l , although the p r e c i s e neuroanatomical s u b s t r a t e f o r t h i s c o n t r o l remains t o be e l u c i d a t e d . 132 CHAPTER 4 CHARACTERIZATION OF AVIAN MID- AND HINDBRAIN SITES THAT PRODUCE LOCOMOTION WITH INTRACEREBRAL INFUSION OF NEUROTRANSMITTER AGONISTS AND ANTAGONISTS (II) : r-AMINOBUTYRIC ACID (GABA) 1 3 3 INTRODUCTION In v e r t e b r a t e s , the c o n t r o l of locomotor behaviour i s dependent upon c e n t r a l nervous system (CNS) c i r c u i t r y at a l l l e v e l s of the neuraxis. Attempts t o c h a r a c t e r i z e t h i s c i r c u i t r y p h y s i o l o g i c a l l y have u t i l i z e d a v a r i e t y of techniques i n c l u d i n g a b l a t i o n , s e l e c t i v e l e s i o n s , e x t r a - and i n t r a c e l l u l a r r ecording, e l e c t r i c a l s t i m u l a t i o n (for review see G r i l l n e r , 1975; Orlovsky and Shik, 1976) and more r e c e n t l y , neurochemical s t i m u l a t i o n ( G a r c i a - R i l l et a l . , 1985; Noga et al., 1988; Sholomenko and Steeves, 1987a). Neuroanatomical s t u d i e s have provided valuable inform a t i o n regarding the o r i g i n , course and terminations of CNS n u c l e i thought t o be in v o l v e d i n locomotion (Kuypers, 1981; Holstege and Kuypers, 1987; Steeves and Jordan, 1984; G a r c i a - R i l l et al., 1983a). Recent s t u d i e s have aided i n the i d e n t i f i c a t i o n and l o c a l i z a t i o n of neurotransmitters and t h e i r receptors which may be i n v o l v e d i n the locomotor process (e.g. Mugnaini and O e r t e l , 1985). The present s t a t e of knowledge, t h e r e f o r e , allows f o r the i n i t i a t i o n of an i n t e g r a t e d approach to the study of locomotor c o n t r o l . The previous chapter (Chapter 3) takes t h i s approach i n the d e s c r i p t i o n of the locomotor e f f e c t s of c h o l i n e r g i c agonist and antagonist i n j e c t i o n s i n t o s i t e s i n the avian mid- and hi n d b r a i n . R esults from t h a t study demonstrated t h a t c h o l i n e r g i c muscarinic agonist s t i m u l a t i o n of a v a r i e t y of locomotor s i t e s i n the h i n d b r a i n of the decerebrate b i r d (Canada goose or Pekin duck) evoked locomotor behaviours. Furthermore, attempts were made to e l u c i d a t e the c h o l i n e r g i c substrate i n v o l v e d i n 134 locomotor c o n t r o l and g e n e r a l i z e these f i n d i n g s t o other v e r t e b r a t e s . T h i s chapter d e s c r i b e s the locomotor e f f e c t s o f i n j e c t i o n o f y-aminobutyric a c i d (GABA), i t s a g o n i s t s and an t a g o n i s t s i n t o these same s i t e s . The study was designed t o survey whether GABAergic neurochemicals were e f f e c t i v e i n these r e g i o n s as a pre l u d e t o more d e t a i l e d p h a r m a c o l o g i c a l c h a r a c t e r i z a t i o n . Since the d i s c o v e r y o f GABA by two independent r e s e a r c h groups i n 1950 (Roberts and F r a n k e l , 1950; Awapara et al., 1950), GABA has been w e l l c h a r a c t e r i z e d as a r a t h e r u b i q u i t o u s i n h i b i t o r y n e u r o t r a n s m i t t e r which a c t s both p r e - and p o s t s y n a p t i c a l l y (Olsen, 1981; Bloom, 1985). Recent s t u d i e s i n the c at ( G a r c i a - R i l l et al., 1985, 1987; E l d r i d g e et al., 1985; Noga et al., 1988) and r a t (Brudzynski et al., 1988) have demonstrated t h a t locomotion can be b l o c k e d by GABA i n f u s i o n i n t o s e l e c t e d locomotor r e g i o n s o f the ne u r a x i s , while i n j e c t i o n o f GABA a n t a g o n i s t s has been shown t o induce or i n c r e a s e locomotor behaviour i n these p r e p a r a t i o n s . In the b i r d , GABA i n j e c t i o n i n t o s i t e s from which locomotion c o u l d be e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n was e f f e c t i v e at b l o c k i n g or i n c r e a s i n g the t h r e s h o l d f o r e l e c t r i c a l l y s t i m u l a t e d locomotion i n the decerebrate p r e p a r a t i o n . In a d d i t i o n , GABA a n t a g o n i s t s e l i c i t e d locomotion when i n j e c t e d i n t o some of these same s i t e s . Because the neurochemicals i n f u s e d are b e l i e v e d t o act on n e u r o t r a n s m i t t e r r e c e p t o r s (Goodchild et al., 1982), i t i s l i k e l y t h a t these locomotor s i t e s c o n t a i n neuronal p o p u l a t i o n s or t e r m i n a l s which are i n v o l v e d i n the locomotor p r o c e s s . Our r e s u l t s i n the b i r d 135 are s i m i l a r t o those d e s c r i b e d f o r mammals ( G a r c i a - R i l l et a l . , 1985, 1987; Noga et al., 1988) and t h e r e f o r e u n d e r l i e our c o n t e n t i o n t h a t a v i a n CNS motor c i r c u i t r y , at l e a s t at mid- and h i n d b r a i n l e v e l s , i s homologous to t h a t of mammalian s p e c i e s . 136 MATERIALS AND METHODS The m a t e r i a l s and methods, i n c l u d i n g the d e c e r e b r a t i o n procedure, e l e c t r i c a l s t i m u l a t i o n methodology, neurochemical i n j e c t i o n parameters and h i s t o l o g i c a l procedures f o r the l o c a l i z a t i o n o f s t i m u l a t i o n s i t e s have been p r e v i o u s l y d e s c r i b e d i n Chapters 2 & 3. 137 RESULTS GABA A g o n i s t s and A n t a g o n i s t s I n t r o d u c t i o n of GABA, or GABAergic a g o n i s t s and a n t a g o n i s t s i n t o e l e c t r i c a l l y s t i m u l a t e d locomotion promoting s i t e s i n the h i n d - and midbrain were e f f e c t i v e at b l o c k i n g and e l i c i t i n g a v a r i e t y of locomotor p a t t e r n s i n decerebrate b i r d s . The neurochemicals i n j e c t e d i n c l u d e d : GABA; p i c r o t o x i n , a non-competitive GABA a n t a g o n i s t ; muscimol, a GABAA r e c e p t o r a g o n i s t and; b i c u c u l l i n e , a GABAA r e c e p t o r c o m p e t i t i v e a n t a g o n i s t . Neurochemical i n j e c t i o n s i t e s , the lowest e f f e c t i v e c o n c e n t r a t i o n and time course o f a c t i v i t y are l i s t e d i n Table 2 and d e s c r i b e d i n the t e x t . A composite diagram of the i n j e c t i o n s i t e s i s shown i n F i g u r e 15. Pontobulbar Locomotor S t r i p (PLS) F i v e of e i g h t animals which r e c e i v e d p i c r o t o x i n i n j e c t i o n s (3-20mM/l. Ojil) i n t o PLS produced long l a s t i n g locomotor p a t t e r n s . The mean l a t e n c y t o onset f o r locomotion f o l l o w i n g the i n i t i a l i n j e c t i o n was 13.5 minutes (range 4-22 minutes) with the p e r i o d s o f a c t i v i t y l a s t i n g between 35 and 60 minutes (at which p o i n t the experiment was t e r m i n a t e d ) . The site-dependent modes of locomotion i n c l u d e d walking alone, running and f l y i n g , and f l y i n g alone. In t h r e e b i r d s , the i n i t i a l locomotor r e a c t i o n t o p i c r o t o x i n i n j e c t i o n i n v o l v e d s m a l l u n i l a t e r a l l e g ex c u r s i o n s ( s i m i l a r t o the b i l a t e r a l s t e p p i n g e l i c i t e d at t h r e s h o l d 138 T A B L E 2 GABAergic agonists and antagonists Animal Site Chemical pH Concentration* Lowest Volume Rale Time Course (min) Injected Effective Latency Period Concentration TTD GABA 7.2-7.4 0.3-O.SM 0.3M I.Oul 0.2ul/min 1-5 2-21 Picrotoxin " 3-20mM 3mM 4-22 35-60 Bicuculline " 10mM 10mM " 15 >30 Muscimol " 6.5-25mM 6.5mM 6-10 30-70 Cnd Picrotoxin " SmM none •• none none Muscimol " 6.25mM none " none none Cnv GABA O.SM 0.5M •• •• <1 0-14 Picrotoxin " 6-20mM SmM " 4-22 30 Muscimol " 6.25mM 6.2SmM " M >B >30 RP GABA O.SM 0.6M » •• «1 12-21 Picrotoxin " 3-SmM SmM •' 10 35 Muscimol 6.25mM none *' •• — — MRF GABA 0.6M 0.6M •• <1 2-12 Picrotoxin " 5-20mM SmM " " 11-13 >30 ABBREVIATIONS: Cnd — dorsal part, medullary central nucleus Cnv — ventral part, medullary central nucleus MRF — mesencephalic reticular formation RP — pontine reticular nucleus TTD — descending trigeminal tract and nucleus 139 F i g u r e 15. Composite diagram o f GABAergic a g o n i s t and ant a g o n i s t neurochemical i n j e c t i o n s i t e s . The diagram o f c o r o n a l s e c t i o n s through v a r i o u s l e v e l s o f the a v i a n n e u r a x i s [numbers i n upper l e f t corner o f each l e v e l , A=anterior, P=posterior ( i n mm)] i l l u s t r a t e s the locomotor e f f e c t s o f each neurochemical i n brainstem r e g i o n s from which locomotion was f i r s t e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n . Where GABA ( f i l l e d square) and muscimol ( f i l l e d c i r c l e ) are shown as f i l l e d , t h e i r e f f e c t s were t o block locomotion. Key A b b r e v i a t i o n s : BICUC - b i c u c u l l i n e , GABA - y-aminobutyric a c i d , MUSC - muscimol, PICRO - p i c r o t o x i n ; LOCO - locomotion (except f o r GABA and muscimol), TH - decreased e l e c t r i c a l t h r e s h o l d i n t e n s i t y f o r locomotion, tTH - i n c r e a s e d e l e c t r i c a l t h r e s h o l d i n t e n s i t y f o r locomotion, NR - no response. A b b r e v i a t i o n s : BC - brachium conjunctivum, CC - c e n t r a l c a n a l , Cnd - c e n t r a l nucleus medulla, d o r s a l p a r t , Cnv - c e n t r a l nucleus medulla, v e n t r a l p a r t , EW - Edinger Westphal nucleus, I I I - occulomotor nucleus, 10 - i n f e r i o r o l i v a r y nucleus, IP -nucleus i n t e r p e d u n c u l a r i s , LC - lo c u s c o e r u l e u s , MLd - l a t e r a l mesencephalic nucleus, d o r s a l d i v i s i o n , MLF - medial l o n g i t u d i n a l f a s c i c u l u s , MRF - medial mesencephalic r e t i c u l a r formation, MV - t r i g e m i n a l motor nucleus, N IV - t r o c h l e a r nerve, N XII - h y p o g l o s s a l nerve, OT - o p t i c tectum., R - raphe nucleus, RP - nucleus pontine r e t i c u l a r formation, Rpc - pontine p a r v o c e l l u l a r r e t i c u l a r nucleus, RPO - nucleus r e t i c u l a r i s p o n t i s o r a l i s , Ru - r e d nucleus, SSP - s u p r a s p i n a l nucleus, SO -s u p e r i o r o l i v a r y nucleus, ST - s u b t r i g e m i n a l nucleus, SV -t r i g e m i n a l sensory nucleus, TPc - s u b s t a n t i a n i g r a , TTD -t r i g e m i n a l descending t r a c t and nucleus, VeL - l a t e r a l v e s t i b u l a r nucleus, VeM - medial v e s t i b u l a r nucleus, VI -abducens nucleus, X - d o r s a l motor nucleus vagus. 140 LDCO JTH TTH IR PIBB A A A A m • B B • HBC • © e o BIQJC • • • O 141 e l e c t r i c a l s t i m u l a t i o n as seen i n F i g . 16A) f o l l o w e d by b i l a t e r a l t r e a d m i l l s t e p p i n g which i n c r e a s e d i n f o r c e as time prog r e s s e d . In these animals, weak and then s t r o n g e r b i l a t e r a l wing f l a p p i n g ( F i g . 16B) a l s o became i n c o r p o r a t e d i n t o the behaviour i n a manner s i m i l a r to the t r a n s i t i o n between walking and f l y i n g i n the normal animal. T h i s change i n locomotor p a t t e r n was s i m i l a r t o t h a t seen i n these animals as e l e c t r i c a l s t i m u l a t i o n i n t e n s i t y was g r a d u a l l y i n c r e a s e d from t h r e s h o l d . GABA a n t a g o n i s t i n j e c t i o n e l i c i t e d walking alone i n one b i r d ( F i g . 17A, p i c r o t o x i n ) , while i n a second, wing f l a p p i n g alone was observed w i t h b i l a t e r a l l e g e x t e n s i o n ( F i g . 17B, b i c u c u l l i n e ) , as has been seen d u r i n g e l e c t r i c a l s t i m u l a t i o n (13A). One animal, not i n c l u d e d i n the locomotion group, d i s p l a y e d only t o n i c e x t e n s i o n ( c o n v u l s a n t - l i k e ) of both wings and l e g s without any d i s c r e t e locomotor movements. T h i s behaviour resembled t h a t seen a f t e r a prolonged p e r i o d of p i c r o t o x i n - i n d u c e d locomotion i n s e v e r a l of the animals. In f o u r of the above p i c r o t o x i n - s t i m u l a t e d b i r d s , t r i g e m i n a l f i e l d s t i m u l a t i o n (TFS) ( e i t h e r a i r p u f f s or s t r o k i n g the s u r f a c e of the head near the eyes) appeared t o i n c r e a s e the i n t e n s i t y of the locomotor behaviour b e i n g performed. Thus, a f t e r p i c r o t o x i n i n j e c t i o n but b e f o r e the onset of locomotion, t h i s s t i m u l a t i o n , which p r i o r t o i n j e c t i o n was i n e f f e c t i v e at e l i c i t i n g locomotion, would e l i c i t s h o r t bouts of walking or f l y i n g behaviour. A l s o , a f t e r p i c r o t o x i n - i n d u c e d locomotion was e s t a b l i s h e d , TFS seemed to i n c r e a s e the f o r c e of walking or would cause a c o n v e r s i o n from walking to f l y i n g . S i m i l a r to the mechanical TFS s t i m u l a t i o n , l o u d n o i s e would o c c a s i o n a l l y e l i c i t F i g u r e 16. Electromyographic records (EMGs) showing locomotor a c t i v i t y e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n and p i c r o t o x i n i n j e c t i o n i n t o the pontobulbar locomotor s t r i p (PLS). A: A l t e r n a t i n g s t e p p i n g r e p r e s e n t e d by EMG p a t t e r n s from the r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles (major h i p f l e x o r muscle) e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n of the PLS. B: Stepping and wing f l a p p i n g EMGs e l i c i t e d by i n j e c t i o n of p i c r o t o x i n i n t o the same s i t e . The top two t r a c e s i l l u s t r a t e the in-phase a c t i v i t y o f the r i g h t (RPECT) and l e f t (LPECT) p e c t o r a l i s muscles, the major wing depressors used f o r f l i g h t . The bottom two t r a c e s are from the l e g ITC f l e x o r muscles as i n A. 143 A RITC M H i I M I I M | M t > * LITC 1 M M « M < M M M M M i 3 sec 144 F i g u r e 17. Electromyographic records (EMGs) showing locomotor a c t i v i t y e l i c i t e d by p i c r o t o x i n and b i c u c u l l i n e i n j e c t i o n i n t o the pontobulbar locomotor s t r i p (PLS). A: A l t e r n a t i n g s t e p p i n g r e p r e s e n t e d by EMG p a t t e r n s from r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles e l i c i t e d by p i c r o t o x i n i n j e c t i o n i n t o the PLS. B: Simultaneous (in-phase) wing f l a p p i n g EMGs from the right.(RPECT) and l e f t (LPECT) p e c t o r a l i s muscles e l i c i t e d by i n j e c t i o n of b i c u c u l l i n e i n t o the PLS of a d i f f e r e n t animal. 145 146 bouts of locomotor a c t i v i t y i n these animals. GABA (0.3-0.5M) i n f u s e d i n t o a l l animals t e s t e d (N=7) r a p i d l y (mean <1.5 minutes; range 1-5 minutes), t r a n s i e n t l y (2-21 minutes; mean 10.7 minutes) and r e v e r s i b l y b l o c k e d locomotion (or i n c r e a s e d e l e c t r i c a l t h r e s h o l d f o r locomotor a c t i v i t y ) e l i c i t e d by e i t h e r e l e c t r i c a l s t i m u l a t i o n or p i c r o t o x i n i n f u s i o n ( F i g . 18A,B). The GABA-induced locomotor blockade decayed with time such t h a t e l e c t r i c a l s t i m u l a t i o n immediately p o s t - i n j e c t i o n was i n e f f e c t i v e at e l i c i t i n g locomotion. However, over time (mean 10.7 min.), the e l e c t r i c a l t h r e s h o l d f o r locomotion decreased u n t i l the blockade wore o f f and a c t i v i t y s i m i l a r t o t h a t seen p r e v i o u s t o GABA i n j e c t i o n r e t u r n e d . In two b i r d s i n which locomotion was produced by e l e c t r i c a l s t i m u l a t i o n alone, GABA i n j e c t i o n (0.5M) r e p l i c a b l y and r e v e r s i b l y i n c r e a s e d t h r e s h o l d above the s t i m u l a t i o n i n t e n s i t y maximum (170/iA) from a p r e - i n j e c t i o n t h r e s h o l d mean of 50(LLA. In these animals, the r e v e r s i b l e nature o f the GABA e f f e c t s were seen a f t e r 3 t r i a l s o f GABA i n f u s i o n (GABA i n j e c t i o n f o l l o w e d by r e c o v e r y ) , i n which the e l e c t r i c a l t h r e s h o l d r e q u i r e d t o i n i t i a t e locomotion r e t u r n e d t o near pre-GABA v a l u e s (70LLA) . In t h r e e b i r d s , the t o n i c extensor a c t i v i t y which was evident a f t e r the prolonged p i c r o t o x i n - i n d u c e d locomotion or the extensor h y p e r t o n i c i t y d e s c r i b e d above disappeared f o r short p e r i o d s f o l l o w i n g GABA i n j e c t i o n i n t o these s i t e s . As the GABA bl o c k appeared t o wear o f f , locomotor movements reappeared f o r a b r i e f p e r i o d before extensor h y p e r t o n i c i t y r e a s s e r t e d i t s e l f . Muscimol, the GABAA a g o n i s t (6.5-25mM/l. Ojil) / produced 147 F i g u r e 18. Electromyographic records (EMGs) showing locomotor a c t i v i t y e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n (A) of the pontobulbar locomotor s t r i p (PLS) which was b l o c k e d by GABA i n f u s i o n i n t o the same s i t e (B). A: A l t e r n a t i n g hindlimb locomotion e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n as demonstrated by the EMGs from r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles. B: The s t e p p i n g locomotion e l i c i t e d i n A was bl o c k e d by i n j e c t i o n o f GABA i n t o the same s i t e . The t r a c e s from r i g h t and l e f t ITC muscles were taken d u r i n g ramp e l e c t r i c a l s t i m u l a t i o n (0-170uA) of t h i s PLS s i t e . 148 I 1 2 sec 149 i r r e v e r s i b l e block of e l e c t r i c a l and p i c r o t o x i n (and b i c u c u l l i n e ) induced locomotor behaviour i n 2 animals (N=2). The laten c y to muscimol-induced block was 5-10 minutes a f t e r the i n i t i a l i n j e c t i o n (0.2ul) and l a s t e d throughout the course of the experiment (30 & 70 minutes). A d d i t i o n a l i n j e c t i o n s of p i c r o t o x i n and e l e c t r i c a l s t i m u l a t i o n were i n e f f e c t i v e at e l i c i t i n g f u r t h e r locomotion from the muscimol i n j e c t i o n s i t e i n e i t h e r animal. In one animal (N=l), b i c u c u l l i n e (GABAA antagonist) i n j e c t i o n (lOmM/ljil) i n t o PLS produced locomotor a c t i v i t y (onset of 15 minutes l a s t i n g f o r 30 minute experimental period) which appeared s i m i l a r t o th a t seen f o l l o w i n g p i c r o t o x i n i n j e c t i o n . C e n t r a l Nucleus Medulla, d o r s a l part (Cnd) I n j e c t i o n of p i c r o t o x i n (5mM) i n t o Cnd (N=3) was i n e f f e c t i v e at promoting chemical-induced locomotion or reducing e l e c t r i c a l s t i m u l a t i o n t h r e s h o l d . Muscimol (6.25mM) (N=l) i n j e c t i o n had no b l o c k i n g e f f e c t on e l e c t r i c a l s t i m u l a t i o n - i n d u c e d locomotor behaviour i n one animal. C e n t r a l Nucleus Medulla, v e n t r a l part (Cnv) I n j e c t i o n s of p i c r o t o x i n i n t o Cnv (5-20mM) i n 4 out of 8 b i r d s e l i c i t e d long l a s t i n g walking (hopping i n 1 b i r d ) (see Figure 19A,B) w i t h an onset of f i r s t a c t i v i t y appearing at a mean time of 13.5 minutes (range 4-22 minutes). In one of the remaining animals, p i c r o t o x i n (25mM) decreased e l e c t r i c a l 150 F i g u r e 19. Electromyographic records (EMGs) showing GABA-reversible locomotor a c t i v i t y e l i c i t e d by p i c r o t o x i n i n j e c t i o n i n t o the c e n t r a l nucleus of the medulla, v e n t r a l p a r t (Cnv). A: A l t e r n a t i n g s t e p p i n g as r e p r e s e n t e d by EMGs from r i g h t (RITC).and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles f o l l o w i n g p i c r o t o x i n i n j e c t i o n i n t o Cnv. B: The s t e p p i n g locomotion e l i c i t e d by p i c r o t o x i n i n j e c t i o n was b l o c k e d by i n f u s i o n of GABA i n t o the same s i t e . The t r a c e s from r i g h t and l e f t ITC muscles were taken d u r i n g ramp e l e c t r i c a l s t i m u l a t i o n of t h i s Cnv s i t e (0-170LIA, arrow under bottom t r a c e i n d i c a t e s 170jxA) . 151 152 t h r e s h o l d f o r locomotion from 100 t o lOpA, while i n yet another, p i c r o t o x i n i n j e c t i o n (lOmM) r e s u l t e d i n long l a s t i n g e x t e n s i o n of both wings and l e g s without any rhythmic p a t t e r n s of locomotion. A l l of the above locomoting animals d i s p l a y e d some degree of both wing and l e g extensor a c t i v i t y f o l l o w i n g the p e r i o d of locomotor behaviour. Only one of e i g h t b i r d s d i s p l a y e d no r e a c t i o n t o p i c r o t o x i n i n j e c t i o n . GABA (0.5M) i n f u s i o n i n t o Cnv (N=3) r a p i d l y (<1 minute), t r a n s i e n t l y (mean = 11.5 minutes; range = 9-14 minutes) and r e v e r s i b l y b l o c k e d or i n c r e a s e d t h r e s h o l d f o r p i c r o t o x i n and e l e c t r i c a l l y s t i m u l a t e d locomotion ( F i g . 19B), while muscimol (6.25mM) (N=2) i r r e v e r s i b l y b l o c k e d (time t o onset o f the bl o c k <9 minutes) both p i c r o t o x i n and e l e c t r i c a l s t i m u l a t i o n - i n d u c e d locomotion i n one of two b i r d s . Pontine R e t i c u l a r Formation (RP) P i c r o t o x i n (5mM) s t i m u l a t e d locomotion (l a t e n c y t o onset 10 minutes: l a s t i n g >35 minutes) or reduced the t h r e s h o l d (3mM) f o r e l e c t r i c a l l y s t i m u l a t e d locomotion (100->50/iA) i n two of four b i r d s when i n j e c t e d i n t o RP (Figure 20). GABA (0.5M) b l o c k e d the p i c r o t o x i n response i n both animals w i t h a f a s t onset (<1 minute). The b l o c k l a s t i n g f o r 12 minutes i n one b i r d and 21 minutes i n the other (Figure 20). S i m i l a r t o the GABA i n j e c t i o n s i n t o Cnv, the bl o c k was t r a n s i t o r y and time dependent, w i t h the most e f f e c t i v e b l o c k ( f o r e l e c t r i c a l s t i m u l a t i o n ) o c c u r r i n g immediately a f t e r completion o f the i n j e c t i o n . GABA (0.5M) was e f f e c t i v e at b l o c k i n g e l e c t r i c a l l y 153 Figure 20. Electromyographic records (EMGs) showing GABA-reversible locomotor a c t i v i t y e l i c t e d by e l e c t r i c a l s t i m u l a t i o n and p i c r o t o x i n i n f u s i o n i n t o the v e n t r a l pontine r e t i c u l a r formation (RP). STIM; EMGs from r i g h t (RPECT) and l e f t (LPECT) p e c t o r a l i s muscles and r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles during e l e c t r i c a l s t i m u l a t i o n of the s i t e shown ( f i l l e d t r i a n g l e ) i n the coronal s e c t i o n at the bottom r i g h t . Threshold e l e c t r i c a l s t i m u l a t i o n evoked a l t e r n a t i n g hindlimb stepping as shown by the a c t i v i t y of the ITC muscles. PICRO (TIP): Ten minutes (T10) a f t e r p i c r o t o x i n i n f u s i o n i n t o the same s i t e , locomotor a c t i v i t y appeared both i n the wings (PECT) and legs (ITC). PICRO (T30): The locomotion continued, as evidenced by the EMGS showing a l t e r n a t i n g stepping a c t i v i t y , at T30. GABA: GABA i n j e c t i o n at T35 blocked the p i c r o t o x i n - i n d u c e d locomotion t r a n s i e n t l y , as seen from the ITC EMG t r a c e s . PICRO: The p i c r o t o x i n - i n d u c e d locomotion returned, however, as seen by the hindlimb EMG t r a c e s at 55 minutes p o s t - i n j e c t i o n (T55). A b b r e v i a t i o n s : BC - brachium conjunctivium, MLF - medial l o n g i d t u d i n a l f a s c i c u l u s , N VI -abducens nerve, N VII - v e s t i b u l a r nerve, R - raphe nucleus, RP - nucleus r e t i c u l a r i s p o n t i s , Rpc - p a r v o c e l l u l a r p a r t , pontine r e t i c u l a r formation, VI - abducens nucleus, VS - t r i g e m i n a l sensory nucleus. 154 induced locomotion ( s t i m u l a t i o n maximum 170/iA) i n two other b i r d s i n which p i c r o t o x i n i n j e c t i o n was i n e f f e c t i v e at e l i c i t i n g locomotion. Muscimol (6.25mM) i n j e c t e d i n t o one s i t e d i d not change the q u a l i t y or the t h r e s h o l d f o r e l e c t r i c a l l y s t i m u l a t e d locomotor a c t i v i t y . P i c r o t o x i n i n j e c t i o n (5mM) f o l l o w i n g muscimol i n f u s i o n was i n e f f e c t i v e at changing the s t i m u l a t i o n parameters necessary t o evoke locomotion from t h i s s i t e . Mesencephalic R e t i c u l a r Formation (MRF) B i l a t e r a l hindlimb s t e p p i n g produced by e l e c t r i c a l s t i m u l a t i o n o f the mesencephalic r e t i c u l a r formation was bl o c k e d t r a n s i e n t l y by GABA i n f u s i o n (0.5M) (l a t e n c y <1 minute; l a s t i n g 8-12 minutes) i n two b i r d s . In one of thr e e animals, i n f u s i o n of p i c r o t o x i n (20mM) decreased the t h r e s h o l d f o r e l e c t r i c a l l y induced walking from 160 to 90LIA over a 70 minute p e r i o d . GABA (0.5M) i n f u s e d i n t o the same s i t e i n c r e a s e d the t h r e s h o l d (220/xA) . In two other b i r d s , p i c r o t o x i n (5mM & lOmM) was e f f e c t i v e at e l i c i t i n g l o n g l a s t i n g f l y i n g behaviour with t o n i c l e g e x t e n s i o n which was a l s o r e v e r s i b l e by GABA i n f u s i o n . I n j e c t i o n o f p i c r o t o x i n (3mM) i n t o a more medial i n j e c t i o n s i t e i n the MRF of one animal had no e f f e c t on e l e c t r i c a l l y induced locomotion. 156 DISCUSSION Pontobulbar Locomotor S t r i p (PLS) The i n j e c t i o n of GABA antagonists p i c r o t o x i n and b i c u c u l l i n e i n t o the PLS evoked locomotion which could be blocked by GABA or muscimol. These r e s u l t s are s i m i l a r t o those found i n the cat, where p i c r o t o x i n i n j e c t i o n e l i c i t e d muscimol/GABA-reversible locomotion (Noga et a l . , 1988) and fu r t h e r v e r i f y our contention of the homology between the avian and mammalian PLS. Noga et al. (1988) suggest t h a t the PLS i s under GABAergic i n h i b i t o r y c o n t r o l and s t a t e as evidence that locomotion could be e l i c i t e d by t r i g e m i n a l f i e l d s t i m u l a t i o n f o l l o w i n g PLS i n j e c t i o n of p i c r o t o x i n , n e i t h e r of which alone a c t i v a t e d the behaviour. They argue from these r e s u l t s t h a t the PLS i s synonymous wit h the descending t r a c t of the t r i g e m i n a l nerve (TTD) tha t sends e f f e r e n t s v i a p r o p r i o s p i n a l pathways t o more caudal TTD and a l s o t o r e t i c u l a r formation neurons. They a l s o argue t h a t s t i m u l a t i o n of PLS (TTD) r e s u l t s i n locomotion through secondary a c t i v a t i o n of the r e t i c u l a r neurons th a t p l a y a d i r e c t r o l e i n the i n i t i a t i o n of locomotion (Noga et a l . , 1988) (for d i s c u s s i o n of e f f e r e n t TTD pathways see Chapter 3). As seen i n the cat, t r i g e m i n a l f i e l d s t i m u l a t i o n ( a i r p u f f s or s t r o k i n g of the head) of the b i r d s h o r t l y f o l l o w i n g p i c r o t o x i n i n f u s i o n but before the onset of locomotion e l i c i t e d by p i c r o t o x i n alone appeared t o i n i t i a t e locomotion. Loud noise a l s o e l i c i t e d bouts of locomotion p r i o r to the onset of neurochemical induced locomotion. In a d d i t i o n , s t r o k i n g of the head r e g i o n f o l l o w i n g the onset of p i c r o t o x i n - i n d u c e d locomotion appeared t o augment the f o r c e of s t e p p i n g and/or f l a p p i n g behaviour. I t appears from these r e s u l t s , t h e r e f o r e , t h a t p e r i p h e r a l s t i m u l a t i o n , a c t i n g through TTD, may i n c r e a s e the o v e r a l l g a i n of the system and f a c i l i t a t e the i n d u c t i o n of locomotor behaviour from PLS/TTD s t i m u l a t i o n . While no p r o j e c t i o n s of GABAergic neurons t o the PLS/TTD have been e l u c i d a t e d i n b i r d , i n the r a t , Mugnaini and O e r t e l (1985) r e p o r t the presence of both GABA-containing c e l l bodies and t e r m i n a l s i n TTD along i t s r o s t r o c a u d a l e x t e n t . McGeer and McGeer (1981) r e p o r t a v a r i e t y of proposed GABAergic pathways, many of which are i n t e r n e u r o n a l i n o r i g i n . However, i n t e r n u c l e a r GABA-containing a f f e r e n t s t o TTD have not been r e p o r t e d (E. McGeer, p e r s o n a l communication). Although the present data i n d i c a t e t h a t TTD GABAergic i n t e r n e u r o n s may subserve the motor e f f e c t s observed i n our experiments, the v e r a c i t y of t h i s s u p p o s i t i o n remains to be determined. The r o l e t h a t GABA p l a y s i n the c o n t r o l of P L S - a s s o c i a t e d locomotion i s p r e s e n t l y unknown, but combined wi t h the h y p o t h e s i s of Noga et a l . (1988) t h a t the PLS/trigeminal/LRF system "provides a s u b s t r a t e f o r sensorimotor r e f l e x i n i t i a t i o n of locomotion", I hypothesize t h a t GABAergic i n t e r n e u r o n s may modulate a f f e r e n t t r i g e m i n a l and p o s s i b l y c e n t r a l l y generated i n f o r m a t i o n by down-regulating or dampening the e f f e c t s of t h i s a f f e r e n t i nput on TTD b e f o r e TTD sends locomotion^producing s i g n a l s t o r e t i c u l a r formation or other l o c o m o t o r - r e l a t e d s t r u c t u r e s . 158 C e n t r a l Nucleus Medulla, d o r s a l part (Cnd) I n j e c t i o n of GABA agonists and antagonists were i n e f f e c t i v e at modulating locomotor behaviour f o l l o w i n g i n j e c t i o n i n t o Cnd. These r e s u l t s c o r r e l a t e w i t h those found by Noga et al. (1988) f o r the cat, where e l e c t r i c a l s t i m u l a t i o n e l i c i t e d , but p i c r o t o x i n i n j e c t i o n f a i l e d t o e l i c i t , locomotion. They suggest th a t the e l e c t r i c a l s t i m u l a t i o n was a c t i v a t i n g f i b e r s t r a v e l l i n g from the PLS/TTD t o more medial brainstem r e t i c u l a r formation s t r u c t u r e s . Our r e s u l t s , however, demonstrate t h a t i n j e c t i o n of c h o l i n e r g i c and e x c i t a t o r y amino a c i d agonists i n t o Cnd e l i c i t s locomotion, i n d i c a t i n g that Cnd i n t r i n s i c neurons are capable of a c t i v a t i n g locomotion. A d i f f e r e n t explanation f o r the la c k of GABAergic e f f e c t s i n t h i s region may l i e i n the d i f f e r e n t i a l d i s t r i b u t i o n of GABAergic receptors and ter m i n a l s i n the brainstem. Although l i t t l e i n f o r m a t i o n i s a v a i l a b l e f o r GABA d i s t r i b u t i o n s i n b i r d s , i n the r a t , Mugnaini and O e r t e l (1985) show only low l e v e l s of GABA te r m i n a l s i n the regions medial t o TTD (Cnd i n b i r d ; FTL i n c a t ) , while higher l e v e l s were found i n TTD. I t i s l i k e l y t h a t w i t h e l e c t r i c a l s t i m u l a t i o n of Cnd, both f i b e r s of passage from TTD and c e l l bodies (dendrites) i n Cnd are being a c t i v a t e d , while chemical s t i m u l a t i o n a c t i v a t e s only Cnd r e c e p t o r s . Thus, while a c e t y l c h o l i n e and the e x c i t a t o r y amino acid s appear t o be i n v o l v e d i n locomotor c o n t r o l v i a Cnd, GABA does not appear to be a c t i v e as a neurotransmitter i n t h i s r e gion of the brainstem i n e i t h e r the b i r d or cat (Noga et a l , , 1988). 159 C e n t r a l Nucleus Medulla, v e n t r a l p a r t (Cnv) GABAergic a n t a g o n i s t s i n j e c t e d i n t o Cnv produced locomotion or caused a decrease i n e l e c t r i c a l s t i m u l a t i o n t h r e s h o l d r e q u i r e d t o i n i t i a t e locomotion. Both e f f e c t s c o u l d be bl o c k e d by GABA or muscimol i n j e c t i o n . These r e s u l t s are s i m i l a r t o those found i n the cat (Noga et a l . , 1988; G a r c i a - R i l l and Skinner, 1987) f o l l o w i n g i n j e c t i o n o f GABA a g o n i s t s / a n t a g o n i s t s . G a r c i a - R i l l and Skinner (1987) r e p o r t e d b l o c k of e l e c t r i c a l l y s t i m u l a t e d locomotion with GABA or muscimol i n f u s i o n i n t o the medial r e t i c u l a r formation, but found t h a t p i c r o t o x i n or b i c u c u l l i n e i n j e c t i o n produced c o n v u l s i o n s at c o n c e n t r a t i o n s g r e a t e r than 5mM. In the b i r d , only one animal o f seven demonstrated convulsant type a c t i v i t y immediately f o l l o w i n g p i c r o t o x i n i n f u s i o n . However, a l l animals i n which p i c r o t o x i n was e f f e c t i v e at producing locomotor behaviour e v e n t u a l l y d i s p l a y e d t o n i c extensor a c t i v i t y o f both wings and le g s t o v a r y i n g degrees. As d i s c u s s e d by Noga et al. (1988), and s i m i l a r to our s t u d i e s i n b i r d s u s i n g e l e c t r i c a l s t i m u l a t i o n (unpublished o b s e r v a t i o n s ) , i t i s p o s s i b l e t h a t a locomotor somatotopic o r g a n i z a t i o n e x i s t s i n the r e t i c u l a r formation. Our ob s e r v a t i o n s have shown t h a t s m a l l l a t e r o m e d i a l t r a n s l o c a t i o n s of the s t i m u l a t i n g e l e c t r o d e w i l l o f t e n change the p a t t e r n of hindlimb walking from c o n t r a l a t e r a l u n i l a t e r a l s t e p p i n g t o b i l a t e r a l s t e p p i n g . These r e s u l t s are, however, seldom seen f o r wing locomotion, where f l a p p i n g i s b i l a t e r a l i n the v a s t m a j o r i t y o f cases. Taken t o g e t h e r with the i n f o r m a t i o n t h a t i n neurochejnical-induced locomotion t r i a l s , some f e l i n e muscle 160 groups showed a l o s s o f p h a s i c a c t i v i t y (Noga et al., 1988), i t appears t h a t the convulsant a c t i v i t y demonstrated a f t e r i n j e c t i o n o f the long a c t i n g p i c r o t o x i n (Franz, 1985) may r e s u l t from d i f f u s i o n and subsequent d i s i n h i b i t o r y a c t i o n o f p i c r o t o x i n on c e n t r e s c o n t r o l l i n g d i f f e r e n t extensor and/or f l e x o r muscles which are normally modulated out of phase. GABAergic c e l l bodies and t e r m i n a l s have been found i n the r a t v e n t r a l r e t i c u l a r formation (Mugnaini and O e r t e l , 1985). E q u i v a l e n t s t r u c t u r e s may serve as the neuroanatomical s u b s t r a t e f o r the observed changes i n locomotor behaviour induced by GABA or i t s a g o n i s t s and a n t a g o n i s t s i n the b i r d . To our knowledge, GABA neuroanatomy and neurochemistry have been e x p l o r e d i n the b i r d u s i n g r e t r o g r a d e t r a n s p o r t o f [3H]GABA only w i t h r e s p e c t t o GABAergic s t r i a t o t e g m e n t a l p r o j e c t i o n s ( H a l l et a l . , 1984) and no r e p o r t s o f GABAergic i n n e r v a t i o n or i n t r i n s i c GABA-containing c e l l bodies i n t h i s r e g i o n are a v a i l a b l e . However, our r e s u l t s suggest the p r o b a b i l i t y t h a t Cnv c e l l s g i v i n g r i s e t o r e t i c u l o s p i n a l f i b e r s (Webster and Steeves, 1988) are under GABAergic i n h i b i t o r y c o n t r o l . Whether t h i s c o n t r o l a r i s e s from i n t r i n s i c or e x t r i n s i c neurons remains t o be determined both f o r b i r d s and mammals. Pontine R e t i c u l a r Formation (RP) Chemical s t i m u l a t i o n of the v e n t r a l RP r e g i o n w i t h the GABA an t a g o n i s t p i c r o t o x i n produced locomotion which was b l o c k e d by GABA i n f u s i o n . While no data i s a v a i l a b l e f o r GABAergic c e l l b o dies or t e r m i n a l s i n the b i r d , i n the r a t , medium t o low 161 l e v e l s of GABA t e r m i n a l s and low to very low l e v e l s of GABAergic c e l l bodies have been found i n the pontine r e t i c u l a r formation (Mugnaini and O e r t e l , 1985) . I t appears u n l i k e l y , t h e r e f o r e , that GABAergic neurons i n t r i n s i c to the pontine r e t i c u l a r formation are re s p o n s i b l e f o r t h i s response. However, c e l l bodies i n the pontine r e t i c u l a r formation appear to be under GABAergic i n h i b i t o r y c o n t r o l . The greater concentration of GABAergic t e r m i n a l s r e l a t i v e to c e l l bodies suggests an e x t r i n s i c source of inpu t . Neural pathways which u n d e r l i e t h i s c o n t r o l are, however, p r e s e n t l y unknown, l e a v i n g any s i g n i f i c a n t locomotor r o l e f o r GABA i n the pontine r e t i c u l a r formation undetermined. Mesencephalic R e t i c u l a r Formation (MRF) In f u s i o n of GABAergic antagonists and agonists i n t o the MRF was e f f e c t i v e i n e l i c i t i n g or b l o c k i n g locomotion i n b i r d s . These r e s u l t s are s i m i l a r t o those found f o l l o w i n g the i n f u s i o n of GABAergic neurochemicals i n t o the MLR both i n the decerebrate cat ( G a r c i a - R i l l et a l . , 1985) and the f r e e l y moving r a t (Brudzynski and Mogenson, 1986) . GABA i s present i n the cuneiform nucleus (CN) and pedunculopontine nucleus (PPN) of the r a t , w i t h higher l e v e l s of both GABA-containing c e l l bodies and ter m i n a l s being found i n the cuneiform nucleus (Mugnaini and O e r t e l , 1985). P o s s i b l e locomotion-related GABAergic p r o j e c t i o n s from the s u b s t a n t i a n i g r a , pars r e t i c u l a t a , nucleus accumbens and entopeduncular nucleus to the PPN/MLR have been reported i n the cat ( G a r c i a - R i l l et al., 1983b, G a r c i a - R i l l and Skinner, 162 1986) and r a t ( G a r c i a - R i l l et al., 1 9 8 6 ) . These r e s u l t s were supported, i n p a r t , by the d e s c r i p t i o n o f nucleus accumbens GABAergic p r o j e c t i o n s t o both PPN and CN i n the r a t (Mogenson et al., 1985; Mogenson and Wu, 1986; Brudzynski et al., 1 9 8 8 ) . As d i s c u s s e d i n the p r e v i o u s chapter, the mammalian CN and PPN, which may repr e s e n t the neuroanatomical s u b s t r a t e s f o r the l a t e r a l and medial mesencephalic locomotor r e g i o n s r e s p e c t i v e l y , ( G a r c i a - R i l l et a l . , 1985; Noga et al., 1988; Brudzynski et al., 1 9 8 8 ) , appear t o be e q u i v a l e n t t o the avian mesencephalic r e g i o n s s t i m u l a t e d i n our study. While t o our knowledge, no s t u d i e s l o c a l i z i n g GABA-containing c e l l bodies or t e r m i n a l s have been c a r r i e d out f o r these r e g i o n s i n the b i r d , comparison of the mammalian r e s u l t s w i t h those found above i n b i r d s p r o v i d e s evidence which supports the presence of an avian e q u i v a l e n t o f the mammalian MLR. However", due to the smal l number of mesencephalic i n j e c t i o n s i t e s and the i n a b i l i t y t o e l i c i t locomotion f o l l o w i n g a s i n g l e i n j e c t i o n o f p i c r o t o x i n i n t o the more medial MRF, f u r t h e r t e s t i n g o f t h i s h y p o thesis i s r e q u i r e d b e f o r e any f i r m e q u i v a l e n c y can be e s t a b l i s h e d . Pharmacological C o n s i d e r a t i o n s P i c r o t o x i n i n j e c t i o n i n t o a v a r i e t y of e l e c t r i c a l l y i d e n t i f i e d locomotor r e g i o n s a l s o e l i c i t e d locomotion which was t r a n s i e n t l y r e v e r s e d with GABA i n f u s i o n , and i n some cases, appeared t o be i r r e v e r s i b l y b l o c k e d by muscimol i n j e c t i o n . Furthermore, s u b t h r e s h o l d ( f o r e l e c t r i c a l l y evoked locomotion) i n j e c t i o n of p i c r o t o x i n decreased the e l e c t r i c a l s t i m u l a t i o n 163 i n t e n s i t y necessary to evoke locomotion. Pharmacologically, these r e s u l t s appear somewhat p e r p l e x i n g , as p i c r o t o x i n has been demonstrated to act at d i f f e r e n t receptor s i t e s than GABA, muscimol and b i c u c u l l i n e (Olsen, 1981). How then does GABA or muscimol reverse the locomotor a c t i o n of p i c r o t o x i n ? P i c r o t o x i n i s a potent antagonist at both the GABAft and GABA b receptor subtypes (Krogsgaard-Larsen et a l . , 1983). The GABAft receptor (defined by i t s s e n s i t i v i t y to the competetitive antagonist b i c u c u l l i n e ) , i s thought to c o n t r o l a c h l o r i d e ionophore (Krogsgaard-Larsen et al., 1983) and has been demonstrated to possess f i v e d i f f e r e n t b i n d i n g s i t e s i n c l u d i n g a GABA agonist/antagonist ( i n c l u d i n g muscimol and b i c u c u l l i n e ) s i t e , a benzodiazepine s i t e , a p i c r o t o x i n s i t e , a depressant s i t e (e.g. b a r b i t u r a t e s ) and a s i t e ( s ) which binds the channel-permeating ions (Barnard et a l . , 1987) . The GABA receptor (defined by i t s s e n s i t i v i t y t o the B agonist b a c l o f e n ) , on the other hand, i s b e l i e v e d to exert i t s e f f e c t s by r e s t r i c t i n g p r e s y n a p t i c (voltage dependent) calcium i n f l u x (Desarmenien et a l . , 1983). As discussed i n Chapter 3, the e l i c i t a t i o n of locomotion by neurochemical i n j e c t i o n i s viewed as a recruitment phenomenon whereby a s u f f i c i e n t number of neurons must be a c t i v a t e d or blocked by the neurochemical to i n i t i a t e / b l o c k the v i s i b l e signs of locomotion (see Chapter 3). In t h i s study, i t i s not p o s s i b l e to determine whether the i n h i b i t o r y a c t i o n of p i c r o t o x i n on GABA receptors i s a c t i n g p r e - s y n a p t i c a l l y , p o s t s y n a p t i c a l l y or both, although the e f f e c t i v e n e s s of b i c u c u l l i n e at e l i c i t i n g locomotion would suggest t h a t GABAft and not GABA b receptors 164 mediate the e f f e c t . I n t r o d u c t i o n of a hig h c o n c e n t r a t i o n of GABA should not d i s p l a c e the bound p i c r o t o x i n , but may exert i t s locomotor i n h i b i t o r y e f f e c t s by b i n d i n g t o GABA r e c e p t o r s which are not occupied by p i c r o t o x i n , thereby short c i r c u i t i n g the p i c r o t o x i n i n h i b i t i o n and b l o c k i n g locomotion. T h i s may a l s o e x p l a i n the h i g h c o n c e n t r a t i o n o f GABA necessary t o r e v e r s e the p i c r o t o x i n - i n d u c e d locomotion. The r a p i d breakdown of GABA i n v i v o (Franz, 1 9 8 5 ) would account f o r the r e t u r n o f locomotion induced by the more p e r s i s t e n t b i n d i n g o f p i c r o t o x i n . The i n h i b i t o r y a c t i o n o f muscimol, a potent, long l a s t i n g GABA^ ago n i s t (Bloom, 1 9 8 5 ) on p i c r o t o x i n - i n d u c e d locomotion presumably u t i l i z e s the same mechanism and u n d e r l i e s the suggestion t h a t GABAft r e c e p t o r s may be the t a r g e t o f the GABA ago n i s t and an t a g o n i s t locomotor e f f e c t s . Furthermore, the e l e c t r i c a l s t i m u l a t i o n i n t e n s i t y necessary t o evoke locomotion f o l l o w i n g s u b t h r e s h o l d ( f o r locomotion) p i c r o t o x i n i n f u s i o n may augment the number of neurons a c t i v a t e d by p i c r o t o x i n , thereby e l i c i t i n g locomotion at reduced e l e c t r i c a l t h r e s h o l d . In f u t u r e s t u d i e s , d i f f e r e n t i a t i o n o f the avian GABA r e c e p t o r subtype u n d e r l y i n g the locomotion observed i n t h i s study may be p o s s i b l e u t i l i z i n g more s p e c i f i c G A B A a + b a g o n i s t s and a n t a g o n i s t s such as the newly r e p o r t e d GABA a n t a g o n i s t p h a c l o f e n (Karlsson et al., 1 9 8 8 ) . Such s t u d i e s would p r o v i d e f u r t h e r v a l u a b l e i n f o r m a t i o n concerning the r o l e of GABA i n the c o n t r o l o f locomotion. 1 6 5 CHAPTER 5 CHARACTERIZATION OF AVIAN MID- AND HINDBRAIN SITES THAT PRODUCE LOCOMOTION WITH INTRACEREBRAL INFUSION OF NEUROTRANSMITTER AGONISTS AND ANTAGONISTS (I I I ) : EXCITATORY AMINO ACIDS AND SUBSTANCE P 166 INTRODUCTION Previous chapters (3 & 4) i n t h i s thesis have demonstrated that s i t e s p e c i f i c intracerebral microinjection of cholinergic and GABAergic neurotransmitter agonists and antagonists are ef f e c t i v e at e l i c i t i n g or blocking locomotor behaviours i n decerebrate bir d s . Furthermore, attempts were made to describe a possible neuroanatomical substrate for those e f f e c t s . This chapter surveys the effects of i n j e c t i o n of glutamate, i t s agonists or antagonists into several locomotor regions previously i d e n t i f i e d by e l e c t r i c a l stimulation (Steeves et al., 1986, 1987; Sholomenko and Steeves, 1987a,b, 1988). Results of Substance P infusion into several s i t e s w i l l also be described. The results demonstrate that microinjection of the glutamate agonist NMDA into various locomotor regions was e f f e c t i v e at evoking locomotor behaviour i n birds . Substance P e l i c i t e d locomotion i n only one region injected. These results w i l l be correlated with i n t e r s p e c i f i c neuroanatomical, immunohistochemical and receptor autoradiographic data i n an attempt to elucidate and compare the neural substrate c o n t r o l l i n g locomotion across a broad range of vertebrate species. 167 MATERIALS AND METHODS The m a t e r i a l s and methods, i n c l u d i n g the d e c e r e b r a t i o n procedure, e l e c t r i c a l s t i m u l a t i o n methodology, neurochemical i n j e c t i o n parameters and h i s t o l o g i c a l i d e n t i f i c a t i o n o f s t i m u l a t i o n / i n j e c t i o n s i t e s have been p r e v i o u s l y d e s c r i b e d i n Chapters 2 & 3. 168 RESULTS E x c i t a t o r y Amino A c i d s A g o n i s t s , A n t a g o n i s t s and Substance P E x c i t a t o r y amino a c i d (EAA) n e u r o t r a n s m i t t e r s , t h e i r a g o n i s t s , a n t a g o n i s t s and Substance P were i n f u s e d i n t o regions i n the h i n d - and midbrain t o determine t h e i r e f f e c t i v e n e s s at evoking or i n h i b i t i n g locomotion at s i t e s from which locomotion can be e l i c i t e d by f o c a l e l e c t r i c a l s t i m u l a t i o n (Steeves et a l , , 1986). NMDA, the glutamate a g o n i s t most e f f e c t i v e at the NMDA re c e p t o r subtype, and glutamate were i n f u s e d t o e x c i t e p u t a t i v e g l u t a m a t e r g i c r e c e p t o r s . Glutamic a c i d d i e t h y l e s t e r (GDEE) , a r e l a t i v e l y n o n - s p e c i f i c EAA an t a g o n i s t was used i n an attempt t o bl o c k EAA r e c e p t o r s (Noga et a l . , 1988; L a i and S i e g e l , 1988; Stone and Burton, 1988). Substance P was a l s o i n j e c t e d i n t o s e v e r a l e l e c t r i c a l l y i d e n t i f i e d locomotor s i t e s t o determine i t s e f f e c t s on locomotor behaviour. The s i t e s i n j e c t e d were d i s t r i b u t e d w i t h i n r e g i o n s of the mid- and h i n d b r a i n which i n c l u d e d the pontobulbar locomotor s t r i p , d o r s a l and v e n t r a l p a r t s o f the medullary r e t i c u l a r formation, pontine r e t i c u l a r formation, mesencephalic r e t i c u l a r formation and medial l o n g i t u d i n a l f a s c i c u l u s . Neurochemical i n j e c t i o n s i t e s , lowest e f f e c t i v e c o n c e n t r a t i o n and time course of a c t i v i t y are l i s t e d i n Table 3 and d e s c r i b e d i n the t e x t . A composite diagram of i n j e c t i o n s i t e s i s shown i n F i g u r e 21. 169 T A B L E 3 Excitatory Amino Acids and Substance P Animal Site Chemical pH Concentrations Lowed Volume Rate Time Courts (min) Injected Effective Latency Period Concentration TTD Glutamate 0.SO.0M none 1.0ul — — NMDA eOmM 80mM 0.2ul <1 10 GDEE 2-B0mM 2mM 0.4-1 ul 4-6 35-55 Substance P " S.44mM none 1.0ul — — Cnd NMDA » 20-83mM 20mM 0.2ul <1.5 3-24 GDEE •• 80mM 80mM o.eui <5 >35 Substance P " 6.44mM none 1.0ul — — Cnv Glutamate •• 1M none I.Oul — — NMDA '• 4-83mM 4mM 0.4 ul <1 3-4 GDEE " 2-B0mM 2mM 0.2ul 4-6 35-50 Substance P " 6.44mM none 1.0ul — — RP NMDA 7.2-7.4 B1-83mM 83mM 0.2ul <1-1.25 4-8 Substance P " 6.44 mM 6.44 mM 1.0ul 4 >15 MRF NMDA " 6-34mM 5mM (RT) 0.2ul 5 15 MLF NMDA •• 20-34mM 34mM 0.4ul <1 8 ABBREVIATIONS: Cnd — dorsal part, medullary central nucleus Cnv — ventral part, medullary central nucleus MLF — medial longitudinal fasciculus MRF — mesencephalic reticular formation RP — pontine reticular nucleus RT — reduced threshold lor electrically stimulated locomotion TTD — descending trigeminal tract and nucleus 170 F i g u r e 21. Composite diagram o f g l u t a m a t e r g i c a g o n i s t and ant a g o n i s t neurochemical i n j e c t i o n s i t e s . The diagram o f c o r o n a l s e c t i o n s through v a r i o u s l e v e l s of the av i a n n e u r a x i s [numbers i n upper l e f t corner o f each l e v e l , A=anterior, P=posterior ( i n mm)] i l l u s t r a t e s the locomotor e f f e c t s o f each neurochemical i n brainstem r e g i o n s from which locomotion was f i r s t e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n . Where glutamic a c i d d i e t h y l e s t e r (GDEE) ( f i l l e d square) i s shown as f i l l e d , i t s e f f e c t was to bl o c k locomotion. Key A b b r e v i a t i o n s : GDEE - glutamic a c i d d i e t h y l e s t e r , GLUT -glutamate, NMDA - N-methyl-D-aspartate, SUBP - substance P; LOCO - locomotion (except f o r GDEE), TH - decreased e l e c t r i c a l t h r e s h o l d i n t e n s i t y f o r locomotion, ^TH - i n c r e a s e d e l e c t r i c a l t h r e s h o l d i n t e n s i t y f o r locomotion, NR - no response. F i g u r e A b b r e v i a t i o n s : AL - ansa l e n t i c u l a r i s , AQ - aqueduct, BC - brachium conjunctivum, CC - c e n t r a l c a n a l , Cnd - c e n t r a l nucleus medulla, d o r s a l p a r t , Cnv - c e n t r a l nucleus medulla, v e n t r a l p a r t , EM - ectomammillary nucleus, EW - Edinger Westphal nucleus, I I I - occulomotor nucleus, 10 - i n f e r i o r o l i v a r y nucleus, IP - nucleus i n t e r p e d u n c u l a r i s , LC - lo c u s coeruleus, MLd - l a t e r a l mesencephalic nucleus, d o r s a l d i v i s i o n , MLF -medial l o n g i t u d i n a l f a s c i c u l u s , MRF - medial mesencephalic r e t i c u l a r formation, MV - t r i g e m i n a l motor nucleus, N I I I -occulomotor nerve, N IV - t r o c h l e a r nerve, N XII - h y p o g l o s s a l nerve, OT - o p t i c tectum, R - raphe nucleus, RP - nucleus pontine r e t i c u l a r formation, Rpc - pontine p a r v o c e l l u l a r r e t i c u l a r nucleus, RPO - nucleus r e t i c u l a r i s p o n t i s o r a l i s , Ru -red nucleus, SSP - s u p r a s p i n a l nucleus, SV - t r i g e m i n a l sensory nucleus, TPc - s u b s t a n t i a n i g r a , TTD - t r i g e m i n a l descending t r a c t and nucleus, X - d o r s a l motor nucleus vagus. 171 LOCO JTH TTH ffi NBA • © e O GDS D B B • GLUT • • • O SUBP A A A A A 3.75 172 Pontobulbar Locomotor S t r i p (PLS) Fo l l o w i n g establishment o f a low i n t e n s i t y s t i m u l a t i o n p o i n t f o r evoking locomotion, i n j e c t i o n o f NMDA (80mM/0.2ul) (N=3) e l i c i t e d r e p e a t a b l e bouts o f locomotion i n 2 of 3 b i r d s i n j e c t e d ( F i g . 22). The locomotion f o l l o w e d NMDA i n j e c t i o n with a r a p i d onset (<lmin) and oc c u r r e d i n vi g o r o u s bouts (l-2min) f o r approximately 10 minutes. Both NMDA and e l e c t r i c a l l y s t i m u l a t e d locomotion were i r r e v e r s i b l y b l o c k e d by GDEE (80mM/0.4ul and 2mM/0.2ul) f o r the d u r a t i o n of the experiment (>40min) (Figure 22C). Glutamate i n f u s i o n (0.5-1.0M/1.Oul) (N=2) i n t o PLS had no e f f e c t on the t h r e s h o l d of e l e c t r i c a l l y s t i m u l a t e d locomotor behaviour. Substance P (N=l) (5.44mM/l.Oul) a l s o had no e f f e c t when i n f u s e d i n t o t h i s s i t e . C e n t r a l Nucleus, d o r s a l p a r t (Cnd) I n j e c t i o n o f NMDA i n t o Cnd (81-83mM/0.2ul) i n 2/5 b i r d s e l i c i t e d r e p e a t a b l e b u r s t s o f running and f l y i n g behaviour (20-40sec) ( F i g . 23B). The locomotion o c c u r r e d w i t h f a s t onset ( 1.0 & 1.5 minutes) and l a s t e d f o r 3 and 14 minutes. P r i o r t o chemical i n j e c t i o n , e l e c t r i c a l s t i m u l a t i o n i n one of these animals produced only b i l a t e r a l s t e p p i n g movements, while i n the other, both running and f l y i n g were d i s p l a y e d at e l e c t r i c a l t h r e s h o l d ( F i g . 26A). One of f i v e of the b i r d s d i s p l a y e d r a p i d onset (<1 min) and long l a s t i n g (24 minutes) walking behaviour f o l l o w i n g NMDA s t i m u l a t i o n (20mM/0.2ul) which was s i m i l a r t o t h a t produced by p r e - i n j e c t i o n e l e c t r i c a l s t i m u l a t i o n . In the F i g u r e 22. Electromyographic records (EMGs) showing locomotor a c t i v i t y e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n and NMDA i n j e c t i o n i n t o a s i t e i n the pontobulbar locomotor s t r i p (PLS) shown i n the c o r o n a l s e c t i o n o f the medulla (bottom r i g h t ) . STIM: A l t e r n a t i n g s t e p p i n g r e p r e s e n t e d by EMG p a t t e r n s from the r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles (major h i p f l e x o r muscle) e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n of the PLS. No a c t i v i t y i s present i n the r i g h t (RPECT) and l e f t (LPECT) p e c t o r a l i s muscles which are the major wing depressor muscles. NMDA: NMDA i n j e c t i o n i n t o the same s i t e evoked b u r s t s o f ste p p i n g and wing f l a p p i n g as shown by the EMGs f o r the p a i r e d wings (PECT) and leg s (ITC) at 90 seconds p o s t - i n j e c t i o n (only 1st b u r s t i s shown). STIM: E l e c t r i c a l s t i m u l a t i o n at 20 minutes p o s t - i n j e c t i o n e l i c i t e d hindlimb s t e p p i n g as shown by the EMGs of r i g h t and l e f t ITC muscles. GDEE: Hindlimb EMG records f o l l o w i n g GDEE i n f u s i o n i n t o the same s i t e which b l o c k e d f u r t h e r NMDA and e l e c t r i c a l s t i m u l a t i o n - i n d u c e d locomotion. A b b r e v i a t i o n s : CC - c e n t r a l c a n a l , Cnd - c e n t r a l medullary nucleus, d o r s a l p a r t , Cnv - c e n t r a l medullary nucleus, v e n t r a l p a r t , 10 - i n f e r i o r o l i v a r y nucleus, MLF - medial l o n g i t u d i n a l f a s c i c u l u s , N X - vagus nerve, ST - s u b t r i g e m i n a l nucleus, TTD -t r i g e m i n a l descending t r a c t and nucleus, X - va g a l motor nucleus. 174 F i g u r e 23. Electromyographic records (EMGs) showing locomotor a c t i v i t y e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n and NMDA i n j e c t i o n i n t o the d o r s a l p a r t o f the c e n t r a l medullary nucleus (Cnd) A: A l t e r n a t i n g s t e p p i n g and wing f l a p p i n g r e p r e s e n t e d by EMG p a t t e r n s from r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles and right. (RPECT) and l e f t (LPECT) p e c t o r a l i s muscles e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n o f Cnd. B: Simultaneous (in-phase) wing f l a p p i n g EMGs (PECT) and a l t e r n a t i n g stepping.(ITC) EMGs evoked by i n j e c t i o n o f NMDA (83mM/0.2ul) i n t o the same s i t e . C: EMG t r a c e s from wings and leg s a f t e r i n j e c t i o n of GDEE (80mM/0.6ul) i n t o the s i t e . Traces were taken d u r i n g e l e c t r i c a l s t i m u l a t i o n (170uA) of the s i t e 4 minutes a f t e r the i n f u s i o n o f NMDA (83mM/0.2ul) . 176 I ! 1 1 sec 177 RITC -ifr *— fKdfeif 4h"" t*' 1")!** LITC 0 . 5 sec 178 RPECT LPECT RITC LITC i 1 2 sec 179 b i r d which d i s p l a y e d r u n n i n g / f l y i n g behaviour both w i t h e l e c t r i c a l and NMDA s t i m u l a t i o n , GDEE (80mM/0. 6jil) i n f u s i o n i n t o t h i s s i t e i r r e v e r s i b l y b l o c k e d f u r t h e r e l e c t r i c a l and NMDA s t i m u l a t e d locomotion ( F i g . 23C). In one b i r d which was p a r a l y z e d f o l l o w i n g establishment of an a l t e r n a t i n g walking e l e c t r i c a l s t i m u l a t i o n s i t e (see Chapter 6), i n j e c t i o n of 20mM/0.2Lil a l i q u o t s o f NMDA re p e a t a b l y i n i t i a t e d bouts (-1.5 minutes long) o f b i l a t e r a l in-phase x f i c t i v e ' l e g movements (hopping) w i t h i n 1 minute p o s t - i n j e c t i o n . The e l e c t r o n e u r o g r a p h i c a c t i v i t y appeared s t r o n g e s t immediately p o s t - i n j e c t i o n and became weaker over time. No bouts were observed a f t e r approximately 6 minutes p o s t - i n j e c t i o n (see Chapter 6, F i g . 33) . Substance P (5.44mM) i n j e c t i o n produced no changes i n e l e c t r i c a l s t i m u l a t i o n t h r e s h o l d when i n j e c t e d i n t o Cnd i n one animal. C e n t r a l Nucleus, v e n t r a l p a r t (Cnv) NMDA i n j e c t i o n i n t o Cnv produced locomotion i n 3 of 3 b i r d s . E l e c t r i c a l s t i m u l a t i o n , c a r r i e d out p r i o r t o chemical s t i m u l a t i o n , produced running and f l y i n g behaviour at t h r e s h o l d i n t e n s i t y ( F i g . 24A). Chemical s t i m u l a t i o n (81mM/0.2Lil;4mM/0.4/il) i n both o f these animals r e s u l t e d i n short bouts (5-15 seconds) o f running and f l y i n g locomotion ( l a t e n c y t o onset <1 min) which continued f o r 3 minutes i n one animal and 4 minutes i n the other and was s i m i l a r t o t h a t seen i n response t o e l e c t r i c a l s t i m u l a t i o n ( F i g . 24B). GDEE at both 180 Figure 24. Electromyographic records (EMGs) showing locomotor a c t i v i t y e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n and NMDA i n j e c t i o n i n t o the v e n t r a l p a r t of the c e n t r a l medullary nucleus (Cnv) A: A l t e r n a t i n g stepping and wing f l a p p i n g represented by EMG patterns from r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles and r i g h t (RPECT) and l e f t (LPECT) p e c t o r a l i s muscles e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n of Cnv. B: Simultaneous (in-phase) wing f l a p p i n g EMGs (PECT) and a l t e r n a t i n g stepping (ITC) EMGs evoked by i n j e c t i o n of NMDA (4mM/0.4ul) i n t o the same s i t e . C: EMG tr a c e s from wings and legs a f t e r i n j e c t i o n of GDEE (2mM/0.2fil) i n t o the s i t e . Traces were taken 4 minutes a f t e r the i n f u s i o n of NMDA (4mM/0.4ul). 181 LITC i 0.5 sec 182 LITC M #' • # I 1 1 sec 183 c RPECT LPECT RITC LITC — 2 sec 184 high (80mM/0.4ul) and low (2mM/0.2ul) concentrations i r r e v e r s i b l y blocked both NMDA and e l e c t r i c a l l y induced locomotion when infused into these s i t e s (Fig 24C). In the t h i r d animal, e l e c t r i c a l stimulation produced continuous b i l a t e r a l walking behaviour which was repeatably (5 t r i a l s ) mimicked by NMDA (83mM/0.2/il) i n j e c t i o n with short latency (<1 minute) and time course (~4 min). Substance P (5.44mM/l. 0(il) i n j e c t i o n at th i s s i t e had no ef f e c t either on the quality of chemically induced locomotion or e l e c t r i c a l stimulation threshold. In two other animals which received Substance P (5.44mM/l. Ojil) i njections into Cnv, no changes were observed i n the locomotor pattern or e l e c t r i c a l threshold needed to i n i t i a t e locomotion. One of the above birds received an i n j e c t i o n of glutamate (lM/l/il) into the same s i t e 20 minutes a f t e r Substance P i n j e c t i o n . Although the glutamate i n j e c t i o n did not induce locomotion or affe c t any changes to stimulation i n t e n s i t y threshold, increases i n breathing frequency and force of expiration were observed which were similar to those seen r e s u l t i n g from e l e c t r i c a l stimulation i n t h i s s i t e . Pontine Reticular Formation (RP) Introduction of NMDA into s i t e s i n the pontine r e t i c u l a r formation e l i c i t e d locomotion i n 3 of 5 birds. One of these animals displayed running/flying both at e l e c t r i c a l stimulation threshold and following NMDA (81mM/0. 4fil) infusion. The latency to onset of running/flying was 1.25 minutes with short bouts (15-25 seconds) of t h i s behaviour occurring for up to 4.25 185 minutes p o s t - i n j e c t i o n . The two other b i r d s with RP NMDA i n j e c t i o n s (83mM/0. 2fil) e l i c i t e d walking only ( s i m i l a r t o p r e - i n j e c t i o n e l e c t r i c a l l y s t i m u l a t e d l o c o m o t i o n ) . One of the above animals r e p e a t a b l y produced walking movements w i t h i n 1 minute p o s t - i n j e c t i o n which l a s t e d approximately 4 minutes. The second animal r e c e i v e d NMDA 15 minutes a f t e r i n j e c t i o n of Substance P (5.44mM / lL i l ) i n t o the s i t e ( F i g . 25A,B) . The NMDA (83mM/0.2 / i l ) i n j e c t i o n s i g n i f i c a n t l y i n c r e a s e d the f o r c e o f the weak b i l a t e r a l a l t e r n a t i n g s t e p p i n g (and i n i t i a t e d f l a p p i n g behaviour) ( F i g . 25C) produced by a Substance P i n j e c t i o n (onset 4 minutes p o s t - i n j e c t i o n l a s t i n g up t o 15 minutes at which time NMDA was i n j e c t e d ) w i t h i n 1 minute. The e f f e c t o f NMDA l a s t e d approximately 9 minutes and decreased i n a time dependent manner. Mesencephalic R e t i c u l a r Formation (MRF) NMDA i n j e c t i o n (5mM/0.2/jl) i n t o the MRF i n one of two b i r d s reduced the t h r e s h o l d f o r e l e c t r i c a l l y s t i m u l a t e d walking from IOOJIA to 60/iA but d i d not e l i c i t locomotion independently, while NMDA i n j e c t i o n i n the second b i r d (paralyzed) (34mM/0. 6jxl) had no e f f e c t e i t h e r on locomotor p a t t e r n or t h r e s h o l d f o r e l e c t r i c a l l y s t i m u l a t e d locomotion. Medial L o n g i t u d i n a l F a s c i c u l u s (MLF) I n f u s i o n of NMDA (34mM/0. 4fil) i n t o a locomotion promoting s t i m u l a t i o n s i t e (walking and f l a p p i n g , F i g . 26A) i n t o the MLF 1 8 6 Figure 25. Electromyographic records (EMGs) showing locomotor a c t i v i t y e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n , Substance P and NMDA i n j e c t i o n i n t o the pontine r e t i c u l a r formation (RP). A: A l t e r n a t i n g stepping evoked by t h r e s h o l d e l e c t r i c a l s t i m u l a t i o n of RP. The bottom two tr a c e s demonstrate the EMG a c t i v i t y i n the r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles, while the top t r a c e s (RPECT & LPECT), taken from the p e c t o r a l i s muscles show no a c t i v i t y . B: EMG a c t i v i t y demonstrating only a l t e r n a t i n g hindlimb stepping i n the b i r d f o l l o w i n g i n j e c t i o n of Substance P i n t o the same s i t e . The rhythmic p a t t e r n of EMG a c t i v i t y seen i n the PECT t r a c e s i s breath i n g . C: Stepping and f l a p p i n g a c t i v i t y evoked f o l l o w i n g the i n j e c t i o n of NMDA i n t o the s i t e p r e v i o u s l y i n j e c t e d w i t h Substance P as shown by the EMGs from the l e g f l e x o r muscles (ITC - bottom 2 traces) and wing depressor muscles (PECT - top 2 t r a c e s ) . 187 A RPECT i M I i i — i ,, i LPECT i MI I I J I ii^ iiiiM)<>i ni ;-|••»>•• RITC LITC 1 sec 188 RPECT I I I I 1 > »'l I I I I I I I M'tH I I t > I I M I M I I I I I I I I H " I I I I I LPECT RITC LITC 189 I 1 2 sec 190 F i g u r e 26. Electromyographic records (EMGs) showing locomotor a c t i v i t y e l i c t e d by e l e c t r i c a l s t i m u l a t i o n and NMDA i n f u s i o n i n t o the medial l o n g i t u d i n a l f a s c i c u l u s (MLF). A: EMGs from the r i g h t (RPECT) and l e f t (LPECT) p e c t o r a l i s and r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles i l l u s t r a t i n g the c o a c t i v a t e d s t e p p i n g (ITC) and f l a p p i n g (PECT) e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n o f the MLF. B: E l e c t r o n e u r o g r a p h i c r e cords (ENGs) taken from the l e f t p e c t o r a l i s muscle (LPECT) and r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s muscles d u r i n g p a r a l y s i s demonstrating the a l t e r n a t i n g p a t t e r n o f x f i c t i v e ' h indlimb a c t i v i t y e l i c i t e d by i n j e c t i o n o f NMDA (34mM/0. 4LI1) i n t o the same s i t e as i n A. 191 1 sec 192 RITC LITC i i 2 sec 193 r e p l i c a b l y (4 times) evoked ^ f i c t i v e ' b i l a t e r a l a l t e r n a t i n g s t e p p i n g w i t h i n one minute p o s t - i n j e c t i o n which l a s t e d f o r approximately 8 minutes i n one animal ( F i g . 2 6B). NMDA i n j e c t i o n (20mM/0.2jil) i n t o a p a r a l y z e d b i r d from which ^ f i c t i v e ' walking c o u l d be e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n of the MLF showed no e f f e c t s . 194 DISCUSSION Neurochemical i n j e c t i o n i n t o s e l e c t e d r e g i o n s of the avian mid- and h i n d b r a i n w i t h EAA a g o n i s t s , a n t a g o n i s t s and Substance P produced a v a r i e t y o f locomotor responses i n decerebrate b i r d s . I n t r o d u c t i o n o f the glutamate a g o n i s t NMDA, but not glutamate i t s e l f , e l i c i t e d locomotion or reduced the c u r r e n t i n t e n s i t y t h r e s h o l d f o r locomotion when i n j e c t e d i n t o e l e c t r i c a l - s t i m u l a t i o n d e f i n e d locomotor r e g i o n s . The induced locomotion c o u l d be bl o c k e d by the i n t r o d u c t i o n o f the glutamate a n t a g o n i s t GDEE i n t o the same s i t e . Substance P a l s o e l i c i t e d locomotion f o l l o w i n g i t s i n j e c t i o n i n t o the pons. These r e s u l t s w i l l be d i s c u s s e d f o r each locomotor r e g i o n and an attempt w i l l be made t o e l u c i d a t e the neuroanatomical pathways which subserve them. The data from our study w i l l be compared t o t h a t found i n other s p e c i e s i n which s i m i l a r i n v e s t i g a t i o n s have been performed. Pontobulbar Locomotor S t r i p (PLS) I n j e c t i o n of NMDA i n t o the PLS, the p h y s i o l o g i c a l l y d e f i n e d locomotor r e g i o n which appears t o be synonymous wi t h the descending t r i g e m i n a l t r a c t and nucleus (TTD) (Noga et a l , , 1988), evoked locomotion i n b i r d s . The e f f e c t i v e i n j e c t i o n s i t e s were h i s t o l o g i c a l l y i d e n t i f i e d as l y i n g on the dorsomedial border between TTD and Cnd. These r e s u l t s are s i m i l a r t o those found by Noga et a l . , (1988) i n the cat, i n which i n j e c t i o n of glutamate e l i c i t e d locomotion or decreased the e l e c t r i c a l 195 t h r e s h o l d i n t e n s i t y necessary t o evoke walking. The av i a n neuroanatomical connections which subserve the observed locomotion are p r e s e n t l y unknown, but i n the r a t , EAA c o r t i c o f u g a l pathways descending t o the brainstem may pr o v i d e g l u t a m a t e r g i c input t o t h i s r e g i o n (Fagg and F o s t e r , 1983). S a l t and H i l l (1981, 1983) demonstrated : t h a t PDA ( c i s - 2 , 3 - p i p e r i d i n e c a r b o x y l a t e ) , a broad spectrum EAA ant a g o n i s t (acts at a l l r e c e p t o r t y p e s ) , but not the s p e c i f i c NMDA an t a g o n i s t , D-a-aminoadipate, c o u l d b l o c k v i b r i s s a l and noxious mechanical but not noxious thermal a f f e r e n t i nput t o c e l l s i n the nucleus c a u d a l i s o f TTD i n the urethane a n a e s t h e t i z e d r a t . They d i d not, however, examine the types of s t i m u l a t i o n which have been shown to e l i c i t locomotion i n the decerebrate p r e p a r a t i o n d i s c u s s e d above (e.g. pinna s t i m u l a t i o n - Aoki and Mori, 1981). Although NMDA r e c e p t o r b i n d i n g s t u d i e s i n mammals show low d e n s i t i e s o f NMDA r e c e p t o r s i n the r e g i o n o f TTD and surrounding l a t e r a l * r e t i c u l a r formation, Monaghan and Cotman (1985) surmise t h a t "there i s a t r e n d f o r motor-associated, v e n t r a l l y l o c a t e d , glutamate-using pathways t o have a lower d e n s i t y o f NMDA s i t e s than the c o r t i c a l , l i m b i c and s e n s o r y - a s s o c i a t e d glutamate u s i n g pathways". However, from these p r e l i m i n a r y f i n d i n g s , i t appears t h a t g l u t a m a t e r g i c pathways, p o s s i b l y impinging on NMDA re c e p t o r s i n the PLS/TTD r e g i o n , may p l a y some r o l e i n the c o n t r o l of locomotor behaviour. Why glutamate i t s e l f was not e f f e c t i v e i n the PLS i s unknown, although i t i s p o s s i b l e t h a t a f t e r i n j e c t i o n , the n e u r o t r a n s m i t t e r i s taken up be f o r e i t can spread t o a s u f f i c i e n t number of neurons t o e l i c i t locomotion. NMDA, on the other hand, i s l e s s s u s c e p t i b l e t o removal (Stone 196 and Burton, 1988) and through d i f f u s i o n through the t i s s u e , may t h e r e f o r e e x e r t i t s a c t i o n over a l a r g e r group of neuronal r e c e p t o r s (see Chapters 3 & 4). The neuroanatomical connections through which NMDA/glutamate serve a locomotor r o l e have yet t o be determined. A l s o , other EAA r e c e p t o r types cannot be r u l e d out, as the c o n c e n t r a t i o n s of NMDA i n j e c t e d , even at the lowest c o n c e n t r a t i o n s necessary t o evoke locomotion, may c r o s s r e a c t w i t h other glutamate r e c e p t o r subtypes (H. McLennan, p e r s o n a l communication). While Noga et al. (1988) found t h a t Substance P i n j e c t i o n i n t o the caudal PLS evoked locomotion i n the cat, i n j e c t i o n s i n t o comparable s i t e s i n the b i r d were i n e f f e c t i v e at e l i c i t i n g locomotor behaviour. Substance P has been l o c a l i z e d i n t r i g e m i n a l g a n g l i o n c e l l s and appears t o be a t r a n s m i t t e r i n v o l v e d i n p a i n p e r c e p t i o n ( K i s h i d a et a l . , 1985; f o r review see Dubner and Bennett, 1983). In the c a t , locomotion e l i c i t e d by Substance P i n j e c t i o n mimics t h a t seen f o l l o w i n g a noxious st i m u l u s ( S a l t and H i l l , 1983) and thus would appear to support the h y p o thesis t h a t PLS/TTD s t i m u l a t i o n - i n d u c e d locomotion i s i n v o l v e d i n the sensorimotor i n i t i a t i o n o f locomotion. In the b i r d , f u r t h e r s t u d i e s are r e q u i r e d t o examine a p o s s i b l e r o l e f o r Substance P i n locomotor c o n t r o l . C e n t r a l Nucleus, d o r s a l p a r t (Cnd) The d o r s a l p a r t of the medullary c e n t r a l nucleus (Cnd) g i v e s r i s e , i n p a r t , t o the r e t i c u l o s p i n a l pathway which 197 descends to a l l s p i n a l cord l e v e l s i n b i r d s (Cabot et al., 1982; Webster and Steeves, 1987). S e l e c t i v e l e s i o n s t u d i e s have demonstrated t h a t the r e t i c u l o s p i n a l pathway plays a major r o l e i n the descending c o n t r o l of locomotion i n a l l v e r t e b r a t e s s t u d i e d (Sholomenko and Steeves, 1987; f o r review see G r i l l n e r , 1976). In b i r d s , the i n f u s i o n of the glutamatergic agonist NMDA i n t o Cnd evoked locomotor behaviour which was blocked by the antagonist GDEE. The e f f e r e n t c o n t r i b u t i o n of Cnd to the r e t i c u l o s p i n a l pathway and i t s importance to the descending c o n t r o l of locomotion i n the b i r d and other species has been discussed p r e v i o u s l y (See Chapters 1 & 2). However, no previous i n j e c t i o n s of glutamate agonists i n t o t h i s region have been reported. More medial i n j e c t i o n s of glutamate and homocysteic a c i d i n t o the g i g a n t o c e l l u l a r tegmental f i e l d have been reported to induce locomotion i n the cat (Noga et al., 1988). In the lamprey, interneurons c o n t a i n i n g e x c i t a t o r y amino acids have been i d e n t i f i e d as s p e c i f i c a l l y impinging upon r e t i c u l o s p i n a l neurons which are i n v o l v e d i n the descending c o n t r o l of locomotion (Dubuc et al., 1988). Furthermore, Dubue and co-workers (Dubuc et al., 1988) have e s t a b l i s h e d the presence of NMDA receptors on lamprey r e t i c u l a r formation neurons. In a d d i t i o n , these EAA c o n t a i n i n g interneurons r e c e i v e t r i g e m i n a l , v e s t i b u l a r and ascending spinobulbar pathways input (Dubuc et al., 1988). Whether comparable interneurons e x i s t i n b i r d s remains to be determined. Receptor b i n d i n g s t u d i e s i n the r a t (Monaghan and Cotman, 1985) report low l e v e l s of NMDA/glutamate receptors i n the hi n d b r a i n r e t i c u l a r formation. Low l e v e l s of receptor do not, 198 however, p r e c l u d e a modulatory r o l e f o r t h i s n e u r o t r a n s m i t t e r (Monaghan and Cotman, 1985) i n the c o n t r o l o f locomotion from f o r e b r a i n s t r u c t u r e s which have been shown t o impinge on t h i s r e g i o n i n mammals (Fagg and F o s t e r , 1983). I t i s unknown at t h i s time whether t e l e n c e p h a l i c t o r e t i c u l a r formation EAA pathways comparable t o those i n mammals a l s o e x i s t i n b i r d s . However, a number of neuroanatomical r e t r o g r a d e t r a n s p o r t s t u d i e s have e s t a b l i s h e d a v a r i e t y o f l o c o m o t o r - r e l a t e d a f f e r e n t s t o t h i s r e g i o n which c o u l d a l s o be the source o f EAA input t o Cnd (see Chapters 3 & 4) . The above r e s u l t s , gathered from a broad p h y l o g e n e t i c range of s p e c i e s , s t r o n g l y i m p l i c a t e an e x c i t a t o r y r o l e f o r glutamate i n the c o n t r o l o f locomotion from r e t i c u l a r formation s t r u c t u r e s . Furthermore, the glutamate NMDA r e c e p t o r subtype appears t o be i n v o l v e d i n t h i s c o n t r o l . However, the gl u t a m a t e r g i c pathways and exact l o c a t i o n of glutamate involvement, be i t d i r e c t l y on r e t i c u l o s p i n a l neurons, in t e r n e u r o n s or both, remains t o be e l u c i d a t e d . C e n t r a l Nucleus, v e n t r a l p a r t (Cnv) S i m i l a r t o Cnd, neurons l o c a l i z e d w i t h i n Cnv g i v e r i s e t o a p o r t i o n of the descending r e t i c u l o s p i n a l p r o j e c t i o n which i s e s s e n t i a l f o r the i n i t i a t i o n and maintenance of locomotion i n b i r d s (Webster and Steeves, 1987; Sholomenko and Steeves, 1987) and other s p e c i e s ( G r i l l n e r , 1985). F o c a l e l e c t r i c a l s t i m u l a t i o n of t h i s r e g i o n e l i c i t s locomotion i n a wide range o f s p e c i e s (Steeves et a l . , 1986). P r e v i o u s papers i n t h i s s e r i e s have 199 e s t a b l i s h e d t h a t Cnv, l i k e Cnd, appears t o f u n c t i o n as an i n t e g r a t o r y and descending output c e n t r e f o r a v a r i e t y of sensory and c e n t r a l l y generated locomotor r e l a t e d i n p u t s . In t h i s study, i n j e c t i o n of NMDA, but not glutamate, i n t o Cnv e l i c i t e d locomotion which was bl o c k e d by GDEE i n f u s i o n . Both G a r c i a - R i l l and Skinner (1987a) and Noga et al. (1988) have found t h a t glutamate i n f u s i o n i n t o the medial r e t i c u l a r formation e l i c i t e d locomotion or decreased e l e c t r i c a l s t i m u l a t i o n t h r e s h o l d i n the ca t , although i n some cases the behaviour was s h o r t - l i v e d ( G a r c i a - R i l l and Skinner, 1987a). We u t i l i z e d NMDA i n an attempt t o s p e c i f y the r e c e p t o r type u n d e r l y i n g the p r o d u c t i o n o f locomotion by the e x c i t a t o r y amino a c i d s . One p o s s i b l e d i f f e r e n c e between our r e s u l t s and those d e s c r i b e d i n cat i s the l o n g e v i t y of locomotor behaviour produced by NMDA i n j e c t i o n . The apparent longer l a s t i n g a c t i o n of NMDA versus glutamate may r e s u l t from the slower uptake, and t h e r e f o r e , i n c r e a s e d e f f i c a c y , o f NMDA (Stone and Burton, 1988). L i k e Cnd, the sources o f g l u t a m a t e r g i c input t o Cnv remain t o be c h a r a c t e r i z e d . However, EAA p r o j e c t i o n s t o Cnv may a r i s e from more r o s t r a l s t r u c t u r e s (e.g. t e l e n c e p h a l o n ) , g l u t a m a t e r g i c r e t i c u l a r formation i n t e r n e u r o n s and ascending s p i n o b u l b a r p r o j e c t i o n s (Dubuc et a l . , 1988; Fagg and F o s t e r , 1983). The p a u c i t y o f glutamate neuroanatomical, n e u r o t r a n s m i t t e r and r e c e p t o r p r o f i l e data f o r b i r d s and other s p e c i e s leaves the p h y s i o l o g i c a l r o l e f o r g l u t a m a t e r g i c c o n t r o l of r e t i c u l a r formation neurons u n r e s o l v e d . However, combined data from neurochemical s t i m u l a t i o n s t u d i e s i n lamprey (Dubuc et al., 1988), b i r d and mammal ( G a r c i a - R i l l et a l . , 1985; G a r c i a - R i l l 200 and Skinner, 1987a; Noga et al., 1988) suggest an important e x c i t a t o r y r o l e f o r glutamate i n the c o n t r o l o f l o c o m o t i o n - r e l a t e d r e t i c u l a r formation s t r u c t u r e s . Pontine R e t i c u l a r Formation (RP) F o c a l e l e c t r i c a l s t i m u l a t i o n o f the v e n t r a l p o ntine r e t i c u l a r formation e l i c i t s locomotion i n the decerebrate b i r d p r e p a r a t i o n (Steeves et al., 1986). Neuroanatomical t r a c i n g s t u d i e s demonstrate t h a t RP gi v e s r i s e t o a component o f the descending r e t i c u l o s p i n a l pathway (Cabot et al., 1982; Webster and Steeves, 1987) In a d d i t i o n , RP p r o j e c t s t o motor r e l a t e d s t r u c t u r e s such as TTD and more caudal r e t i c u l a r formation n u c l e i (Webster and Steeves, i n p r e p a r a t i o n ) . In b i r d s , as i n mammals, RP r e c e i v e s a f f e r e n t i nput from a wide v a r i e t y o f motor r e l a t e d sources i n c l u d i n g T T D , tectum, cerebellum, Cnv, Cnd and v e s t i b u l a r n u c l e i (Hunt et a l . , 1977; Wold, 1978; Webster and Steeves, i n p r e p a r a t i o n ) . Thus, from a neuroanatomical viewpoint, the pontine r e t i c u l a r formation i s i n an i d e a l p o s i t i o n t o r e g u l a t e motor c o n t r o l . F o l l o w i n g the establishment of e l e c t r i c a l l y s t i m u l a t e d locomotion, i n f u s i o n of NMDA i n t o the v e n t r a l RP e l i c i t e d locomotor behaviour i n s e v e r a l animals. EAA pathways are known t o impinge on the pontine tegmentum from f o r e b r a i n s t r u c t u r e s i n the r a t (Fagg and F o s t e r , 1983) and NMDA-sensitive b i n d i n g s i t e s have been l o c a l i z e d i n the pons o f the r a t (Monaghan and Cotman, 1985). Our data suggest t h a t s i m i l a r NMDA b i n d i n g s i t e s e x i s t i n the b i r d . However, i t has yet t o be determined whether the EAA 201 p r o j e c t i o n s a r i s e from the f o r e b r a i n , pontine i n t e r n e u r o n s , ascending i n p u t s from b r a i n s t e m / s p i n a l c o r d or other r e g i o n s . Our r e s u l t s suggest t h a t p ontine r e t i c u l a r formation neurons are under g l u t a m a t e r g i c e x c i t a t o r y c o n t r o l . I n t e r e s t i n g l y , i n j e c t i o n o f Substance P i n t o RP produced weak b i l a t e r a l walking behaviour which appeared t o be s i g n i f i c a n t l y enhanced by NMDA i n f u s i o n i n t o the same s i t e . R einer (personal communication) d e s c r i b e s Substance P - c o n t a i n i n g f i b r e s l o c a l i z e d w i t h i n the paramedian pontine r e t i c u l a r formation near the v e n t r a l m i d l i n e . The o r i g i n o f these f i b r e s , however, i s u n s p e c i f i e d . Thus, s p e c u l a t i o n on the r o l e which Substance P has i n the c o n t r o l or modulation o f locomotor behaviours i n the pontine r e t i c u l a r formation would be premature. Mesencephalic R e t i c u l a r Formation (MRF) Two locomotion-evoking e l e c t r i c a l s t i m u l a t i o n s i t e s have been p r e v i o u s l y d e s c r i b e d i n the av i a n mesencephalon (See Chapter 2 and Sholomenko and Steeves, 1987b), The more l a t e r a l s i t e i s s i t u a t e d i n c l o s e p r o x i m i t y t o the i n t e r c o l l i c u l a r nucleus (ICo) of the a v i a n tectum (see Chapter 2, F i g u r e 1). A second, more medial s i t e , i s found i n the medial mesencephalic r e t i c u l a r formation v e n t r o l a t e r a l t o the red nucleus and medial t o the nucleus o f the ansa l e n t i c u l a r i s (mMRF) (see Chapter 2, F i g u r e 1). The mMRF r e c e i v e s a major a f f e r e n t p r o j e c t i o n from the deep t e c t a l l a y e r s (Hunt et a l . , 1977) and sends e f f e r e n t s t o tectum, h i g h c e r v i c a l s p i n a l c o r d and medial medullary 202 r e t i c u l a r formation (Reiner and Karten, 1982; Webster and Steeves, i n p r e p a r a t i o n ) . These e l e c t r o p h y s i o l o g i c a l and h o d o l o g i c a l c o n s i d e r a t i o n s , t h e r e f o r e , p o s s i b l y i m p l i c a t e t h i s r e g i o n as an avi a n e q u i v a l e n t o f the mammalian MLR d e s c r i b e d by G a r c i a - R i l l and Skinner (198 6). However, t h i s e q u i v a l e n c y i s l i m i t e d by the apparent l a c k o f an avian e q u i v a l e n t o f the mammalian a c e t y l c h o l i n e - c o n t a i n i n g PPN neurons (Steeves and Taccogna, i n p r e p a r a t i o n ) . Controversy s t i l l e x i s t s as t o whether the c h o l i n e r g i c PPN neurons a c t u a l l y u n d e r l i e the e f f e c t s o f MLR s t i m u l a t e d locomotion. Rye and co-workers (Rye et a l . , 1987, 1 9 8 8 ) , f o l l o w i n g a thorough examination of the c y t o a r c h i t e c t o n i c , c y t o c h e m i c a l and h o d o l o g i c a l r e l a t i o n s h i p s of the pedunculopontine nucleus i n the r a t , p o s t u l a t e d t h a t the PPN was not p a r t o f the MLR and s u b s t i t u t e d i n i t s p l a c e a r e g i o n termed the midbrain e x t r a p y r a m i d a l area. Only a complete p r o f i l e o f t h i s r e g i o n , p o s s i b l y based on a combination of i n t r a c e l l u l a r r e c o r d i n g , immunohistochemistry, i n t r a c e l l u l a r dye i n j e c t i o n and neurochemical s t i m u l a t i o n w i l l a l l e v i a t e t h i s c o n t r o v e r s y . In t h i s study, NMDA was found t o decrease the t h r e s h o l d f o r e l e c t r i c a l l y s t i m u l a t e d locomotion when i n f u s e d i n t o the mMRF i n one animal. S i m i l a r l y , i n the ca t , G a r c i a - R i l l et al. (1985) found t h a t glutamate i n f u s i o n i n t o the PPN/mMLR decreased the t h r e s h o l d f o r e l e c t r i c a l l y s t i m u l a t e d locomotion but d i d not, i n i s o l a t i o n , e l i c i t locomotion. Mogenson and Brudzynski (1986) found t h a t glutamate i n j e c t i o n i n t o the PPN/MLR i n c r e a s e d locomotor a c t i v i t y i n the f r e e l y moving r a t . Reasons f o r the apparent d i s c r e p a n c y between the f i n d i n g s i n b i r d and cat versus those found i n r a t are p r e s e n t l y not known, although v a r i a t i o n 203 i n the s p a t i a l d i s t r i b u t i o n o f c e l l s w i t h i n the locomotor re g i o n s i n these s p e c i e s may account f o r some of the d i f f e r e n c e s (Mogenson and Brudzynski, 1986). In combination with our data from c h o l i n e r g i c i n j e c t i o n s t u d i e s d i s c u s s e d i n Chapter 3, and the congruence of those data i n the b i r d w i t h those found i n mammals, i t appears p o s s i b l e t h a t avian e q u i v a l e n t s o f the MLRs e x i s t i n b i r d s . F u r t h e r study w i l l be r e q u i r e d t o l e n d g r e a t e r s t r e n g t h t o t h i s h y p o t h e s i s . Medial L o n g i t u d i n a l F a s c i c u l u s (MLF) Previou s s t u d i e s demonstrated t h a t locomotion c o u l d be e l i c i t e d both by e l e c t r i c a l and neurochemical (carbachol) s t i m u l a t i o n o f the MLF at pontomedullary l e v e l s . The i n j e c t i o n of NMDA i n t o t h i s r e g i o n a l s o e l i c i t e d r e p e a t a b l e locomotion i n the decerebrate b i r d , although only i n one animal. As d i s c u s s e d p r e v i o u s l y (Chapters 2 & 3), the MLF i s g e n e r a l l y r e c o g n i z e d as a f i b r e t r a c t which c o n t a i n s a v a r i e t y o f i n t r a n u c l e a r p r o j e c t i o n s . Thus, i t was s u r p r i s i n g t h a t neurochemical i n j e c t i o n i n t o t h i s r e g i o n e l i c i t e d locomotion. However, s e r o t o n i n - c o n t a i n i n g neurons which s t a i n p o s i t i v e l y f o r a c e t y l c h o l i n e s t e r a s e have been l o c a l i z e d i n c l o s e p r o x i m i t y t o the i n j e c t e d s i t e and may be a c t i v a t e d by neurochemical i n j e c t i o n (Dube and Parent, 1981). Whether these a v i a n neurons a l s o possess EAA r e c e p t o r s i s p r e s e n t l y unknown, but a v a r i e t y of r e p o r t s from mammalian s p e c i e s do not r e p o r t EAA r e c e p t o r s l o c a l i z e d i n t h i s r e g i o n (Monaghan and Cotman, 1985; Monaghan et al., 1985; Greenamyre et a l . , 1 9 8 4 ) . At present, t h e r e f o r e , EAA 204 e x c i t a t i o n o f locomotion from t h i s s i t e l a c k s even a p u t a t i v e neuroanatomical s u b s t r a t e . The r e s u l t o f NMDA i n j e c t i o n - i n d u c e d locomotion i n t h i s r e g i o n , however, suggests p o s s i b i l i t i e s f o r f u t u r e study. Pharmacological C o n s i d e r a t i o n s In t h i s study, d i r e c t i n t r a c e r e b r a l i n f u s i o n o f NMDA, but not glutamate, proved e f f e c t i v e at e l i c i t i n g locomotion i n s e v e r a l e l e c t r o p h y s i o l o g i c a l ^ d e f i n e d avian locomotor r e g i o n s . In some cases, the locomotion c o u l d be bl o c k e d by GDEE i n f u s i o n i n t o the same s i t e . The n e u r o t r a n s m i t t e r glutamate has been demonstrated t o be e f f e c t i v e at th r e e d i f f e r e n t proposed r e c e p t o r subtypes i n the CNS. The r e c e p t o r s are c l a s s i f i e d a c c o r d i n g t o t h e i r d i f f e r e n t i a l a f f i n i t y f o r the a g o n i s t s NMDA, kai n a t e and q u i s q u a l a t e ( f o r review, see Stone and Burton, 1988/ Watkins and Olverman, 1987), although the a g o n i s t f o r each r e c e p t o r c r o s s r e a c t s t o some extent w i t h the other two re c e p t o r s (D. Magnusson, p e r s o n a l communication). At the NMDA rec e p t o r , NMDA has been found t o be 10-1000 times more potent than glutamate and i s r e l a t i v e l y s p e c i f i c f o r i t s r e c e p t o r (Watkins and Olverman, 1987). The potency i s dependent on the type of p r e p a r a t i o n and may r e f l e c t the r a t e o f NMDA (slow) versus glutamate (fast) uptake (Stone and Burton, 1988). Recent r e s u l t s suggest t h a t the s i t e o f NMDA a c t i o n may be both p r e -and p o s t s y n a p t i c ( f o r review, see Stone and Burton, 1988). The a c t i o n of NMDA i s complex and i n v o l v e s a magnesium and v o l t a g e dependent i n c r e a s e i n membrane p e r m e a b i l i t y probably t o cal c i u m 205 and sodium i o n s . T h i s i s d i s t i n c t from the a c t i o n o f the non-NMDA r e c e p t o r s (e.g. q u i s q u a l a t e and k a i n a t e ) , whose a c t i v a t i o n r e s u l t s i n a voltage-independent i n c r e a s e i n membrane conductance mediated by sodium (q u i s q u a l a t e and k a i n a t e - f a s t d i r e c t l i n k t o ionophore ( q u i s q u a l a t e response p o t e n t i a t e d by z i n c , k a i n a t e not p o t e n t i a t e d by zinc) or v i a an i n t r a c e l l u l a r second messenger system (quisqualate) ( f o r review, see Choi, 1988 & Stone and Burton, 1988). GDEE, u t i l i z e d i n t h i s study as a glutamate a n t a g o n i s t , was o r i g i n a l l y found t o decrease neuronal s e n s i t i v i t y at glutamate r e c e p t o r s p r e f e r e n t i a l l y over a s p a r t a t e r e c e p t o r s (Haldeman and McLennan, 1972). Subsequent t o the f i n d i n g s o f Haldeman and McLennan (1972), GDEE has been demonstrated t o b l o c k a l l t h r e e glutamate r e c e p t o r subtypes, but with g r e a t e r e f f i c a c y at the qu i s q u a l a t e r e c e p t o r than at the NMDA and ka i n a t e r e c e p t o r s (Watkins and Olverman, 1987; Stone and Burton, 1988). The above glutamate pharmacology i l l u s t r a t e s the inhe r e n t problems i n a s s e s s i n g the exact type and l o c a t i o n of r e c e p t o r s r e s p o n s i b l e f o r a c t i v a t i n g (or bl o c k i n g ) neurons i n v o l v e d i n locomotion e l i c i t e d by NMDA i n f u s i o n . The NMDA c o n c e n t r a t i o n s at the i n j e c t i o n p o i n t (see Table 3 and r e s u l t s ) were high e r than p h y s i o l o g i c a l c o n c e n t r a t i o n s , and probably a c t i v a t e d a l l t h r e e glutamate r e c e p t o r subtypes (D. Magnusson, p e r s o n a l communication), although the NMDA c o n c e n t r a t i o n , f o l l o w i n g d i f f u s i o n i n t o the t i s s u e s , would presumably have decreased as i t spread throught the CNS t i s s u e . I t i s not p o s s i b l e t o determine which type(s) o f glutamate r e c e p t o r was a c t i v a t e d i n the present experiments. H o p e f u l l y , f u t u r e a v i a n s t u d i e s t h a t 206 i d e n t i f y the neuroanatomical l o c a t i o n of glutamate r e c e p t o r subtypes w i l l a s s i s t i n determining which p o s s i b l e r e c e p t o r s u n d e r l i e the observed r e s u l t s . Future s t u d i e s u t i l i z i n g more s p e c i f i c NMDA r e c e p t o r a n t a g o n i s t s , such as 2-amino-5-phosphonopentanoic a c i d (AP5) (Watkins and Olverman, 1987), may a l s o be u s e f u l i n t h i s regard. 207 CHAPTER 6 AVIAN LOCOMOTOR PATTERNS IN THE ABSENCE OF PHASIC AFFERENT INPUT - THE 'FICTIVE' PREPARATION 208 INTRODUCTION In v e r t e b r a t e s , both the i n i t i a t i o n and ongoing c o n t r o l of locomotion are dependent upon a h i e r a r c h i c a l o r g a n i z a t i o n o f b r a i n and s p i n a l c o r d n e u r a l networks. These c i r c u i t s , i n t u r n , are modulated by p e r i p h e r a l a f f e r e n t feedback d u r i n g locomotion and the i n t e r a c t i o n between the two i s r e s p o n s i b l e f o r normal p r o d u c t i o n of locomotor p a t t e r n ( f o r reviews see G r i l l n e r , 1985; M c C l e l l a n , 198 6). Pr e v i o u s a v i a n s t u d i e s have demonstrated t h a t b i r d s possess brainstem and s p i n a l c o r d locomotor networks analogous t o those found i n h i g h e r and lower v e r t e b r a t e s (Jacobson & Hollyday, 1982; Steeves et a l . , 1986, 1987a,b; Ten Cate, I960, 1962). These networks i n c l u d e : 1) s p i n a l c o r d ^pattern generators', which i n the absence of descending l; s u p r a s p i n a l i n p u t , can produce x s p i n a l s t e p p i n g ' (Jacobson & Hollyday, 1982; Sholomenko & Steeves, 1987; ten Cate, 1960, 1962), 2) descending pathways p r o j e c t i n g through the v e n t r o l a t e r a l f u n i c u l i t h a t are e s s e n t i a l f o r i n i t i a t i n g locomotion and o r i g i n a t e i n the h i n d b r a i n r e t i c u l a r formation (Sholomenko & Steeves, 1987; Steeves et a l . , 1987), and 3) d i s c r e t e brainstem r e g i o n s , which when e l e c t r i c a l l y or c h e m i c a l l y s t i m u l a t e d i n a decerebrate p r e p a r a t i o n , evoke walking and/or f l y i n g (Sholomenko & Steeves, 1987a,b, 1988; Steeves et a l . , 1987). In t h i s chapter, I examine whether f o r e l i m b and hindlimb p h a s i c a f f e r e n t input i s a p r e r e q u i s i t e f o r the p r o d u c t i o n of av i a n locomotor p a t t e r n s by the c e n t r a l nervous system. P h a s i c a f f e r e n t feedback was e l i m i n a t e d through p a r a l y z a t i o n of the 209 animal and then brainstem locomotor r e g i o n s were s t i m u l a t e d i n the decerebrate b i r d w h i le r e c o r d i n g e l e c t r o n e u r o g r a p h i c a c t i v i t y from locomotor-muscle nerves. The term x f i c t i v e ' has been used to d e s c r i b e t h i s type of n e u r a l a c t i v i t y a s s o c i a t e d w i t h locomotion or motor output d u r i n g neuromuscular p a r a l y s i s (Perret et al., 1972). My r e s u l t s demonstrate t h a t a l l unparalyzed locomotor p a t t e r n s can be e l i c i t e d i n a p a r a l y z e d decerebrate animal i n c l u d i n g : 1) a l t e r n a t i n g (out of phase) l e g stepping, 2) b i l a t e r a l ( i n phase) jumping (or hopping), 3) c o a c t i v a t e d walking and f l y i n g ( i . e . combined lumbar and c e r v i c a l c o r d a c t i v i t y ) and 4) b i l a t e r a l ( i n phase) wing f l a p p i n g . 210 MATERIALS AND METHODS Surgery S u r g i c a l procedures have been p r e v i o u s l y d e s c r i b e d i n d e t a i l i n Chapter 2. D e c e r e b r a t i o n l e v e l s of spontaneous versus non-spontaneous animals are d e s c r i b e d i n Chapter 7. Brainstem S t i m u l a t i o n E i t h e r i n t r a c e r e b r a l e l e c t r i c a l or chemical s t i m u l a t i o n of l o c a l i z e d r e g i o n s w i t h i n the brainstem was used t o evoke locomotion from a non-spontaneously locomoting p r e p a r a t i o n ( f o r methods, s t i m u l a t i o n parameters and procedures, see Chapters 2-5 t h i s t h e s i s ) . A f t e r e s t a b l i s h i n g an optimum brainstem s t i m u l a t i o n s i t e and r e c o r d i n g EMG a c t i v i t y from the PECT and ITC muscles d u r i n g evoked locomotion, each animal (17 Canada geese, 7 Pekin ducks) was p a r a l y z e d with t u b o c u r a r i n e c h l o r i d e ( i n i t i a l l y 0.15mg/kg; supplemented as required) and u n i d i r e c t i o n a l v e n t i l a t i o n (UDV) was i n i t i a t e d . Electroneurograms (ENGs) were recorded with p l a t i n u m hook e l e c t r o d e s p l a c e d on nerves d i r e c t l y i n n e r v a t i n g the main body of ITC and PECT muscles. S i g n a l s were a m p l i f i e d (10,000x), f i l t e r e d and recorded i n the same manner as the EMG s i g n a l s . ENG r e c o r d i n g s were made d u r i n g spontaneous or evoked locomotion and EMG muscle a c t i v i t y was monitored t o ensure t h a t t h e r e was no movement i n response t o s t i m u l a t i o n . At the c o n c l u s i o n of each experiment, the p o s i t i o n of the m i c r o p i p e t t e t i p was marked with an e l e c t r o l y t i c l e s i o n made by p a s s i n g a d i r e c t c a t h o d a l c u r r e n t of 3mA f o r 5 seconds at the s t i m u l a t i o n s i t e . H i s t o l o g i c a l i d e n t i f i c a t i o n of s t i m u l a t i o n s i t e s has been p r e v i o u s l y d e s c r i b e d (Chapter 2 ) . 212 RESULTS Spontaneous Locomotion S e v e r a l h i g h decerebrate b i r d s (N=6) e x h i b i t e d spontaneous p e r i o d s of walking i n response t o a moving t r e a d m i l l b e l t ( F i g . 27A). When the t r e a d m i l l b e l t was stopped, s e v e r a l o f the b i r d s maintained an u p r i g h t s t a n d i n g p o s t u r e . A f t e r p a r a l y z a t i o n , 2 of the 6 animals showed spontaneous a l t e r n a t i n g ITC n e u r a l a c t i v i t y p a t t e r n s c h a r a c t e r i s t i c of walking ( i . e . ^ f i c t i v e ' l ocomotion). As shown f o r one of these animals i n F i g . 27, the long l a s t i n g (>30min) spontaneous a l t e r n a t i n g ITC a c t i v i t y p a t t e r n s were s i m i l a r both b e f o r e ( F i g . 27A, EMGs) and a f t e r ( F i g . 27B, ENGs) p a r a l y z a t i o n . However, the step frequency was reduced somewhat i n both b i r d s d u r i n g * f i c t i v e ' walking r e l a t i v e t o the p r e p a r a l y z e d s t a t e ( F i g . 28, spontaneous). The other 4 animals d i d not demonstrate spontaneous x f i c t i v e ' locomotion i n response t o the moving t r e a d m i l l b e l t . However, two of these animals animals d i d show short b u r s t s (5-10 seconds) of Xf i c t i v e ' walking i n response t o exteroceptive s t i m u l a t i o n of the head. In the f i n a l 2 b i r d s , x f i c t i v e ' locomotion was i n i t i a t e d and maintained only i n response t o f o c a l e l e c t r i c a l s t i m u l a t i o n of brainstem locomotor r e g i o n s . E l e c t r i c a l l y S t i m u l a t e d Locomotion E l e c t r i c a l s t i m u l a t i o n of brainstem regions i n c l u d i n g the pontobulbar locomotor s t r i p , medullary r e t i c u l a r formation, 213 F i g u r e 27. B i l a t e r a l a l t e r n a t i n g w a l k i n g , a c t i v i t y i n a spontaneously locomoting b i r d b e f o r e (A) and a f t e r (B) p a r a l y z a t i o n . The t r a n s e c t i o n l e v e l (dotted l i n e ) which allows p o s t - d e c e r e b r a t i o n spontaneous locomotion i s shown i n the s a g g i t a l s e c t i o n . A: EMG t r a c e s from l e f t and r i g h t ITC (*) muscles d u r i n g spontaneous t r e a d m i l l walking. B: Subsequent ENG t r a c e s from l e f t and r i g h t ITC nerves i n the p a r a l y z e d animal showing spontaneous walking p a t t e r n a c t i v i t y . * The i l i o t i b i a l i s c r a n i a l i s (ITC) muscle i s synonymous with the mammalian s a r t o r i u s muscle. 214 A - PRE-PARALYZED EMG B_ - PARALYZED ENG LEFT ITC RIGHT ITC ^ . i i i ^ l i ! ^ i H l ^ i . r t , . I ^ I .iin..^ I 1 2 sec 215 F i g u r e 28. Histogram of e l e c t r i c a l s t i m u l a t i o n - i n d u c e d and• spontaneous step frequency d u r i n g p r e - p a r a l y z e d (small c r o s s hatch) and p a r a l y z e d x f i c t i v e ' ( l a r g e c r o s s hatch) s t e p p i n g . P r e - p a r a l y z e d data have been normalized t o 100%. P a r a l y z e d ^ f i c t i v e ' v a l u e s are shown as % change r e l a t i v e t o p r e - p a r a l y z e d data. A c t u a l p r e - p a r a l y z e d and p a r a l y z e d x f i c t i v e ' step f r e q u e n c i e s f o r each b i r d (numbers above record) were averaged from t r i a l s evoked at brainstem s t i m u l a t i o n t h r e s h o l d c u r r e n t i n t e n s i t i e s (numbers i n b r a c k e t s ) . 216 200 - i 175 -150 -125 -100 -75-50-25-0 3 ZZ I I I I m % 1° I I £ w _ | • U II §3 it t i 1: I 11 CNJ *— m~ fill • II 11 1 - 2 3 4 5 6 7 8 CO I I « C3 1 i 1 2 ANIMAL I (Electrical brainstem s t i m u l a t i o n ) (Spontaneous) - PRE-PARALYZED S3 = PARALYZED 217 r o s t r a l p o n t i n e r e t i c u l a r f o r m a t i o n , m e s e n c e p h a l i c r e t i c u l a r f o r m a t i o n a n d m e d i a l l o n g i t u d i n a l f a s c i c u l u s p r o d u c e d l o c o m o t i o n i n 16 a n i m a l s . The v a r i e t y o f p a t t e r n s i n t h e e v o k e d l o c o m o t i o n d e p e n d e d n o t o n l y on t h e c o n d i t i o n o f t h e d e c e r e b r a t e a n i m a l , b u t a l s o on t h e l o c a t i o n o f t h e s t i m u l a t i n g e l e c t r o d e w i t h i n t h e b r a i n s t e m . However, t h e e l i c i t e d l o c o m o t o r p a t t e r n was c o n s t a n t f o r any g i v e n s i t e . I n one a n i m a l , f o r e x a m p l e , a t m o d e r a t e l y weak c u r r e n t s t r e n g t h s (25-100 uA), m o n o p o l a r e l e c t r i c a l s t i m u l a t i o n o f t h e d e s c e n d i n g t r i g e m i n a l n u c l e u s (TTD) ( K a r t e n & Hodos, 1967) e v o k e d w a l k i n g ( F i g . 2 9 A ) . A f t e r p a r a l y z a t i o n , as d e t e r m i n e d b y c o m p a r i n g a l t e r n a t i n g f i r i n g p a t t e r n s o f t h e ITC m u s c l e s a n d n e r v e s , a s i m i l a r w a l k i n g p a t t e r n was e l i c i t e d . H owever, a h i g h e r s t i m u l a t i o n i n t e n s i t y was r e q u i r e d t o i n i t i a t e w a l k i n g f o l l o w i n g p a r a l y z a t i o n ( F i g . 2 9 B ) . Of t h e e i g h t a n i m a l s w h i c h w a l k e d i n b o t h t h e u n p a r a l y z e d a n d p a r a l y z e d s t a t e s , t h e a v e r a g e d f r e q u e n c y o f s t e p p i n g o b s e r v e d a t t h r e s h o l d s t i m u l a t i o n l e v e l s d e c r e a s e d i n s e v e n b i r d s f r o m t h e u n p a r a l y z e d (1.6Hz±0.27) t o t h e p a r a l y z e d (1.0Hz±0.18) c o n d i t i o n ( F i g . 28, e l e c t r i c a l b r a i n s t e m s t i m u l a t i o n ) . A l t h o u g h t h e mean s t e p p i n g f r e q u e n c i e s o f t h e 2 g r o u p s w ere n o t s i g n i f i c a n t l y d i f f e r e n t (ANOVA, p = . 0 7 5 ) , t h e mean t h r e s h o l d s t i m u l a t i o n i n t e n s i t y n e c e s s a r y t o e v o k e w a l k i n g i n t h e p a r a l y z e d b i r d (176±25LIA) was s i g n i f i c a n t l y g r e a t e r (p<0.005) t h a n i n t h e u n p a r a l y z e d a n i m a l s (67±8LIA) . E l e c t r i c a l s t i m u l a t i o n o f o t h e r l o c o m o t o r r e g i o n s c h a r a c t e r i s t i c a l l y e l i c i t e d w a l k i n g b e h a v i o u r a t l p w e r s t i m u l a t i o n i n t e n s i t i e s , w i t h r e c r u i t m e n t o f w i n g a c t i v i t y as i n t e n s i t y i n c r e a s e d . T h i s p a t t e r n i s shown f o r one a n i m a l 218 F i g u r e 29. B i l a t e r a l a l t e r n a t i n g walking a c t i v i t y evoked by f o c a l e l e c t r i c a l s t i m u l a t i o n of the h i n d b r a i n b e f o r e (A) and a f t e r (B) p a r a l y z a t i o n . The t r a n s e c t i o n l e v e l i s shown by the d o t t e d l i n e The medullary s t i m u l a t i o n s i t e , marked by the t r i a n g l e i n the t r a n s v e r s e and s a g g i t a l s e c t i o n s , was l o c a t e d i n the descending t r a c t of the t r i g e m i n a l nerve (PLS). A: EMG t r a c e s from l e f t and r i g h t l e g ITC muscles d u r i n g e l e c t r i c a l l y s t i m u l a t e d walking. B: ENG t r a c e s from l e f t and r i g h t ITC nerves showing evoked walking p a t t e r n s i n the p a r a l y z e d b i r d . 219 A - PRE- P A R A L Y Z E D E M G LEFT ITC « RIGHT ITC I + M M T ' O T ' M 2 sec T T D STIMULATION SITE LEVEL OF DECEREBRATION B - P A R A L Y Z E D E N G LEFT ITC 4+ RIGHT ITC , n O 4 n I < | i » UK i 1 2 sec 220 stimulated within the region of the mesencephalic r e t i c u l a r formation (MRF) p r i o r to paralyzation (Fig. 30A). At a stimulation i n t e n s i t y of 40LIA, walking movements were i n i t i a t e d and increased i n frequency as i n t e n s i t y was increased. When current i n t e n s i t y reached approximately 90/iA, b i l a t e r a l wing flapping was also observed. The same sequence of events was not observed i n the paralyzed animal (Fig. 30B), but coactivation of the nerves from both legs and one wing was observed. As seen previously during walking alone, the e l e c t r i c a l current strengths necessary to evoke rhythmical locomotor a c t i v i t y were at least two times greater i n the paralyzed versus unparalyzed animal. In-phase wing flapping alone, c h a r a c t e r i s t i c of f l y i n g behavior, was also often e l i c i t e d i n response to e l e c t r i c a l stimulation of s p e c i f i e d brainstem regions (Fig. 31A). After paralyzation, a si m i l a r pattern of evoked motor a c t i v i t y could be recorded from the PECT nerves (Fig. 31B). The average current strength necessary, however, to evoke x f i c t i v e ' f l y i n g i n the paralyzed birds (155±27/iA) was s i g n i f i c a n t l y greater (ANOVA, p=0.036) than that required to i n i t i a t e locomotion i n the unparalyzed birds (71±26fiA, n=6) (Fig. 32) . In spite of t h i s increased stimulus intensity, the six animals which e l i c i t e d both unparalyzed flapping and Xf i c t i v e ' flapping displayed a s i g n i f i c a n t reduction (ANOVA, p=0.018) in evoked flapping frequency after paralysis was i n i t i a t e d (from 2.9Hz±0.4 to 1.6Hz±0.2) (Fig. 32). 221 F i g u r e 30. C o - a c t i v a t i o n of l e g and wing a c t i v i t y evoked by f o c a l e l e c t r i c a l s t i m u l a t i o n of the midbrain b e f o r e (A) and a f t e r (B) p a r a l y z a t i o n . The t r a n s e c t i o n l e v e l i s shown by the d o t t e d l i n e . The midbrain s t i m u l a t i o n s i t e , marked by the t r i a n g l e i n both s a g g i t a l and t r a n s v e r s e s e c t i o n s , was l o c a t e d i n the medial mesencephalic r e t i c u l a r formation. A: EMG t r a c e s from l e f t and r i g h t P e c t o r a l i s (PECT) (top 2 t r a c e s ) and l e f t and r i g h t ITC (bottom 2 t r a c e s ) muscles d u r i n g e l e c t r i c a l l y s t i m u l a t i o n showing t r a n s i t i o n from walking alone to simultaneous w a l k i n g / f l y i n g . B: ENG t r a c e s from l e f t P e c t o r a l i s (top t r a c e ) and l e f t and r i g h t ITC (bottom 2 t r a c e s ) nerves showing c o a c t i v a t e d evoked locomotor p a t t e r n s of the l e g s and wing d u r i n g e l e c t r i c a l s t i m u l a t i o n i n the p a r a l y z e d animal. Due t o r e c o r d i n g d i f f i c u l t y , only a s i n g l e wing t r a c e i s shown i n these data. However, wings are normally phase l o c k e d and b i l a t e r a l l y synchronous d u r i n g both p a r a l y z e d and a c t u a l locomotion (see F i g u r e 31). 222 A - PRE-PARALYZED E M G STIMULATION SITE LEVEL OF DECEREBRATION B - P A R A L Y Z E D E N G LEFT PECT LEFT ITC m I t t « i *<••» RIGHT ITC H j \ 2i»c 223 F i g u r e 31. B i l a t e r a l synchronous f l y i n g a c t i v i t y evoked by f o c a l e l e c t r i c a l s t i m u l a t i o n of the h i n d b r a i n b e f o r e (A) and a f t e r (B) p a r a l y z a t i o n . The t r a n s e c t i o n l e v e l i s shown by the d o t t e d l i n e . The medullary s t i m u l a t i o n s i t e , marked by the t r i a n g l e i n the t r a n s v e r s e and s a g g i t a l s e c t i o n s , was l o c a t e d i n the d o r s a l p a r t of the medullary c e n t r a l nucleus ( r e t i c u l a r f o r m a t i o n ) . A: EMG t r a c e s from l e f t and r i g h t P e c t o r a l i s muscles d u r i n g e l e c t r i c a l l y s t i m u l a t e d f l y i n g . B: ENG t r a c e s from l e f t and r i g h t P e c t o r a l i s nerves showing evoked f l y i n g p a t t e r n s i n the p a r a l y z e d b i r d . 224 A - PRE-PARALYZED EMG LEFT PECT RIGHT PECT TTD 1 sec STIMULATION SITE O C V LEVEL OF DECEREBRATION B-PARALYZED ENG LEFT PECT RIGHT PECT 2 sec 225 F i g u r e 32. Histogram of brainstem s t i m u l a t i o n - i n d u c e d wingbeat frequency d u r i n g p r e - p a r a l y z e d (small c r o s s hatch) and p a r a l y z e d x f i c t i v e ' ( l a r g e c r o s s hatch) f l a p p i n g . P r e - p a r a l y z e d data have been normalized to 100%. P a r a l y z e d ^ f i c t i v e ' v a l u e s f o r each b i r d are shown as % change r e l a t i v e t o p r e - p a r a l y z e d data. A c t u a l p r e - p a r a l y z e d and p a r a l y z e d * f i c t i v e ' wingbeat f r e q u e n c i e s (numbers above record) were averaged from f l a p p i n g evoked at t h r e s h o l d s t i m u l a t i o n i n t e n s i t i e s (numbers i n b r a c k e t s ) . 226 I W I N G B E A T F R E Q U E N C Y Chemically S t i m u l a t e d Locomotion D i r e c t i n t r a c e r e b r a l i n f u s i o n o f s p e c i f i c n e u r o t r a n s m i t t e r a g o n i s t s and a n t a g o n i s t s a l s o evoked locomotion i n p a r a l y z e d decerebrate b i r d s (see Chapters 3-5). Carbachol i n j e c t i o n (N = 3) (l(jl at 0.2ml/min/ 27mM i n PBS) i n t o the ventromedial brainstem r e t i c u l a r formation [nucleus r e t i c u l a r i s g i g a n t o c e l l u l a r i s (Rgc)] evoked a l t e r n a t i n g b u r s t i n g a c t i v i t y i n the r i g h t and l e f t l e g ITC nerves o f a p a r a l y z e d goose ( F i g . 33A). Carbachol a c t i v a t i o n o f locomotor a c t i v i t y was a l s o r e c o r d e d i n more d i s t a l l e g muscle nerves (e.g. gastrocnemius) i n d i c a t i n g t h a t the p a r a l y z e d locomotor p a t t e r n was not r e s t r i c t e d t o j u s t the major h i p muscles. Carbachol-induced locomotor p a t t e r n s mimicked those seen i n response t o f o c a l e l e c t r i c a l s t i m u l a t i o n of the brainstem both b e f o r e and a f t e r p a r a l y z a t i o n . I n j e c t i o n of N-methyl-d-aspartate (NMDA) i n 2 animals (0.4fil at 0.2jj.l/min, 5 or 30mM i n PBS) i n t o the caudal r e t i c u l a r formation [nucleus c e n t r a l i s medulla oblongata, pars d o r s a l i s (Cnd)] produced s i m i l a r locomotor responses i n both unparalyzed and p a r a l y z e d b i r d s . F i g u r e 33B shows NMDA a c t i v a t i o n of the l e f t and r i g h t l e g ITC nerves o f a decerebrate p a r a l y z e d goose i n an in-phase hopping or jumping locomotor p a t t e r n . T h i s locomotor p a t t e r n was i d e n t i c a l t o t h a t observed i n a d i f f e r e n t u n p aralyzed b i r d a f t e r an i n t r a c e r e b r a l i n j e c t i o n o f NMDA. Both a l t e r n a t i n g (e.g. walking) and in-phase (e.g. hopping) locomotor p a t t e r n s can be e l i c i t e d by i n t r a c e r e b r a l i n j e c t i o n of NMDA i n t o the brainstem, however, i t i s not p r e s e n t l y c l e a r what 228 F i g u r e 33. B i l a t e r a l * f i c t i v e ' hindlimb nerve a c t i v i t y e l i c i t e d by neurochemical m i c r o i n j e c t i o n o f ca r b a c h o l and NMDA. A: ENG t r a c e s from the l e f t and r i g h t ITC nerves showing a l t e r n a t i n g A f i c t i v e ' walking a c t i v i t y f o l l o w i n g i n j e c t i o n o f car b a c h o l ( I . O L I I : 0.2Lil/min : lOOmM) , a c h o l i n e r g i c a g o n i s t , i n t o the pons. The s i t e i n j e c t e d ( t r i a n g l e ) , as shown i n the t r a n s v e r s e s e c t i o n A, was l o c a t e d w i t h i n the pontine g i g a n t o c e l l u l a r r e t i c u l a r formation. B: ENG t r a c e s from the l e f t and r i g h t ITC nerves showing synchronous in-phase a c t i v i t y ( ^ f i c t i v e ' hopping or g a l l o p i n g ) a f t e r i n j e c t i n g NMDA (0.2fil : 20mM) , a gl u t a m a t e r g i c a g o n i s t , i n t o the d o r s a l p a r t o f the medullary c e n t r a l nucleus ( t r i a n g l e i n t r a n s v e r s e s e c t i o n B ). 229 A - CARBACHOL - PARALYZED ENG LEFT ITC RIGHT ITC 3 s e c \ UJ fl-v PaM T T D C n d A CARBACHOL STIMULATION SITE B NMDA STIMULATION SITE B - NMDA - PARALYZED ENG LEFT ITC RIGHT ITC 3 sec 230 determines which p a t t e r n w i l l be expressed (Sholomenko & Steeves, i n p r e p a r a t i o n ) . The time course of NMDA a c t i v a t i o n i n both unparalyzed and p a r a l y z e d animals was s i m i l a r , with the onset of locomotor a c t i v i t y b e g i n n i n g w i t h i n 2 minutes of the i n t r a c e r e b r a l i n j e c t i o n . Locomotion (both unparalyzed and paralyzed) ceased 10-15 minutes p o s t - i n j e c t i o n . 231 DISCUSSION The main f i n d i n g of the present study i s t h a t p a r a l y z e d decerebrate b i r d s ( i . e . * f i c t i v e ' p r e p a r a t i o n s ) are capable of producing a l l the same locomotor p a t t e r n s as unparalyzed animals, r e g a r d l e s s of whether the ^ f i c t i v e ' locomotion i s generated: 1) spontaneously ( F i g . 2 7 B ) , 2) i n response t o f o c a l e l e c t r i c a l s t i m u l a t i o n ( F i g . 29B, 30B, 31B), or 3) i n response t o d i r e c t i n t r a c e r e b r a l chemical i n f u s i o n i n t o brainstem locomotor r e g i o n s ( 3 3 A , B ). To my knowledge, t h i s i s the f i r s t example of * f i c t i v e ' b i p e d a l locomotion i n a v e r t e b r a t e . Furthermore, the c o a c t i v a t i o n o f both modes of locomotor behaviour (stepping and f l a p p i n g ) ( F i g . 30A,B) i l l u s t r a t e s t h a t r e l a t i v e l y complex a c t i v a t i o n of brainstem and s p i n a l locomotor networks can a l s o occur i n the absence of p h a s i c p e r i p h e r a l feedback. I t i s c l e a r from the present o b s e r v a t i o n s t h a t rhythmic motor a c t i v i t y resembling walking and f l y i n g can be e l i c i t e d i n the absence of p h a s i c p e r i p h e r a l input, thereby i m p l i c a t i n g the CNS i n g e n e r a t i n g a c o n s i d e r a b l e a r r a y of avian motor p a t t e r n s . I t has been argued t h a t the ^ f i c t i v e ' locomotion observed i n response t o f o c a l e l e c t r i c a l s t i m u l a t i o n or i n t r a c e r e b r a l i n f u s i o n of n e u r o t r a n s m i t t e r r e l a t e d chemicals i n t o brainstem locomotor r e g i o n s i s due, i n p a r t , t o the a c t i v a t i o n of c e n t r a l pathways t h a t normally r e l a y somatosensory i n f o r m a t i o n d u r i n g locomotion [e.g. TTD or pontobulbar locomotor s t r i p (PLS) e l e c t r i c a l s t i m u l a t i o n (Noga et a l . , 1988) ( F i g . 2 9 )]. Thus, t h i s x f i c t i v e ' locomotor a c t i v i t y i n p a r a l y z e d animals may not 232 r e f l e c t an in h e r e n t CNS locomotor p a t t e r n g e n e r a t i n g c a p a c i t y . However, t h i s p o s s i b i l i t y i s e l i m i n a t e d , at l e a s t f o r avian walking, by the perseverance of ^ f i c t i v e ' l e g locomotor a c t i v i t y a f t e r p a r a l y s i s i n the spontaneously moving animal ( F i g . 27B), where the somatosensory pathways d e s c r i b e d by Noga et al. (1988) are not a c t i v a t e d by p h a s i c sensory i n p u t . In s p i t e o f the c a p a c i t y o f the CNS to independently generate locomotor rhythm, i t i s important t o note t h a t p r o p r i o c e p t i v e feedback does a l t e r motor p a t t e r n . One f a c e t of t h i s i n f l u e n c e was evidenced by the g r e a t e r s t i m u l a t i o n i n t e n s i t y r e q u i r e d t o a c t i v a t e locomotion i n the p a r a l y z e d b i r d s , i n a d d i t i o n t o the lower locomotor f r e q u e n c i e s observed i n the p a r a l y z e d animals when locomotion was f i n a l l y a c t i v a t e d . The h i g h e r t h r e s h o l d o f the p a r a l y z e d b i r d s might suggest t h a t sensory input serves t o lower the t h r e s h o l d f o r a c t i v a t i n g movement by i n c r e a s i n g the o v e r a l l g a i n of the animal's a c t i v a t i o n l e v e l . While the occurrence of spontaneous * f i c t i v e ' w alking i n p a r a l y z e d b i r d s may argue a g a i n s t t h i s suggestion, only 2 of the 6 animals t h a t e x h i b i t e d spontaneous locomotion p r i o r t o p a r a l y z a t i o n e l i c i t e d spontaneous p a t t e r n s d u r i n g p a r a l y z a t i o n . I t i s p o s s i b l e t h a t the a c t i v a t i o n s t a t e of the 2 spontaneous f i c t i v e animals was h i g h enough t o allow spontaneous A f i c t i v e ' walking, while the a c t i v a t i o n l e v e l i n the remaining 4 was reduced t o l e v e l s t h a t p r e c l u d e d spontaneous a c t i v i t y f o l l o w i n g p a r a l y z a t i o n . T h i s i s supported by the f i n d i n g t h a t e x t e r o c e p t i v e s t i m u l a t i o n (non-noxious) c o u l d e l i c i t s h o r t bouts o f ^ f i c t i v e ' walking i n 2 of the 4 b i r d s t h a t d i d not e l i c i t spontaneous locomotion d u r i n g p a r a l y s i s . A f u r t h e r p i e c e o f 233 evidence, namely, the r a r e occurrence of f i c t i v e f l y i n g , i n d i r e c t l y supports the argument t h a t a f f e r e n t input serves to i n c r e a s e the animals a c t i v a t i o n l e v e l . From r e s u l t s presented here, where b i l a t e r a l f l a p p i n g i s c o n s i s t e n t l y observed t o occur at s t i m u l a t i o n i n t e n s i t i e s g r e a t e r than t h a t r e q u i r e d t o e l i c i t walking, i t appears, as seen i n the cat (Shik et a l . , 1966, 1967), t h a t the t h r e s h o l d f o r f o r e l i m b a c t i v a t i o n i s g r e a t e r than t h a t f o r h i n d l i m b s . Thus, where s t i m u l a t i o n up to 170/nA i s o f t e n s u f f i c i e n t t o e l i c i t walking and/or wing f l a p p i n g when a f f e r e n t feedback i s present, s i m i l a r s t i m u l a t i o n , although a c t i v a t i n g l e g s , i s r a r e l y s u f f i c i e n t t o a c t i v a t e the f o r e l i m b p a t t e r n generators i n the p a r a l y z e d b i r d . The degree t o which a f f e r e n t input i s important i n e s t a b l i s h i n g t h i s " a c t i v a t i o n l e v e l " , and, i n t u r n , the r o l e a f f e r e n t input p l a y s i n determining the frequency of locomotor output remains u n c l e a r . However, s t u d i e s i n which the degree of a f f e r e n t input i s c o n t r o l l e d may help to s o l v e these i s s u e s . A f f e r e n t input c o u l d be a p p l i e d , f o r example, by a l t e r n a t e l y l i f t i n g the l e g s of the animal (graded excursions) d u r i n g the a p p r o p r i a t e extensor phase of ^ f i c t i v e ' locomotion, thereby p r o v i d i n g the b i r d w i t h v a r y i n g degrees of sensory feedback. T h i s paradigm would presumably i n c r e a s e the a c t i v a t i o n l e v e l of the p r e p a r a t i o n and c o u l d be u t i l i z e d t o e x p l o r e the e f f e c t s of p e r i p h e r a l feedback on locomotion both i n the spontaneous and e l e c t r i c a l l y s t i m u l a t e d p r e p a r a t i o n s . In conclusion,, t h i s study has demonstrated t h a t the c e n t r a l nervous system possesses n e u r a l networks which can produce a wide a r r a y of a v i a n locomotor p a t t e r n s i n the absence of 234 a f f e r e n t feedback. T h i s study, however, has a l s o a l l u d e d t o the importance of a f f e r e n t feedback i n the p r o d u c t i o n of a normal walking p a t t e r n . Removal of a l l feedback appears to lower the " a c t i v a t i o n l e v e l " o f the animal such t h a t g r e a t e r s t i m u l a t i o n i s r e q u i r e d t o i n i t i a t e locomotion and t h a t , once i n i t i a t e d , the locomotor movements are reduced c o n s i d e r a b l y . 235 CHAPTER 7 TRANSECTION LEVEL DETERMINES SPONTANEOUS MOTOR ACTIVITY IN THE DECEREBRATE AVIAN PREPARATION INTRODUCTION In v e r t e b r a t e s , s e l e c t i v e b r a i n t r a n s e c t i o n at d i f f e r e n t l e v e l s of the n e u r a x i s y i e l d s v a r y i n g degrees of spontaneous locomotor a c t i v i t y ( f o r review see Shik and Orlovsky, 1976; Wetzel and S t u a r t , 1976; Armstrong, 1986). Table 1 b r i e f l y summarizes the motor c a p a b i l i t i e s of c a t s f o l l o w i n g t r a n s e c t i o n of the n e u r a x i s at the v a r i o u s l e v e l s shown i n F i g u r e 1. These r e s u l t s , with minor v a r i a t i o n , are e q u a l l y a p p l i c a b l e t o a v a r i e t y of mammals i n c l u d i n g dogs (Magnus, 1924), r a b b i t s (Hinsey et a l . , 1930) and r a t s (Woods, 1964; Skinner and G a r c i a - R i l l , 1984). D i f f e r e n c e s i n an animal's a b i l i t y t o produce spontaneous locomotion between the premammillary versus postmammillary acute p r e p a r a t i o n ( t r a n s e c t i o n B and C i n F i g u r e 1) have been a t t r i b u t e d t o the s p a r i n g of s t r u c t u r e s c o n t a i n e d w i t h i n the p r e s e r v e d wedge of n e u r a l t i s s u e (Wetzel and S t u a r t , 1976; Shik and Orlovsky, 1976). Neural s t r u c t u r e s found i n t h i s r e g i o n which c o u l d be r e s p o n s i b l e f o r the i n i t i a t i o n o f spontaneous locomotor behaviour i n c l u d e the subthalamic locomotor r e g i o n (SLR) (Waller,1940), subthalamic neurons, p o s t e r i o r hypothalamic n u c l e i and p o s t e r i o r t h a l a m i c m i d l i n e neurons (Wetzel and S t u a r t , 1976). Our s t u d i e s w i t h b i r d s have demonstrated t h a t the avian n e u r a l s u b s t r a t e s f o r locomotion appear s i m i l a r t o those found i n a v a r i e t y of both h i g h e r and lower v e r t e b r a t e s (Steeves et a l . , 1986, 1987; Sholomenko and Steeves, 1987a,b; Sholomenko, t h i s t h e s i s ; M c C l e l l a n , 198 6), thus i m p l y i n g a h i g h degree of 237 TABLE 4. TRANSECTION ANIMAL ACUTE CHRONIC LEVEL PREPARATION PREPARATION THALAMIC CAT SPONTANEOUS LOCOMOTION SPONTANEOUS LOCOMOTION Fig 1-Line A ( M A G N U S . 1 9 2 4 ) ( H I N S E Y E T A L , 1 9 3 0 ) ( S H I K E T A L . , 1 9 6 6 ) - S T I M U L A T I O N OF MLR OR S L R E L I C I T S W A L K I N G ( S H I K E T A L . , 1 9 6 6 A ) ( D E N N E Y - B R O W N , 1 9 6 6 ) ( B A R D AND M A C H T , 1 9 5 8 ) PRECOLLICULAR CAT SPONTANEOUS LOCOMOTION SPONTANEOUS LOCOMOTION PREMAMMILLARY ( H I N S E Y E T A L . , 1 9 3 0 ) ( H I N S E Y E T A L . , 1 9 3 0 ) Fig 1-Line B ( S H I K E T A L . , 1 9 6 6 ) - S T I M U L A T I O N OF MLR E L I C I T S L O C O M O T I O N ( S H I K ET A L . , 1 9 6 6 ) PRECOLLICULAR CAT NO SPONTANEOUS SPONTANEOUS LOCOMOTION POST- LOCOMOTION ( B A R D AND M A C H T , 1 9 5 8 ) MAMMILLARY ( H I N S E Y E T A L . , 1 9 3 0 ) ( V I L L A B L A N C A , 1 9 6 2 ) Fig 1-Line C ( S H I K E T A L . , 1 9 6 6 , 1 9 6 7 ) - S T I M U L A T I O N OF MLR E L I C I T S L C O M O T I O N ( S H I K E T A L . , 1 9 6 6 , 1 9 6 7 ) PRECOLLICULAR CAT NO SPONTANEOUS LOCOMOTION IN RESPONSE POST-OCCULO- LOCOMOTION TO STRONG MOTOR NERVE ( B A R D AND M A CHT, 1 9 5 8 ) EXTEROCEPTIVE Fig 1-Line D STIMULATION ( B A R D AND M A CHT, 1 9 5 8 ) MIDCOLLICULAR CAT NO SPONTANEOUS ALTERNATING LIMB PRE-OCCULO- LOCOMOTION MOVEMENTS IN AN MOTOR NERVE ( B A R D AND M A C H T , 1 9 5 8 ) ANIMAL WHICH IS Fig 1-Line E LYING PRONE ( B A R D AND M A C H T , 1 9 5 8 ) 238 Figure 34. Diagram of a saggital section through the cat brainstem showing neuraxis transection l e v e l s and locomotor s i t e s important for the study of locomotor control. Transection le v e l s are designated by l e t t e r s A-E. Transection l e v e l : A -thalamic, B - p r e c o l l i c u l a r premammillary (hypothalamic of Hinsey et al., 1930)/ C - p r e c o l l i c u l a r postmammillary, D -p r e c o l l i c u l a r post-occulomotor, E - m i d c o l l i c u l a r pre-occulomotor. Locomotor s i t e s include the subthalamic locomotor region (SLR) and mesencephalic locomotor region (MLR). The hatched l i n e s surrounding RPC and RGC represent the pontine and medullary r e t i c u l a r formation that are thought to be the major motor information projection systems to the spinal cord. Abbreviations: CM - mammillary body, CO - optic chiasm, IC -i n f e r i o r c o l l i c u l u s , MLR - mesencephalic locomotor region, P -pons, R - red nucleus, RGC - medullary gigantocellular r e t i c u l a r nucleus, RPC - caudal pontine r e t i c u l a r nucleus, SC - superior c o l l i c u l u s , SLR - subthalamic locomotor region, T - trapezoid body, Th - thalamus. III - occulomotor nerve. See text for additional explanation. This figure i s redrawn from: 1) Shik et al., 1968, 2) Orlovsky, 1970a, 3) G r i l l n e r and Shik, 1973 and 4) Wetzel and Stuart, 1976. 239 c o n s e r v a t i o n of motor c i r c u i t r y across a broad p h y l o g e n e t i c range. To f u r t h e r t h i s comparison and i n an attempt t o d e l i n e a t e the s t r u c t u r e s r e q u i r e d f o r the i n i t i a t i o n of spontaneous locomotion, we have examined the e f f e c t s of d i f f e r e n t l e v e l s of b r a i n t r a n s e c t i o n on spontaneous locomotor c a p a b i l i t y i n b i r d s . Our r e s u l t s i n d i c a t e t h a t i n the acute a v i a n p r e p a r a t i o n , as i n mammals, animals w i t h a r o s t r a l t r a n s e c t i o n of the n e u r a x i s (post-habenular/preoptic) d i s p l a y spontaneous locomotion, while more caudal t r a n s e c t i o n (post-habenular/postoptic) e l i m i n a t e s any spontaneous behaviour. D i e n c e p h a l i c s t r u c t u r e s which are c o n t a i n e d between these two t r a n s e c t i o n l e v e l s presumably u n d e r l i e t h i s d i f f e r e n c e . 240 MATERIALS AND METHODS Surgery S u r g i c a l and EMG/ENG r e c o r d i n g procedures have been p r e v i o u s l y d e s c r i b e d (Chapter 2) with the e x c e p t i o n t h a t f o l l o w i n g a craniotomy, a s u c t i o n d e c e r e b r a t i o n was performed along a plane extending d o r s a l l y from the caudal margin o f the habenular nucleus t o e i t h e r : 1) the r o s t r a l or 2) the caudal edge of the o p t i c chiasm v e n t r a l l y . S t i m u l a t i o n procedures f o r low decerebrate animals have been d e s c r i b e d i n d e t a i l p r e v i o u s l y (Sholomenko and Steeves, 1987; t h i s t h e s i s , Chapters 2-5). In hig h decerebrate animals, a f t e r r e c o r d i n g the spontaneous electromyographic (EMG) a c t i v i t y from the l e g s and/or wings, the b i r d s were deeply a n a e s t h e t i z e d and s a c r i f i c e d w i t h an intravenous i n j e c t i o n o f KC1 (2M). H i s t o l o g i c a l procedures have been d e s c r i b e d i n d e t a i l i n Chapter 2, w i t h the e x c e p t i o n t h a t the l e v e l o f d e c e r e b r a t i o n was determined by the r e c o n s t r u c t i o n of s e r i a l c o r o n a l s e c t i o n s a c c o r d i n g t o the s t e r e o t a x i c a t l a s e s o f Karten and Hodos (1967) and Zweers (1971). Assessment o f Spontaneous versus Non-spontaneous Two c r i t e r i a were used t o d i s t i n g u i s h spontaneous from non-spontaneous b i r d s . F i r s t was the b i r d ' s response t o foot web p i n c h s t i m u l a t i o n f o l l o w i n g removal of a n a e s t h e t i c . Strong 241 r e f l e x withdrawal was common to a l l b i r d s , however, those b i r d s responding t o web pinch w i t h stepping motions and/or wing f l a p p i n g met the f i r s t c r i t e r i a f o r spontaneous pr e p a r a t i o n s . Second, i f the b i r d s a l s o d i s p l a y e d prolonged walking behaviour (longer than 1 minute continuous) i n response to the t a c t i l e and l e g movement s t i m u l a t i o n produced when the t r e a d m i l l was turned on, they were considered spontaneous. Animals which d i d not meet the above c r i t e r i a were c l a s s i f i e d as non-spontaneous. These animals r e q u i r e d e i t h e r e l e c t r i c a l or neurochemical brainstem s t i m u l a t i o n to e l i c i t locomotor behaviours. 242 RESULTS Ten animals (6 Canada geese, 4 Pekin ducks) with the r o s t r a l transection depicted i n Figure 35 (line A) displayed spontaneous locomotion. Several of the animals displayed s i g n i f i c a n t extensor tonus, resembling that of a standing posture, when the treadmill belt was motionless. When the belt was turned on, the legs were moved caudally and the animals. would begin to make alternating stepping movements (Figure 36A,B). The stepping seldom appeared of s u f f i c i e n t force to be self-supporting, although t o t a l force was d i f f i c u l t to determine. Of the t o t a l of ten spontaneous animals, only one b i r d displayed wing flapping behaviour combined with stepping i n response to the treadmill be l t stimulation alone (Figure 37). The flapping occurred i n short bursts (approximately 10 seconds long) and subsided over several hours, even though belt-stimulated stepping continued for several hours. In the spontaneous birds, t a c t i l e stimulation of parts of the body other than the feet (e.g. stroking around the eye or caudal back region) increased the force of hindlimb stepping when the animal was walking. Similarly, i f an animal ceased stepping i n response to the belt, t a c t i l e stimulation would often r e - i n i t i a t e the stepping behaviour. Two birds which were paralyzed displayed alternating hindlimb ^ f i c t i v e ' stepping a c t i v i t y i n the absence of any phasic afferent input (treadmill belt off) from the periphery 243 F i g u r e 35. Diagram of the t r a n s e c t i o n l e v e l s of the avian b r a i n which permit or e l i m i n a t e spontaneous locomotion i n the decerebrate b i r d . The diagram i s of a near m i d l i n e ( l a t e r a l 0.5mm) s a g g i t a l s e c t i o n of the pigeon b r a i n (Karten and Hodos, 1967). L i n e A d e s i g n a t e s the approximate l e v e l of s e c t i o n which allows spontaneous locomotion i n the decerebrate b i r d . Animals w i t h a t r a n s e c t i o n at the approximate l e v e l of L i n e B do not show spontaneous locomotor a c t i v i t y . A b b r e v i a t i o n s : AHP - area hypothalami p o s t e r i o r i s , AM - Nucleus a n t e r i o r m e d i a l i s hypothalami, A n l - Nucleus a n n u l a r i s , APH -Area parahippcampalis, BO - o l f a c t o r y bulb, CA - a n t e r i o r commissure, Cb - cerebellum, CCV - v e n t r a l c e r e b e l l a r commissure, CHCS - c o r t i c o h a b e n u l a r and c o r t i c o s e p t a l t r a c t , CO - o p t i c chiasm, CoS - s e p t a l commissural nucleus, CP - p o s t e r i o r commissure, CS - c e n t r a l s u p e r i o r nucleus (Betcherew), CT -t e c t a l commissure, DBC - d e c u s s a t i o n brachium conjunctivum, DMA - dorsomedial a n t e r i o r t h a l a m i c nucleus, DMP - dorsomedial p o s t e r i o r t h a l a m i c nucleus, DSD - s u p r a o p t i c d o r s a l d e c u s s a t i o n , DSV - s u p r a o p t i c v e n t r a l d e c u s s a t i o n , EW - Edinger-Westphal nucleus, FD - d o r s a l f u n i c u l u s , FLM - medial l o n g i t u d i n a l f a s c i c u l u s , FV - v e n t r a l f u n i c u l u s , GC - cuneate and g r a c i l e n u c l e i , GCt - c e n t r a l gray, HA - accessory h y p e r s t r i a t u m , Hb -habenular nucleus, Hp - hippocampus, HV - v e n t r a l h y p e r s t r i a t u m , IM - i n t e r m e d i a t e nucleus, IP - i n t e r p e d u n c u l a r nucleus, LFM -supreme f r o n t a l lamina, LH - h y p e r s t r i a t a l lamina, LMD - d o r s a l medullary lamina, LPO - p a r o l f a c t o r y lobe, MNV - mesencephalic t r i g e m i n a l nerve nucleus, N - neostriatum, NC - caudal neostriatum, NIII - occulomotor nerve, nIV - t r o c h l e a r nucleus, nX - motor nucleus vagus, nXII - h y p o g l o s s a l nucleus, 01 -i n f e r i o r o l i v a r y nucleus, OMd - d o r s a l p a r t , oculomotor nucleus, OMv - v e n t r a l p a r t , oculomotor nucleus, Ov - o v o i d nucleus, P -p i n e a l , PaM - paramedian nucleus, PMH - p o s t e r i o r p a r t , medial hypothalamic nucleus, PMI - paramedian i n t e r n a l t h a l a m i c nucleus, POA - a n t e r i o r p r e o p t i c nucleus, POM - medial p r e o p t i c nucleus, PVM - p e r i v e n t r i c u l a r m a g n o c e l l u l ar nucleus, Rgc -g i g a n t o c e l l u l a r r e t i c u l a r nucleus, RP - pontine caudal r e t i c u l a r nucleus, RPgc - g i g a n t o c e l l u l a r p a r t , caudal pontine r e t i c u l a r nucleus, Ru - r e d nucleus, S - nucleus s o l i t a r i u s , SCE -e x t e r n a l c e l l u l a r stratum, SCI - i n t e r n a l c e l l u l a r stratum, SL -l a t e r a l s e p t a l nucleus, SM - medial s e p t a l nucleus, TO -o l f a c t o r y t u b e r c l e , TSM - septomesencephalic t r a c t , TU - t u b e r a l nucleus, V - v e n t r i c l e , vm - ventromedial i n t e r n a l c e r e b e l l a r nucleus 244 245 F i g u r e 36. B i l a t e r a l a l t e r n a t i n g walking a c t i v i t y i n a spontaneously locomoting b i r d b e f o r e (A) and a f t e r (B) p a r a l y z a t i o n . The t r a n s e c t i o n l e v e l (dotted l i n e ) which allows p o s t - d e c e r e b r a t i o n spontaneous locomotion i n shown i n the s a g g i t a l s e c t i o n . A: EMG t r a c e s from l e f t and r i g h t ITC (*) muscles d u r i n g spontaneous t r e a d m i l l walking. B: Subsequent ENG t r a c e s from l e f t and r i g h t ITC nerves i n the p a r a l y z e d animal showing spontaneous walking p a t t e r n a c t i v i t y . * The i l i o t i b i a l i s c r a n i a l i s (ITC) muscle i s synonymous with the mammalian s a r t o r i u s muscle. 246 A - PRE-PARALYZED EMG B - PARALYZED ENG LEFT ITC RIGHT ITC 2 sec 247 F i g u r e 3 7 . Electromyographic records (EMGs) showing spontaneous s t e p p i n g and f l y i n g a c t i v i t y . The EMGs were taken from the r i g h t (RPECT) and l e f t (LPECT) p e c t o r a l i s muscles and r i g h t (RITC) and l e f t (LITC) i l i o t i b i a l i s c r a n i a l i s muscles d u r i n g a bout of spontaneous a c t i v i t y . The PECT muscle i s the major wing depressor e s s e n t i a l f o r f l i g h t and the ITC muscle i s the major h i p f l e x o r i n b i r d s . 248 RPECT LPECT RITC LITC 249 (Figure 36B) (see a l s o Chapter 6). Both animals were s t r o n g l y spontaneously a c t i v e p r i o r to p a r a l y z a t i o n . F i v e other spontaneous animals which were subsequently p a r a l y z e d d i d not demonstrate any spontaneous x f i c t i v e ' locomotion a f t e r c u r a r i z a t i o n and r e q u i r e d e i t h e r e l e c t r i c a l or chemical s t i m u l a t i o n to i n i t i a t e X f i c t i v e ' locomotor p a t t e r n s (see Chapters 2-6). Non-spontaneous animals (N=122) r e q u i r e d e i t h e r : 1) f o c a l e l e c t r i c a l s t i m u l a t i o n of brainstem locomotor r e g i o n s (Steeves et al., 1986, 1987; Sholomenko and Steeves, 1987; Sholomenko, t h i s t h e s i s ) or 2) i n t r a c e r e b r a l m i c r o i n j e c t i o n of n e u r o t r a n s m i t t e r a g o n i s t s or a n t a g o n i s t s i n t o the same locomotor re g i o n s (Sholomenko and Steeves, 1987b; Sholomenko, t h i s t h e s i s ) to evoke locomotor behaviours (see Chapters 2-6). The approximate l e v e l of t r a n s e c t i o n averaged from a random sample of 10 of these b i r d s i s d i s p l a y e d i n F i g u r e 35 ( l i n e B). D e c e r e b r a t i o n L e v e l Spontaneous The t r a n s e c t i o n l e v e l i n s i x of the spontaneous b i r d s extended from the caudal border of the habenular nucleus d o r s a l l y t o the r o s t r a l border of the a n t e r i o r p r e o p t i c nucleus v e n t r a l l y . T h i s t r a n s e c t i o n e l i m i n a t e d the e n t i r e t e l e n c e p h a l o n i n a d d i t i o n t o p o r t i o n s of the a n t e r o d o r s a l thalamus (DMA). T r a n s e c t i o n s which e l i m i n a t e d more dorsocaudal s t r u c t u r e s (N=2), 250 i n c l u d i n g the p o s t e r i o r t h a l a m i c n u c l e i and o v o i d nucleus, extended d o r s a l l y from caudal to the p o s t e r i o r commissure (CP) t o a n t e r i o r t o the a n t e r i o r p r e o p t i c nucleus v e n t r a l l y . Two spontaneous b i r d s had t r a n s e c t i o n s which a b l a t e d more caudal s t r u c t u r e s i n c l u d i n g the e x t e r n a l (SCE) and p o r t i o n s of the i n t e r n a l (SCI) c e l l u l a r stratum. One of these b i r d s , with a t r a n s e c t i o n t h a t separated the e n t i r e thalamus, p o s t e r i o r commissure and p a r t of the medial p o s t e r i o r hypothalamic nucleus from the brainstem, e l i c i t e d prolonged hindlimb s t e p p i n g i n response t o t r e a d m i l l b e l t s t i m u l a t i o n . In a l l of the above spontaneous animals, the subthalamic nucleus (nucleus of the ansa l e n t i c u l a r i s (nAL)), the s u b s t a n t i a n i g r a (TPc), the l a t e r a l s p i r i f o r m nucleus and caudal p o r t i o n s of the hypothalamic n u c l e i [e.g. p o s t e r i o r (AHP) and l a t e r a l (LHA) hypothalamic areas] remained i n t a c t . Non-spontaneous Neuroanatomic a n a l y s i s of the ten b i r d s chosen at random (9 Canada geese, 1 Pekin duck) from those which d i d not e l i c i t spontaneous locomotion a f t e r the d e c e r e b r a t i o n ( F i g . 35B) demonstrated t h a t the m a j o r i t y of animals had more caudal t r a n s e c t i o n l e v e l s than those found i n spontaneous b i r d s . In two animals, the t r a n s e c t i o n e l i m i n a t e d the most r o s t r a l p o r t i o n s of the S p l , but l e f t the bulk of the nucleus i n t a c t . These t r a n s e c t i o n s completely removed the subthalamic nucleus (nAL) and the b u l k of the p o s t e r i o r and l a t e r a l hypothalamic n u c l e i . 251 A l s o , t r a n s e c t i o n l e v e l s i n t h r e e animals damaged the most r o s t r a l r e g i o n o f the s u b s t a n t i a n i g r a . S l i g h t l y more r o s t r a l l e s i o n s a l s o e l i m i n a t e d spontaneous locomotion i n 4 b i r d s . In these animal, the s u b s t a n t i a n i g r a and Spl n u c l e i were spared, but again the nAL and l a t e r a l and p o s t e r i o r hypothalamic n u c l e i were e x c i s e d . One b i r d which d i d not perform spontaneously a f t e r d e c e r e b r a t i o n had a t r a n s e c t i o n which was of the same l e v e l as those seen i n animals which e x h i b i t e d spontaneous locomotion. A l l o f the n u c l e i which remained i n the spontaneous b i r d s were a l s o present i n t h i s animal. Reasons why t h i s animal d i d not e l i c i t spontaneous locomotion cannot be determined, but i t i s p o s s i b l e t h a t the c o n d i t i o n o f the p r e p a r a t i o n d e t e r i o r a t e d more r a p i d l y than i n the other b i r d s . 252 DISCUSSION The r e s u l t s f r o m t h i s s t u d y d e m o n s t r a t e t h a t i n b i r d s , as i n mammals , p r e s e r v a t i o n o f a r e g i o n l y i n g w i t h i n t h e b o u n d a r i e s o f t h e c a u d a l d i e n c e p h a l o n a l l o w s s p o n t a n e o u s l o c o m o t i o n i n t h e a c u t e d e c e r e b r a t e p r e p a r a t i o n . M o r e c a u d a l t r a n s e c t i o n s e l i m i n a t e t h e s p o n t a n e i t y b u t do n o t a l t e r t h e a b i l i t y t o g e n e r a t e l o c o m o t o r r h y t h m s i n r e s p o n s e t o e l e c t r i c a l a n d / o r c h e m i c a l s t i m u l a t i o n o f b r a i n s t e m l o c o m o t o r r e g i o n s ( S h o l o m e n k o a n d S t e e v e s , 1 9 8 7 ; S h o l o m e n k o , t h i s t h e s i s ) . The n u c l e i w h i c h were p r e s e n t i n t h e wedge o f t i s s u e l y i n g b e t w e e n t h e two t r a n s e c t i o n l e v e l s i n c l u d e d t h e n u c l e u s o f t h e a n s a l e n t i c u l a r i s ( n A L ) , n u c l e i o f t h e l a t e r a l a n d p o s t e r i o r h y p o t h a l a m i c a r e a s (LHA & PHA) a n d t h e mos t r o s t r a l p o r t i o n o f t h e c o m p a c t p a r t o f t h e s u b s t a n t i a n i g r a ( T P c ) . The n A L , b a s e d p r i m a r i l y on h o d o l o g i c a l c o n s i d e r a t i o n s , i s c o m p a r a b l e t o t h e m a m m a l i a n s u b t h a l a m i c n u c l e u s ( B r a u t h e t al., 1 9 7 8 ) , w h i l e t h e T P c h a s b e e n c o m p a r e d t o t h e mammal ian s u b s t a n t i a n i g r a ( B r a u t h e t al., 1978; R e i n e r e t al., 1 9 8 4 ) . The LHA a n d PHA a l s o l i e w i t h i n t h e wedge o f t i s s u e w h i c h was r e m o v e d i n t h e n o n - s p o n t a n e o u s b u t p r e s e r v e d i n t h e s p o n t a n e o u s b i r d s . A l l b u t t h e most r o s t r a l p o r t i o n o f S p l , w h i c h r e c e i v e s t h e m a j o r o u t f l o w o f t h e a v i a n b a s a l g a n g l i a ( R e i n e r e t al., 1 9 8 4 ) , was s p a r e d i n b o t h r o s t r a l a n d c a u d a l l e s i o n s . S e v e r a l e x p l a n a t i o n s h a v e b e e n p u t f o r t h i n an a t t e m p t t o a c c o u n t f o r t h e d i f f e r e n t l e v e l s o f a c t i v i t y i n p r e c o l l i c u l a r - p r e m a m m i l l a r y v e r s u s p r e c o l l i c u l a r - p o s t m a m m i l l a r y 253 t r a n s e c t e d mammals. Wetzel and Stuart (1976) suggest that the s t r u c t u r e s remaining i n the p r e c o l l i c u l a r - p r e m a m m i l l a r y but removed from the p r e c o l l i c u l a r - p o s t m a m m i l l a r y p r e p a r a t i o n , c o n s i s t i n g of the p o s t e r i o r hypothalamic, subthalamic and v e n t r a l p o s t e r i o r thalamic neurons, increase the general e x c i t a b i l i t y of more caudal neurons. S i m i l a r l y , Armstrong, i n a recent review (1986), p o s t u l a t e d that c e l l s contained w i t h i n the preserved s l i c e of t i s s u e , p o s s i b l y a r i s i n g from the p o s t e r i o r hypothalamus, zona i n c e r t a or H and H2 f i e l d s of F o r e l , provide a t o n i c (as opposed to rhythmic or patterned) e x c i t a t o r y input to downstream motor r e l a t e d s t r u c t u r e s . G a r c i a - R i l l and Skinner (1986), on the other hand, suggested t h a t t o n i c GABAergic subthalamic nucleus p r o j e c t i o n s t o the s u b s t a n t i a n i g r a (SN), which are spared a f t e r the p r e c o l l i c u l a r - p r e m a m m i l l a r y t r a n s e c t i o n , i n h i b i t an i n h i b i t o r y GABAergic p r o j e c t i o n from the SN to the pedunculopontine nucleus (mesencephalic locomotor r e g i o n ) . Thus, locomotor patterns are e f f e c t i v e l y d i s i n h i b i t e d (released) and can express themselves i n the acute premammillary cat p r e p a r a t i o n . Their hypothesis accounts f o r the l o s s of spontaneous a c t i v i t y a f t e r a more caudal t r a n s e c t i o n through the e l i m i n a t i o n of the i n h i b i t i o n from the SN to the PPN. This theory i s supported by the f i n d i n g t h a t i n j e c t i o n of GABA antagonists i n t o the SN blocks spontaneous stepping i n the premammillary p r e p a r a t i o n and th a t stepping can be r e i n s t a t e d by i n j e c t i o n of GABA or muscimol i n t o the SN ( G a r c i a - R i l l and Skinner, 1986). However, s p e c i f i c damage to the subthalamic nucleus, which should, according to G a r c i a - R i l l and. Skinner 254 (1986), through d i s i n h i b i t i o n o f an i n h i b i t o r y pathway, i n h i b i t locomotion, only r e l e a s e s motor behaviours such as hemiballismus or chorea (Hammond et a l . , 1979). Another d i e n c e p h a l i c r e g i o n , d e f i n e d e l e c t r o p h y s i o l o g i c a l ^ as the subthalamic locomotor r e g i o n (SLR), w i l l e l i c i t locomotion when e l e c t r i c a l l y s t i m u l a t e d . I t i s i n t i m a t e l y i n v o l v e d i n locomotor c o n t r o l and l i e s d o r s o m e d i a l l y t o the subthalamic nucleus (Shik and Orlovsky, 1976; Orlovsky and Shik, 1976). Mogenson and co-workers (Mogenson, 1984; Mogenson et a l . , 1985; Brudzynski et al., 1988) used a v a r i e t y of neuroanatomical t r a c i n g techniques t o d e s c r i b e the SLR as l y i n g w i t h i n the zona i n c e r t a (ZI) and l a t e r a l hypothalamic area (LHA) i n the r a t . T h i s r e g i o n has been found t o p r o j e c t t o the pedunculopontine, cuneiform and r e t i c u l a r formation n u c l e i , which themselves are s t r o n g l y i m p l i c a t e d i n motor c o n t r o l (Swanson et al., 1984; Mogenson et a l . , 1985; Orlovsky, 1970a,b; Steeves and Jordan, 1984; G a r c i a - R i l l and Skinner, 1986). T h e i r evidence c o r r o b o r a t e s t h a t found by Orlovsky (1969), who used e l e c t r o p h y s i o l o g i c a l techniques t o d e s c r i b e d i r e c t p r o j e c t i o n s from the SLR both t o the MLR and r e t i c u l a r formation. Evidence t h a t the SLR-evoked locomotion r e s u l t s not from s t i m u l a t i o n o f axons of passage but from s t i m u l a t i o n o f neuronal r e c e p t o r s comes from the f i n d i n g t h a t i n f u s i o n o f the GABAergic a n t a g o n i s t p i c r o t o x i n i n t o the SLR induced locomotion i n the p r e c o l l i c u l a r - p r e m a m m i l l a r y c at ( E l d r i d g e et a l . , 1985). Of i n t e r e s t i s the hypothesis t h a t the SLR r e g i o n u n d e r l i e s goal d i r e c t e d behaviour (Shik and Orlovsky,. 1976; Mogenson, 1984; 255 Mogenson et al., 1985) and may be the locu s through which the l i m b i c system i n f l u e n c e s locomotor behaviours (Mogenson et al., 1985/ Brudzynski et al., 1988). Thus, the SLR, i n a d d i t i o n t o the subthalamic nucleus i s a p o s s i b l e source o f the spontaneous locomotor a c t i v i t y i n hig h decerebrate animals. In b i r d s , the s t r u c t u r e s conserved i n the h i g h decerebrate animal p a r a l l e l those found i n mammals. The nAL (subthalamic nucleus) p r o j e c t s t o the av i a n e q u i v a l e n t of the s u b s t a n t i a n i g r a (TPc) (Reiner et al., 1984), p a l l i d u m ( p a l e o s t r i a t u m primativum) and l a t e r a l s p i r i f o r m nucleus (Brauth et al., 1978). I t i s p r e s e n t l y unknown whether t h i s pathway i s e q u i v a l e n t t o the GABAergic p r o j e c t i o n suggested i n mammals (McGeer et al., 1984; G a r c i a - R i l l and Skinner, 1986), but i t i s i n t e r e s t i n g t o note t h a t the s t r u c t u r e s t o which nAL p r o j e c t are a l l m o t o r - r e l a t e d i n b i r d s (Reiner et al., 1984). I t remains u n c l e a r , however, whether the nAL i s the n e u r a l s u b s t r a t e r e s p o n s i b l e f o r spontaneous locomotion i n hig h decerebrate b i r d s . The a f f e r e n t and e f f e r e n t p r o j e c t i o n s of the hypothalamic n u c l e i which are p r e s e r v e d i n the h i g h decerebrate spontaneous p r e p a r a t i o n (LHA and PHA) have not been e l u c i d a t e d i n b i r d s , although Berk and Hawkin (1985) make b r i e f mention t h a t i n pigeon, as i n r a t (Mogenson et al., 1985), the parahippocampal r e g i o n p r o j e c t s t o the l a t e r a l hypothalamic area. Thus, although no a v i a n e q u i v a l e n t o f the SLR/zona i n c e r t a has yet been r e c o g n i z e d i n b i r d s (Karten and Hodos, 1967; Zweers, 1971), some homology appears t o e x i s t between l i m b i c t o hypothalamic 256 connections i n b i r d s and mammals (Berk and F i n k e l s t e i n , 1983) which may u n d e r l i e the p r e s e r v a t i o n of spontaneous locomotion a f t e r a r o s t r a l d ecerebration. In b i r d s , as i n mammals, more info r m a t i o n i s r e q u i r e d t o determine which nucleus or combination of n u c l e i i n the conserved d i e n c e p h a l i c t i s s u e i s e s s e n t i a l f o r spontaneous locomotion. Furthermore, the exact r o l e of t h i s region, although p o s s i b l y limbic-motor r e l a t e d , remains t o be determined. The l a c k of informat i o n regarding the avian pathways which l e a d to spontaneous locomotion a f t e r a r o s t r a l t r a n s e c t i o n of the neuraxis make i t impossible to d e l i n e a t e the neural substrate f o r the a c t i v i t y at t h i s time (Armstrong, 198 6). However, taken together w i t h our previous r e s u l t s demonstrating t h a t b i r d s possess locomotor c i r c u i t r y which i s very s i m i l a r to that of mammals, we hypothesize that the neural substrate and mechanisms i n v o l v e d i n avian high decerebrate spontaneous locomotion w i l l be the same as those found i n the mammalian pr e p a r a t i o n . 257 CHAPTER 8 SUMMARY DISCUSSION 258 In t h i s t h e s i s , I have attempted to take an i n t e g r a t e d or multimodal approach to the study of a v i a n c e n t r a l nervous system motor c o n t r o l mechanisms. T h i s approach compares my r e s u l t s from s e l e c t i v e l e s i o n , brainstem e l e c t r i c a l s t i m u l a t i o n and brainstem neurochemical i n j e c t i o n s t u d i e s i n b i r d s w i t h those from other v e r t e b r a t e s . R e s u l t s from avian and v e r t e b r a t e neuroanatomy, e l e c t r o p h y s i o l o g y , immunohistochemistry and r e c e p t o r autoradiography have been i n c o r p o r a t e d i n an attempt t o f u r t h e r c h a r a c t e r i z e the neuroanatomical pathways i n v o l v e d i n locomotor c o n t r o l i n b i r d s . My p r e v i o u s s t u d i e s i n c l u d e d the use of low t h o r a c i c s p i n a l c o r d l e s i o n s t o examine two aspects of motor f u n c t i o n i n b i r d s . The f i r s t was t o c o r r o b o r a t e the f i n d i n g t h a t pigeons w i t h an i s o l a t e d lumbosacral s p i n a l c o r d c o u l d e l i c i t ^ s p i n a l s t e p p i n g ' (ten Cate, 1960, 1962). T h i s r e s u l t was supported by the f i n d i n g t h a t f o l l o w i n g complete t r a n s e c t i o n of the low t h o r a c i c s p i n a l cord, geese demonstrated the a b i l i t y t o N s p i n a l step' '. T h i s s t e p p i n g has been a t t r i b u t e d to the presence of s p i n a l c o r d locomotor p a t t e r n generators (LPG) t h a t are capable of producing a rhythmic p a t t e r n i n the absence of descending i n f l u e n c e s (Sholomenko and Steeves, 1987). S i m i l a r s p i n a l LPGs have been found i n v i r t u a l l y a l l v e r t e b r a t e s examined, p o s s i b l y e x c l u d i n g primates ( E i d e l b e r g et al., 1981; Graham-Brown, 1911, 1914; Wetzel and S t u a r t , 1976; E i d e l b e r g , 1981). As w i l l be d i s c u s s e d below, the o s c i l l a t o r s are modulated both by pathways descending from the brainstem (Kuypers, 1982) and by a f f e r e n t p e r i p h e r a l i n f o r m a t i o n ( G r i l l n e r , 1985; M c C l e l l a n , 1986). 259 t The second aspect of my study was to determine the major brainstem descending pathways which impinge on the LPGs and are r e s p o n s i b l e f o r the i n i t i a t i o n and ongoing c o n t r o l of the rhythmic locomotor o s c i l l a t i o n s . S e l e c t i v e l e s i o n s of the avian low t h o r a c i c s p i n a l c o r d demonstrated t h a t the e s s e n t i a l pathways a r i s e from the brainstem r e t i c u l a r formation n u c l e i (Steeves et al., 1986; Sholomenko and Steeves, 1987). T h i s f i n d i n g , t h a t the r e t i c u l o s p i n a l pathways are necessary f o r v o l u n t a r y locomotor p a t t e r n s , i s the same as t h a t found f o r a l l v e r t e b r a t e s s t u d i e d (Lawrence and Kuypers, 1968a,b; Orlovsky and Shik, 1976; E i d e l b e r g et al., 1981; Steeves and Jordan, 1984; Armstrong, 1986). E l e c t r i c a l s t i m u l a t i o n and neurochemical i n j e c t i o n (Chapters 2-5) s t u d i e s were then undertaken and have p r o v i d e d a d d i t i o n a l , although n e c e s s a r i l y incomplete, i n f o r m a t i o n r e g a r d i n g the important r o l e of the r e t i c u l a r formation i n locomotor c o n t r o l . I have i d e n t i f i e d f o u r a v i a n brainstem r e t i c u l a r formation r e g i o n s from which locomotion can be e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n (Steeves et al., 1986; Sholomenko and Steeves, 1987; Steeves et al., 1987). E f f e c t i v e l o c o m o t i o n - i n d u c i n g s t i m u l a t i o n s i t e s were l o c a l i z e d t o the d o r s a l (Cnd) and v e n t r a l (Cnv) p a r t s of the c e n t r a l medullary r e t i c u l a r n u c l e i , the v e n t r a l p o ntine r e t i c u l a r nucleus (RP) and the medial mesencephalic r e t i c u l a r formation (mMRF). Two of these r e g i o n s , Cnd and Cnv, c o n t r i b u t e s u b s t a n t i a l l y t o the descending r e t i c u l o s p i n a l pathways, while RP and mMRF, s t r u c t u r e s which w i l l be d i s c u s s e d subsequently, p r o j e c t only s p a r s e l y t o the s p i n a l c o r d (Cabot et al., 1982; 260 Webster and Steeves, 1988). The view t h a t neurons i n Cnd and Cnv gi v e r i s e t o the f i n a l common b r a i n s t e m - s p i n a l locomotor pathway i s supported by l i n e s o f evidence other than l e s i o n and e l e c t r i c a l s t i m u l a t i o n data. F i r s t , Cnd and Cnv neurons r e t r o g r a d e l y l a b e l l e d from the s p i n a l c o r d have been c o - l o c a l i z e d w i t h l o c o m o t i o n - i n d u c i n g s t i m u l a t i o n s i t e s i n the same b i r d (Steeves et al., 1987). Second, neuroanatomical r e s u l t s demonstrate t h a t many l o c o m o t i o n - r e l a t e d brainstem n u c l e i send p r o j e c t i o n s t o Cnd and Cnv, thus p l a c i n g Cnd and Cnv i n an i d e a l p o s i t i o n t o i n t e g r a t e and p r o j e c t motor i n f o r m a t i o n t o the cor d . The motor r e l a t e d n u c l e i p r o j e c t i n g t o Cnd/Cnv, many of which have been i d e n t i f i e d e l e c t r o p h y s i o l o g i c a l l y , i n c l u d e the RP, mMRF, g i g a n t o c e l l u l a r (Rgc) and p a r v o c e l l u l a r (Rpc) r e t i c u l a r formation, t r i g e m i n a l descending t r a c t and nucleus r e g i o n (TTD), nucleus i n t e r c o l l i c u l a r i s (ICo), tectum, a r c h i s t r i a t u m intermedium, cerebellum, Area v e n t r a l i s of T s a i , r e d nucleus and nucleus raphe magnus (Webster, p e r s o n a l communication; Arends et al., 1984; Arends and Dubbeldam, 1984; Hunt and Kunzle, 1976; Wild, 1984; W i l d et a l . , 1985; Reiner and Karten, 1982). F i n a l l y , d i r e c t i n t r a c e r e b r a l neurochemical m i c r o i n j e c t i o n of s p e c i f i c n e u r o t r a n s m i t t e r a g o n i s t s / a n t a g o n i s t s i n t o these r e g i o n s can e l i c i t or b l o c k locomotion (Chapters 3-5). Neurochemicals which were e f f e c t i v e at e l i c i t i n g locomotion from these r e g i o n s i n c l u d e c h o l i n e r g i c a g o n i s t s (Cnd & Cnv), GABAergic a n t a g o n i s t s (Cnv only) and g l u t a m a t e r g i c a g o n i s t s (Cnd and Cnv). The s t u d i e s u t i l i z i n g neurochemical i n j e c t i o n were combined 261 with available anatomical, immunohistochemical and receptor autoradiographic data from the l i t e r a t u r e i n an attempt to d i f f e r e n t i a t e afferents to Cnd and Cnv which excite or i n h i b i t these n u c l e i . My analysis revealed several p o t e n t i a l sources of motor related cholinergic input to Cnd and Cnv. Possible cholinergic afferents to Cnd arise from the subtrigeminal nucleus, TTD, RP, laterodorsal tegmental nucleus, and nucleus isthmi, pars p a r v o c e l l u l a r i s , while those to Cnv may arise from TTD, Rpc, Rgc and the nucleus mesencephalicus, pars profundus. In addition, both Cnd and Cnv contain i n t r i n s i c cholinergic neurons. While i t i s possible that TTD and other r e t i c u l a r formation (e.g. RP, Rgc, Rpc) structures give r i s e to the cholinergic motor connection, as e l e c t r i c a l stimulation of these regions gives r i s e to locomotion, i n s u f f i c i e n t information i s available to unequivocally i d e n t i f y any single pathway, or combination of pathways, as being responsible for my r e s u l t s . It i s clear, however, from the effects of carbachol on locomotion and the a b i l i t y of atropine, a muscarinic antagonist, to block the locomotor e f f e c t s e l i c i t e d by carbachol, that neurons i n both Cnd and Cnv appear to be under cholinergic control v i a muscarinic receptors. While cholinergic agonist i n j e c t i o n e l i c i t s locomotion i n both Cnv and Cnd, GABAergic antagonists induce locomotion only when injected into Cnv. Equally, GABA i n j e c t i o n blocks locomotion only when infused into Cnv. It i s l i k e l y , therefore, that neurons i n Cnd and Cnv play s l i g h t l y d i f f e r e n t roles i n locomotor control, receiving and integrating input from 262 d i f f e r e n t p a r t s o f the n e u r a x i s . The d i f f e r e n t neuroanatomical i n p u t s t o these n u c l e i (described, i n p a r t , above) and t h e i r d i f f e r e n t c y t o a r c h i t e c t u r e (Karten and Hodos, 1967) would suggest t h a t t h i s i s the case. , Locomotion i s a l s o e l i c i t e d through the i n f u s i o n of the gl u t a m a t e r g i c a g o n i s t NMDA i n t o both Cnd and Cnv i n the avian p r e p a r a t i o n . While g l u t a m a t e r g i c input t o both n u c l e i remains t o be c h a r a c t e r i z e d i n b i r d s , i n other s p e c i e s , r e t i c u l o s p i n a l neurons have been demonstrated t o r e c e i v e g l u t a m a t e r g i c input from s e v e r a l sources. In lamprey, i n t r i n s i c r e t i c u l a r formation e x c i t a t o r y amino a c i d - c o n t a i n i n g i n t e r n e u r o n s , which themselves c o n t a i n NMDA r e c e p t o r s , have been r e p o r t e d t o r e c e i v e t r i g e m i n a l , v e s t i b u l a r and ascending s p i n o b u l b a r i n p u t . The ne u r o t r a n s m i t t e r s u t i l i z e d by the l a t t e r pathways are undetermined (Dubuc et a l . , 1988). However, the int e r n e u r o n s have been demonstrated t o impinge on r e t i c u l o s p i n a l neurons (Dubuc et a l . , 1988). In mammals, descending e x c i t a t o r y amino a c i d e r g i c (EAA) input t o t h i s r e g i o n from t e l e n c e p h a l i c s t r u c t u r e s has been r e p o r t e d (Fagg and F o s t e r , 1983). A l s o i n mammals, neurochemical s t i m u l a t i o n u s i n g glutamate has been shown t o be e f f e c t i v e at e l i c i t i n g locomotion when i n f u s e d i n t o the r e t i c u l a r formation ( G a r c i a - R i l l and Skinner, 1987a; Noga et a l . , 1988). In b i r d s , t o my knowledge, t h e r e i s no i n f o r m a t i o n about g l u t a m a t e r g i c pathways or e x c i t a t o r y amino a c i d - c o n t a i n i n g neuronal elements. However, combined with the above data from a v a r i e t y o f s t u d i e s , my r e s u l t s suggest an important e x c i t a t o r y r o l e f o r glutamate in. the c o n t r o l o f l o c o m o t i o n - r e l a t e d 263 r e t i c u l a r formation s t r u c t u r e s . The above f i n d i n g s f o r Cnd and Cnv, which are s i m i l a r t o those found f o r synonymous s t r u c t u r e s i n other v e r t e b r a t e s ( G r i l l n e r , 1976; Armstrong, 1986), suggest t h a t r e t i c u l o s p i n a l neurons i n Cnd and Cnv serve as the brainstem l i a i s o n t o s p i n a l c o r d LPGs. The hi g h e r l e v e l s of c o n t r o l , the n e u r a l s t r u c t u r e s which impinge on and modulate the r e t i c u l a r formation n u c l e i , have not been c h a r a c t e r i z e d t o the same extent. However, s e v e r a l r e g i o n s have been i d e n t i f i e d which may exert c o n t r o l over Cnd and Cnv (Chapters 2-7). Two r o s t r a l r e t i c u l a r formation n u c l e i , RP and mMRF are l i k e l y c a n d i d a t e s . Both RP and mMRF p r o j e c t mainly t o Cnd, Cnv and only s p a r s e l y t o the s p i n a l c o r d (Webster, p e r s o n a l communication; Webster and Steeves, 1988). RP r e c e i v e s a f f e r e n t input from s e v e r a l motor r e l a t e d sources i n c l u d i n g TTD, tectum, cerebellum, v e s t i b u l a r n u c l e i and Cnd/Cnv. From a neuroanatomical standpoint, t h e r e f o r e , RP i s i d e a l l y s i t u a t e d t o i n t e g r a t e a broad range o f sensory i n f o r m a t i o n and i n f l u e n c e downstream motor s t r u c t u r e s . Evidence t h a t t h i s r e g i o n has a r o l e i n locomotor c o n t r o l comes from e l e c t r i c a l s t i m u l a t i o n s t u d i e s which demonstrate t h a t RP e l e c t r i c a l s t i m u l a t i o n e l i c i t s locomotion i n b i r d s (Chapters 1-5). Furthermore, locomotion can be evoked by the i n j e c t i o n o f GABAergic a n t a g o n i s t s and the glutamate a g o n i s t NMDA, but not c h o l i n e r g i c a g o n i s t s , i n t o RP (Chapters 4,5). While i t i s not yet p o s s i b l e t o determine the pathways through which these n e u r o t r a n s m i t t e r s e x e r t t h e i r e f f e c t s on RP, the data (see Chapters 1-5) suggest t h a t RP, l i k e 264 s e v e r a l other locomotor-related s t r u c t u r e s , appears t o e l i c i t locomotor behaviour mainly through i t s p r o j e c t i o n s to Cnd and Cnv. The neurotransmitters which subserve the c o n t r o l a l s o remain t o be determined, although, from the Cnd/Cnv data above, a c e t y l c h o l i n e and glutamate are p o s s i b l e candidates. S i m i l a r to RP, the mMRF p r o j e c t s mainly t o the brainstem r e t i c u l a r formation n u c l e i and only s p a r s e l y to the s p i n a l cord. The mMRF has strong r e c i p r o c a l connections w i t h the deep t e c t a l l a y e r s ( i n t e r c o l l i c u l a r nucleus, ICo) which appear to form one l i n k of the avian b a s a l g a n g l i a loop (Chapter 2). E l e c t r i c a l s t i m u l a t i o n of the mMRF e l i c i t s locomotion i n b i r d s (Chapter 2). In a d d i t i o n , neurochemical i n j e c t i o n s t u d i e s (Chapters 4,5) demonstrate t h a t the e l e c t r i c a l t h r e s h o l d f o r locomotion can be reduced by the i n f u s i o n of p i c r o t o x i n and NMDA i n t o t h i s r egion. Based on the above c o n s i d e r a t i o n s and those from mammalian stu d i e s ( G a r c i a - R i l l et a l . , 1985/ Mogenson and Brudzynski, 1986/ see Chapter 5), i t appears p o s s i b l e that the mMRF may form one component of an avian equivalent t o the mammalian medial MLR. A second component, p o t e n t i a l l y equivalent t o the mammalian l a t e r a l MLR, i s found i n the region of the midbrain i n t e r c o l l i c u l a r nucleus (ICo). ICo r e c e i v e s input from the s p i n a l cord (Hunt and Kunzle, 1976/ Webster and Steeves, i n p r e p a r a t i o n ) , deep t e c t a l l a y e r s , and cuneate and g r a c i l e n u c l e i (Wild et a l . , 1987). I t p r o j e c t s to TTD, Rgc, Cnv and high c e r v i c a l cord (Reiner and Karten, 1982/ Webster and Steeves, i n p r e p a r a t i o n ) . E l e c t r i c a l s t i m u l a t i o n of t h i s region (Chapter 2) e l i c i t s locomotion i n 265 b i r d s . I t s c o n n e c t i v i t y suggests t h i s nucleus may be e q u i v a l e n t t o the mammalian cuneiform nucleus (Cabot et a l . , 1982), a r e g i o n which has been i m p l i c a t e d through e l e c t r i c a l s t i m u l a t i o n s t u d i e s as the l a t e r a l MLR (Shik et a l . , 1967; Noga et al., 1988). The mammalian l a t e r a l MLR i s b e l i e v e d t o e x e r t motor c o n t r o l e f f e c t s v i a p r o j e c t i o n s t o the medullary r e t i c u l a r f ormation (Steeves and Jordan, 1984; Noga et a l . , 1988). F u r t h e r evidence c o r r e l a t i n g ICo with the mammalian MLR a r i s e s from neurochemical i n f u s i o n s i n t o t h i s r e g i o n which, as seen i n c a t s , demonstrates t h a t the GABAergic a n t a g o n i s t p i c r o t o x i n e l i c i t s locomotor behaviour (Chapter 4; G a r c i a - R i l l et a l . , 1983). While these r e s u l t s are i n d i c a t i v e o f a c o r r e l a t i o n between the mammalian and a v i a n MLRs, f u r t h e r t e s t i n g i s r e q u i r e d b e f o r e any f i r m e q u i v a l e n c y can be e s t a b l i s h e d . P a r t of the study r e q u i r e d t o e s t a b l i s h e q u i v a l e n c y o f the av i a n MLRs with t h e i r mammalian c o u n t e r p a r t s i s to d e s c r i b e the n u c l e i at the next l e v e l o f the h i e r a r c h y which c o n t r o l the a c t i v i t y o f the mesencephalic r e g i o n s . T r a n s e c t i o n s t u d i e s have shown t h a t n u c l e i i n the mammalian caudal diencephalon are i m p l i c a t e d i n such c o n t r o l , as p r e s e r v a t i o n o f these n u c l e i a f t e r s e l e c t i v e t r a n s e c t i o n allows spontaneous locomotor a c t i v i t y i n decerebrate p r e p a r a t i o n s ( f o r review, see Armstrong, 1986). The spontaneous a c t i v i t y i s a t t r i b u t e d t o the c o n t r o l which the p r e s e r v e d n u c l e i e x e r t over more caudal l o c o m o t i o n - r e l a t e d n u c l e i . To determine whether such r e g i o n s e x i s t i n b i r d s , d e c e r e b r a t i o n s were performed at v a r y i n g l e v e l s o f the neuraxis (Chapter 7). R o s t r a l d e c e r e b r a t i o n s which p r e s e r v e d a wedge of 266 neural t i s s u e c o n t a i n i n g the subthalamic nucleus (nucleus of the ansa l e n t i c u l a r i s ) , the l a t e r a l and p o s t e r i o r hypothalamic n u c l e i and the most r o s t r a l p o r t i o n of the s u b s t a n t i a n i g r a y i e l d e d spontaneous locomotion i n b i r d s . A b l a t i o n of t h i s region r e s u l t e d i n b i r d s which- would locomote only w i t h e l e c t r i c a l or neurochemical brainstem s t i m u l a t i o n . Previous s t u d i e s i n mammals have demonstrated that s i m i l a r d i e n c e p h a l i c s t r u c t u r e s (e.g. subthalamic nucleus, subthalamic locomotor region (SLR), zona i n c e r t a and l a t e r a l hypothalamic area) are preserved i n spontaneous decerebrate (acute premammillary p r e c o l l i c u l a r preparation) animals (Waller et a l . , 1940). Also i n mammals, these s t r u c t u r e s appear t o send p r o j e c t i o n s t o the more caudal locomotor regions (for review see Chapter 1). My r e s u l t s , t h e r e f o r e , suggest that counterparts of these mammalian d i e n c e p h a l i c regions are al s o found i n b i r d s . Whether they serve as the locus through which the avian l i m b i c system governs goal d i r e c t e d locomotor behaviours, as has been suggested f o r mammals (Mogenson et a l . , 1985), awaits f u r t h e r i n f o r m a t i o n . My s t u d i e s have revealed a novel locomotor region i n b i r d s which l i e s w i t h i n the confines of the pontine and r o s t r a l medullary medial l o n g i t u d i n a l f a s c i c u l u s (MLF) (Chapter 2). E l e c t r i c a l s t i m u l a t i o n of t h i s region repeatably e l i c i t s locomotor p a t t e r n s . Furthermore, the locomotion can be repeatably evoked by neurochemical i n f u s i o n (carbachol and NMDA), p r o v i d i n g c o n c l u s i v e evidence that neurotransmitter receptors (not en passant f i b r e s ) were s t i m u l a t e d to produce t h i s e f f e c t (Goodchild et a l . , 1982). S e r o t o n i n e r g i c c e l l 267 bodies, which s t a i n p o s i t i v e l y f o r a c e t y l c h o l i n e s t e r a s e (Dube and Parent, 1981; Taccogna et a l . , i n preparation) and l i e i n clos e p r o x i m i t y t o the i n j e c t i o n s i t e have been i m p l i c a t e d i n the locomotor response. These c e l l s have been shown to p r o j e c t t o the region of the midbrain p r e t e c t a l nucleus near s i t e s from which locomotion can be e l i c i t e d (Dube and Parent, 1981). The r o l e of these c e l l s i n locomotor c o n t r o l and the neuroanatomical substrate through which the c o n t r o l may be exerted, however, has yet t o be determined. Sensory i n f o r m a t i o n a l s o appears to have a r o l e i n motor c o n t r o l . At l e a s t one locus f o r the i n t e g r a t i o n of t h i s sensory informa t i o n may l i e w i t h i n the t r i g e m i n a l descending t r a c t and nucleus (TTD), a region which receives a v a r i e t y of sensory, as w e l l as c e n t r a l a f f e r e n t s and which, i n t u r n , p r o j e c t s to the hi n d b r a i n r e t i c u l a r formation n u c l e i (Cnd & Cnv) (see Chapters 1, 3-5). My st u d i e s i n b i r d s , s i m i l a r to those i n many ver t e b r a t e s (for review see M c C l e l l a n , 1986), have demonstrated th a t e l e c t r i c a l s t i m u l a t i o n of the pontobulbar locomotor s t r i p (PLS) e l i c i t s locomotion i n b i r d s . The e l e c t r i c a l l y induced locomotion (walking at threshold) can be maintained over considerable periods (>10 minutes) (Steeves et a l . , 1987; Funk et a l . , submitted), thereby i n d i c a t i n g that the locomotion i s not simply a t r a n s i t o r y escape behaviour. In b i r d s , as i n mammals, the s t i m u l a t i o n locus l i e s w i t h i n the TTD and s t r o n g l y i m p l i c a t e s t h i s region as being synonymous w i t h the PLS (Jordan, 1986; G a r c i a - R i l l and Skinner, 1986; Noga et a l . , 1988)(Figs. 1, Chapters 3-5). Furthermore, locomotion can be evoked by the 268 i n t r o d u c t i o n o f a v a r i e t y o f neurochemicals (carbachol, p i c r o t o x i n and NMDA) i n t o TTD. As d i s c u s s e d i n Chapter 3, intra-TTD c h o l i n e r g i c a g o n i s t i n j e c t i o n e l i c i t e d long l a s t i n g locomotion i n b i r d s . The most l i k e l y i n p u t s f o r t h i s c h o l i n e r g i c i n n e r v a t i o n a r i s e from the pontine and medullary r e t i c u l a r formation, the descending v e s t i b u l a r nucleus, the nucleus raphe p a l l i d u s and the p a r a b r a c h i a l r e g i o n , while TTD i t s e l f encompasses CHAT-containing neurons. Although s e v e r a l p o t e n t i a l pathways f o r c h o l i n e r g i c c o n t r o l of TTD have been p o s t u l a t e d , the locomotor e f f e c t s produced with i n j e c t i o n o f GABAergic and g l u t a m a t e r g i c a n t a g o n i s t s / a g o n i s t s remain without a s t r o n g neuroanatomical s u b s t r a t e (Chapters 4,5). GABA-containing c e l l bodies and r e c e p t o r s have been l o c a l i z e d t o TTD i n mammals (Mugnaini and O e r t e l , 1985), sugges t i n g t h a t GABA p l a y s some r o l e , p o s s i b l y s e l e c t i v e l y down-regulating a f f e r e n t i n f o r m a t i o n , i n locomotor c o n t r o l v i a TTD. A r o l e f o r glutamate, p o s s i b l y a n t a g o n i s t i c t o t h a t of GABA, may a l s o be p o s t u l a t e d (Chapter 5 ) . The above r e s u l t s i n b i r d s , taken t o g e t h e r with those from other v e r t e b r a t e s ( G r i l l n e r , 1976; Armstrong, 1986; G a r c i a - R i l l and Skinner, 1986; Jordan, 1986; Noga et al., 1988), suggest t h a t the PLS and TTD r e g i o n are e q u i v a l e n t . F u r t h e r , i t s neuroanatomical connections suggest t h a t the TTD r e g i o n i s i n t i m a t e l y a s s o c i a t e d w i t h a v a r i e t y of sensory i n p u t s and may t h e r e f o r e serve as an i n t e g r a t o r y c e n t r e through which sensory i n f o r m a t i o n a f f e c t s ongoing locomotor output. T h i s hypothesis 269 supports the hypothesis of Noga et al. (1988), which s t a t e s that P L S / t r i g e m i n a l / l a t e r a l r e t i c u l a r formation system "provides a substrate f o r sensorimotor r e f l e x i n i t i a t i o n of locomotion". Further support f o r and extension of t h i s hypothesis comes from a study designed t o determine whether phasic p e r i p h e r a l a f f e r e n t i n f o r m a t i o n i s e s s e n t i a l f o r the production of locomotor patterns i n b i r d s . In the study (Chapter 6), I examined the r o l e of phasic a f f e r e n t feedback by comparing the locomotor p a t t e r n s of animals p r i o r t o and f o l l o w i n g p a r a l y z a t i o n . My r e s u l t s i n d i c a t e t h a t a f f e r e n t input i s not e s s e n t i a l f o r the production of the wide array of avian locomotor patterns i n both high decerebrate spontaneously locomoting or i n s t i m u l a t e d (chemical and e l e c t r i c a l s t i m u l a t i o n ) p a r a l y z e d animals. One of the r e s u l t s of the study was th a t locomotion could be e l i c i t e d by t r i g e m i n a l f i e l d s t i m u l a t i o n i n b i r d s which previous to p a r a l y z a t i o n were spontaneous, but a f t e r p a r a l y z a t i o n d i d not show any spontaneous a c t i v i t y . This f i n d i n g , together w i t h the r e s u l t that increased s t i m u l a t i o n i n t e n s i t y was necessary to i n i t i a t e locomotion a f t e r p a r a l y z a t i o n i n low decerebrate animals, suggests that a f f e r e n t input may not only i n i t i a t e r e f l e x locomotion, but may a l s o serve t o set the animal's o v e r a l l a c t i v a t i o n l e v e l f o r locomotion. I f p e r i p h e r a l a f f e r e n t input serves to set the a c t i v a t i o n l e v e l , TTD may w e l l serve as the i n t e g r a t o r y centre f o r the i n f o r m a t i o n . The r e s u l t s of s t u d i e s presented i n t h i s t h e s i s s t r o n g l y suggest that b i r d s , l i k e mammals (Armstrong, 1986), possess a 270 h i e r a r c h i c a l system of locomotor control. The lowest l e v e l of the hierarchy i s found i n the spinal cord LPG. The LPG appears to be controlled both by afferent input and, more importantly for voluntary locomotion, v i a r e t i c u l o s p i n a l input from the brainstem r e t i c u l a r formation nuclei (Cnd & Cnv). In turn, control i s exerted on the r e t i c u l o s p i n a l neurons by sensory (e.g. v i a TTD) and ce n t r a l l y generated input (e.g. RP, ICo, mMRF,). Central structures themselves (e.g. subthalamic region, basal ganglia) appear to be under higher lev e l s of control from limbic and telencephalic regions. The general impression of my studies strongly suggests that avian locomotion-related structures and t h e i r interconnections are s i m i l a r to those found i n both higher and lower vertebrates. This s i m i l a r i t y i s discussed throughout the thesis (Chapters 1-7). While the neural c i r c u i t r y which comprises the hierarchy appears to have been highly conserved during the evolutionary process, considerably more information i s required for the understanding of neural locomotor control mechanisms. However, i n l i g h t of the currently available techniques, I believe that the study of motor control has reached a point from which i t w i l l now be possible to define and manipulate a l l of the major neural pathways involved i n locomotor control. Techniques such as neuroanatomical tracing, both retrograde and anterograde, make i t possible to define e f f e c t i v e l y the afferent and efferent connections of every brain region (Steeves and Jordan, 1984; G a r c i a - R i l l et a l . , 1983a; Webster and Steeves, 1988). Immunohistochemistry allows description of some of the 271 n e u r o t r a n s m i t t e r s u t i l i z e d by these pathways (Taccogna et al., i n p r e p a r a t i o n ) . Receptor autoradiography d e f i n e s some of the n e u r o t r a n s m i t t e r r e c e p t o r s present both p r e - and p o s t s y n a p t i c a l l y ( D i e t l et al., 1988). E l e c t r o p h y s i o l o g y allows us t o r e c o r d from i n d i v i d u a l neurons t o examine t h e i r a c t i v i t y (Orlovsky, 1970a, 1972b,c; G a r c i a - R i l l and Skinner, 1988) and in vivo m i c r o d i a l y s i s ( P h i l l i p s et a l . , 1988) determines changes i n n e u r o t r a n s m i t t e r c o n c e n t r a t i o n s from s p e c i f i c r e g i o n s d u r i n g a c t i v i t y . F i n a l l y , s e l e c t i v e i n t r a c e r e b r a l neurochemical i n f u s i o n o f n e u r o t r a n s m i t t e r a g o n i s t s and a n t a g o n i s t s allows us to manipulate the process (Chapters 3-5/ G a r c i a - R i l l et al., 1985; Noga et al., 1988). While these techniques, though powerful, a l l o w f o r the d e f i n i t i o n and ma n i p u l a t i o n o f locomotor pathways, only the i n t e g r a t i o n o f i n f o r m a t i o n gathered from these techniques w i l l i n c r e a s e the understanding o f locomotor c o n t r o l and have p r e d i c t i v e v a l u e f o r f u t u r e s t u d i e s . In t h i s t h e s i s , I have attempted t o u t i l i z e t h i s i n t e g r a t e d approach i n the study o f av i a n locomotion. The a t t r i b u t e s which the b i r d possesses, i n c l u d i n g i t s b i p e d a l locomotion, two separate modes of locomotion and absence of a c o r t i c o s p i n a l t r a c t make the b i r d an e x c e l l e n t animal model f o r the study o f many aspects of locomotion. 272 CHAPTER 9 LIST OF REFERENCES 273 Adams R.D. P a i n In: H a r r i s o n ' s P r i n c i p l e s o f I n t e r n a l Medicine, N i n t h E d i t i o n , K . J . I s s e l b a c h e r , R.D. Adams, E. Braunwald, R.G. P e t e r s d o r f and J.D. Wilson (Eds.). McGraw-Hill, New York, 1980. pp. 13-20. A f e l t Z. 1972. F u n c t i o n a l s i g n i f i c a n c e o f v e n t r a l descending t r a c t s o f the s p i n a l c o r d of the c a t . Ac t a N e u r o b i o l . Exper. 34:393-407. Ajima A. and T. Kato. 1988. B r a i n d i a l y s i s : d e t e c t i o n of a c e l t y c h o l i n e r e l e a s e i n the s t r i a t u m , hippocampus and f r o n t a l c o r t e x of f r e e l y moving r a t s . B i o g e n i c Amines 5(6):461-464. Aoki M. and S. Mori. 1981. Locomotion e l i c i t e d by pinna s t i m u l a t i o n i n the acute p r e c o l l i c u l a r - p o s t - m a m m i l l a r y decerebrate c a t . B r a i n Res.214:424-428. Arends J.J.A. and J.L. Dubbeldam. 1984. The s u b n u c l e i and primary a f f e r e n t s of the descending t r i g e m i n a l system i n the m a l l a r d (Anas pl a t y r h y n c h o s L . ) . Neuroscience 13 (3) : 781-795.. Arends J.J.A. and J.L. Dubbeldam. 1982. E x t e r o c e p t i v e and p r o p r i o c e p t i v e a f f e r e n t s of the t r i g e m i n a l and f a c i a l motor n u c l e i i n the m a l l a r d (Anas pl a t y r h y n c h o s L . ) . J . Comp. Neurol. 209:313-329. Arends J.J.A., A. Woelders-Blok and J.L. Dubbeldam. 1984. The e f f e r e n t connections of the n u c l e i o f the descending t r i g e m i n a l t r a c t i n the m a l l a r d (Anas p l a t y r h y n c h o s L . ) . Neuroscience 13 (3) :797-817. Armstrong D.M. 1986. S u p r a s p i n a l c o n t r i b u t i o n s t o the i n i t i a t i o n and c o n t r o l of locomotion i n the c a t . Prog. N e u r o b i o l . (Oxf) 26 (4) :273-361. Arshavsky Y.I. and G.N. Orlovsky. Role of the cerebellum i n the c o n t r o l o f rhythmic movements. In: Neural C o n t r o l of V e r t e b r a t e Locomotion. S . G r i l l n e r , R.M. Herman, P.S.G. S t e i n , H. F o r s s b e r g and D.G. S t u a r t (Eds.). MacMillan Press, London, 1986. pp. 677-689. A s i n K.E. and H.C. F i b i g e r . 1983. An a n a l y s i s of neuronal elements w i t h i n the median nucleus of the raphe t h a t mediate l e s i o n - i n d u c e d i n c r e a s e s i n locomotor a c t i v i t y . B r a i n Res. 268:211-223. Awapara J . , A . J . Landua, R. F u e r s t and B. Seale. 1950. Free y-aminobutyric a c i d i n b r a i n . Jour. B i o l . Chem. 187 (1) :35-39. Bard P. and M.B. Macht. 1958. The behavior of c h r o n i c a l l y d ecerebrate c a t s . In: N e u r o l o g i c a l B a s i s of Behavior. G. Wostenholme and CM. O'Connor (Eds.). L i t t l e Brown, Boston, pp. 55-71. Barnard E.A., M.G. D a r l i s o n and P. Seeburg. 1987. M o l e c u l a r b i o l o g y of the GABAA r e c e p t o r : the r e c e p t o r / c h a n n e l s u p e r f a m i l y . 274 TINS 10 (12) :502-508. Becker J.B., F. Adams and T.E. Robinson. 1988. I n t r a v e n t r i c u l a r m i c r o d i a l y s i s : a new method f o r determining monoamine m e t a b o l i t e c o n c e n t r a t i o n s i n the c e r e b r o s p i n a l f l u i d o f f r e e l y moving r a t s . J . N e u r o s c i . Methods 24(3): 271-278. Berk M.L. and J.A. F i n k e l s t e i n . 1983. Long descending p r o j e c t i o n s of the hypothalamus i n pigeon, (Columba l i v i a ) . J . Comp. Neu r o l . 220:127-136. Berk M.L. and R.F. Hawkin. 1985. Ascending p r o j e c t i o n s of the mammillary r e g i o n i n the pigeon: emphais on t e l e n c e p h a l i c c o n n ections. J . Comp. Neu r o l . 239 (3):330-340. Besson J.M. and D. Le Bars. E f f e c t s of intravenous a d m i n i s t r a t i o n of morphine and r e l a t e d compounds on the a c t i v i t y of d o r s a l horn neurons i n the s p i n a l c o r d . In: Advances i n Pai n Research and Therapy Volume 3, Proceedings of the Second World Congress on P a i n . Bonica J . J . , J.C. L i e b e s k i n d and D.G. A l b e - F e s s a r d (Eds.), Raven Press, New York. 1979. pp. 441-458. Bloom F.E., Neurohumoral t r a n s m i s s i o n and the c e n t r a l nervous system. In: The Pharmacological B a s i s of T h e r a p e u t i c s . A.G. Gilman, L.S. Goodman, T.W. R a i l and F. Murad (Eds.), Macmillan P u b l i s h i n g , New York. 1985 pp. 236-259. Brauth S.E., J.L. Ferguson and C A . K i t t . 1978. P r o s e n c e p h a l i c pathways r e l a t e d t o the p a l e o s t r i a t u m of the pigeon (Columba  l i v i a ) . B r a i n Res. 147:205-221. Brudzynski S.M., P.E. Houghton, R.D. Bronlee and G.J. Mogenson. 1986. Involvement of neuronal c e l l bodies of the mesencephalic locomotor r e g i o n i n the i n i t i a t i o n of locomotor a c t i v i t y i n f r e e l y behaving r a t s . B r a i n Res. B u l l . 16 (3) :377-382. Brudzynski, S.M. and G.J. Mogenson. 1986. Decrease i n locomotor a c t i v i t y by i n j e c t i o n s of c a r b a c h o l i n the a n t e r i o r hypothalamic p r e o p t i c area of the r a t . B r a i n Res 376(1): 38-46. Brudzynski S.M., M. Wu and G.J. Mogenson. 1988. Modulation of locomotor a c t i v i t y induced by i n j e c t i o n s of c a r b a c h o l i n t o the tegmental pedunculopontine nucleus and adjacent areas i n the r a t . B r a i n Res. 451:119-125. Buckley N.J., T.I. Bonner and M.R. Brann. 1988. L o c a l i z a t i o n of a f a m i l y of m u s c a r i n i c r e c e p t o r mRNAs i n r a t b r a i n . J . N e u r o s c i . 8 (12) :4646-4652. Budakova N.N. and M.L. Shik. 1970. E f f e c t of brain-stem s t i m u l a t i o n evoking locomotion on ascending r e f l e x e s i n the mesencephalic c a t . B u l l . Exp. B i o l . Med. 69:5-8. Cabot J.B., A. Reiner and N. Bogan. 1982. Avian b u l b o s p i n a l pathways: anterograde and r e t r o g r a d e s t u d i e s of c e l l s of o r i g i n , f u n i c u l a r t r a j e c t o r i e s and laminar t e r m i n a t i o n s , i n Progress i n B r a i n Research V o l . 57, Anatomy of descending pathways to the 275 s p i n a l cord. H.G.J.M.Kuypers and G.F. Martin (eds.) E l s e v i e r Biomedical Press, Amsterdam, pp. 79-108. Carpenter D.O. and T.S. Reese. Chemistry and physiology of synapti c t r a n s m i s s i o n . In: B a s i c Neurochemistry, G.J. S i e g e l , R.W. A l b e r s , B.W. Agranoff and R. Katzman (Eds.). L i t t l e , Brown and Co., Boston., 1981. pp. 161-181. Carpenter M.B. 1978. Core Text of Neuroanatomy. W i l l i a m and W i l k i n s , Baltimore, pp. 249-254. Carpenter M.B. and J . S u t i n . Human Neuroanatomy.(8th E d i t i o n ) W i l l i a m s and W i l k i n s , Baltimore, 1983. Charpentier J . A n a l y s i s and measurement of pain i n animals A new conception of p a i n . In: Pain, Proceedings of the I n t e r n a t i o n Symposium on Pain organized by the Laboratory of Psychophysiology, F a c u l t y of Sciences, P a r i s . S o u l a i r a c A., J . Cahn and J . Charpentier (Eds.). Academic Press, London and New York, 1968. pp. 171-200. C h i l d s J.A. and K. Gale. 1983. Neurochemical evidence f o r a nigrotegmental gabaergic p r o j e c t i o n . B r a i n Res. 258:109-114. Choi D.W. 1988. Glutamate n e u r o t o x i c i t y and diseases of the nervous system. Neuron 1:623-634. C h r i s t i e M.J. and R.A. North. 1988. Co n t r o l of i o n conductances by muscarinic r e c e p t o r s . TIPS Suppl. Feb. 1988:30-39. Cohen A.H. and P. Wallen. 1980. The neuronal c o r r e l a t e of locomotion i n f i s h . " F i c t i v e swimming" induced i n an i n v i t r o p r e p a r a t i o n of the lamprey s p i n a l cord. Exp. B r a i n Res. 41:11-18. Denny-Brown D. The Cerebral C o n t r o l of Movement. Thomas:Springfield. 1966 Desan P.H., E.R. Gruberg and F. Eckenstein. 1984. A c h o l i n e r g i c p r o j e c t i o n from the nucleus i s t h m i t o the o p t i c tectum i n t u r t l e and f r o g . Soc. Neurosci. A b s t r . 10:575. Desarmenien M., F. Santangelo, G. O c c h i p i n t i , R. S c h l i c h t e r , J.P. L o e f f l e r , E. Desaulles, B.A. Demeneix and P. F e l t z . E l e c t r o p h y s i o l o g i c a l study of GABAA versus GABAB receptors on e x c i t a t i o n - s e c r e t i o n c o u p l i n g . In: Advances i n Biochemical Psychopharmacology V o l . 37, CNS Receptors From Molecular Pharmacology t o Behavior. Raven Press, New York. 1983, pp. 93-105. D i e t l M.M., R. Cortes and J.M. P a l a c i o s . 1988. Neurotransmitter receptors i n the avian b r a i n . I I . Muscarinic c h o l i n e r g i c r e c e p t o r s . B r a i n Research 439:360-365.. Diet z V. 1987. Role of p e r i p h e r a l a f f e r e n t s and s p i n a l r e f l e x e s i n normal and impaired human locomotion. Rev. Neurol. (Paris) 276 143 (4) :241-254. Dube L. and A. Parent. 1981. The monoamine-containing neurons i n the a v i a n b r a i n : I. A study of the b r a i n stem of the chicken ( G a l l u s domesticus) by means of f l u o r e s c e n c e and a c e t y l c h o l i n e s t e r a s e h i s t o c h e m i s t r y . J . Comp. Neu r o l . 196 :695-708. Dubner R. and G.J. Bennett. 1983 . S p i n a l and t r i g e m i n a l mechanisms of n o c i c e p t i o n . Ann. Rev. N e u r o s c i . 6:381-418. Dubuc R., Y. Ohta and S. G r i l l n e r . 1988. E x c i t a t o r y amino a c i d and GABA t r a n s m i s s i o n to r e t i c u l o s p i n a l neruons i n the lamprey. N e u r o s c i . Absts. 14(1):264. Edwards S.B. 1975. A u t o r a d i o g r a p h i c s t u d i e s of the p r o j e c t i o n s of the midbrain r e t i c u l a r formation: descending p r o j e c t i o n s of nucleus c u n e i f o r m i s . J . Comp. Neu r o l . 161:341-358. Edwards S.B. 1980. The deep l a y e r s of the s u p e r i o r c o l l i c u l u s : t h e i r r e t i c u l a r c h a r a c t e r i s t i c s and s t r u c t u r a l o r g a n i z a t i o n . In: The R e t i c u l a r Formation R e v i s i t e d . J.A. Hobson and M.A.B. B r a z i e r (Eds.), Raven P r e s s . New York, pp 193-209. E i d e l b e r g E. 1981. Consequences of s p i n a l c o r d l e s i o n s upon motor f u n c t i o n , with s p e c i a l r e f e r e n c e to locomotor a c t i v i t y . Prog. N e u r o b i o l . 17:185-202. E i d e l b e r g E., J.L. Story, J.G. Walden, and B.L. Meyer. 1981a. Anatomical c o r r e l a t e s of r e t u r n of locomotor f u n c t i o n a f t e r p a r t i a l s p i n a l c o r d l e s i o n s E i d e l b e r g E., J.G. Walden, and L.H. Nguyen. 1981b. Locomotor C o n t r o l i n Macaque Monkeys. B r a i n 104;647-663. E l d r i d g e F.L., D.E. M i l l h o r n , J.P. K i l e y and T.G. Waldrop. 1985. S t i m u l a t i o n by c e n t r a l command of locomotion, r e s p i r a t i o n and c i r c u l a t i o n d u r i n g e x e r c i s e . R e s p i r a t i o n P h y s i o l o g y 59:313-337. Emmers R. P a i n : A S p i k e - I n t e r v a l Coded Message i n the B r a i n . Raven Press, New York, 1981. pp. 1-134. Fagg G.E. and A.C. F o s t e r . 1983. Amino a c i d n e u r o t r a n s m i t t e r s and t h e i r pathways i n the mammalian c e n t r a l nervous system. Neuroscience 9 (4):701-719. Fanardzhyan V.V. and D.S. S a r k i s y a n . 1984. Neuronal mechanisms of i n t e r a c t i o n s between the r e d nucleus and other b r a i n stem s t r u c t u r e s . Neurophysiology 16(5):506-517. D.N. Franz. C e n t r a l nervous system s t i m u l a n t s . In: Goodman and Gilman's The Pharmacological B a s i s of T h e r a p e u t i c s , Seventh E d i t i o n , A.G. Gilman, L.S. Goodman, T.W. R a i l and F. Murad. (Eds.). Macmillan, New York. 1985 . pp. 582-588. Freedman S.B., E.A. Harley and L.L. Iversen. B i o c h e m i c a l measurement of m u s c a r i n i c r e c e p t o r e f f i c a c y and i t s r o i e i n 277 r e c e p t o r r e g u l a t i o n . TIPS Suppl. Feb. 1988:54-60. Freusberg A. 1874. Reflexbewegungen beim Hund. Pfug e r s . Arch, ges. P h y s i o l . 9; 358-391. (from Wetzel M.C. and D.G. S t u a r t . 1976. Ensemble c h a r a c t e r i s t i c s of cat locomotion and i t s n e u r a l c o n t r o l . P r o g .Neurobiol. 7:1-98.) Freusberg A. and F r . G o l t z . 1874a Ueber gefasserwesternde nerven. P f l u g e r s . Arch. ges. P h y s i o l . 9; 174-197 (from Wetzel M.C. and D.G. S t u a r t . 197 6. Ensemble c h a r a c t e r i s t i c s of cat locomotion and i t s n e u r a l c o n t r o l . Prog. N e u r o b i o l . 7:1-98. Funk G.D., W.K. Milsom, G.N. Sholomenko and J.D. Steeves. Role o f the t e l e n c e p h a l o n i n the s y n c h r o n i z a t i o n of locomotor and r e s p i r a t o r y f r e q u e n c i e s d u r i n g walking i n Canada geese, (submitted). F u r g o t t R.F. and P.D. Cherry. 1984. The m u s c a r i n i c r e c e p t o r of v a s c u l a r endothelium t h a t subserves v a s o d i l a t i o n . TIPS Suppl. Jan. 1984:45-48. G a r c i a - R i l l E. 1983. Connections of the mesencephalic locomotor r e g i o n (MLR) I I I . i n t r a c e l l u l a r r e c o r d i n g s . Br. Res. B u l l . 10:73-81. G a r c i a - R i l l E. 1986. The b a s a l g a n g l i a and the locomotor r e g i o n s . B r a i n Res. Reviews 11:47-63. G a r c i a - R i l l E., C.R. Houser, R.D. Skinner, W. Smith and D.J. Woodward. 1987. Locomotion-inducing s i t e s i n the v i c i n i t y o f the. pedunculopontine nucleus. B r a i n Res. B u l l . 18:731-738. G a r c i a - R i l l E. and R.D. Skinner. 1985. The mesencephalic locomotor r e g i o n . I. Descending c h o l i n e r g i c p r o j e c t i o n s t o the medial tegmental f i e l d . Anat. Rec. 211:65A. G a r c i a - R i l l E., and R.D. Skinner. The b a s a l g a n g l i a and the mesencephalic locomotor r e g i o n . In: Neural C o n t r o l of V e r t e b r a t e Locomotion. S . G r i l l n e r et a l . , (Eds). MacMillan Press, London 1986. pp. 77-103. G a r c i a - R i l l E. and R.D. Skinner. 1987a. The mesencephalic locomotor r e g i o n . I. A c t i v a t i o n of the medullary p r o j e c t i o n s i t e . B r a i n Research 411:1-12. G a r c i a - R i l l E. and R.D. Skinner. 1987b. The mesencephalic locomotor r e g i o n . I I . P r o j e c t i o n s t o r e t i c u l o s p i n a l neurons. B r a i n Research 411:13-20. G a r c i a - R i l l E. and R.D. Skinner. 1988. Modulation of rhythmic f u n c t i o n i n the p o s t e r i o r midbrain. Neuroscience 27 (2):639-654. G a r c i a - R i l l , E., R D. Skinner, C. Conrad, D. Mosley and C. Campbell. 1986. P r o j e c t i o n s of the mesencephalic locomotor r e g i o n i n the r a t . B r a i n Res B u l l . 1 7 ( 1 ) : 33-40. G a r c i a - R i l l E., R.D. Skinner, and J.A. F i t z g e r a l d . 1983a. 278 A c t i v i t y i n the mesencephalic locomotor region during locomotion. E x p t l . Neurol. 82:609-622. G a r c i a - R i l l E., R.D. Skinner and J.A. F i t z g e r a l d . 1985. Chemical a c t i v a t i o n of the mesencephalic locomotor region (MLR). B r a i n Res. 330:43-54. G a r c i a - R i l l E., R.D. Skinner, S.A. Gilmore, and R. Owings. 1983b. Connections of the mesencephalic locomotor region (MLR) I I . a f f e r e n t s and e f f e r e n t s . Br. Res. B u l l . 10:63-71. G a r c i a - R i l l E., R.D. Skinner, M.B. Jackson, and M.M. Smith. 1983a. Connections of the mesencephalic locomotor region (MLR) 1. s u b s t a n t i a n i g r a a f f e r e n t s . Br. Res. B u l l . , V o l . 10:57-62. Glover J.C., G. P e t u r s d o t t i r and J.K.S. Jansen. 1986. Fluorescent dextran-amines used as axonal t r a c e r s i n the nervous system of the chicken embryo. J . Neurosci. Methods 18 (3) :243-254. Goldsmith M. and D. van der Kooy. 1988. Separate n o n - c h o l i n e r g i c descending p r o j e c t i o n s and c h o l i n e r g i c ascending pathways from the nucleus tegmenti pedunculopontinus. B r a i n Res. 445:386-391. Goodchild A.K., R.A.L. Dampney and R. Bandler. 1982. A method f o r evoking p h y s i o l o g i c a l responses by s t i m u l a t i o n of c e l l bodies, but not axons of passage, w i t h i n l o c a l i z e d regions of the c e n t r a l nervous system. J . Neurosci. Methods 6:351-363. Graham-Brown T. 1911. The i n t r i n s i c f a c t o r s i n the act of progression i n the mammal. Royal Society of London, Proceedings, B, V o l . 84:308-319. Graham-Brown T. 1914. On the nature of the fundamental a c t i v i t y of the nervous centres; together w i t h an a n a l y s i s of the c o n d i t i o n i n g of rhythmic a c t i v i t y i n progression, and a theory of the e v o l u t i o n of f u n c t i o n i n the nervous system. J . P h y s i o l . 48:1-46. Greenamyre J.T., A.B. Young and J.B. Penney. 1984. Q u a n t i t a t i v e autoradiographic d i s t r i b u t i o n of L-[3H]glutamate-binding s i t e s i n r a t c e n t r a l nervous system. J . Neuroscience 4(8):2133-2144. G r i l l n e r S. 1975. Locomotion i n v e r t e b r a t e s : c e n t r a l mechanisms and r e f l e x i n t e r a c t i o n . P h y s i o l o g i c a l Reviews 45:249-304. G r i l l n e r S. 1985. N e u r o b i o l o g i c a l bases of rhythmic motor acts i n v e r t e b r a t e s . Science 228 (4696):143-149. G r i l l n e r S. and S. Kashin. 1976. On the generation and performance of swimming i n f i s h , i n Neural C o n t r o l of Locomotion. Herman R.M., S . G r i l l n e r , P.S.G. S t e i n and D.G. Stuart (eds.). Plenum P u b l i s h i n g , New York. pp. 181-201. G r i l l n e r S. and M.L. Shik. 1973. On the descending c o n t r o l of the lumbosacral s p i n a l cord from the "mesencephalic locomotor 279 r e g i o n " . A c t a P h y s i o l . Scand. 87:320-333. G r i l l n e r S. and P. Zangger. 1979. On the c e n t r a l g e n e r a t i o n of locomotion i n the low s p i n a l c a t . Exp. B r a i n Res. 34:241-261. H a e r t i g E.W. and J.H. Masserman. 1940. Hypothalamic l e s i o n s and pneumonia i n c a t s . With notes on behaviour changes. J . Neu r o p h y s i o l . 3:293-299. Haldeman S. and H. McLennan. 1972. The a n t a g o n i s t i c a c t i o n o f glutamic a c i d d i e t h y l e s t e r towards amino a c i d - i n d u c e d and s y n a p t i c e x c i t a t o n s of c e n t r a l neurones. B r a i n Res. 45:393-400. H a l l K., S.E. Brauth and C A . K i t t . 1984. Retrograde t r a n s p o r t of [ H]GABA i n the s t i a t o t e g m e n t a l system of the pigeon. B r a i n Res. 310:157-163. Hammond C , J . Feger, B. B i o u l a c and J.P. Souteyrand. 1979. Experimental h e m i b a l l i s m i n the monkey produced by u n i l a t e r a l k a i n i c a c i d l e s i o n i n corpus L u y s i i . B r a i n Res. 171:577-580. H i l l e B. I o n i c Channels o f E x c i t a b l e Membranes. Sinauer A s s o c i a t e s Inc. Sunderland, Mass. 1984. Hinsey J . C , S.W. Ranson, and R.F. McNattin. 1930. The r o l e of the hypothalamus and mesencephalon i n locomotion. Arch. N e u r o l . P s y c h i a t . 23:1-43. Holstege J.C. and H.G.J.M. Kuypers. 1987. Brainstem p r o j e c t i o n s to s p i n a l motoneurons: an update. Neuroscience 23 (3) :809-821. Hongo T., E. Jankowska and A. Lundberg. 1969a. The r u b r o s p i n a l t r a c t . I. E f f e c t s o f alpha-motoneurons i n n e r v a t i n g hindlimb muscles i n c a t s . E x p t l . B r a i n Res. 7:344-364. Huerta M.F. and J.K. H a r t i n g . T e c t a l c o n t r o l of s p i n a l c o r d a c t i v i t y : neuroanatomical demonstration of pathways connecting the s u p e r i o r c o l l i c u l u s with the c e r v i c a l s p i n a l c o r d grey. In: Progress i n B r a i n Research, Volume 57, Anatomy of Descending Pathways t o the S p i n a l Cord, Kuypers, H.J.CM. and Martin, G-F. (Eds.). E l s e v i e r Biomedical Press, Amsterdam, 1982. pp. 293-328. Hongo T., E. Jankowska and A. Lundberg. 1969b. The r u b r o s p i n a l t r a c t . I I . F a c i l i t a t i o n of i n t e r n e u r o n a l t r a n s m i s s i o n i n r e f l e x paths t o motoneurones. E x p t l . B r a i n Res. 7:365-391. Hunt S.P. and N. Brecha. 1984. The av i a n o p t i c tectum: a s y n t h e s i s o f morphology and b i o c h e m i s t r y . In: Comparative Neurology o f the O p t i c Tectum, Vanegas H. (ed.), Plenum P r e s s . New York. pp. 619-648. Hunt S.P and H. Kunzle. 1976. Observations on the p r o j e c t i o n s and i n t r i n s i c o r g a n i z a t i o n o f the pigeon o p t i c tectum: an a u t o r a d i o g r a p h i c study based on anterograde and re t r o g r a d e , axonal and d e n d r i t i c flow. J . Comp. Neurol. 170:153-172. Hunt S.P., P. S t r e i t , H. Kunzle and M. Cuenod. 1977. 280 C h a r a c t e r i z a t i o n of the pigeon i s t h m o - t e c t a l pathway by s e l e c t i v e uptake and retrograde movement of r a d i o a c t i v e compounds and by g o l g - l i k e horeseradish peroxidase l a b e l l i n g . B r a i n Res. 129:197-212. Ingram, W.R. and S.W. Ranson. 1932. E f f e c t s of l e s i o n s i n the red n u c l e i i n c a t s . Arch. Neural. P s y c h i a t . 28:482-513. Jacobson R.D., and M. Hollyday. 1982. E l e c t r i c a l l y evoked walking and f i c t i v e locomotion i n the chick J . Neurophysiol. 48:257-270. Jankowska E., M.G.M. Jukes, S. Lund and A. Lundberg. 1967. The e f f e c t o f DOPA on the s p i n a l cord. IV. H a l f - c e n t r e o r g a n i z a t i o n of interneurones t r a n s m i t t i n g e f f e c t s from the f l e x o r r e f l e x a f f e r e n t s . Acta P h y s i o l . Scand. 70:389-402. J e l l R.M., C. E l l i o t t and L.M. Jordan. 1985. I n i t i a t i o n of locomotion from the mesencephalic locomotor r e g i o n : e f f e c t s of s e l e c t i v e brainstem l e s i o n s . B r a i n Res. 328:121-128. Jones D.L. and G.J. Mogenson. 1980. Nucleus accumbens to globus p a l l i d u s GABA p r o j e c t i o n s : e l e c t r o p h y s i o l o g i c a l and i o n t o p h o r e t i c i n v e s t i g a t i o n s . B r a i n Res. 188:93-105. Jones B.E., M. Pare and A. Beaudet. 1986. Retrograde l a b e l l i n g of neurons i n the b r a i n stem f o l l o w i n g i n j e c t i o n s of ( H)choline i n t o the s p i n a l cord, i n press. Jones D.L. and G.J. Mogenson. 1980. Nucleus accumbens to globus p a l l i d u s GABA p r o j e c t i o n subserving ambulatory a c t i v i t y . Amer. J . P h y s i o l . 238:R65-R69. Jordan L.M. I n i t i a t i o n of locomotion from the mammalian brainstem. In:Neural C o n t r o l of Vertebrate Locomotion, (eds. S . G r i l l n e r , R.M. Herman, P.S.G. S t e i n , H. Forssberg and D.G. S t u a r t ) , MacMillan Press, London, 1986, pp. 21-37. Karls s o n G., M. Pozza and H. Olpe. 1988. Phaclofen: a GABAB b l o c k e r reduces long-duration i n h i b i t i o n i n the neocortex. Eur. J . Pharmacol. 148:485-486. Karten H.J. and W. Hodos. 1967. A s t e r e o t a x i c a t l a s of the b r a i n of the pigeon (Columba l i v i a ) . John Hopkins Press, Baltimore. 193p. Kashin S.M., A.G. Fel'dman and G.N. Orlovsky. 1974. Locomotion of f i s h evoked by e l e c t r i c a l s t i m u l a t i o n of the b r a i n . B r a i n Res. 82:41-47. Katayama Y., D.S. DeWitt, D.P. Becker and R.L. Hayes. 1984. Behavioural evidence f o r a c h o l i n o c e p t i v e pontine i n h i b i t o r y area: descending c o n t r o l of s p i n a l motor output and sensory in p u t . BrainRes. 296:241-262. Kazennikov O.V., V.A. Selionov, M.L. Shik and G.V. Yakovleva. 1983b. Stepping movements induced i n cats by s t i m u l a t i o n of the 281 d o r s o l a t e r a l f u n i c u l u s of the s p i n a l cord. B u l l . Exp. B i o l . Med. 96:1036 -1039 . Kazennikov O.V., M.L. Shik and G.V. Yakovleva. 1983a. Responses of upper c e r v i c a l s p i n a l neurons i n ca t s t o s t i m u l a t i o n of the brain-stem locomotor r e g i o n at d i f f e r e n t f r e q u e n c i e s . Neurophysiology 15(4):256- 261. Kazennikov O.V., M.L. Shik and G.V. Yakovleva. 1980. Two pathways f o r t h e b r a i n stem "locomotor i n f l u e n c e " on the s p i n a l c o r d . Sechenov P h y s i o l . J . USSR. 66 (8) :1259-1263. Kimura H., P.L. McGeer, J.H. Peng and E.G. McGeer. 1981. The c e n t r a l c h o l i n e r g i c system s t u d i e d by c h o l i n e a c e t y l t r a n s f e r a s e immunohistochemistry i n the c a t . J . Comp. Neurol. 200:151-201. K i n j o N., Y. A t s u t a , R.D. Skinner, M.O. Webber and E. G a r c i a - R i l l . 1988. E l e c t r i c a l l y and c h e m i c a l l y induced locomotion f o l l o w i n g a c t i v a t i o n o f the mediovental medulla i n the r a t . N e u r o s c i . Absts. 14(1) :182. K i s h i d a R., J.L. Dubbeldam and R.C. G o r i s . 1985. Primary sensory g a n g l i o n c e l l s p r o j e c t i n g t o the p r i n c i p a l t r i g e m i n a l nucleus i n the m a l l a r d , Anas p l a t y r h y n c h o s . J . Comp. Neu r o l . 240:171-179. Krogsgaard-Larsen P., E. F a l c h , M.J. Peet, J.D. Leah and D.R. C u r t i s . M o l e c u l a r pharmacology o f the GABA r e c e p t o r s and GABA a g o n i s t s . In: Advances i n Bioch e m i c a l Psychopharmacology V o l . 37, CNS Receptors From M o l e c u l a r Pharmacology t o Behavior. Raven Press, New York. 1983, pp. 1-13. Kuypers, H.J.G.M. 1964. The descending pathways t o the s p i n a l cord, t h e i r anatomy and f u n c t i o n . In Progress i n B r a i n Research, O r g a n i z a t i o n o f the S p i n a l Cord, V o l . I I , J.C. E c c l e s and J.P. Schade (eds.). E l s e v i e r B i o m e d i c a l Press, Amsterdam, pp. 178-200. Kuypers H.G.J.M. 1981. Anatomy of the descending pathways. In: Handbook of Phys i o l o g y , the Nervous System, V o l . I I , Motor C o n t r o l , p a r t I, J.M. Brookhart, V.B. Mountcastle, V.B. Brooks and S.R. Geiger. (Eds.), pp.597-666. Kuypers, H.J.G.M. 1982. A new look at the o r g a n i z a t i o n o f the motor system. In: Progress i n B r a i n Research, Anatomy of Descending Pathways t o the S p i n a l Cord, Volume 57, Kuypers, H.J.G.M. and Ma r t i n , G.F. (eds.). E l s e v i e r B iomedical Press, Amsterdam, 1982 . pp 81-403. Laughton N.B. 1924. Studie s on the nervous r e g u l a t i o n of p r o g r e s s i o n i n mammals. Am. J . P h y s i o l . 70:358-384. Lawrence D.G. and H.G.J.M. Kuypers. 1968a. The f u n c t i o n a l o r g a n i z a t i o n o f b i l a t e r a l pyramidal l e s i o n s . B r a i n 91:1-14. Lawrence D.G. and H.G.J.M. Kuypers. 1968b. The f u n c t i o n a l o r g a n i z a t i o n o f the motor c o r t e x i n monkey I I . The e f f e c t s of l e s i o n s o f the descending brainstem pathways. B r a i n 91:15-36. 282 Leonard R.B., P. Rudomin, M.H. Droge, A.E. Grossman and W.p. W i l l i s . 1979. Locomotion i n the decerebrate s t i n g r a y . Neuroscience L e t t e r s 14:315-319. Loeser J.D. and R.G. Black . 1975. A taxonomy of p a i n . Pa i n 1 (1) :81-84. Magnus R. 1924. Die K o r p e r s t e l l u n g . J u l i u s S p r i n g e r : B e r l i n Marsden CD., P.A. Merton, and H.B. Morton. 1981. Maximal tw i t c h e s from s t i m u l a t i o n o f the motor c o r t e x i n man. J . Physiol.(Lond.) 312:5P. M a r t i n J.P. The Bas a l G a n g l i a and Posture. Pittman, London. 1967 Mats u s h i t a M., M. Ikeda and N. Okado. 1982. The c e l l s o f o r i g i n o f the t r i g e m i n o t h a l a m i c , t r i g e m i n o s p i n a l and t r i g e m i n o c e r e b e l l a r p r o j e c t i o n s i n the c a t . Neuroscience 7:1439-1454. Mat s u s h i t a M., N. Okado, M. Ikeda and Y. Hosoya. 1981. Descending p r o j e c t i o n s from the s p i n a l and mesencephalic n u c l e i of the t r i g e m i n a l nerve t o the s p i n a l c o r d i n the c a t . a study with the h o r s e r a d i s h peroxidase technique. J . Comp. Neu r o l . 196:173-187. M c C l e l l a n A.D. Command systems f o r i n i t i a t i n g locomotion i n f i s h and amphibians: p a r a l l e l s t o i n i t i a t i o n systems i n mammals. In: Neural C o n t r o l o f V e r t e b r a t e Locomotion (eds. S. G r i l l n e r , R.M. Herman, P.S.G. S t e i n , H. F o r s s b e r g and D.G. S t u a r t ) , MacMillan Press, London, 1986, pp. 3-20. M c C l e l l a n A.D. and S. G r i l l n e r . 1984. A c t i v a t i o n of v f i c t i v e swimming' by e l e c t r i c a l m i c r o s t i m u l a t i o n of brainstem locomotor r e g i o n s i n an in vitro p r e p a r a t i o n o f the lamprey c e n t r a l nervous system. B r a i n Res. 300:357-361. McGeer P.L. and E.G. McGeer. Amino a c i d n e u r o t r a n s m i t t e r s . In: B a s i c Neurochemistry. G.J. S i e g e l , R.W. A l b e r s , B.W. Agranoff and R. Katzman (Eds.). L i t t l e , Brown and Co., Boston. 1981. pp. 233-253. McGeer E.G., W.A. Sta i n e s and P.L. McGeer. 1984. Neu r o t r a n s m i t t e r s i n the b a s a l g a n g l i a . Can. J . Neu r o l . S c i . 11:89-99. Mogenson G.J. Limbic-motor i n t e g r a t i o n - with emphasis on on i n i t i a t i o n of e x p l o r a t o r y and g o a l - d i r e c t e d locomotion. In: Modulation o f Sensorimotor A c t i v i t y During A l t e r a t i o n s i n Be h a v i o u r a l S t a t e s , R. Bandler (Ed.). L i s s , New York. 1984. pp. 121-137. Mogenson G.J., L.W. Swanson and M. Wu. 1985. Evidence t h a t p r o j e c t i o n s from s u b s t a n t i a innominata t o zona i n c e r t a and mesencephalic locomotor r e g i o n c o n t r i b u t e t o locomotor a c t i v i t y . B r a i n R e s . 334 (1) :65-76. 283 Mogenson G.J. and M. Wu. 1986. S u b p a l l i d a l p r o j e c t i o n s t o the mesencephalic locomotor r e g i o n i n v e s t i g a t e d with a combination of b e h a v i o r a l and e l e c t r o p h y s i o l o g i c a l r e c o r d i n g t e chniques. B r a i n Res. B u l l . 16 (3):383-390. Mogenson G.J. and M. Wu. 1988. D i f f e r e n t i a l e f f e c t s on locomotor a c t i v i t y o f i n j e c t i o n s o f p r o c a i n e i n t o mediodorsal thalamus and pedunculopontine nucleus.. B r a i n Res. B u l l . 20:241-246. Monaghan D.T. and C.W. Cotman. 1985. D i s t r i b u t i o n o f N - M e t h y l - D - a s p a r t a t e - s e n s i t i v e L-[3H]Glutamate-binding s i t e s i n r a t b r a i n . J . N e u r o s c i . 5 (11) :2909-2919. Monaghan D.T., D. Yao and C.W. Cotman. 1985. L-[ 3H]Glutamate binds t o k a i n a t e - , NMDA- and AMPA-sensitive b i n d i n g s i t e s : an a u t o r a d i o g r a p h i c a n a l y s i s . B r a i n Res. 340:378-383. Mori S., K. Kawahara, T. Sakamoto, M. Aoki and T. Tomiyama. 1982. S e t t i n g and r e s e t t i n g of l e v e l o f p o s t u r a l muscle tone i n decerebrate c at by s t i m u l a t i o n of b r a i n stem. J . N e u r o p h y s i o l . 48 (3) -.737-748. Mori S., H. Nishimura, C. Kurakami, T. Yamamura and M. A o k i . 1978. C o n t r o l l e d locomotion i n the mesencephalic c a t : d i s t r i b u t i o n o f f a c i l i t a t o r y and i n h i b i t o r y r e g i o n s w i t h i n the pontine tegmentum. J . N e u r o p h y s i o l . 41 (6) :1580-1591. Mori S., M.L. Shik and A.S. Yagodnitsyn. 1977. Role of pontine tegmentum f o r locomotor c o n t r o l i n mesencephalic c a t . J . Ne u r o p h y s i o l . 40 (2) :284-295. Mugnaini E. and W.H. O e r t e l . An a t l a s o f the d i s t r i b u t i o n of GABAergic neurons and t e r m i n a l i n the r a t CNS as r e v e a l e d by GAD immunohistochemistry. In Handbook of Chemical Neuroanatomy V o l . 4: GABA and Neuropeptides i n the CNS, P a r t I. A. B j o r k l u n d and T. H o k f e l t (Eds.). E l s e v i e r Science P u b l i s h e r s B.V., 1985. pp 436-662. Noga B.R., J . K e t t l e r and L.M. Jordan. 1988. Locomotion produced i n mesencephalic c a t s by i n j e c t i o n o f p u t a t i v e t r a n s m i t t e r substances and a n t a g o n i s t s i n t o the medial r e t i c u l a r formation and the pontomedullary locomotor s t r i p . J . N e u r o s c i . 8 (6) :2074-2086. Olsen R.W. 1981. GABA-benzodiazepine-barbiturate r e c e p t o r i n t e r a c t i o n s . J . Neurochem. 37(1):1-13. Orlovsky G.N. 1969. Spontaneous and induced locomotion i n the t h a l a m i c c a t . B i o p h y s i c s 14:1154-1162 ( E n g l i s h t r a n s l a t i o n of B i o f i z i c a 14:1095-1102.) Orlovsky G.N. 1970a. Connexions o f the r e t i c u l o s p i n a l neurones w i t h the "locomotor s e c t i o n s " o f the b r a i n stem. B i o f i z i k a 15, No.1:171-177. Orlovsky G.N. 1970b. Work of the r e t i c u l o - s p i n a l neurones d u r i n g 284 locomotion. B i o f i z i k a 15, No. 4:728-737. Orlovsky G.N. 1972a. The e f f e c t o f d i f f e r e n t descending systems on f l e x o r and extensor a c t i v i t y d u r i n g locomotion. Br. Res. 40:359-371. Orlovsky G.N. 1972b. A c t i v i t y o f v e s t i b u l o s p i n a l neurons d u r i n g locomotion. B r a i n Res. 46:85-98. Orlovsky G.N. 1972c. A c t i v i t y o f r u b r o s p i n a l neurons d u r i n g locomotion. B r a i n Res. 46:99-112. Orlovsky G.N. and M.L. Shik. 1976. C o n t r o l o f locomotion: a n e u r o p h y s i o l o g i c a l a n a l y s i s o f the cat locomotor system. In: I n t e r n a t i o n a l Review of Phys i o l o g y , Neurophysiology I I , V o l . 10. R. P o r t e r (ed.). U n i v e r s i t y Park Press, B a l t i m o r e , pp 281-317. P a l k o v i t s M. and D.M. Jacobowitz. 1974. Topographic a t l a s of catecholamine and a c e t y l c h o l i n e s t e r a s e - c o n t a i n i n g neurons i n the r a t b r a i n . J . Comp. Neurol. 157:29-42. P a r i s J.M. and S.A. Lorens. 1987. Intra-median raphe i n f u s i o n s of muscimol and the substance P analogue DiMe-C7 produce h y p e r a c t i v i t y : r o l e o f s e r o t o n i n neurons. B e h a v i o u r a l B r a i n Research 26:139-151. P h i l l i p s A.G., A. Lesage, C D . Blaha and H.C F i b i g e r . 1988 . C h o l o c y s t o k i n i n r e v e r s e s a neur o t e n s i n - i n d u c e d i n c r e a s e i n dopamine r e l e a s e i n the nucleus accumbens: an in vivo a n a l y s i s . Neuroscience A b s t r a c t s 14(2):1212. P e r r e t C , M. M i l l a n v o y e and J.M. Cabelguen 1972. Message spinaux ascendants pendant une locomotion f i c t i v e chez l e chat c u r a r i s e . J . P h y s i o l . , P a r i s 65:153A. Pompeiano 0. 1984. A comparison of the response c h a r a c t e r i s t i c s of v e s t i b u l o s p i n a l and medullary r e t i c u l o s p i n a l neurons t o l a b y r i n t h and neck i n p u t s . In: Brainstem C o n t r o l o f S p i n a l Cord F u n c t i o n . C D . Barnes (Ed.). Academic Press, New York. pp. 87-140. Ramon-Moliner E. and J.A. Dansereau. 1974. The p e r i b r a c h i a l r e g i o n o f cat I I : d i s t r i b u t i o n o f a c e t y t h i o c h o l i n e s t e r a s e and monoaminoxidase with s p e c i a l r e f e r e n c e t o the l o c u s c o e r u l e u s . C e l l T i s s . Res. 149:191-204. Reiner A., N.C Brecha and H.J. Karten. 1982 . B a s a l g a n g l i a pathways t o the tectum: the a f f e r e n t and e f f e r e n t connections of the l a t e r a l s p i r i f o r m nucleus of pigeon. J . Comp. Neurol. 208:16-36. Reiner A., S.E. Brauth and H.J. Karten. 1984. E v o l u t i o n of the amniote b a s a l g a n g l i a . TINS 7 (9):320-325. Reiner A. and H.J. Karten. 1982. Laminar d i s t r i b u t i o n of the c e l l s of o r i g i n o f the descending t e c t o f u g a l pathways i n the pigeon (Columba l i v i a ) . J . Comp. Neurol. 204:165-187. 285 Reiner A., H.J. Karten and A.R. S o l i n a . 1983. Substance P: l o c a l i z a t i o n w i t h i n p a l e o s t r i a t a l - t e g m e n t a l pathways i n the pigeon. Neuroscience 9(l):61-85. Reiner P.B. and S.R. V i n c e n t . 1987. Topographic r e l a t i o n s o f c h o l i n e r g i c and no r a d r e n e r g i c neurons i n the f e l i n e pontomesencephalic tegmentum: an immunohistochemical study. B r a i n Res. B u l l . 19:705-714. Roberts E. and S. F r a n k e l . 1950. y-Aminobutyric a c i d i n b r a i n : i t s formation from glutamic a c i d . J . B i o l . Chern. 187:55-63. Rye D.B., C.B.'Saper, H.J. Lee and B.H. Wainer. 1987. Pedunculopontine tegmental nucleus o f the r a t : c y t o a r c h i t e c t u r e , cytochemistry, and some ex t r a p y r a m i d a l connections of the mesopontine tegmentum. J . Comp.Neurol. 259:483-528. Rye D.B., H.J. Lee, C.B. Saper and B.H. Wainer. 1988. Medullary and s p i n a l e f f e r e n t s o f the pedunculopontine tegmental nucleus and adjacent mesopontine tegmentum i n the r a t . J . Comp. Neurol. 269:315-341. Sabol K.E. and C.R. Freed. 1988. B r a i n acetominophen measurement by in vivo d i a l y s i s , in vivo e l e c t r o c h e m i s t r y and t i s s u e assay -a study o f the d i a l y s i s technique i n the r a t . J . N e u r o s c i . Methods 24 (2):163-168. S a l t T.E. and R.G. H i l l . 1981. E x c i t a t o r y amino a c i d s as t r a n s m i t t e r candidates o f v i b r i s s a e a f f e r e n t f i b r e s t o the r a t t r i g e m i n a l nucleus c a u d a l i s . N e u r o s c i . L e t t . 22:183-187. S a l t T.E. and R.G. H i l l . 1983. Pharmacological d i f f e r e n t i a t i o n between responses o f r a t medullary d o r s a l horn neurons t o noxious mechanical and noxious thermal cutaneous s t i m u l i . B r a i n Res. 263:167-171. S c a r n a t i E., A. G a s b a r r i , E. Campana and C. P a c i t t i . 1987. The o r g a n i z a t i o n o f nucleus tegmenti pedunculopontinus neurons p r o j e c t i n g t o b a s a l g a n g l i a and thalamus: a r e t r o g r a d e f l u o r e s c e n t double l a b e l l i n g study i n the r a t . Neuroscience L e t t e r s 79:11-16. S e l i o n o v V.A. and M.L. Shik. 1984. Medullary locomotor s t r i p and column i n the c a t . N e u r o s c i . 13 (4):1267-1278. Shefchyk S.J., R.M. J e l l and L.M. Jordan. 1984. R e v e r s i b l e c o o l i n g o f the brainstem r e v e a l s areas r e q u i r e d f o r mesencephalic locomotor r e g i o n evoked t r e a d m i l l locomotion. Exp. B r a i n Res. 56:257-262. Shefchyk S.J. and L.M. Jordan. 1985. E x c i t a t o r y and i n h i b i t o r y p o s t s y n a p t i c p o t e n t i a l s i n alpha-motoneurons produced d u r i n g f i c t i v e locomotion by s t i m u l a t i o n of the mesencephalic locomotor r e g i o n . J . Ne u r o p h y s i o l . 53:1345-1355. > S h e r r i n g t o n C S . 1910. F l e x i o n - R e f l e x of the limb c r o s s e d 286 e x t e n s i o n r e f l e x , and r e f l e x s t e p p i n g and s t a n d i n g . J . P h y s i o l . 40;28-121. S h e r r i n g t o n C.S. 1915. Simple apparatus f o r o b t a i n i n g a decerebrate p r e p a r a t i o n of the c a t . J.. P h y s i o l . V o l . 49. Proceedings of the P h y s i o l o g i c a l S o c i e t y , J u l y 3, 1915. pp l i i - l i v . Shik M.L. and G.N. Orlovsky. 1976. Neurophysiology of locomotor automatism. P h y s i o l o g i c a l Reviews 56 (3) :465-501. Shik M.L., G.N. Orlovsky and F.V. S e v e r i n . 1967. S t r u c t u r e s of the b r a i n stem r e s p o n s i b l e f o r evoked locomotion. Sechenov P h y s i o l o g i c a l J . of USSR L I I I ( 9 ) : 6 6 0 - 6 6 9 . Shik M.L., G.N. Orlovsky, and F.V. S e v e r i n . 1968. Locomotion of the mesencephalic cat e l i c i t e d by s t i m u l a t i o n of the pyramids. B i o p h y s i c s 13 (1):143-152. Shik M.L., F.V. S e v e r i n , and G.N. Orlovsky. 1966. C o n t r o l of walking and running by means of e l e c t r i c a l s t i m u l a t i o n of the midbrain. B i o f i z i c a 11 (4) :756-765. Shik M.L. and A.S. Yagodnitsyn. 1977. The pontobulbar "locomotor s t r i p " . Neurophysiology 9:95-97. Shik M.L. and A.S. Yagodnitsyn. 1978. U n i t responses i n the "locomotor s t r i p " o f the cat h i n d b r a i n to m i c r o s t i m u l a t i o n . Neurophsyiology 10:373-379. Sholomenko G.N. and J.D. Steeves. 1986. Descending s u p r a s p i n a l pathways necessary f o r hindlimb locomotion i n b i r d s . N e u r o s c i . A b s t r . 16:882. Sholomenko G.N. and J.D. Steeves. 1987a. A c t i v a t i o n of locomotion by m i c r o i n j e c t i o n of n e u r o t r a n s m i t t e r a g o n i s t s and a n t a g o n i s t s i n t o the avian brainstem. N e u r o s c i . A b s t r . 17 (2):826. Sholomenko, G.N. and J.D. Steeves. 1987b. E f f e c t s of s e l e c t i v e s p i n a l c o r d l e s i o n s on h i n d limb locomotion i n b i r d s . Exp. N e u r o l . 95(2): 403-418. S i e g e l A. and J.P. F l y n n . 1968. D i f f e r e n t i a l e f f e c t s of e l e c t r i c a l s t i m u l a t i o n and l e s i o n s of the hippocampus and adjacent r e g i o n s upon a t t a c k behavior i n c a t s . B r a i n Res. 7:252-267. S i r o t a M.G. and M.L. Shik. 1973. The cat locomotion e l i c i t e d through the e l e c t r o d e implanted i n the midbrain. Sechenov. P h y s i o l . J . USSR 59:1314-1321 ( i n Russian) ( c . f . S h i k M.L. and G.N. Orlovsky. 1976. Neurophysiology of locomotor automatism. P h y s i o l o g i c a l Reviews, V o l . 5, No. 3:465-501). Skinner R.D., E. G a r c i a - R i l l , S. G r i f f i n , R.Nelson and J.A. F i t z g e r a l d . 1984. I n t e r s t i t i a l nucleus of C a j a l (INC) p r o j e c t i o n s t o the r e g i o n of P r o b s t ' s t r a c t . B r a i n Res. B u l l . 287 13:613- 621. Spencer D.G., L.A.A. van Rooijen, E. Horvath, W. Dompert and J . Traber. 1988. Neurochemical and b e h a v i o u r a l e f f e c t s of mu s c a r i n i c a c e t y l c h o l i n e r e c e p t o r a g o n i s t s . TIPS Suppl. Feb. 1988:61-68. Steeves J.D. and L.M. Jordan. 1980. L o c a l i z a t i o n o f a descending pathway i n the s p i n a l c o r d which i s necessary f o r c o n t r o l l e d t r e a d m i l l locomotion. Neuroscience L e t t e r s 20:283-288. Steeves J.D. and L.M. Jordan. 1984. A u t o r a d i o g r a p h i c demonstration o f the p r o j e c t i o n s from the mesencephalic locomotor r e g i o n . B r a i n Res. 307:263-276. Steeves J.D., G.N. Sholomenko and D.M.S. Webster. R e t i c u l a r formation s t i m u l a t i o n evokes walking and f l y i n g i n b i r d s . In: Neurobiology o f V e r t e b r a t e Locomotion, (eds. S . G r i l l n e r , R.M. Herman, P.S.G. S t e i n , D.G. S t u a r t ) . MacMillan Press, London, 1986. pp. 51-54. Steeves J.D., G.N. Sholomenko and D.M.S. Webster. 1987. S t i m u l a t i o n o f t h e pontomedullary r e t i c u l a r formation i n i t i a t e s locomotion i n decerebrate b i r d s . B r a i n Res. 401:205-212. Stone T.W. and N.R. Burton. 1988. NMDA r e c e p t o r s and l i g a n d s i n the v e r t e b r a t e CNS. Progress i n N e u r o b i o l . 30:333-368. Swanson L.W., G.J. Mogenson, C.R. Gerfen and P. Robinson. 1984. Evidence f o r a p r o j e c t i o n from the l a t e r a l p r e o p t i c area and s u b s t a n t i a innominata t o the ^Mesencephalic locomotor r e g i o n ' i n the r a t . B r a i n Res. 285:161-178. Tarchanoff J . 1895. Movement f o r c e s des canards d e c a p i t e s . Compt. Rend. Soc. B i o l . 47:454. T a y l o r P. G a n g l i o n i c s t i m u l a t i n g and b l o c k i n g agents. In: The Pharmacological b a s i s o f The r a p e u t i c s (7th E d i t i o n ) , Gilman A.G., L.S. Goodman, T.W. R a i l and F. Murad (Eds.), MacMillan P u b l i s h i n g , New York, 1985. pp. 215-221. T a y l o r P. Neuromuscular b l o c k i n g agents. In: The Pharmacological b a s i s of Th e r a p e u t i c s (7th E d i t i o n ) , Gilman A.G., L.S. Goodman, T.W. R a i l and F. Murad (Eds.), MacMillan P u b l i s h i n g , New York, 1985. pp. 222-235. Ten Cate J . 1960. Locomotor movements i n the s p i n a l pigeon. J . E x p t l . B i o l . 37:609-613. Ten Cate J . 1962. I n n e r v a t i o n of locomotor movements by.the lumbosacral c o r d i n b i r d s and mammals. J . E x p t l . B i o l . 39:239-242. V i l l a b l a n c a J . 1962. Electroencephalogram i n the permanently i s o l a t e d f o r e b r a i n i n the c a t . Science 138:44-46. V i n c e n t S.R. and P.B. Reiner. 1987. The immunohistochemical 288 l o c a l i z a t i o n of c h o l i n e a c e t y l t r a n s f e r a s e i n the cat b r a i n . B r a i n Res. B u l l . 18:371-415. Vogt B.A. 1988. L o c a l i z a t i o n of c o r t i c a l muscarinic receptor subtypes. TIPS Suppl. Feb, 1988:49-53. Waldrop T.G., R.M. Bauer and G.A. Iwamoto. 1988. M i c r o i n j e c t i o n of GABA antagonists i n t o the p o s t e r i o r hypothalamus e l i c i t s locomotor a c t i v i t y and a c a r d i o r e s p i r a t o r y a c t i v a t i o n . B r a i n Res. 444:84-94. Wall P.D. 1975. E d i t o r i a l . Pain l ( l ) : l - 2 . Wall P.D. and R.A. Sternbach. 1976. E d i t o r i a l . Pain 2 ( l ) : l - 4 . Waller W.H. 1940. Progression movement e l i c i t e d by subthalamic s t i m u l a t i o n . J . Neurophysiol. 3:300-307. Watkins J.C. and H.J. Olverman. 1987. Agonists and antagonists f o r e x c i t a t o r y amino a c i d r e c e p t o r s . TINS 10 (7) :265-272. Watson M., T.W. Vi c k r o y , W.R. Roeske and H.I. Yamamura. 1984. S u b c l a s s i f i c a t i o n of muscarinic receptors based upon the s e l e c t i v e antagonist p i r e n z e p i n e . TIPS Suppl. Jan. 1984:9-11. Webster D.M.S. and J.D. Steeves. 1986. O r i g i n of avian s u p r a s p i n a l pathways p r o j e c t i n g to the s p i n a l cord. Neurosci. A b s t r . 16:882. Webster D.M.S. and J.D. Steeves. 1988. O r i g i n s of brainst e m - s p i n a l p r o j e c t i o n s i n the duck and goose. J . Comp. Neurol. 273:573-583. Weinstein G.N., C. Anderson and J.D. Steeves. 1984. F u n c t i o n a l c h a r a c t e r i z a t i o n of limb muscles i n v o l v e d i n locomotion i n the Canada goose, Branta canadensis. Can. J . Zool. 62:1596-1604. Wetzel M.C. and D.G. St u a r t . 1976. Ensemble c h a r a c t e r i s t i c s of cat locomotion and i t s neural c o n t r o l . Progress i n Neurobiology 7:1-98. Wild J.M. 1985. The avian somatosensory system. I . primary s p i n a l a f f e r e n t input t o the s p i n a l cord and brainstem i n the pigeon (Columba l i v i a ) . J . Comp. Neurol. 240:377-395. Wild J.M. 1987. The avian somatosensory system: connections of regions of body r e p r e s e n t a t i o n i n the f o r e b r a i n of the pigeon. B r a i n Res. 412:205-223. Wild J.M., J.J.A. Arends and H.P. Z e i g l e r . 1984. A t r i g e m i n a l sensorimotor c i r c u i t f o r pecking, grasping and feeding i n the pigeon {Columba l i v i a ) . B r a i n Res. 300:146-151. Wilson V.J. and M. Yoshida. 1968. V e s t i b u l o s p i n a l and r e t i c u l o s p i n a l e f f e c t s on hindlimb, f o r e l i m b , and neck alpha motoneurons of the c a t . Proc. Nat. Ass. S c i . USA. 60:836-840. 289 Wold J.E. 1978. The v e s t i b u l a r n u c l e i of the domestic hen (Gallus domesticus) IV. the p r o j e c t i o n t o the s p i n a l cord. B r a i n Behav. E v o l . 15:41-62. Woods J.W. 1964. Behaviour of c h r o n i c decerebrate r a t s . J . N e u r o p h y s i o l . 28:85-99. Woodson W. and H. Kunzle. 1982. D i s t r i b u t i o n and s t r u c t u r a l c h a r a c t e r i z a t i o n of neurons g i v i n g r i s e t o descending s p i n a l p r o j e c t i o n s i n the t u r t l e , Pseudemys s c r i p t a elegans. J . Comp. Neur o l . 212:336-348. Yu J . and E. E i d e l b e r g . 1981. E f f e c t s of v e s t i b u l o s p i n a l neurons upon locomotor f u n c t i o n i n c a t s . Br. Res. 220:179-183. Zweers G.A. 1971. A s t e r e o t a x i c a t l a s of the brainstem of the m a l l a r d (Anas platyrhynchos). Van Gorcum and Company, N.V. Assen, The Netherlands ppl-148. 290 APPENDIX I Neurochemical Injection Parameters for Alter ing Locomot ion Experimenters Animal Site Chemical pH Concentrations Injected Lowest Effective Concentration Volume Rate Time Course (min) Latency Period Garcia-Rill decerebrate PPN acetylcholine 4-7 1mM-1M none I.Sul <1-2ul/min •t al., 1985 oat (mMLR) GABA 4-7 0.6-1M O.SM (T) 1.5ul •• <1 3-5 picrotoxin 1-5mM SmM 1.S-3ul •• 3.4 20-60 bicuculline SmM SmM I.Sul •• na na muacimo! 6mM 5mM (Til) I.Sul •• <5 >15 diazepam " 1-5mM 5mM (T) I.Sul na 1-5 ttrychnine " 5-10mM none I.Sul — — glutamate 4-7.5 8mU-1M 1.0M (RT) 1.5ul •• — — norepinephrine 4-7 50mm-1M none I.Sul •• — — dopamine *' 100mM-1M none I.Sul — — Garcia-Rill decerebrate PPN picrotoxin 4-7 1-5mM ImM 1.5ul 1ul/min tee above results a Skinner. cat 19B7a NRV acetylcholine '• 0.2-1M O.SM •• lul/min 1-3 5-60s NRG carbachol 1uM-1mM 1-10uM " 1-5 1-5 NRV atropine " 1uM-1mM 1uM-1mM •• •• na 1-2h NRV edrophonium 0.1-0.6M 100mM " 2-3 1-5 NRV etarine " 10uM-1mM 100-500uM " 2-3 1-5 NRG methacholino " 10-100uM 10-100uM 1-5 1-5 NRV GABA 0.2-0.5M 0.2M 1S-30S 2-5 NRG picrotoxin " 1-5mM 5mM (C) — — NRG bicuculline 100uM-5mM 5mM (C) — — NRG muscimol 1-5mM 1mM na 1-3h NRG glutamate •• 0.5-1M 0.5M (NR) •• — — NRG aspartate 0.5-1M 0.5M (NR) " — — NRV Substance P 0.5-5mM 0.5mM •• •• 1S-30S 15-30. Eldridga et decerebrate SLR picrotoxin na 8mM 8mM Sul na 15 45 al.. 1886 cat SLR muscimol na 44 mM 44mM 6ul na 20 na Noga et al, deoerebrate MRF GABA 7.2-7.5 S-IOmM na Sul 1ul/min S 10-16 1S88 cat MRF picrotoxin 6-10mM SmM (RT) 6ul •• — 15-25 MRF muscimol 5-10mM na Sul " 5 irr. block MRF glutamate •• 5-100mM lOOmM Sul 30 100 MRF DL-HCA " 0.1M 0.1M (RT) Sul " — 15-25 PLS picrotoxin •• 6-10mM 10mM lul •• 2-20 20-80 PLS glutamate •• 6-100mM 100mM Sul •• na 15-20 PLS GDEE " 6-100mM 100mM Sul " 12 IS PLS DL-HCA lOOmM lOOmM 7ul 4 na PLS Substance P • 0.74mM 0.74 mM Sul 1-2 30 Brudzynski A intact AH/LPA carbachol na 65mM S5mM 0.2ul 0.4ul/min 5 na Mogenoon, rat atropine na 11.1mM 11mM 0.4ul •• na na 1S88 Brudzyntki A intact AH/LPA carbachol 8.5-7.0 27.4mM 27.4mM 0.2ul 0.4ul/min <1 S Mogenson, rat AH/LPA atropine 11.1mM 11.1mM <1 S 1988b N.Acc amphetamine 583mM 583mM " <1 S Brudzyntki et intact PPN carbachol B.0-7.0 27.4mM 27.4mM 0.2 ul 0.4ul/min 3-5 >7 al.. 1988 rat PPN atropine 11.1mM 11.1mM '• na na N.Acc amphetamine •• 683mM 683mM •• na na Lai and decerebrate NMC acetylcholine na 1.1M na O.Sul 0.5ul/min na na Siegel. 1888 cat A carbachol na 44-1 OOmM na " •• na na NPM atropine na 7mM na M na na glutamate na 0.05-0.4M na - - na na NMDA na 1-4-6.8mM na " na na kainate na O.BuM-O.BmM na •• '• na na quisqualate na 0.2-0.SmU na " •• na na GDEE na 200mM na •• na na APV na SOmM na na na 2 9 1 Experiment*™ Animal Site Chemical pH Concentrat ion! Lowest Volume Rale Injected Effective Concentration Time Course (min) Latency Period Sholomenko Acetylcholine Agonists and Antagonists Thesis. 1989 TTD Carbachol 7.2-7.4 2SmM-100mM 2SmM 1.0ul 0.2ul/min 2.2-12 7-45 Scopolamine " 2SmM none •• — — Nicotine " 26mM none " — — Atropine 2SmM 25mM 8 25 Pilocarpine " SOmM none " •• 7 8 Cnd Carbachol 11mM-100mM 11 mM M 3.3-6 33 Scopolamine 2SmM none •• — — Atropine 30mM 30mM " <5 >26 Cnv Carbachol 7.2-7.4 27-100mM 27mM •• 2.2-6 7-45 Scopolamine " 25mM none " — — Nicotine " 100mM none " — — Atropine " 3-50mM 20mM >7.5 21-40 Pilocarpine " SOmM none " — — RP Carbachol " 64mM none " •• — — MRP Carbachol " lOOmM 100mM (RT) " •• — 45 MLF Carbachol " 27-10OmM 27 •• 8-10 23-40 Sholomenko GABAergic agonists and antagonists Thesis. 19SS TTD GABA 7.2-7.4 0.3-0.SM O.SM I.QuI 0.2ul/min 1-S 2-21 Picrotoxin 3-20mM 3mM 4-22 35-60 Bicuculline 10mM 10mM " 15 >30 Muscimol 6.5-25mM 6.5mM " " 5-10 30-70 Cnd Picrotoi in " 5mM none •• none none Muscimol " 6.2SmM none " " none none Cnv GABA O.SM O.SM •• <1 9-14 Picrotoxin " 6-20mM 6mM •• 4-22 30 Muscimol " 8.25mM 6.25mM " >9 >30 RP GABA O.SM O.SM •• <1 12-21 Picrotoxin " 3-6mM 3mM " •• 10 36 Muscimol " 6.2SmM none — — MRF GABA O.SM O.SM •• <1 2-12 Picrotoxin " 6-20mM SmM 11-13 >30 Sholomenko Excitatory Amino Acids and Substance P Thesis. 1989 TTD- Glutamate " 0.5-1.OM none I.Oul — — NMDA SOmM SOmM 0.2ul <1 10 GDEE 2-80mM 2mM 0.4-1 ul 4-6 35-55 Substance P " 6.44mM none I.Oul — — Cnd NMDA 20-83mM 20mM 0.2ul <1.S 3-24 GDEE SOmM SOmM 0.6ul <5 >35 Substance P 8.44mM none I.Oul — — Cnv Glutamate 1M none I.Oul — NMDA 4-83mM 4mM 0.4ul <1 3-4 GDEE 2-80mM 2mM 0.2ul 4-6 35-50 Substance P " S.44mM none I.Oul — — RP NMDA 7.2-7.4 81-83mM 83mM 0.2ul <1-1.25 4-9 Substance P 6.44mM 6.44 mM I.Oul 4 >1S MRF NMDA 6-34mM 6mM (RT) 0.2ul 5 15 MLF NMDA 20-34mM 34mM 0.4 ul <1 8 292 ABBREVIATIONS: Appendix I A H — a n t e r i o r h y p o t h a l a m u s C — c o n v u l s a n t C n d — d o r s a l pa r t , m e d u l l a r y c e n t r a l n u c l e u s C n v — v e n t r a l pa r t , m e d u l l a r y c e n t r a l n u c l e u s h — h o u r s I — i r r e v e r s i b l e i r r . b l o c k — i r r e v e r s i b l e b lock L P A — l a t e r a l p r e o p t i c a r e a M L F — m e d i a l l o n g i t u d i n a l f a s c i c u l u s m M L R — m e d i a l m e s e n c e p h a l i c l o c o m o t o r r e g i o n (see P P N ) mr f — m e d i a l r e t i c u l a r f o r m a t i o n ( m e d u l l a ) M R F — m e s e n c e p h a l i c r e t i c u l a r f o r m a t i o n n a — n o t a v a i l a b l e N .Acc — n u c l e u s a c c u m b e n s N M C — m a g n o c e l l u l a r re t i cu la r f o r m a t i o n N P M — p a r a m e d i a n n u c l e u s N R — no r e p e a t a b l e r e s p o n s e N R G — g i g a n t o c e l l u l a r r e t i c u l a r f o r m a t i o n N R V — v e n t r a l r e t i cu la r f o r m a t i o n P L S — p o n t o b u l b a r l o c o m o t o r s t r ip — e q u i v a l e n t to the d e s c e n d i n g t r i g e m i n a l t r a c t a n d n u c l e u s t o g e t h e r w i th t he a d j a c e n t p a r v o c e l l u l a r r e t i c u l a r f o r m a t i o n -P P N — p e d u n c u l o p o n t i n e n u c l e u s ( see m M L R ) R P — p o n t i n e r e t i c u l a r n u c l e u s RT — r e d u c e d t h r e s h o l d for e l e c t r i c a l l y s t i m u l a t e d l o c o m o t i o n s — s e c o n d s S L R — s u b t h a l a m i c l o c o m o t o r r e g i o n T T D — d e s c e n d i n g t r i g e m i n a l t rac t a n d n u c l e u s , 293 APPENDIX II RESPIRATORY AND CARDIOVASCULAR VALUES IN DECEREBRATE AND INTACT CANADA GEESE DECEREBRATE INTACT V £ (mt/min/Kg) V T (ml/Kg) f (min' 1) y m i n ) V 0 2 (m l /m in /Kg ) V ^ f m l / m i n / K g ) V°c> Ent% Hesrtrate (min BP. Rest n 314±28 13 29.9*1.5 13 10.7*1.0 13 11.3*0.8 8 7.6*0.7 8 0.6710.03 8 40.4*0.7 S 198*24 12 69*4 .0 4 Exercise 448*27 # 34.1*2.3 13.4*0.8 57.5*5.0 15.9*1.1 # 12.6*0.6 # 0.8*0.03 # 40.6*0.6 28.9*3.3 %Ctange n 42.2 13 14.6 25.2 46.2 68.3 207*21.5 108.3*6.6 4.5 21.7 13 13 13 9 S 9 2 9 12 Rest n 417*30 21 28.2*1.3 21 14.8*0.80 21 13.5*1.5 9 13.1*1.3* 9 0.98*0.04 • 9 42.3 2 264 * 28 4 164*7 3 Ex6TCtS6 887*65 # • 26.5*2.4 34.9*3.0 # * 55.2*2.3 32.7*3.8 #* 29.9*3.8 #* 0.91*0.03 42.7 28.3*4.0 340*28 ' 162*9 ' %Change n 119 •4.6 137 128 47.7 11.0 S t o p e V E ^ V 0 2 s t o p , v E / v C 0 2 Blood Oases P e O 2 ( m m H 0 ' P B C O 2 ( m m H 0 ) 24.43 (R = 0.787) 24.74 (R = 0.988) 93.4 * 7.1 8 94.9 * 7.3 24.9*1.2 12 23.9*0.9 7.50*0.02 12 7.54*0.02 8 12 12 18.48 (R = 0.985) 18.67 (R = 0.982) 89.0 27.5 7.52 1 103.0 1 24.5 1 7.56 Appendix M: Respiratory vatues in decerebrate and mtsct Canada Qoese recorded prior to and over the last 4 minutes of a 10minuta (decerebrate) and 6 minute (intact) walking period, (mean ± S.E.) n = number of experiments % Change; percent change from rest to exercise. # ; significant difference between rest and exercise wtthm a group. *I Significant difference between the two groups during rest and exercise. mtnute vanttatton tidal volume f^; breathing frequency f^; stride frequency V j ^ ; C » 2 production V02' ^ 2 P"30^*00 T^; body ternperature. R*t Pearson's Correlation Coefficient I*, not tested lor significant difference 2 9 4 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0098328/manifest

Comment

Related Items