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Avian brainstem and descending spinal projections associated with locomotion Webster, Deirdre M.S. 1989

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AVIAN BRAINSTEM AND DESCENDING SPINAL PROJECTIONS ASSOCIATED WITH LOCOMOTION by DEIRDRE M.S. WEBSTER B.S.R. (P.T.), The University of B r i t i s h Columbia, 1978 M.Sc, The University of B r i t i s h Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Zoology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA MAY 198 9 (c) DEIRDRE M.S. WEBSTER 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 /<?go/«?^y  The University of British Columbia Vancouver, Canada Date ^ C L ^ 3i>, DE-6 (2/88) ABSTRACT 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 s t u d i e s i n the d e c e r e b r a t e b i r d have p r e v i o u s l y i d e n t i f i e d brainstem regions that play a r o l e i n the i n i t i a t i o n of locomotion. This t h e s i s was designed t o i d e n t i f y t h e n e u r o a n a t o m i c a l c o n n e c t i o n s of thes e 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 . The experiments were conducted on the Pekin duck, Anas platyrhynchos; Canada goose, Branta canadensis; Sulphur-crested cockatoo, Cacatua galerita; and Eastern r o s e l l a , Platycercus eximius. A v a r i e t y of retrograde t r a c e r chemicals (e.g. wheat germ a g g l u t i n i n - h o r s e r a d i s h peroxidase and fl u o r e s c e n t t r a c e r True Blue) were i n j e c t e d i n t o e i t h e r the c e r v i c a l or lumbar s p i n a l cord alone or i n conjunction w i t h a more r o s t r a l s u b t o t a l l e s i o n of the s p i n a l cord t o determine (1) the o r i g i n s of s p i n a l p r o j e c t i o n s to the s p i n a l cord, and (2) the f u n i c u l a r o r g a n i z a t i o n of thes e pathways at the lumbar l e v e l . The d i s t r i b u t i o n of re t r o g r a d e l y l a b e l l e d neurones was s i m i l a r i n a l l avian species examined. There were no d i r e c t t e l e n c e p h a l i c p r o j e c t i o n s to the s p i n a l cord. Descending brai n s t e m - s p i n a l s p i n a l pathways from p r e v i o u s l y i d e n t i f i e d "locomotor s i t e s " i n the v e n t r o m e d i a l m e d u l l a (nucleus r e t i c u l a r i s m e d u l l a r i s c e n t r a l i s and 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 ) p r o j e c t e d to the lumbar l e v e l v i a the v e n t r o l a t e r a l f u n i c u l u s , whereas d o r s o l a t e r a l "locomotor s i t e s " (nucleus 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 , nucleus and descending t r a c t of the t r i g e m i n a l nerve) only p r o j e c t e d as f a r as the c e r v i c a l s p i n a l cord. The a f f e r e n t p r o j e c t i o n s to these i d e n t i f i e d brainstem locomotor regions were determined by d i s c r e t e i n j e c t i o n s of r e t r o g r a d e f l u o r e s c e n t t r a c e r s i n t o the v e n t r o m e d i a l or d o r s o l a t e r a l pontomedullary locomotor s i t e s . A f f e r e n t input o r i g i n a t e d p r i n c i p a l l y from the p o n t o m e d u l l a r y r e t i c u l a r formation and the mesencephalon. The s i m i l a r i t y of avian s p i n a l and brainstem connections t o those p r e v i o u s l y d e s c r i b e d f o r mammals i n d i c a t e s considerable conservation of the o r i g i n s and o r g a n i z a t i o n of descending locomotor pathways between mammalian and non-mammalian species. i i i TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i ABBREVIATIONS ix ACKNOWLEDGEMENTS x i I INTRODUCTION 1 II ORIGINS OF BRAINSTEM-SPINAL PROJECTIONS IN THE DUCK AND GOOSE 7 INTRODUCTION 8 MATERIALS AND METHODS 9 RESULTS 12 Medulla-pons 14 Mesencephalon-hypothalamus 28 DISCUSSION 29 III ORIGINS OF BRAINSTEM-SPINAL PROJECTIONS IN PARROTS 36 INTRODUCTION 37 MATERIALS AND METHODS 39 RESULTS 42 Lumbar Injection 42 Cervical Injection 52 DISCUSSION 55 i v IV FUNICULAR TRAJECTORIES 60 INTRODUCTION 61 MATERIALS AND METHODS 65 RESULTS 67 Brainstem-spinal projections 70 Funicular Projection Summary 80 DISCUSSION 83 V BRAINSTEM LOCOMOTOR SITES 92 INTRODUCTION 93 MATERIALS AND METHODS 94 RESULTS. 97 DISCUSSION 101 VI AFFERENT PROJECTIONS TO PONTOMEDULLARY LOCOMOTOR REGIONS 103 INTRODUCTION 104 MATERIALS AND METHODS 107 RESULTS 108 Afferents to the Ventromedial Medulla 10 9 Afferents to the Dorsolateral Medulla 121 DISCUSSION 127 VII GENERAL DISCUSSION 140 REFERENCES 160 v LIST OF TABLES I Summary of lumbar i n j e c t i o n s i t e s , subtotal spinal cord lesions and subsequent locomotor function i n ducks and geese 68 II 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 retrogradely l a b e l l e d neurones i n the avian brainstem a f t e r l o c a l i z e d lumbar spinal cord i n j e c t i o n 71 III Distributions of retrogradely l a b e l l e d neurones i n the avian brainstem following subtotal lesions of the spinal cord 72 v i LIST OF FIGURES 1. S p i n a l cord i n j e c t i o n s i t e s 13 2. D i s t r i b u t i o n of T B - l a b e l l e d neurones a f t e r a c e r v i c a l i n j e c t i o n i n the duck 15 3. D i s t r i b u t i o n of T B - l a b e l l e d neurones a f t e r lumbar i n j e c t i o n , i n the duck 19 4. Photomicrographs of retrograde l a b e l l i n g 24 5. Diagrams of brainstem se c t i o n s i n the pa r r o t 43 6. Photomicrographs of T B - l a b e l l e d neurones i n s a g i t t a l s e c t i o n 44 7. D i s t r i b u t i o n of l a b e l l i n g a f t e r lumbar i n j e c t i o n 45 8. Photomicrographs of T B - l a b e l l e d neurones 48 9. Photomicrographs of WGA-HRP r e a c t i v e neurones 53 10. Photomicrographs of T B - l a b e l l e d neurones a f t e r a TB i n j e c t i o n at Cl-2 i n the cockatoo 54 11. Diagrams of f u n i c u l a r i n j e c t i o n s and l e s i o n s 69 12. Photomicrographs of l a b e l l e d neurones a f t e r f u n i c u l a r i n j e c t i o n or l e s i o n 73 13. D i s t r i b u t i o n of T B - l a b e l l e d neurones a f t e r s u b t o t a l l e s i o n of the s p i n a l cord 75 14. Summary diagrams of the o r g a n i z a t i o n of descending p r o j e c t i o n s w i t h i n the s p i n a l cord 82 15. Electromyographic and potentiometer records..' 98 16. Photomicrographs of e l e c t r o l y t i c l e s i o n s marking 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 99 v i i 17. I l l u s t r a t i o n s of l o c a t i o n s of T B - l a b e l l e d neurones and locomotor s t i m u l a t i o n s i t e s 100 18. Diagrams of the duck b r a i n t o i l l u s t r a t e the plane of i n j e c t i o n and s e c t i o n 110 19. D i s t r i b u t i o n of T B - l a b e l l e d neurones a f t e r Cnv i n j e c t i o n I l l 20. D i s t r i b u t i o n of T B - l a b e l l e d neurones a f t e r Rgc i n j e c t i o n 113 21. Photomicrographs of r e t r o g r a d e l y l a b e l l e d neurones a f t e r i n j e c t i o n of TB i n t o the Cnv or Rgc... 116 22. D i s t r i b u t i o n of l a b e l l e d neurones a f t e r TTD/Rpc i n j e c t i o n 122 24. Photomicrographs of r e t r o g r a d e l y l a b e l l e d neurones a f t e r i n j e c t i o n i n t o the TTD region 124 v i i i ABBREVIATIONS Ac nucleus accumbens AC anterior commissure A i intermediate archistriatum AL ansa l e n t i c u l a r i s Ala nucleus alatus AQ aqueduct BC brachium conjunctivum Cbl i n t e r n a l c e r e b e l l a r nucleus CbL l a t e r a l c e r e b e l l a r nucleus CbM medial c e r e b e l l a r nucleus cc c e n t r a l canal CE external cuneate nucleus Cnd nucleus c e n t r a l i s medullaris, pars d o r s a l i s Cnv nucleus c e n t r a l i s medullaris, pars v e n t r a l i s Ctz corpus trapezoidium D nucleus of Dacharwitz DBC decussation of the brachium conjunctivum DLF do r s o l a t e r a l funiculus DMF dorsomedial funiculus DY diamidino yellow dihydrochloride EM ectomammillary nucleus EW Edinger-Westphal nucleus FB Fast Blue FDA Fluoroscein conjugated dextran amines FRL l a t e r a l mesencephalic r e t i c u l a r formation FRM medial mesencephalic r e t i c u l a r formation GC dorsal column n u c l e i Get c e n t r a l gray GLv nucleus geniculatus l a t e r a l i s v e n t r a l i s ICo nucleus i n t e r c o l l i c u l a r i s 10 i n f e r i o r o l i v a r y nucleus IP nucleus interpeduncularis IS i n t e r s t i t i a l nucleus LH l a t e r a l hypothalmus LoC locus coeruleus MLd l a t e r a l mesencephalic nucleus, dorsal 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 MV motor nucleus of the trigeminal nerve nTS nucleus tractus s o l i t a r i u s N III-IIX c r a n i a l nerves III-XII 01 i n f e r i o r o l i v a r y nucleus 0M occipitomesencephalic t r a c t 0T optic tectum PA paleostriatum augmentatum PC nucleus of the pos t e r i o r commissure PGL nucleus p a r a g i g a n t o c e l l u l a r i s l a t e r a l i s PP paleostriatum primitivum PrV p r i n c i p a l trigeminal nucleus PVH nucleus p e r i v e n t r i c u l a r i s hypothalami PVM nucleus p a r a v e n t r i c u l a r i s i x R raphe n u c l e i RDA rhodomine conjugated dextran amines Rgc 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 RL nucleus r e t i c u l a r i s l a t e r a l i s Rm raphe magnus Rmc magnocellular r e t i c u l a r formation Rob raphe obscurus Rp raphe p a l l i d u s RP nucleus r e t i c u l a r i s pontis caudalis RPgc nucleus r e t i c u l a r i s pontis caudalis, pars g i g a n t o c e l l u l a r i s Rpc nucleus 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 RPO nucleus r e t i c u l a r i s pontis o r a l i s Rt nucleus rotundus Ru red nucleus Scd subcoeruleus nucleus, dorsal d i v i s i o n SCE stratum c e l l u l a r e externum SCI stratum c e l l u l a r e internum SCN suprachiasmatic nucleus Scv subcoeruleus nucleus, ventral d i v i s i o n SITS 4-acetamido,4'-isothiacyano stilbene-2,2'-di-sulphonic a c i d SM supramammillary nucleus SO superior o l i v a r y nucleus SpL l a t e r a l spiroform nucleus SSP supraspinal nucleus ST subtrigeminal nucleus SV p r i n c i p a l sensory nucleus of the trigeminal nerve TB True Blue T l nucleus tuberis i n f u n d i b u l i TPc pedunculo-pontine nucleus, pars compacta TS tractus s o l i t a r i u s TTD nucleus and t r a c t of the descending trigeminal nerve TTDc TTD, caudalis TTDi TTD, i n t e r p o l a r i s TTDo TTD, o r a l i s 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 VeLd l a t e r a l v e s t i b u l a r nucleus, dorsal component VeLv l a t e r a l v e s t i b u l a r nucleus, v e n t r a l component VeM medial v e s t i b u l a r nucleus VLF v e n t r o l a t e r a l funiculus VMF ventromedial funiculus III-XII n u c l e i c r a n i a l nerves III-XII X ACKNOWLEDGEMENTS Many people have been i n s t r u m e n t a l i n h e l p i n g me t o complete t h i s t h e s i s . F i r s t I wish to express my thanks to Dr. John Steeves f o r the opportunity to embark on t h i s research, and f o r the guidance and ass i s t a n c e he has provided throughout the p r o j e c t . I a l s o wish to thank f e l l o w student Gerry Sholomenko f o r t e c h n i c a l support and many h e l p f u l d i s c u s s i o n s and A r t h u r Vanderhorst, Armin Tepper, C r i s Taccogna, James Pernu, P h i l l i p Hannan, Dr. B i l l Milsom, Dr. Steve Vincent, Dr. T a l i Conine and Moya Palmer who have helped i n a v a r i e t y of ways. My thanks a l s o go to others i n the l a b and the department who have c o n t r i b u t e d i n one way or another t o my s u r v i v a l . Thanks are al s o due t o Dr. L e s l i e Rogers and Dr. Jack Pettigrew f o r p r o v i d i n g the f a c i l i t i e s to study the p a r r o t , and to the School of R e h a b i l i t a t i o n Medicine, UBC f o r a s s i s t a n c e w i t h t r a v e l costs to A u s t r a l i a . S p e c i a l thanks go to Mary Raphael f o r help w i t h the t y p i n g and t o many o t h e r f r i e n d s who have p r o v i d e d support and encouragement over the years. F i n a l l y , I dedicate t h i s work to the memory of my parents, e s p e c i a l l y my mother Maura who saw i t commenced but not f i n i s h e d . x i I INTRODUCTION 1 Locomotion i s a complex behaviour which i n v o l v e s the i n t e g r a t i o n and t i m i n g of nervous system a c t i v i t i e s . I t i s c l e a r t h a t the b a s i c motor programme i s produced i n the s p i n a l c o r d , s i n c e s p i n a l t r a n s e c t e d mammals ( S h e r r i n g t o n , 1910) , r e p t i l e s (Lennard and S t e i n , 1977) and b i r d s (ten Cate, 1960; Sholomenko and Steeves, 1987) are cap a b l e of c o o r d i n a t e d locomotor movements. Ever since Brown (1911, 1914) p o s t u l a t e d the existence of autonomous s p i n a l cord locomotor networks or "pattern generators", e f f o r t s have been made t o i d e n t i f y the descending pathways t h a t form t he command system f o r the i n i t i a t i o n and maintenance of a c t i v i t y i n these s p i n a l cord locomotor networks. Methods used to d e l i n e a t e these descending p r o j e c t i o n s have i n c l u d e d l e s i o n s w i t h i n the b r a i n and s p i n a l c o r d , and e l e c t r i c a l or chemical s t i m u l a t i o n of s e l e c t i v e regions along the neuraxis. Together they have shown tha t s u p r a s p i n a l c o n t r o l i s not a r i g i d chain of command but a loose h i e r a r c h i a l system of m u l t i p l e pathways w i t h p a r a l l e l p r o c e s s i n g ( G a l l i s t e l , 1980). While many s u p r a s p i n a l pathways, t o g e t h e r w i t h p e r i p h e r a l feedback, are e s s e n t i a l f o r modulating the b a s i c motor p a t t e r n and normal locomotor f u n c t i o n (Orlovsky and Shik, 1976) , the medullary r e t i c u l o s p i n a l p r o j e c t i o n s appear t o be of paramount importance i n the i n i t i a t i o n of locomotion (Steeves and Jordan, 1980; E i d e l b e r g et a l . , 1981a; Shefchyk et a l . , 1984). Locomotor r e g i o n s have been i d e n t i f i e d w i t h i n the brainstem; when st i m u l a t e d e l e c t r i c a l l y they evoke locomotion 2 i n the d e c e r e b r a t e c a t . One of t h e s e , the mesencephalic locomotor region (MLR), l i e s beneath the i n f e r i o r c o l l i c u l u s (Shik et a l . , 1966). Anatomically, the MLR has been i d e n t i f i e d as the caudal cuneiform nucleus i n the cat (Shik et a l . , 1967; Steeves et a l . , 1975; Jordan, 198 6) or the pedunculopontine nucleus i n the cat and r a t ( G a r c i a - R i l l , 1986; G a r c i a - R i l l and Skinner, 1985). There i s convincing evidence that both regions are i n v o l v e d i n the a c t i v a t i o n of l o c o m o t i o n . The 2-deoxyglucose method has shown that the cuneiform nucleus i s a c e n t r e of a c t i v i t y d u r i n g MLR-evoked f i c t i v e l o c o m o t i o n ( K e t t l e r and Jordan, 1984; reviewed by Jordan, 198 6) . The r o l e of the PPN has been demonstrated by the f a c t t h a t locomotion can be induced by i n f u s i o n of neurotransmitter agonists or antagonists t h a t are a s s o c i a t e d w i t h i t s a f f e r e n t GABAergic ( G a r c i a - R i l l et a l , 1985), or c h o l i n e r i c e f f e r e n t connections ( G a r c i a - R i l l and Skinner, 1987b). Another locomotor region, the pontobulbar locomotor s t r i p (PLS), l i e s i n the l a t e r a l brainstem (Shik and Yagodnitsyn, 1977). Whether the PLS c o n s i s t s of a f i b r e t r a c t or a column of neuronal somata that can be a c t i v 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 i s s t i l l being i n v e s t i g a t e d . The PLS may c o n s i s t of descending p r o j e c t i o n s from the mesencephalic t r i g e m i n a l nucleus, (Garcia-R i l l et a l . , 1983a), and/or f i b r e s from the d o r s o l a t e r a l parvo-c e l l u l a r r e t i c u l a r formation (Selionov and Shik, 1984) or the descending t r i g e m i n a l system (Noga et a l . , 1988). While c o n s i d e r a b l e p r o g r e s s has been made towards understanding the descending systems c o n t r o l l i n g the i n i t i a t i o n 3 of locomotion i n the cat, and t o a l e s s e r extent i n the monkey and r a t , l e s s d a t a are a v a i l a b l e r e g a r d i n g non-mammalian ve r t e b r a t e species. Brainstem s t i m u l a t i o n has been found to evoke locomotion i n lamprey (McClellan and G r i l l n e r , 1984), t e l e o s t f i s h (Kashin et a l . , 1974, 1981), and t u r t l e (Kazennikov et a l . , 1981). In these species most s t i m u l a t i o n s i t e s appear to be l o c a t e d w i t h i n the l a t e r a l region of the pontomedullary r e t i c u l a r formation (Kazennikov et a l . , 1981; M c C l e l l a n , 198 6) and a l s o the m i d l i n e of the mesencephalon (Kashin et a l . , 1974, 1981; Leonard et a l . , 1979). Evoked locomotion has a l s o been demonstrated i n b i r d s , by s t i m u l a t i o n of the cut c e r v i c a l cord (Jacobson and Hollyday, 1982), the t r i g e m i n a l region, or medial r e t i c u l a r formation (Steeves and Weinstein, 1984; Steeves et a l . , 1986, 1987). The b i r d provides an i n t e r e s t i n g model f o r the study of l o c o m o t i o n . F i r s t , d i f f e r e n t p a t t e r n s of l o c o m o t i o n are performed w i t h the wings and hindlimbs and these modes of locomotion are amenable to separate and i n t e r a c t i v e study. Second, many b i r d s are strong overground walkers and t h e i r locomotor p a t t e r n more c l o s e l y resembles t h a t of humans than quadrapedal v e r t e b r a t e s . T h i r d , most b i r d s appear to have a p o o r l y developed cortex, no c o r t i c o s p i n a l t r a c t (Portmann and S t i n g e l i n , 1961), and are a b l e t o walk and f l y d e s p i t e e x t i r p a t i o n of the cortex (Visser and Rademaker, 1934, c i t e d by ten Cate, 1975). This suggests t h a t normal avian locomotion i s under the predominant, i f not e x c l u s i v e , d i r e c t c o n t r o l of the b r a i n s t e m . C o n s i d e r i n g t h a t non-mammalian v e r t e b r a t e s 4 show a remarkable v a r i e t y of locomotor patterns without the b e n e f i t of a c o r t i c o s p i n a l t r a c t , i t has been suggested t h a t the brainstem p r o j e c t i o n s may play a dominant r o l e i n the i n i t i a t i o n and maintenance of locomotion i n a l l v e r t e b r a t e s , while the primary r o l e of the mammalian c o r t i c o s p i n a l system i s f o r the c o n t r o l of d i s t a l l i m b m u s c u l a t u r e (Lawrence and Kuypers, 1968a). This separation of f u n c t i o n has been c l e a r l y shown i n monkeys where t o t a l t r a n s e c t i o n of the c o r t i c o s p i n a l t r a c t r e s u l t s i n l i t t l e or no impairment of locomotion but severe l o s s of hand f u n c t i o n (Lawrence and Kuypers, 1968a). The descending s p i n a l pathways have been described i n mammals using degenerative (e.g., Nyberg-Hansen and Brodal, 1964) and retrograde and anterograde t r a c i n g techniques (e.g., Kuypers and Maisky, 1975; f o r review see Kuypers and Ma r t i n , 1982; Kuypers, 1981) . Such data i s a l s o a v a i l a b l e f o r s e v e r a l r e p t i l i a n species (e.g., ten Donkelaar, 1976a,b; ten Donkelaar, et a l . , 1980). However, there i s l i t t l e corresponding data f o r the b i r d (chicken: Wold, 1978; Okada and Oppenheim, 1985; Gross and Oppenheim, 1985; pigeon: Wild et a l . , 1979; Cabot et a l . , 1982; Berk and F i n k e l s t e i n , 1983). A l t h o u g h t h e c o n n e c t i o n s of the a v i a n t r i g e m i n a l system have been w e l l documented (Arends et a l . , 1984; W i l d et a l . , 1985), the descending inputs t o the medial medullary r e t i c u l a r formation have not been described. The purpose of t h i s t h e s i s i s t o determine the o r i g i n s and connections of the descending c e n t r a l nervous system (CNS) pathways presumed t o be i n v o l v e d i n locomotor c o n t r o l i n non-5 d e x t e r o u s p a l m a t e ( w e b - f o o t e d ) w h i t e P e k i n duck (Anas platyrhynchos) and t h e Canada goose (Branta canadensis). To dete r m i n e t h e g e n e r a l i t y o f t h e s e f i n d i n g s , t h e d e s c e n d i n g p r o j e c t i o n s w i l l a l s o be examined i n t h e z y g o d a c t y l ( p a i r e d o p p o s i n g t o e s ) S u l p h u r - c r e s t e d c o c k a t o o , as t h i s b i r d d emonstrates c o n s i d e r a b l e p e d a l d e x t e r i t y . Emphasis i s p l a c e d upon t h e p o n t o m e d u l l a r y r e t i c u l o s p i n a l pathways, s i n c e t h e y have been i d e n t i f i e d as an e f f e c t i v e s i t e f o r t h e i n i t i a t i o n o f l o c o m o t i o n i n a b o t h mammalian and non-mammalian v e r t e b r a t e s . S i n c e p r e v i o u s s t u d i e s have shown a r e m a r k a b l e s i m i l a r i t y i n t h e o r g i n s o f b r a i n s t e m - s p i n a l p r o j e c t i o n s i n a l l v e r t e b r a t e s p e c i e s (Kuypers and M a r t i n , 1982), my h y p o t h e s i s i s t h a t t h e r e w i l l be no d i f f e r e n c e i n t h e o r g a n i z a t i o n o f t h e d e s c e n d i n g p r o j e c t i o n s , o r t h e i r b r a i n s t e m a f f e r e n t s i n b i r d s compared t o o t h e r v e r t e b r a t e s . The f o l l o w i n g q u e s t i o n s w i l l be a d d r e s s e d : (1) What p r o j e c t i o n s descend t o t h e c e r v i c a l v e r s u s t h e lumbar s p i n a l c ord? (2) A r e t h e r e any d i f f e r e n c e s between t h e d e s c e n d i n g b r a i n s t e m - s p i n a l p r o j e c t i o n s o f web-footed b i r d s compared t o d e x t e r o u s b i r d s ? (3) How a r e t h e d e s c e n d i n g p r o j e c t i o n s o r g a n i z e d w i t h i n t h e lumbar s p i n a l cord? (4) Do t h e neurones w i t h i n t h e e f f e c t i v e r a d i u s o f a f o c a l e l e c t r i c a l s t i m u l a t i n g c u r r e n t , t h a t evokes l o c o m o t i o n i n a d e c e r e b r a t e b i r d , send t h e i r axons d i r e c t l y t o t h e cord? (5) What r o s t r a l CNS r e g i o n s p r o j e c t t o b r a i n s t e m l o c o m o t o r s i t e s w i t h i n t h e c a u d a l m e d u l l a ? These q u e s t i o n s a r e a d d r e s s e d i n sequence i n t h e f o l l o w i n g f i v e c h a p t e r s and i n t e g r a t e d i n a d i s c u s s i o n i n t h e f i n a l c h a p t e r . 6 II ORIGINS OF BRAINSTEM-SPINAL PROJECTIONS IN THE DUCK AND GOOSE 7 I N T R O D U C T I O N The o r i g i n and extent of p r o j e c t i o n s descending t o the s p i n a l c o r d have been w e l l documented i n s e v e r a l s p e c i e s i n c l u d i n g the cat (Kuypers and Maisky, 1975; Tohyama et a l . , 1979a,b; M i t a n i et a l . , 1988), r a t (Leichnetz et a l . , 1978; Zemlan and P f a f f , 1979; Jones and Yang, 1985), opossum (Crutcher et a l . , 1978; M a r t i n et a l . , 1979, 1981a), monkey ( C a s t i g l i o n i et a l . , 1978; K n e i s l e y et a l . , 1978; C a r l t o n et a l . , 1985), and r e p t i l e s such as l i z a r d and t u r t l e (ten Donkelaar et a l . , 1980). Most studi e s i n the b i r d , however, have been r e s t r i c t e d t o a d e s c r i p t i o n of a s i n g l e descending system (Wold, 1978; Wild et a l . , 1979; Berk and F i n k e l s t e i n , 1983; Arends et a l . , 1984) or have used immature animals (Okada and Oppenheim, 1985; Gross and Oppenheim, 1985; Glover and P e t u r s d o t t i r , 1988) . One study i n the a d u l t p i g e o n i d e n t i f i e d the o r i g i n s of brai n s t e m - s p i n a l pathways, but t h e i r r o s t r o c a u d a l extent was not c l e a r l y d e l i n e a t e d (Cabot et a l . , 1982) . As p a r t of an ongoing i n v e s t i g a t i o n i n t o the n e u r a l c o n t r o l of locomotion i n b i r d s , the present study had two o b j e c t i v e s . F i r s t , t o determine the o r i g i n s of descending pathways to the c e r v i c a l or lumbar s p i n a l cord. Second, to a s c e r t a i n whether the b r a i n s t e m was the s o l e source o f descending p r o j e c t i o n s t o the s p i n a l cord i n the duck and goose. Some avian s t u d i e s have r e f e r r e d to the presence of a t e l e n c e p h a l o s p i n a l pathway, analogous t o the mammalian c o r t i c o s p i n a l t r a c t (Zecha, 1964; Karten, 1971). This f i n d i n g , 8 however, has not been re-examined s i n c e t h e advent o f modern r e t r o g r a d e t r a c i n g t e c h n i q u e s . T h i s s t u d y d e s c r i b e s t h e d i s t r i b u t i o n o f n e u r o n a l c e l l b o d i e s which were r e t r o g r a d e l y l a b e l l e d f o l l o w i n g i n j e c t i o n o f True B l u e (TB) i n t o t h e c e r v i c a l o r lumbar c o r d . TB was chosen f o r t h i s s t u d y b e c a u s e i t (1) has b e e n r e p o r t e d t o be t r a n s p o r t e d over l o n g d i s t a n c e s , (2) i s not t r a n s p o r t e d t r a n -s y n a p t i c a l l y , and (3) does not r e a d i l y d i f f u s e out o f n e u r o n a l somata ( B e n t i v o g l i o e t a l . , 1979; Sawchenko and Swanson, 1981) . MATERIALS AND METHODS E i g h t e e n a d u l t b i r d s (14 w h i t e P e k i n d u c k s , Anas platyrhynchos, and f o u r Canada geese, Branta canadensis) o f b o t h sexes were used f o r t h i s s t u d y . A n a e s t h e s i a was i n d u c e d w i t h a p p r o x i m a t e l y 5% H a l o t h a n e d i s s o l v e d i n 95% oxygen and 5% carbon d i o x i d e and s u b s e q u e n t l y m a i n t a i n e d a t a H a l o t h a n e l e v e l o f 2%. The depth o f a n a e s t h e s i a was a s s e s s e d by e v i d e n c e o f l o s s o f a r e f l e x response t o f o r c e f u l p r e s s u r e on t h e f o o t , and a slow r e f l e x response o f t h e n i c t a t i n g membrane t o t a c t i l e s t i m u l a t i o n . A l o c a l a n a e s t h e t i c ( X y l o c a i n e h y d r o c h l o r i d e , 2%) was i n f i l t r a t e d s u b c u t a n e o u s l y i n t h e c e r v i c a l o r lumbar r e g i o n p r i o r t o removal o f t h e f e a t h e r s and i n c i s i o n o f t h e s k i n , and s u b s e q u e n t l y i n f i l t r a t e d i n t o t h e u n d e r l y i n g f a s c i a and p e r i o s t e u m . F o l l o w i n g i n c i s i o n o f t h e f a s c i a on e i t h e r s i d e o f t h e m i d l i n e , t h e m u s c l e s were r e t r a c t e d and a p a r t i a l laminectomy was performed a t t h e c e r v i c a l o r lumbar l e v e l . Then t h e v e r t e b r a l column was f i x e d i n an e x t e r n a l frame t o reduce 9 movement o f t h e c o r d due t o r e s p i r a t o r y movements. A 5% aqueous s u s p e n s i o n o f TB (Dr. I l l i n g , GmbH+Co KG, FRG) was i n j e c t e d , w i t h a 1 0 - u l H a m i l t o n s y r i n g e , i n t o a c e r v i c a l o r l u m b a r segment o f t h e s p i n a l c o r d , a t an approximate r a t e o f 0.2 u l / m i n . In t h e i n i t i a l e x p e r i m e n t s , b i l a t e r a l i n j e c t i o n s were made t o o b t a i n maximum l a b e l l i n g and i d e n t i f y d i f f e r e n t i a l p r o j e c t i o n s t o each l e v e l o f t h e c o r d . L a t e r , u n i l a t e r a l i n j e c t i o n s were u s e d t o d e t e r m i n e t h e l a t e r a l i t y o f t h e d e s c e n d i n g pathways. At t h e c e r v i c a l l e v e l o f t h e s p i n a l c o r d , TB was i n j e c t e d (3-5 u l / s i d e ) b i l a t e r a l l y a t C l - 2 , C2-3, or C7, (3 ducks) o r u n i l a t e r a l l y a t C l - 2 (2 ducks, 1 g o o s e ) . At t h e lumbar l e v e l o f t h e s p i n a l c o r d , TB was i n j e c t e d (5-10 u l / s i d e ) a t L I , b i l a t e r a l l y (5 ducks, 1 goose), or u n i l a t e r a l l y (4 ducks, 2 g e e s e ) . I n f o u r o f t h e l a t t e r group o f b i r d s , t h e c o n t r a l a t e r a l s p i n a l c o r d was a l s o h e m i s e c t e d , 10 mm r o s t r a l t o t h e s i t e o f i n j e c t i o n , t o negate any s p r e a d t o t h e o p p o s i t e s i d e . A f t e r c o m p l e t i n g each i n j e c t i o n o f TB, t h e n e e d l e was l e f t i n p l a c e f o r an a d d i t i o n a l 5 minutes b e f o r e w i t h d r a w a l . The s i t e was t h e n c o v e r e d w i t h Gelfoam, and where p o s s i b l e t h e v e r t e b r a l bone, w h i c h h a d b e e n p r e v i o u s l y removed, was r e p l a c e d . The muscles and s k i n were th e n s u t u r e d . P o s t -o p e r a t i v e c a r e i n c l u d e d r o u t i n e p r o p h y l a c t i c a d m i n i s t r a t i o n o f a n a l g e s i c s (Demerol, 0.2 u l , 3 t i m e s / d a y d e c r e a s i n g t o 0.2 u l 1/day over 5 days) and a n t i b i o t i c s ( a m p i c i l l i n , 100 mg/day f o r 5 days) . The b i r d s were kept i n a t e m p e r a t u r e c o n t r o l l e d room w i t h n a t u r a l l i g h t i n g and f e d ad libitum. 1 0 The p o s t - i n j e c t i o n s u r v i v a l time f o r the lumbar i n j e c t e d animals ranged from 10 to 32 days. The median was 20 days, as no f u r t h e r improvement i n l a b e l l i n g was obtained w i t h longer i n t e r v a l s . An 8-10 day s u r v i v a l p e r i o d was used f o r b i r d s w i t h a c e r v i c a l i n j e c t e d b i r d s . Each animal was placed under deep anaesthesia (sodium p e n t o b a r b i t a l , 75 mg/kg, IP) and perfused t r a n s c a r d i a l l y w i t h 1 l i t r e of 0.1 M phosphate-buffered s a l i n e at pH 7.4. This was f ollowed by p e r f u s i o n w i t h 1 l i t r e of 4% paraformaldehyde i n 0.1 M phosphate b u f f e r (pH 7.4), at 4°C. The b r a i n and the i n j e c t e d region of the s p i n a l cord were removed, p o s t f i x e d f o r a f u r t h e r 12 hours, and then immersed i n a c r y o p r o t e c t ant (25% s u c r o s e , 10% g l y c e r o l i n 0.05 M phosphate b u f f e r ) , at 4°C, f o r 48 hours. The brainstem and the s p i n a l cord i n j e c t i o n s i t e s were sectioned at 30 um t h i c k n e s s u s u a l l y i n the transverse plane (one b r a i n was sectioned i n the s a g i t t a l plane) on a f r e e z i n g microtome. S i m i l a r l y the telencephalon was sectioned i n a l l c e r v i c a l and 2 lumbar experiments. Two out of every three s e c t i o n s were f l o a t e d i n 0.2 M T r i s b u f f e r , mounted a l t e r n a t e l y onto two s e r i e s of g e l a t i n i z e d s l i d e s , and a i r d r i e d i n the dark. One set of s e c t i o n s was examined under a L e i t z Orthoplan microscope w i t h e p i f l u o r e s c e n c e and an A2 f i l t e r b l o c k ( e x c i t a t i o n band pass 270-300 nm; b a r r i e r band pass 410-580 nm) . The remaining set of s e c t i o n s was s t a i n e d w i t h c r e s y l v i o l e t or t h i o n i n e . The d i s t r i b u t i o n of f l u o r e s c e n t TB somata was p l o t t e d on camera l u c i d a o u t l i n e s drawn at 270 um i n t e r v a l s and photographs were taken of s e l e c t e d areas. The l o c a t i o n s of 11 the l a b e l l e d c e l l s were then i d e n t i f i e d anatomically with the aid of avian atlases (Karten and Hodos, 1967; Zweers, 1971) and other avian references ( B r e a z i l e , 1979; Katz and Karten, 1983a,b; Kuenzel and van Tienhoven, 1982; Wold, 1976). The nomenclacture used i n t h i s thesis i s generally according to Karten and Hodos (1967) although some terms have been anglicized. RESULTS Each i n j e c t i o n s i t e consisted of a vacuolated core of n e c r o s i s and a f l u o r e s c e n t a c e l l u l a r region surrounded by c e l l s f i l l e d with TB. The r o s t r a l extent of d i f f u s i o n from the TB i n j e c t i o n s i t e s ranged from 1-3 mm. Following i n j e c t i o n of TB into the Cl-2 segment, examination of the tissue sections showed that the i n j e c t i o n was r e s t r i c t e d to the spinal cord, with no d i f f u s i o n of TB into the caudal brainstem. In addition, the u n i l a t e r a l c e r v i c a l cord i n j e c t i o n s r e s u l t e d i n only l i m i t e d spread of TB to the c o n t r a l a t e r a l side of the spinal cord (Fig. 1A). In the lumbar cord injections, the d i f f u s i o n of TB was l i m i t e d to one side of the cord i n 5 of 6 experiments with u n i l a t e r a l injections (Fig. IB) and the r o s t r a l d i f f u s i o n of TB was always at least 5 mm caudal to the c o n t r a l a t e r a l hemisection. The d i s t r i b u t i o n of TB-labelled neurones was comparable following a b i l a t e r a l or a u n i l a t e r a l i n j e c t i o n . Obviously more TB-labelled neurones were present after a b i l a t e r a l i n j e c t i o n , but both sides of the brainstem were not equally labelled, since the locus of i n j e c t i o n was not always symmetrical. Figure 1. Photomicrographs of s p i n a l cord sections showing True Blue (TB) i n j e c t i o n s i t e s . A: U n i l a t e r a l c e r v i c a l 5 u l (Cl-2) i n j e c t i o n i n a duck. B: U n i l a t e r a l 5 u l lumbar (LI) i n j e c t i o n i n a goose. The ventromedial margin of the v e n t r a l horn i s o u t l i n e d . M a g n i f i c a t i o n : A = 37 x; B = 23 x. 13 In the f o l l o w i n g account, only the r e s u l t s of the u n i l a t e r a l i n j e c t i o n s are d escribed, s i n c e they provide additional information regarding the l a t e r a l i t y of pathways. There were a few differences i n the sources of projections to the two l e v e l s of the cord. The number of TB fluorescent c e l l s was always gre a t e r a f t e r a c e r v i c a l i n j e c t i o n , r e f l e c t i n g either a greater number of projections to the c e r v i c a l l e v e l and/or the unavoidable l a b e l l i n g of f i b r e s t r a v e r s i n g the c e r v i c a l i n j e c t i o n s i t e en route to more caudal lev e l s of the spinal cord. The locations of neuronal somata l a b e l l e d with TB following u n i l a t e r a l i n j e c t i o n at c e r v i c a l (Cl-2) or lumbar (LI) l e v e l s of the spinal cord are summarized in Figures 2 and 3, respectively. The b i r d used for each figure i s a duck. However, v i r t u a l l y i d e n t i c a l data were obtained for the goose. In t h i s and subsequent chapters, c e l l size measurements refer to the maximum diameter of the soma. Medulla-pons In the dorsal region of the medulla, caudal to the obex, l a b e l l e d c e l l s (6-10 um) were found b i l a t e r a l l y within the dorsal column nuclei (GC) and external cuneate nucleus (CE) , but only after a c e r v i c a l i n j e c t i o n . The CE neurones extended to more r o s t r a l l e v e l s of the medulla (Fig. 2A). More medially, l a b e l l e d neurones were also found within the nucleus of the s o l i t a r y t r a c t (nTS). After a c e r v i c a l i n j e c t i o n , these TB c e l l s were d i s t r i b u t e d throughout the e n t i r e r o s t r o c a u d a l extent of the nTS; however, after a lumbar i n j e c t i o n , TB c e l l s were sparse and l i m i t e d to the r o s t r a l part of the complex. 14 Figure 2. Diagrams of r e p r e s e n t a t i v e brainstem s e c t i o n s from a duck, summarizing the d i s t r i b u t i o n of r e t r o g r a d e l y l a b e l l e d neurones a f t e r a u n i l a t e r a l i n j e c t i o n of TB i n t o the Cl-2 c e r v i c a l s p i n a l c o r d . The diagrams A-N d e p i c t t r a n s v e r s e s e c t i o n s i n a caudal t o r o s t r a l d i r e c t i o n . The r o s t r o c a u d a l l e v e l i s i n d i c a t e d on the s a g i t t a l o u t l i n e . For every 30 um brainstem s e c t i o n i l l u s t r a t e d , each small f i l l e d c i r c l e (•) denotes a s i n g l e TB l a b e l l e d c e l l , w hile each l a r g e open c i r c l e (0) r e p r e s e n t s 5 TB l a b e l l e d c e l l s . 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 are i n d i c a t e d on the l e f t and r i g h t s i d e s of each diagram, r e s p e c t i v e l y . TB c e l l s l o c a t e d w i t h i n the c o n t r a l a t e r a l i n t e r n a l c e r e b e l l a r n u c l e u s are not i l l u s t r a t e d . Comparable d i s t r i b u t i o n s of r e t r o g r a d e l y l a b e l l e d neurones were observed a f t e r a u n i l a t e r a l i n j e c t i o n at the C2-3 l e v e l i n the goose. For a b b r e v i a t i o n s see l i s t on page (v). 15 91 N )l I 9 3 3 V 17 18 Figure. 3. Diagrams of representative brainstem sections from a duck, summarizing the d i s t r i b u t i o n of r e t r o g r a d e l y l a b e l l e d neurones following a u n i l a t e r a l i n j e c t i o n of TB into the LI lumbar spinal cord. The diagrams A-J depict transverse sections i n a caudal to r o s t r a l d i r e c t i o n . Each small f i l l e d c i r c l e (•) denotes a single TB l a b e l l e d c e l l , while each large open c i r c l e (0) represents 5 TB l a b e l l e d c e l l s observed within a single brainstem section, at t h i s l e v e l of the neuraxis. I p s i l a t e r a l and contra l a t e r a l projections are shown on the l e f t and right sides of each drawing, respectively. The few l a b e l l e d c e l l s within the i p s i l a t e r a l hypothalamus are not i l l u s t r a t e d . Comparable d i s t r i b u t i o n s of retrogradely l a b e l l e d neurones were observed a f t e r u n i l a t e r a l i n j e c t i o n s at the LI l e v e l i n the goose. 19 20 21 Another column of predominantly c o n t r a l a t e r a l l a b e l l e d c e l l s , p r o j e c t i n g to both l e v e l s of the cord, was l o c a t e d v e n t r a l to the d o r s a l nucleus of the vagus nerve (X) , and i d e n t i f i e d as the nucleus a l a t u s (Ala, Gross and Oppenheim, 1985). This p r o j e c t i o n was very prominent a f t e r a c e r v i c a l i n j e c t i o n . A f t e r lumbar i n j e c t i o n , t h e s e TB c e l l s were o c c a s i o n a l l y observed caudal to the obex; however, they were c o n s i s t e n t l y seen 1.5-2.0 mm r o s t r a l t o the obex, where there were 3-7 c e l l s / s e c t i o n (Figs. 2A, 3B) . I n a d d i t i o n , l a r g e r -diameter (25-30 um) m u l t i p o l a r TB c e l l s were l o c a t e d along the vagus nerve (N X ) , w i t h i n the l a t e r a l brainstem, and these a l s o p r o j e c t e d t o both l e v e l s of the cord. Although a few l a b e l l e d neurones were found i n the hypoglossal nucleus ( X I I ) , f o l l o w i n g a CI i n j e c t i o n , they were not present a f t e r the C2-3 i n j e c t i o n and t h e r e f o r e probably r e f l e c t damage to the hypoglossal nerve. Within the v e n t r o l a t e r a l medulla, commencing 2 mm caudal to the obex, m u l t i p o l a r TB neurones (40-60 um) formed a compact group (5 c e l l s / s e c t i o n ) , c h i e f l y w i t h i n the c o n t r a l a t e r a l brainstem. Further r o s t r a l l y , these l a b e l l e d c e l l s appeared to be continuous w i t h more numerous TB neurones (25-40 um; 10 c e l l s / s e c t i o n ) i n the nucleus r e t i c u l a r i s c e n t r a l i s m e d u l l a r i s , pars d o r s a l i s (Cnd). Together these two groups of 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 extended over a r o s t r o c a u d a l d i s t a n c e of approximately 3.5 mm and had a s i m i l a r d i s t r i b u t i o n a f t e r c e r v i c a l or lumbar i n j e c t i o n (Figs. 2A, 3A) . Commencing at the l e v e l of the obex, along the ventro-l a t e r a l margin of the medulla, f l u o r e s c e n t neurones (20-30 um) 22 were f o u n d w i t h i n t h e n u c l e u s r e t i c u l a r i s l a t e r a l i s (RL) , p r i n c i p a l l y on t h e c o n t r a l a t e r a l s i d e . Together w i t h t h e more r o s t r a l l a b e l l e d n e u r o n e s (30-35 um) o f t h e n u c l e u s p a r a g i g a n t o c e l l u l a r i s l a t e r a l i s (PGL); TB l a b e l l i n g i n t h i s r e g i o n extended a p p r o x i m a t e l y 2.5 mm r o s t r a l t o t h e obex, i n e i t h e r c e r v i c a l o r lumbar i n j e c t e d a n i m a l s ( F i g s . 2A-C, 3A-C). Two o t h e r l a t e r a l r e g i o n s were o n l y l a b e l l e d f o l l o w i n g c e r v i c a l i n j e c t i o n . These c o n s i s t e d o f a few c e l l s l o c a t e d w i t h i n t h e t r a c t o f t h e d e s c e n d i n g n u c l e u s o f t h e t r i g e m i n a l n e rve (TTD) and t h e s u b t r i g e m i n a l n u c l e u s (ST). W i t h i n t h e c e n t r a l r e g i o n o f t h e m e d u l l a , l a t e r a l t o t h e h y p o g l o s s a l n e r v e , s m a l l t o medium s i z e d neurones (10-20 um) were l a b e l l e d i n t h e n u c l e u s c e n t r a l i s m e d u l l a r i s , p a r s y e n t r a l i s (Cnv). F o l l o w i n g a c e r v i c a l i n j e c t i o n , a p p r o x i m a t e l y 30 TB c e l l s / s e c t i o n were found v e n t r o m e d i a l l y ( F i g s . 2A,B), p r i m a r i l y i p s i l a t e r a l t o t h e i n j e c t i o n . I n a d d i t i o n , many c e l l s (10-15 um) were l a b e l l e d more d o r s a l l y , l a t e r a l and d o r s a l t o t h e s u p r a s p i n a l n u c l e u s (SSP) on b o t h s i d e s ( F i g . 2A) . A f t e r lumbar i n j e c t i o n s , s i g n i f i c a n t l y fewer l a b e l l e d neurones were p r e s e n t and o n l y i n t h e v e n t r a l r e g i o n o f Cnv ( F i g s . 3A,B). More r o s t r a l l y , w i t h i n t h e 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 (Rgc), many l a r g e neurones (30-50 um) w i t h prominent d e n d r i t e s were l a b e l l e d a f t e r b o t h c e r v i c a l and l u m b a r i n j e c t i o n s ( F i g s . 2D, 3D, 4A,B) . A p p r o x i m a t e l y 75% o f t h e TB n e u r o n e s f o u n d w i t h i n t h e Rgc were i p s i l a t e r a l t o t h e i n j e c t i o n . 23 F i g u r e 4. R e p r e s e n t a t i v e p h o t o m i c r o g r a p h s s h o w i n g r e t r o g r a d e l y l a b e l l e d TB b r a i n s t e m - s p i n a l neurones i n the duck f o l l o w i n g u n i l a t e r a l c e r v i c a l i n j e c t i o n ( A , D , E , F ) or u n i l a t e r a l l u m b a r i n j e c t i o n ( B , C ) . A : I p s i l a t e r a l 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 (Rgc); B: I p s i l a t e r a l Rgc; C : I p s i l a t e r a l l a t e r a l v e s t i b u l a r n u c l e u s (VeL) ; D: C o n t r a l a t e r a l de scend ing v e s t i b u l a r n u c l e u s (VeD); E : I p s i l a t e r a l l o c u s c o e r u l e u s ; F : I p s i l a t e r a l n u c l e u s p a r a v e n t r i c u l a r i s (PVM) . S c a l e b a r s : 100 um. 24 25 At the medullary-pontine junction, there was a further example of topographic l a b e l l i n g of r e t i c u l o s p i n a l neurones. Cervical but not lumbar injections l a b e l l e d multipolar neurones (40 um) i n the dorsal region of the medulla on both sides, adjacent to the medial longitudinal fasciculus (MLF). These neurones were located within the dorsal h a l f of the nucleus r e t i c u l a r i s pontis caudalis (RP; F i g . 2D), where i t borders the Rgc. In a d d i t i o n , the small c e l l s of the nucleus p a r v o c e l l u l a r i s r e t i c u l a r i s (Rpc), located l a t e r a l and dorsal to the Cnv and the Rgc, were l a b e l l e d only after c e r v i c a l injections (Fig. 2B) . In more r o s t r a l regions of the RP, a t o p o g r a p h i c a l o r g a n i z a t i o n was not apparent; comparable l a b e l l i n g was seen aft e r c e r v i c a l or lumbar i n j e c t i o n (Figs. 2E,F, 3E,F). Along the midline of the medulla and pons, TB-labelled c e l l s (15-20 um) were l o c a t e d w i t h i n the nucleus raphe obscurus, a nucleus that i s intermingled with the f i b r e s of the MLF. These raphe-spinal c e l l s were sparse following a lumbar i n j e c t i o n and even afte r c e r v i c a l i n j e c t i o n numbered only 30 c e l l s (over a rostrocaudal distance of 2.0 mm). More l a b e l l e d neurones (10 cells/section) were found i n the nucleus raphe p a l l i d u s (Rp), extending from the caudal i n f e r i o r o l i v a r y nucleus (10) to the abducens nucleus (VI) . However, a greater number of TB-labelled c e l l s (15-40 um) was observed i n the nucleus raphe magnus (Rm), p a r t i c u l a r l y at the l e v e l of the VI nerve. Here, TB c e l l s numbered 20-25/section f o l l o w i n g c e r v i c a l injections and 10-15/section aft e r lumbar injections ( F i g s . 2D,E, 3D,E). Although the raphe n u c l e i are midline structures, there appeared to be more l a b e l l e d c e l l s on the side i p s i l a t e r a l to the i n j e c t i o n . Within the vestibular complex, numerous TB neurones (35-40 um) were present i p s i l a t e r a l l y w i t h i n the l a t e r a l v estibular nucleus (VeL: F i g . 4C) after c e r v i c a l or lumbar i n j e c t i o n ( F i g s . 2D,E, 3D,E). L a b e l l i n g was e x c l u s i v e l y i p s i l a t e r a l i n the d o r s a l s u b d i v i s i o n (VeLd), whereas c o n t r a l a t e r a l l a b e l l i n g was a l s o found i n the v e n t r a l subdivision (VeLv). After c e r v i c a l injections, many l a b e l l e d neurones (25-30 um) were also located within the c o n t r a l a t e r a l descending vestibular nucleus (VeD: F i g . 4D) , but there were few l a b e l l e d a f t e r lumbar injections (Figs. 2D,E, 3D,E). The medial vestibular nucleus (VeM) contained only a sparse number of TB c e l l s . These were found primarily on the i p s i l a t e r a l side and only after a c e r v i c a l i n j e c t i o n (Fig. 2D). Labelled neurones were also found within the c o n t r a l a t e r a l i n t e r n a l c e r e b e l l a r nucleus (homologous with the mammalian f a s t i g i a l nucleus), but only after a high c e r v i c a l i n j e c t i o n . In the pons, r o s t r a l to the f a c i a l nucleus (VII), the d i s t r i b u t i o n of TB l a b e l l i n g was similar after c e r v i c a l or lumbar i n j e c t i o n s . A few i p s i l a t e r a l l y l a b e l l e d neurones were located dorsal and l a t e r a l to VII. Labelled neurones (20-30 um) were also found within the caudal h a l f of the locus coeruleus (LoC; F i g . 4E) and within the dorsal subcoeruleus nucleus, (Scd) primarily on the i p s i l a t e r a l side. Labelled c e l l s (30-40 um) within the ventral subcoeruleus nucleus (Scv), however, 27 were p r i m a r i l y c o n t r a l a t e r a l (Figs. 2G, 3G). Fluorescent TB neurones (40-50 um) w i t h i n the i p s i l a t e r a l nucleus r e t i c u l a r i s p o n t i s c a u d a l i s , p ars g i g a n t o c e l l u l a r i s (RPgc) were more prevalent than the small number w i t h i n the c o n t r a l a t e r a l RPgc. A few l a b e l l e d neurones (30 um) were al s o l o c a t e d b i l a t e r a l l y w i t h i n the nucleus r e t i c u l a r i s p o n t i s o r a l i s (RPO), but these were sparse even a f t e r c e r v i c a l i n j e c t i o n (Figs. 2H, 3H) . Mesencephalon-hypothalamus Within the mesencephalon, TB neurones (15-20 um) were found i n the i p s i l a t e r a l i n t e r s t i t i a l nucleus (IS) a f t e r both c e r v i c a l and lumbar i n j e c t i o n . L a b e l l e d c e l l s (25-40 um) were d i s t r i b u t e d throughout the c o n t r a l a t e r a l red nucleus (Ru, F i g s . 2 J , 3J) a f t e r lumbar as w e l l as c e r v i c a l i n j e c t i o n s . In a d d i t i o n , there were three b i l a t e r a l l y l a b e l l e d regions, but they were seen only a f t e r c e r v i c a l i n j e c t i o n s . These regions were composed of many small diameter c e l l s (10-15 um) w i t h i n the nucleus i n t e r c o l l i c u l a r i s (ICo), and medium s i z e d neurones (20-30 um) w i t h i n the l a t e r a l and medial components of the mesencephalic r e t i c u l a r formation (FRL, FRM; F i g s . 21,J). At the l e v e l of the hypothalamus, f o l l o w i n g c e r v i c a l i n j e c t i o n , T B - l a b e l l e d c e l l s were found i n the i p s i l a t e r a l l a t e r a l h y p o t h a l a m i c n u c l e u s (LH), the s t r a t u m c e l l u l a r e externum (SCE), and the .nucleus p e r i v e n t r i c u l a r i s hypothalami (PVH) . There were 40 l a b e l l e d neurones/section w i t h i n the i p s i l a t e r a l nucleus p a r a v e n t r i c u l a r i s (PVM; F i g . 4F), but only a few TB neurones on the c o n t r a l a t e r a l side (Figs. 2M,N) . In a d d i t i o n , a few TB somata were al s o evident d o r s a l to the 28 supramammillary nucleus (SM) and i n the suprachiasmatic nucleus (SCN). After lumbar injections, a few l a b e l l e d c e l l s were found i n only the i p s i l a t e r a l LH, PVH, and PVM. This formed the r o s t r a l l i m i t of l a b e l l i n g , as TB c e l l s were never found i n the thalamus or telencephalon following either c e r v i c a l or lumbar i n j e c t i o n s . DISCUSSION The major findings of t h i s study were as follows: (1) The origins of descending brainstem-spinal projections i n adult ducks and geese were similar to those described for the pigeon (Wild et a l . , 1979; Cabot et a l . , 1982), hen (Wold, 1978), h a t c h l i n g chicken (Gross and Oppenheim, 1985; Nelson and Steeves, unpublished observations), and mammals (Kuypers and Maisky, 1975; Crutcher et a l . , 1978; Kneisley et a l . , 1978; Martin et a l . , 1979, 1981a; Zemlan et a l . , 1984); (2) there i s an apparent topographical organization of the medullary-pontine r e t i c u l a r formation, not previously reported i n birds; and (3) there i s no evidence for direc t telencephalospinal pathways i n the avian species examined. This discussion commences with a b r i e f comparison of the present r e s u l t s with those of previous s t u d i e s on b i r d s , e s p e c i a l l y where there appears to be c o n f l i c t i n g data. The results are then discussed i n r e l a t i o n to previously i d e n t i f i e d locomotor regions and the descending control of spinal cord motor networks. When compared with previous studies i n birds,- there were a few differences i n the s i t e s of o r i g i n of descending spinal i n p u t s . In the duck and goose, no evidence was found f o r a p r o j e c t i o n from the v e n t r o l a t e r a l t h a l a m i c n u c l e u s t o the c e r v i c a l cord as reported f o r the pigeon (Cabot et a l . , 1982). Nor were s p i n a l p r o j e c t i o n s observed from the l a t e r a l mammillary nucleus, or the nucleus of the l a t e r a l t u b e r c l e , as reported f o r the chick (Okada and Oppenheim, 1985/ Gross and Oppenheim, 1985). However, Gross and Oppenheim (1985) have suggested t h a t the s p i n a l p r o j e c t i o n from the l a t e r a l t u b e r c l e may be a t r a n s i e n t one, prominent i n the chick embryo (Okada and Oppenheim, 1985), yet sparse i n the h a t c h l i n g chick (Gross and Oppenheim, 1985) . I t i s a l s o p o s s i b l e t h a t t h e s e d i s c r e p a n c i e s may be due to d i f f e r e n c e s i n the age and species of animal st u d i e d , and/or t o experimental methodology. A major focus o f t h i s study was t o determine which b r a i n s t e m - s p i n a l pathways descend t o the lumbar c o r d . The p r e s e n t r e s u l t s demonstrated a few p r o j e c t i o n s ( p r i m a r i l y c o n t r a l a t e r a l ) to the lumbar s p i n a l cord from the r o s t r a l nTS, i n c l o s e p r o x i m i t y t o N X. A d i s t i n c t group of r e t r o g r a d e l y l a b e l l e d neurones v e n t r a l t o X a l s o p r o j e c t e d t o the lumbar l e v e l . Gross and Oppenheim (1985) termed t h i s the n u c l e u s a l a t u s (Ala) i n the c h i c k . The term A l a i s used i n t h i s t h e s i s to d escribe the c e l l s v e n t r a l t o X (note t h a t the nucleus a l a t u s i n the r a t r e f e r s t o the raphe-Rmc complex, Watkins et a l . , 1980). P r e v i o u s a v i a n s t u d i e s have o n l y r e p o r t e d nTS f i b r e s descending as f a r as the t h o r a c i c cord (Smolen et a l . , 1979; Wold, 1978), whereas s t u d i e s i n the cat have demonstrated a few b i l a t e r a l nTS p r o j e c t i o n s to the lumbar cord (Kuypers and Maisky, 1975; Loewy and Burton, 1978). The l a b e l l i n g i n the nTS, i n the present study, suggested a t o p o g r a p h i c a l organization, with lumbar projecting neurones confined to the r o s t r a l nTS and c e r v i c a l p r o j e c t i n g neurones d i s t r i b u t e d throughout the entire rostrocaudal extent of nTS. In the cat, however, the converse has been reported with lumbar projections emanating from the caudal nTS (Loewy and Burton, 1978). Projections from VeD have also been reported to descend only as far as the thoracic cord of adult hens and pigeons (Wold, 1978) . However, the results of t h i s study confirm the more recent finding i n the hatchling chicken that, although few, the VeD does send projections to lumbar levels (Gross and Oppenheim, 1985). Furthermore, the present study also suggests that t h i s lumbar projection i s c o n t r a l a t e r a l . These results are consistent with those reported i n the cat that the mammalian equivalent of the VeD, the i n f e r i o r vestibular nucleus, has lumbosacral projections (Peterson and Coulter, 1977; Hayes and Rustioni, 1981), although they were reported to be b i l a t e r a l projections. In the present study, the VeM was defined as an area forming the v e n t r o l a t e r a l walls of the fourth v e n t r i c l e (Figs. 2D,E). From t h i s region, i n the duck and goose, projections were found to the i p s i l a t e r a l c e r v i c a l cord but not to the lumbar cord. However, Gross and Oppenheim (1985) have described a lumbospinal projection from the l a t e r a l border of the VeM (the submedial nucleus) i n the chick. Previous studies have not s p e c i f i e d whether a l l medullary-pontine r e t i c u l o s p i n a l pathways project as far as the lumbar cord. In addition to confirming that the Rpc has, at most, a very sparse projection to lumbosacral levels (Cabot et a l . , 1982), t h i s study has also demonstrated that two other regions of the pontomedullary r e t i c u l a r formation do not descend to lumbar spinal l e v e l s . The f i r s t region consists of a b i l a t e r a l group of small-diameter neurones within the Cnv, dorsal and l a t e r a l to SSP. The second b i l a t e r a l projection, r e s t r i c t e d to the c e r v i c a l cord, originates from the dorsal portion of the caudal avian RP at the l e v e l of the medullary-pontine r e t i c u l a r formation. Anatomically, studies i n the opossum (Martin et a l . , 1981a) and r a t (Zemlan et a l . , 1984) have demonstrated a pr e f e r e n t i a l b i l a t e r a l projection from the dorsal part of the Rgc (which appears to be equivalent to the dorsal aspect of the caudal RP i n birds) to the c e r v i c a l spinal cord. In t u r t l e s , the equivalent region has also been shown to project only to the c e r v i c a l cord, but primarily to the contra l a t e r a l side (Newman et a l . , 1983). Thus the o r g a n i z a t i o n of the r e t i c u l o s p i n a l projections i n the b i r d appear to be sim i l a r to those of mammals at the medullary-pontine junction. At t h i s l e v e l i n mammals, a topographic organization of the r e t i c u l a r formation has been p r e v i o u s l y demonstrated p h y s i o l o g i c a l l y (Peterson et a l . , 1975; Peterson, 1979). Zemlan and coworkers (1984) have suggested that the dorsal d i v i s i o n of the Rgc may be primarily involved i n forelimb motor control. With regard to functionally defined locomotor regions of the avian brainstem (Steeves et a l . , 1987), the present study 32 has c o n f i r m e d t h e e x t e n t o f p r o j e c t i o n s f r o m t h r e e a v i a n r e t i c u l a r f o r m a t i o n r e g i o n s t o t h e s p i n a l c o r d . F i r s t , as n o t e d i n t h e p r e s e n t s t u d y and t h a t o f Gross and Oppenheim (1985) , t h e v e n t r a l p a r t o f t h e m e d u l l a r y Rgc, i n b i r d s and i n mammals (Kuypers and Ma i s k y , 1975; M a r t i n e t a l . , 1979, 1981a; Zemlan e t a l . , 1984; J o n e s and Yang, 1985) has a p r e d o m i n a n t l y i p s i l a t e r a l s p i n a l p r o j e c t i o n t h a t descends t h e l e n g t h o f t h e c o r d . E l e c t r o p h y s i o l o g i c a l and a n a t o m i c a l s t u d i e s have s u g g e s t e d t h a t t h e Rgc p l a y s a prominent r o l e i n t h e i n i t i a t i o n o f l o c o m o t i o n i n a l l v e r t e b r a t 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 e t a l . , 1979), t e l e o s t f i s h ( K a s h i n e t a l . , 1974), t u r t l e (Kazennikov e t a l . , 1981), c a t ( O r l o v s k y , 1970a,b; M o r i e t a l . , 1978; Shimamura and K o g u r e , 1983; G a r c i a - R i l l and S k i n n e r , 1987a) and b i r d s (Steeves and W e i n s t e i n , 1984; Steeves e t a l . , 1987). Second, t h e s e r e s u l t s have c o n f i r m e d t h e f i n d i n g o f Cabot e t a l . (1982) o f a modest b i l a t e r a l p r o j e c t i o n t o t h e c e r v i c a l c o r d from t h e Rpc but l i t t l e o r none t o t h e lumbar c o r d . These a n a t o m i c a l f i n d i n g s a re s i m i l a r t o t h o s e n o t e d f o r mammals (Kuypers and Ma i s k y , 1975; M a r t i n e t a l . , 1979; Tohyama e t a l . , 1979a). The l a t e r a l r e t i c u l a r f o r m a t i o n , w i t h i n t h e v i c i n i t y o f t h e Rpc has been i m p l i c a t e d i n t h e i n i t i a t i o n o f l o c o m o t i o n i n b i r d s (Steeves e t a l . , 1986, 1987), t u r t l e (Kazennikov e t a l . , 1981) and mammals (Shik and Y a g o d n i t s y n , 1977). The loc o m o t o r r o l e o f Rpc i n b i r d s , t h e r e f o r e , may be t o a c t as a r e l a y , f o r m i n g p a r t o f a column o f i n t e r - c o n n e c t e d neurones d e s c e n d i n g t o more c a u d a l s p i n a l l e v e l s ( S e l i o n o v and S h i k , 1984). I t s 33 influence may also be mediated v i a projections to ventromedial r e t i c u l o s p i n a l neurones, as shown i n mammals (Noga et a l . , 1988). Although propriospinal neurones have been implicated as an es s e n t i a l component of t h i s functional pathway (Grant et a l . , 1980; Selionov and Shik, 1984), they were not examined i n the present study. Third, the present study also confirms the findings of Cabot et a l . (1982) that f i b r e s from the ICo and FRL only project to the c e r v i c a l cord. Functionally, and to some degree anatomically, the ICo, together with the FRL, may be the avian eq u i v a l e n t of the mammalian cuneiform nucleus (Reiner and Karten, 1982) . Preliminary data indicate that stimulation of the ICo-FRL region w i l l evoke locomotion i n birds (Sholomenko and Steeves, i n preparation). In mammals, the mesencephalic locomotor region (MLR) , which i n part i s comprised of the caudal cuneiform nucleus (Steeves and Jordan, 1984) or the pedunculopontine nucleus ( G a r c i a - R i l l , 1983; G a r c i a - R i l l and Skinner, 1987b), appears to d i r e c t l y activate the Rgc, but does not project d i r e c t l y to the spinal cord. However, some studies report that the anatomically d e f i n e d cuneiform nucleus, including a more r o s t r a l area, does project to the c e r v i c a l cord ( C a s t i g l i o n i et a l . , 1978; Tohyama et a l . , 1979a; Martin et a l . , 1981a). Whether a comparable organization exists i n b i r d s , with the locomotor region of the avian FRL being anatomically d i s t i n c t from the spinal projecting region, has yet to be determined. In summary, t h i s study has confirmed and extended the d e s c r i p t i o n of the o r i g i n s of descending b r a i n s t e m - s p i n a l projections i n the adult bird , beyond those previously reported for the pigeon (Cabot et a l . , 1982). Furthermore, the present study has c l e a r l y d e l i n e a t e d those pathways that descend d i r e c t l y to the lumbar cord. With few exceptions, they are simi l a r to those reported for the hatchling chicken (Gross and Oppenheim, 1985). The brainstem-spinal pathways i n birds and mammals are remarkably si m i l a r i n o r i g i n and extent, including some lim i t e d topographical representations. In addition to the vestibular system, the r e t i c u l o s p i n a l system also has a limi t e d topographical organization i n birds and mammals. F i n a l l y , t h i s study has v e r i f i e d that there are no di r e c t telencephalospinal pathways i n ducks and geese. The next question to be addressed i s whether the descending pathways to the lumbar spinal cord i n a b i r d with greater hindlimb motor control are sim i l a r to those that have been described above. 35 I l l ORIGINS OF BRAINSTEM-SPINAL PROJECTIONS IN PARROTS 36 I N T R O D U C T I O N The c e l l u l a r o r i g i n s of descending pathways t o the s p i n a l cord have been described f o r the lamprey (Ronan, 1981), f i s h (Kimmel et a l . , 1982), amphibians (ten Donkelaar, 1982), r e p t i l e s (ten Donkelaar et a l . , 1980), b i r d s (Wild et a l . , 1979; Cabot et a l . , 1982; Gross and Oppenheim, 1985; Okada and Oppenheim, 1985; Webster and Steeves, 1988), and mammals (Kuypers and Maisky, 1975; Crutcher et a l . , 1978; Kn e i s l e y et a l . , 1978; Martin et a l . , 1979; Zemlan and P f a f f , 1979; Hayes and R u s t i o n i , 1981; Nudo and Masterton, 1988) There are major d i f f e r e n c e s between mammals and non-mammalian v e r t e b r a t e species, e s p e c i a l l y w i t h regard t o the o r i g i n s of descending su p r a s p i n a l p r o j e c t i o n s t h a t i n f l u e n c e d i s t a l limb muscles. In mammals, these pathways c o n s i s t of the r u b r o s p i n a l t r a c t and the c o r t i c o s p i n a l t r a c t (Lawrence and Kuypers, 1968a,b). The r u b r o s p i n a l t r a c t , which has developed d u r i n g e v o l u t i o n (Nudo and Masterton, 1988; ten Donkelaar, 1988), shows greater somatotopic o r g a n i z a t i o n i n mammalian species such as the monkey ( C a s t i g l i o n i et a l . , 1978; L a r s e n and Yumiya, 1980) and cat (Pompeiano and Brodal, 1957) , than the opossum ( M a r t i n et a l . , 1974, 1981b). F u n c t i o n a l l y t he r u b r o s p i n a l pathway has been found to i n f l u e n c e groups of w r i s t and f i n g e r muscles (Lawrence and Kuypers, 1968b; Houk et a l 1988), and d i r e c t motoneuronal connections are present at the c e r v i c a l (C8-T1) l e v e l i n the monkey and cat (Robinson et a l . , 1987; Holstege, 1987; McCurdy et a l . , 1987). Thus although both the opossum and cat grasp by a p p o s i t i o n of both fo r e l i m b s , the 37 cat exhibits fine control of the wrist. The c o r t i c o s p i n a l t r a c t i s unique to mammals and i t i s most developed i n species that d i s p l a y d i g i t a l d e x t e r i t y (Heffner and Masterton, 1975) . Primates (Petras, 1969; for reviews see Armand, 1982; Porter, 1985) and certain dexterous non-primates, such as the raccoon (Petras and Lehman, 1966; Buxton and Goodman, 1967; Petras, 1969; Wirth et a l . , 1974), possess d i r e c t corticomotoneuronal connections, whereas the cat does not (McCurdy et a l . , 1987). Combined recording and l e s i o n s t u d i e s i n primates have demonstrated that c o r t i c o s p i n a l neurones control i n d i v i d u a l finger movements, while rubrospinal neurones p r e f e n t i a l l y c o n t r o l coordinated hand movements, associated with reaching and grasping (Houk et a l , 1988) . In contrast, birds have neither a somatotopic organization of the rubrospinal t r a c t (Wild et a l . , 197 9; Cabot et a l . , 1982; Gross and Oppenheim, 1985; Webster and Steeves, 1988) nor a c o r t i c o s p i n a l t r a c t (Gross and Oppenheim, 1985; Webster and Steeves, 1988). However some avian species, notably the parrot and owl, which depict the extremes of two divergent l i n e s of avian brain evolution (Portmann and Stingelin, 1961), possess telencephalic efferents that have been reported to project to the c e r v i c a l spinal cord (Kalisner, c i t e d by Pearson, 1972; Zecha, 1964; Verhaart, 1971; Karten, 1971). In both parrots and owls, the basal branch of the septo-mesencephalic t r a c t (TSM), emerging from the d o r s a l telencephalon, has been' reported to descend to the r o s t r a l c e r v i c a l cord (Zecha, 1964; Verhaart, 1971; Karten, 1971) . The 38 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 (OM), which o r i g i n a t e s i n the a r c h i s t r i a t u m , has a l s o been r e p o r t e d t o t e r m i n a t e i n the c e r v i c a l cord of the pa r r o t and parakeet (Verhaart, 1971). In a d d i t i o n t o species d i f f e r e n c e s i n the r o s t r o c a u d a l extent of these pathways, f u n c t i o n a l d i f f e r e n c e s have a l s o been reported. C o r t i c a l and a r c h i s t r i a t a l l e s i o n s r e s u l t i n more profound d e f i c i t s i n p a r r o t s ( K a l i s h e r , c i t e d by Benowitz, 1980; P h i l l i p s , 1968) than p i g e o n s , ducks ( P h i l l i p s , 1964) or chickens (Akerman et a l . , 1962) and incl u d e l e g weakness and slower recovery of locomotor f u n c t i o n ( P h i l l i p s , 1968). While both p a r r o t s and owls have a f o o t s t r u c t u r e (zygodactyl and semizygodactyl r e s p e c t i v e l y ) , which f a c i l i t a t e s grasp (Raikow, 1985) , the pa r r o t appears to be p a r t i c u l a r l y dexterous and some species have been observed to e x h i b i t a s i g n i f i c a n t dominant "footedness" (Friedmann and Davis, 1938; Rogers, 1980) . One of these, the " l e f t - f o o t e d " Sulphur-crested cockatoo (Cacatua galerita) was t h e r e f o r e s e l e c t e d f o r t h i s study. The s p e c i f i c o b j e c t i v e s were t o describe the o r i g i n s of descending p r o j e c t i o n s t o the lumbar cord i n a dexterous p a r r o t and compare them t o the descending pathways p r e v i o u s l y described f o r non-dexterous avian species (Cabot et a l . , 1982; Wild et a l . , 1979; Webster and Steeves, 1988). P r e l i m i n a r y data have been reported (Webster and Steeves, 1987) . MATERIALS AND METHODS S i x adult sulphur-crested cockatoos, of both sexes, were used f o r t h i s s t u d y . In a d d i t i o n , one E a s t e r n r o s e l l a (Platycercus eximius) was used f o r comparative purposes. Each 39 b i r d was anaesthetised with an intramuscular i n j e c t i o n of Ketamine (30 mg/Kg) plus Rompun (10 mg/Kg) i n a r a t i o of 2:1. The surgical procedures, preceeded by i n f i l t r a t i o n of a l o c a l anaesthetic, were as described previously i n Chapter II of t h i s thesis and by Webster and Steeves (1988). E s s e n t i a l l y 2-5 u l of True Blue (TB, 5%) or wheat germ agglutinin-horseradish peroxidase (WGA-HRP, 2%) was injected into each side of the lumbar (LI) spinal cord i n 3 birds. U n i l a t e r a l injections were also attempted at the LI cord l e v e l (2 birds) and at the high c e r v i c a l (Cl-2) l e v e l (1 b i r d ) , however, i n a l l cases the u n i l a t e r a l i n j e c t i o n spread i n t o the opposite side of the spinal cord. The survival time ranged from 8-10 days afte r a lumbar cord i n j e c t i o n of TB, and from 36-72 hours after an i n j e c t i o n of WGA-HRP. Under deep anaesthesia (sodium pentobarbital, 75 mg/kg, IP), the birds were perfused with warm saline (0.9%), followed by cold 4% paraformaldehyde i n 0.1 M phosphate buffer (pH 7.4). The birds injected with WGA-HRP were subsequently perfused with 10% sucrose i n 0.1 M phosphate buffer and after removal, the brain and injected segments were immersed i n a 30% sucrose solution. The brain and i n j e c t i o n s i t e of TB injected birds were post-fixed for a further 12 hours and then immersed i n a cryoprotectant (25% sucrose, 10% glycerol i n 0.05 M phosphate buffer) for 48 hours. The telencephalon and brainstem were sectioned i n the transverse and/or longitudinal plane, at 40 um thickness, on a freezing microtome. Alternate TB sections were mounted from 40 0.01 M T r i s buffer onto g e l a t i n i z e d s l i d e s , a i r dried i n the dark and examined under epifluorescence, using an A2 f i l t e r block ( e x c i t a t i o n , 270-380 nm; suppression, 410-580 nm) . Sections of representative l e v e l s of the neuraxis were l a t e r s t a i n e d with t h i o n i n e , using a m o d i f i c a t i o n that allowed simultaneous d i f f e r e n t i a l s t a i n i n g of f i b r e s and c e l l s ( T o l i v i a and T o l i v i a , 1985). The WGA-HRP sections were co l l e c t e d i n s e r i a l order i n mesh trays containing 0.1 M phosphate buffer and then reacted with tetramethylbenzidene (TMB; Mesusalem, 1978) using a modified procedure (Gibson et a l . , 1984). This consisted of an i n i t i a l prewash i n acetate buffer (pH 3.3, 15 minutes) followed by c y c l i c a l immersion, for 2 minutes, i n each of the reaction solutions (Solution 1: 20 ml of acetate buffer, pH 3.3, i n 280 ml d i s t i l l e d water. Solution 2: 10 mg of TMB dissolved i n 5 ml of 100% ethanol, sonicated for 5 minutes; 2 ml of 3% hydrogen peroxide, 20 ml acetate buffer and 375 ml d i s t i l l e d water. Solution 3: 0.2 gm sodium n i t r o f e r r i c y a n i d e , 20 ml acetate buffer, and 380 ml d i s t i l l e d water) . The entire cycle was repeated ten times. Solution 3 was f i l t e r e d a f t e r each use, the f i l t e r paper was replaced a f t e r the t h i r d cycle and a l l the solutions were renewed afte r the f i f t h cycle. F i n a l l y , the sections were washed for a t o t a l of 45 minutes i n three changes of acetate buffer (pH 3.3) at 4°C. The sections were immediately mounted onto g e l a t i n i z e d s l i d e s and a i r dried. Alternate sections were coverslipped and examined under d a r k f i e l d and b r i g h t f i e l d i l l u m i n a t i o n . Other 41 s e c t i o n s were subsequently counterstained w i t h n e u t r a l red (1% f o r 2.5 minutes). The l o c a t i o n s of r e t r o g r a d e l y l a b e l l e d somata were p l o t t e d on b r a i n s t e m s e c t i o n o u t l i n e s drawn at approximately 240 um i n t e r v a l s , photographed and i d e n t i f i e d w i t h reference to the a t l a s of Karten and Hodos (1967). RESULTS Lumbar I n j e c t i o n Comparable r e s u l t s were found i n the three Sulphur-crested cockatoos and the s i n g l e Eastern r o s e l l a that were i n j e c t e d w i t h TB. Since a l l of the u n i l a t e r a l i n j e c t i o n s r e s u l t e d i n d i f f u s i o n of TB i n t o the opposite side of the s p i n a l cord, the r e s u l t s were i n c o n c l u s i v e regarding l a t e r a l i t y of p r o j e c t i o n s . T h e r e f o r e o n l y the r e s u l t s of b i l a t e r a l i n j e c t i o n s are presented as they i l l u s t r a t e the maximum l a b e l l i n g obtained from the high lumbar regi o n . The p a r a s a g i t t a l s e c t i o n s depicted i n Figure 5 i l l u s t a t e that retrograde T B - l a b e l l e d somata w i t h i n the brainstem were l o c a t e d p r i n c i p a l l y w i t h i n three regions: l a t e r a l l y , w i t h i n the l a t e r a l v e s t i b u l a r nucleus (VeL, F i g s . 5A,B, 6A,B); c e n t r a l l y , w i t h i n the pontomedullary r e t i c u l a r formation (Figs. 5B,C, 6C,D); and r o s t r a l l y , w i t h i n the red nucleus (Ru, F i g . 5C). The l o c a t i o n of l a b e l l e d c e l l s w i t h i n s p e c i f i c n u c l e i were more e a s i l y i d e n t i f i e d i n t r a n s v e r s e s e c t i o n s . Thus s e c t i o n s of the c o n t r a l a t e r a l side of the brainstem from the same b i r d depicted i n Figures 5A-C, are shown i n a caudal t o r o s t r a l sequence i n Figures 7A-L and are described below. A l l c e l l counts t h e r e f o r e r e f e r to l a b e l l e d c e l l s / s e c t i o n / s i d e . C e l l s i z e i n d i c a t e s maximum soma diameter. 42 F i g u r e 5 . Diagrams A-C represent p a r a - s a g i t t a l brainstem s e c t i o n s i n l a t e r a l to medial sequence, summarizing the d i s t r i b u t i o n of r e t r o g r a d e l y l a b e l l e d neurones, a f t e r a b i l a t e r a l i n j e c t i o n of TB into the high lumbar spinal cord. Each f i l l e d c i r c l e (•) represents 5 TB l a b e l l e d c e l l s . 43 Figure 6. Photomicrographs of TB-labelled neurones i n para-s a g i t t a l sections following a high lumbar i n j e c t i o n . A: Lateral vestibular nucleus (VeLv), ventral component. B: VeLd, dorsal aspect. C: Nucleus c e n t r a l i s d o r s a l i s (Cnd). D: Nucleus r e t i c u l a r i s pontis caudalis (RP). Scale bars: 50 um. 4 4 Figure 7 . The d i s t r i b u t i o n of T B - l a b e l l e d somata, a f t e r a h i g h lumbar i n j e c t i o n , shown i n t r a n s v e r s e s e c t i o n s . The diagrams A-L are i n caudal t o r o s t r a l sequence and represent the other h a l f of the brainstem of the p a r r o t shown i n F i g . 5 . Each f i l l e d c i r c l e (•) r e p r e s e n t s 5 T B - l a b e l l e d c e l l s f o l l o w i n g a h i g h lumbar i n j e c t i o n . In a d d i t i o n , each open t r i a n g l e (A) represents a region t h a t was only l a b e l l e d a f t e r a TB i n j e c t i o n i n t o the C l - 2 l e v e l of the s p i n a l cord (TB-l a b e l l e d c e l l s found w i t h i n t he c o n t r a l a t e r a l i n t e r n a l c e r e b e l l a r nucleus a f t e r a C l - 2 i n j e c t i o n are not i l l u s t r a t e d ) . 45 46 47 F i g u r e 8. Photomicrographs of r e t r o g r a d e l y T B - l a b e l l e d neurones i n transverse section aft e r a high lumbar i n j e c t i o n . A : Nucleus c e n t r a l i s d o r s a l i s of the medulla (Cnd). B: Sub-t r i g e m i n a l nucleus .(ST). C: Nucleus r e t i c u l a r i s giganto-c e l l u l a r i s (Rgc). D: L a t e r a l v e s t i b u l a r nucleus, v e n t r a l component (VeLv). E: Ventral subcoeruleus nucleus (Scv). F: Red nucleus (Ru). Arrows depict the v e n t r o l a t e r a l margin of the medulla and N III i n (B) and (F), respectively. Scale bars: 25 um (A and B); 50 um i n a l l other figures. 48 49 Caudal to the obex, large TB-labelled neurones (30-40 um) were found w i t h i n the nucleus c e n t r a l i s m e d u l l a r i s , pars d o r s a l i s (Cnd). These c e l l s , which were o r i e n t a t e d i n a vent r o l a t e r a l to dorsomedial d i r e c t i o n , formed a compact group (10-20 c e l l s / s e c t i o n ) that extended over a r o s t r o c a u d a l distance of 2.0 mm (Figs. 7A,B, 8A) . In contrast, only a few l a b e l l e d somata were found w i t h i n the nucleus c e n t r a l i s v e n t r a l i s (Cnv, F i g . 7B). Approximately 5 l a b e l l e d c e l l s / s e c t i o n were found w i t h i n the nucleus r e t i c u l a r i s l a t e r a l i s (RL, F i g . 7B). In addition, a cl u s t e r of medium sized l a b e l l e d c e l l s (5-7 cells/section) was found at the dorsal edge of the RL, bordering the t r a c t of the trigeminal nucleus. These c e l l s appeared to be i n the subtrigeminal nucleus (ST, Figs. 7C,D, 8B) . In the dorsal region of the medulla, 5-10 retrogradely l a b e l l e d neurones were located ventral to the dorsal nucleus of the vagus (X), i n the nucleus of the s o l i t a r y t r a c t (nTS), and along the course of the vagus nerve (Figs. 7A,B) . Within the r o s t r a l medulla, a few large (30-35 um) l a b e l l e d neurones were l o c a t e d w i t h i n the nucleus para-g i g a n t o c e l l u l a r i s l a t e r a l i s (PGL, F i g . 7C) . Numerous (15-25/section) larger neurones (35-40 um) were l a b e l l e d within the 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), over a distance of approximately 2 mm (Figs. 7C-E, 8C) . Along the midline, there were only a few l a b e l l e d c e l l s within the raphe obscurus, whereas there were 10-20 l a b e l l e d c e l l s / s e c t i o n within the raphe p a l l i d u s and raphe magnus (Figs. 7B-F). 50 Numerous retrogradely l a b e l l e d neurones (15-30/section) were present within the dorsal (VeLd) and ventral subdivision (VeLv) of the l a t e r a l vestibular nucleus (Figs. 7E,F, 8D). In contrast, only a few l a b e l l e d neurones were found within the descending vestibular nucleus (VeD, F i g . 7D). In the pons, l a b e l l e d c e l l s (20-30/section) were found within the nucleus pontis caudalis (RP, Figs. 7F,G), and the nucleus r e t i c u l a r i s pontis caudalis, pars g i g a n t o c e l l u l a r i s (RPgc, F i g . 7H,I), but comparatively few (5 i n total) were located within the more r o s t r a l l y situated r e t i c u l a r i s pontis o r a l i s (RPO). Neurones (30-35 um) were also l a b e l l e d within the locus coeruleus (LoC), dorsal subcoeruleus (Scd, Figs. 7H,I), and ventral subcoeruleus nuclei (Scv, Figs. 7G-I, 8E). At the l e v e l of the mesencephalon, l a b e l l e d neurones (20-30 um, 5/section) were found within the i n t e r s t i t i a l nucleus (IS) and l a t e r a l l y w i t h i n the nucleus of the p o s t e r i o r commissure (PC) (Fig. 7J) . Large and medium sized TB-labelled c e l l s (30-45 um) were present i n the d o r s o l a t e r a l , and ventr o l a t e r a l parts of the caudal red nucleus (Ru), and more r o s t r a l l y within the dorsomedial region of the Ru. There were 10-50 l a b e l l e d neurones/section (Figs. 7J, 8F) depending upon the locus of i n j e c t i o n . Within the diencephalon, l a b e l l e d c e l l s were found within the l a t e r a l hypothalamic region (LH, F i g . 7K) , the nucleus p e r i v e n t r i c u l a r i s hypothalami (PVH), and nucleus para-v e n t r i c u l a r i s (PVM, F i g . 7L) . There were no TB-labelled c e l l s within the telencephalon. 51 The d i s t r i b u t i o n of l a b e l l e d c e l l s following a WGA-HRP lumbar i n j e c t i o n was simi l a r to the d i s t r i b u t i o n following a TB in j e c t i o n described above. Since neither i n j e c t i o n was e n t i r e l y u n i l a t e r a l , no further d e t a i l regarding l a t e r a l i t y of pathways was obtained; however examples of WGA-HRP reactive neurones within the contral a t e r a l Scv (Fig. 9A) and Ru (Fig. 9B) are shown. L a b e l l e d c e l l s were present w i t h i n the medial and l a t e r a l regions of the Ru after lumbar i n j e c t i o n s . Cervical Injection The same regions of the brainstem were l a b e l l e d following i n j e c t i o n of TB at the Cl-2 l e v e l , as those l a b e l l e d after lumbar i n j e c t i o n s . Numerous l a b e l l e d neurones were found i n the raphe magnus (Rm, F i g . 10A), LoC, Scd, Scv, PVM (Fig. 10B) and Ru (Fig. IOC). However, fewer l a b e l l e d c e l l s were found i n the r e t i c u l a r formation and v e s t i b u l a r n u c l e i . Since t h e i r p r o j e c t i o n s . course through the ventromedial and ventr o l a t e r a l f u n i c u l i (Cabot et a l . , 1982; Chapter IV of t h i s t h e s i s ) , t h i s diminished l a b e l l i n g was probably a consequence of the dorsal location of the i n j e c t i o n s i t e . Regions that were l a b e l l e d after the c e r v i c a l but not lumbar injections (Fig. 7, open triangles) included the nucleus p a r v o c e l l u l a r i s (Rpc), dorsal part of the Cnv, TTD, medial vestibular nucleus (VeM) and int e r n a l cerebellar nucleus (Cbl). Both the medial and l a t e r a l regions of the Ru were l a b e l l e d after a c e r v i c a l i n j e c t i o n (Fig. IOC). There were no l a b e l l e d c e l l s within the telencephalon after high c e r v i c a l i n j e c t i o n . 52 F i g u r e 9. Photomicrographs of WGA-HRP r e a c t i v e neurones ( i n d i c a t e d by arrows) f o l l o w i n g a high lumbar i n j e c t i o n i n the cockatoo. A: V e n t r a l subcoeruleus nucleus (Scv); B: Red nucleus (Ru) . Scale bars: 50 um. O r i e n t a t i o n : d o r s a l (d) , medial (m) , a p p l i e s to both A and B. 53 Nlll Figure 10. Photomicrographs of r e t r o g r a d e l y l a b e l l e d neurones f o l l o w i n g a TB i n j e c t i o n at C l - 2 i n the cockatoo. A: Raphe magnus (Rm). B: Nucleus p a r a v e n t r i c u l a r i s (PVM). C: Red nucleus (Ru). Scale bars: 50 um. 54 DISCUSSION The major finding of t h i s study was that the origins of descending braihstem-spinal projections i n adult parrots were simi l a r to those previously described for the pigeon (Wild et a l . , 1979; Cabot et a l . , 1982), hatchling chick (Gross and Oppenheim, 1985), duck, and goose (Webster and Steeves, 1988). No novel projections to the high lumbar spinal cord were found that might account for the enhanced a b i l i t y of parrots or cockatoos to grasp objects with t h e i r feet. According to Kuypers (1964, 1982), the descending motor pathways i n mammals form two f u n c t i o n a l groups; a "medial system" (the vestibular and r e t i c u l o s p i n a l pathways) and a " l a t e r a l system" (the rubrospinal and c o r t i c o s p i n a l tracts) that control the motoneurones of a x i a l and d i s t a l muscles, respectively. Although such a model i s r e l a t i v e l y s i m p l i s t i c , i t serves as a useful basis for discussion. The medial system, phylogenetically (ten Donkelaar, 1982) and developmentally (ten Donkelaar, 1982; Martin et a l . , 1982; Okada and Oppenheim, 1985) older than the l a t e r a l system, i s present i n a l l vertebrate species examined. S i m i l a r i t i e s i n origins, rostrocaudal extent of projections to the spinal cord, and somatotopic o r g a n i z a t i o n of these pathways have been reported i n several vertebrate species (for review see, Kuypers and Martin, 1982). D i f f e r e n t i a l projections to the c e r v i c a l or lumbar s p i n a l cord, as seen i n the present study, have p r e v i o u s l y been demonstrated f o r the v e s t i b u l o s p i n a l projections of the pigeon (Wold, 1978; Cabot et a l . , 1982), and 55 the r e t i c u l o s p i n a l projections of the duck and goose (Webster and Steeves, 1988). The l a t e r a l system i s only represented i n the b i r d by the rubrospinal t r a c t . In contrast to placental mammals, there i s no somatotopic organization of the rubrospinal projections to the spinal cord i n the opossum (Martin et a l . , 1981a) nor i n b i r d s (Wild et a l . , 1979; Cabot et a l . , 1982; Gross and Oppenheim, 1985; Webster and Steeves, 1988). A s i m i l a r finding in the present study for the prehensile parrot was interesting, since enhanced development of the rubrospinal projections i s a s s o c i a t e d with i n c r e a s e d d i s t a l limb motor c o n t r o l , p a r t i c u l a r l y i n animals which lack corticomotoneuronal connections (Holstege, 1987). A small degree of c o l l a t e r a l -i z a t i o n of rubrospinal projections to the lumbar and c e r v i c a l enlargements i s also thought to contribute to improved d i s t a l motor control and has been shown to be lower i n the monkey and cat (Huisman et a l . , 1982) than i n the opossum (Martin et a l . , 1981b) or r a t (Huisman et a l . , 1981). Whether the p a r r o t exhibits a low degree of rubrospinal c o l l a t e r a l s or any d i r e c t motoneuronal connections at the lumbar l e v e l has not been ascertained. The development of the mammalian co r t i c o s p i n a l t r a c t i s a s s o c i a t e d with the a c q u i s i t i o n of dexterous motor s k i l l s (Heffner and Masterton, 1975, 1983). Interestingly, i t extends only to c e r v i c a l levels i n sheep (Bagley, 1922), to thoracic l e v e l s i n the opossum (B a u t i s t a and Matzke, 1965), and throughout the cord i n the rat (Brown, 1971), cat (Chambers and 56 Liu, 1957) and primates (Liu and Chambers, 1964). Independent of body size, the number of fibres and d i r e c t motoneuronal connections are other c h a r a c t e r i s t i c s a s s o c i a t e d with the development of d i g i t a l dexterity (Heffner and Masterton, 1975, 1983) . While d i r e c t corticomotoneuronal connections are present i n a l l primates (Heffner and Masterton, 1983; Armand, 1982), they have been found i n only a few non-primate mammals, such as the raccoon and kinkajou (Petras and Lehman, 1966) . This suggests that such connections have evolved independently together with d i g i t a l dexterity i n primates and carnivores (i. e . as a homoplasy), rather than as a shared mammalian c h a r a c t e r i s t i c ( i . e . homology) which has been l o s t i n most mammalian species. In the present study, no evidence was found for a d i r e c t projection to the c e r v i c a l or lumbar spinal cord from the telencephalon. Two previous s t u d i e s , using anterograde degeneration tr a c i n g techniques, have reported both medial and l a t e r a l telencephalic projections to the c e r v i c a l cord i n birds (Zecha, 1964; Karten, 1971). Zecha (1964), using Haggqvist and Nauta-Gygax methods i n the pigeon, parakeet and parrot, stated that a projection from the caudal telencephalon descending i n the dorsolateral tegmentum, reached the dorsolateral funiculus at r o s t r a l l e v e l s of the c e r v i c a l cord. He compared i t to the corticotegmental t r a c t noted i n sheep (Bagley, 1922), but did not specify i f i t was found i n a l l of the avian species studied. It i s i n t e r e s t i n g to note that the corticotegmental t r a c t terminates i n the dorsal column nuclei of the medulla i n 57 the goat (Haartsen and Verhaart, 1967). Verhaart (1971), using Zecha's material, re-traced Zecha's projection from the caudal telencephalon i n t o the v e n t r o l a t e r a l not the d o r s o l a t e r a l funiculus i n a parrot and named t h i s projection the o c c i p i t o -mesencephalic t r a c t (OM). Zecha (1964) also described a projection from the dorsal telencephalon i n the p a r r o t and parakeet that descended ventromedially, along the same course as the mammalian pyramid, and terminated c h i e f l y i n the c o n t r a l a t e r a l dorsal column. Verhaart (1971) termed t h i s pathway the basal branch of the TSM and traced i t as far as the CI ventral funiculus i n the parrot. Karten (1971) reported that i n the owl t h i s branch of the TSM decussated at the bulbospinal junction, continued caudally i n the dorsal funiculus of the c e r v i c a l spinal cord, and appeared comparable to a component of the mammalian pyramidal t r a c t . The present study f a i l e d to f i n d evidence f o r 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 with retrograde t r a c i n g techniques, even though the c e r v i c a l TB i n j e c t i o n encompassed the dorsal and dorsolateral regions of the spinal cord, where telencephalospinal fibres had been previously reported (Zecha, 1964; Karten, 1971) . Thus, the present study suggests that i f telencephalic projections to the c e r v i c a l cord exist i n the cockatoo, they must terminate at the CI l e v e l . This p o s s i b i l i t y needs to be investigated using an anterograde tracer such as phaleolis vulgaris-leucoagglutinin (PHA-L). Even i f they exist, the question remains regarding what e f f e c t a projection to the c e r v i c a l cord could have upon the parrot's prehensile a b i l i t y . 58 Whether increased numbers of projections to the lumbar cord can account for increased pedal dexterity of the cockatoo has not been determined. Although an attempt was made to quantify the source of descending projections i n t h i s study, the small number of birds, together with differences i n the locus of the i n j e c t i o n , r e s u l t e d i n some v a r i a b i l i t y and therefore these results have not been presented. For the same reasons, the u n i l a t e r a l injections were i n s u f f i c i e n t to provide information regarding l a t e r a l i t y of function that has been previously observed i n t h i s species (Rogers, 1980). While t h i s study has shown that the brainstem-spinal projections i n the parrot and cockatoo are s i m i l a r to those previously demonstrated i n webfooted birds, the foot structure o b v i o u s l y i s not. The p a r r o t has a zygodactyl foot and prehension i s f a c i l i t a t e d by a tendon arrangement that allows simultaneous f l e x i o n and extension of the paired opposing toes (Raikow, 1985). Whether the sensory system i s also s p e c i a l i z e d to enhance reaching and grasping with the foot awaits further study. The physiological function of s p e c i f i c pathways may also be d i f f e r e n t , or perhaps there may be d i f f e r e n c e s i n the r o s t r a l regions of the brain. The parrot, i n contrast to other birds, has an a b i l i t y to manipulate objects with i t s tongue and previous studies have shown that t h i s function i s apparently subserved by increased afferent input (Wild, 1981). Since brainstem-spinal projections to the lumbar spinal cord appear to be comparable i n the avian species examined, the next topic to be addressed i s t h e i r funicular organization. 59 IV FUNICULAR TRAJECTORIES 6 0 INTRODUCTION The spinal cord neuronal networks responsible for the di r e c t generation of locomotor patterns, require supraspinal input for locomotion to be i n i t i a t e d and sustained (for review see G r i l l n e r , 1981; Armstrong, 1986). In a v a r i e t y of vertebrate species, selective lesions of r e s t r i c t e d regions within the central nervous system have been used to i d e n t i f y the pathways providing t h i s descending drive. Lawrence and Kuypers (1968a,b) have c l e a r l y shown that the " l a t e r a l system", c o n s i s t i n g of the c o r t i c o s p i n a l and r u b r o s p i n a l t r a c t s (Kuypers, 1964), i s not e s s e n t i a l f o r locomotion; neither section of the c o r t i c o s p i n a l t r a c t s , nor the subsequent lesioning of the rubrospinal t r a c t s prevents normal overground locomotion i n monkeys. Similar results have been obtained following ablation of the somatosensory cortex i n the dog, raccoon (Buxton and Goodman, 1967) and cat (Eidelberg and Yu, 1981) or l e s i o n of the red nucleus i n the cat (Orlovsky, 1972). Furthermore, kinematic a n a l y s i s of the re s u l t i n g locomotor pattern showed only mild and transient changes f o l l o w i n g c o r t i c o s p i n a l t r a n s e c t i o n i n the cat (Eidelberg and Yu, 1981). Thus, while both c o r t i c o s p i n a l and rubrospinal projections may be necessary for modulation of the locomotor pattern (Wetzel and Stuart, 1976), t h e i r predominant role appears to be the control of d i s t a l limb musculature and d e f i c i t s i n hand function are the hallmark of t h e i r destruction (Lawrence and Kuypers, 1968a,b). In c o n t r a s t , l e s i o n s of the "medial system", the r e t i c u l o s p i n a l and vestibulospinal t r a c t s , produce profound d e f i c i t s i n locomotor f u n c t i o n (for review see E i d e l b e r g , 1981) . Extensive lesions, involving both r e t i c u l o s p i n a l and vestibulospinal projections, within either the medial medulla (Lawrence and Kuypers, 1968b), the v e n t r a l regions of the c e r v i c a l (Lawrence and Kuypers, 1968b; Steeves and Jordan, 1980) or thoracic spinal cord (Eidelberg et a l . , 1981b) resu l t in complete loss of locomotor function i n the monkey (Lawrence and Kuypers, 1968b), cat (Steeves and Jordan, 1980; Eidelberg et a l . , 1981a) and b i r d (Sholomenko and Steeves, 1987). Direct l e s i o n s of the v e s t i b u l a r n u c l e i produce only t r a n s i e n t impairment of locomotion, with some attenuation of extensor tone (Orlovsky, 1972; Yu and Eidelberg, 1981; J e l l et a l . , 1985), suggesting that the r e t i c u l o s p i n a l pathways are fundamental for the provision of descending locomotor drive. P r e s e r v a t i o n of p r o j e c t i o n s w i t h i n e i t h e r the ve n t r o l a t e r a l (VLF) (monkey: Eidelberg et a l . , 1981b; cat: Steeves and Jordan, 1980; E i d e l b e r g et a l . , 1981a); b i r d : Sholomenko and Steeves, 1987; lamprey: McClellan, 1988), or ventromedial (VMF) f u n i c u l i of the spinal cord (cat: A f e l t , 1974; monkey: Eidelberg et a l . , 1981a,b; b i r d : Sholomenko and Steeves, 1987) or the intermediate region of the l a t e r a l columns ( s t i n g r a y : Williams et a l . , 1984) appear to be suffi c e n t for the retention of locomotion. The funicular organization of brainstem-spinal pathways has been described for a variety of species at the c e r v i c a l (rat: Leichnetz et a l . , 1978; cat: Kuypers and Maisky, 1977; Tohyama et a l . , 1979a,b; pigeon: Cabot et a l , 1982) and/or 62 thoracolumbar lev e l s (opossum: Martin et a l . , 1979, 1981a; rat: Zemlan and Pfaff, 1979; Zemlan et a l . , 1984; Martin et a l . , 1985; S i r k i n and Feng, 1987; cat: Mitani et a l . , 1988; monkey: Carlton et a l . , 1985; t u r t l e , l i z a r d , snake: ten Donkelaar, 1976, 1982; Wolters et a l . , 1982; and pigeon: Wild et a l . , 1979) . Some pathways retain a consistent trajectory throughout t h e i r descent (e.g. rubrospinal), while other pathways undergo a gradual s h i f t i n location during t h e i r rostrocaudal descent down the spinal cord. For example the projections from the mammalian medullary r e t i c u l a r formation descend within the VMF and VLF at c e r v i c a l and thoracic levels and the dorsolateral funiculus (DLF) at lumbosacral levels of the cord (Petras, 1967; Holstege et a l . , 1969; Mitani et a l . , 1988). P r o j e c t i o n s to the lumbar cord of b i r d s have been described previously (Ch. II, also Gross and Oppenheim, 1985; Webster and Steeves, 1988) and in c l u d e the pontomedullary r e t i c u l o s p i n a l , raphespinal, vestibulospinal, coeruleospinal, rubrospinal, i n t e r s t i t i o s p i n a l and hypothalamospinal t r a c t s . The funicular t r a j e c t o r i e s of some brainstem-spinal projections have been described for the pigeon to the l e v e l of the b r a c h i a l enlargement (Cabot et a l . , 1982). Since only the p o s i t i o n of the r u b r o s p i n a l t r a c t of the pigeon has been described at lumbar lev e l s of the spinal cord (Wild et a l . , 197 9) , further study seemed warranted. The purpose of t h i s study was to determine the funicular t r a j e c t o r i e s of brainstem-spinal projections within the lumbar cord of the duck and goose and to i d e n t i f y those within the 63 l a t e r a l and ventral columns, i n the duck and goose, so that l i k e l y sources of pathways reported to be e s s e n t i a l for the i n i t i a t i o n of locomotion could be determined (Sholomenko and Steeves, 1987). Retrograde tracing i n conjunction with sub-total spinal cord lesions at a more r o s t r a l l e v e l of the cord i n order to r e s t r i c t transport to a s p e c i f i c funiculus was chosen as the most suitable technique to determine the funicular t r a j e c t o r i e s of a l l the descending brainstem-spinal projections. In addition to True Blue (TB) , other retrograde tracer dyes were used so that the results could be compared to previous data obtained for the duck and goose (Ch. II of t h i s thesis; Webster and Steeves, 1988) . These included Fast Blue (FB) (Bentivoglio et a l . , 1980), Diamidino Yellow Dihydrochloride (DY) (Keizer et a l . , 1983); and fluorescein conjugated dextran-amines (FDA) (Glover et a l . , 1986) which are a l l transported more quickly than TB. Although anterograde t r a c i n g has the advantage of delineating the course and termination of spinal pathways, each source would need a separate i n j e c t i o n . Furthermore, the e f f e c t i v e size of the i n j e c t i o n area i s d i f f i c u l t to control using t r i t i a t e d amino acids. While the i n j e c t i o n region and the neurones l a b e l l e d are c l e a r l y i d e n t i f i e d using the anterograde tracer phaseolus vulgaris leucoagglutinin (PHA-L) (Gerfen and Sawchenko, 1984) i t s uptake zone i s r e s t r i c t e d (less than 1 mm), and multiple injections would be required to label regions such as the r e t i c u l a r formation. 64 MATERIALS AND METHODS Twenty three b i r d s (11 white Pekin ducks, Anas platyrhynchos, and 12 Canada geese, Branta canadensis) , of both sexes, were used f o r t h i s study. The f i r s t s e r i e s of experiments employed a l o c a l i z e d i n j e c t i o n of a fluorescent tracer into either the DMF, DLF, VLF, or VMF at the l e v e l of the f i r s t lumbar segment (LI; n=5). One b i r d , which served as a control, was injected with 4 u l of FB into one side of the cord and 4 u l of DY into the other side. The second series of experiments involved a u n i l a t e r a l or b i l a t e r a l i n j e c t i o n (5-10 ul/side) combined with a more r o s t r a l s u b t o t a l l e s i o n of the t h o r a c i c cord (n=17). The s u b t o t a l lesions consisted of either: a transection of the dorsal h a l f of the cord, a hemisection, a section of the cord sparing only the VMF, a section of the cord sparing the only the VLF u n i l a t e r a l l y or b i l a t e r a l l y . In a few cases a d i f f e r e n t retrograde tracer was injected into each side of the spinal cord combined with b i l a t e r a l symmetrical or asymmetrical sub-t o t a l lesions. These experiments are l i s t e d as separate cases in Table I and examples from each group of experiments are i l l u s t r a t e d i n Figure 11. Under general anaesthesia (induced with 20% nitrous oxide and 4% Halothane i n 95% oxygen and 5% carbon dioxide, and maintained at a Halothane l e v e l of 2%), plus i n f i l t r a t a t i o n of a l o c a l anaesthetic, a p a r t i a l laminectomy was performed at the thoracolumbar j u n c t i o n i n the manner d e s c r i b e d p r e v i o u s l y (Chapter II of t h i s thesis; Webster and Steeves, 1988) . The caudal thoracic spinal cord was exposed, p a r t i a l l y lesioned, 65 using m i c r o s c i s s o r s and/or a hypodermic needle and then covered with Gelfoam. Subsequently the r o s t r a l region of the lumbar spinal cord (LI) was exposed and injected, on one or both sides, with a fluorescent retrograde tracer (TB, FB, DY, or FDA) . In a l l cases, the i n j e c t i o n s i t e was always 10 mm caudal to the l e v e l of the subtotal spinal cord l e s i o n . Post-operative, care included, administration of analgesics (Demerol, 0.2 ml/8 hourly) and a n t i b i o t i c s (ampicillin, 100 mg/day) for f i v e days. A l l birds were kept i n a heated room, with natural l i g h t i n g and fed ad l i b i t u m . Those unable to walk were kept i n an enclosed pen, so that strenuous movements were r e s t r i c t e d but water and food were fr e e l y accessible. When locomotor function returned, the enclosure was removed. The survival time varied with the tracer used and ranged from 5 to 43 days (Table 1). Each animal was placed under deep anaesthesia (sodium pentobarbital, 75 mg/kg, IP) and perfused t r a n s c a r d i a l l y with 1 l i t r e of saline (0.9%, pH 7.4) followed by 1 l i t r e of 4% paraformaldehyde i n 0.1 M phosphate buffer (pH 7.4). The brain and appropriate spinal cord segments were removed, postfixed for a further 12 hours, and then immersed in a cryoprotectant (25% sucrose, 10% g l y c e r o l i n 0.05 M phosphate buffer) for 48 hours. The le s i o n and i n j e c t i o n s i t e s were sectioned i n the transverse or longitudinal plane. The brainstem was sectioned transversely at 30 um thickness on a freezing microtome. Every t h i r d section was floated i n 0.2 T r i s buffer, mounted onto ge l a t i n i z e d s l i d e s , and a i r dried i n the dark. Each section was 6 6 examined under a L e i t z Orthoplan microscope with e p i -fluorescence using the appropriate f i l t e r s for TB, FB, DY, and FDA. The d i s t r i b u t i o n of l a b e l l e d c e l l s was drawn onto brain section outlines (0.27 mm i n t e r v a l s ) . When necessary, sections were stained with thionine to aid i d e n t i f i c a t i o n . RESULTS In the f i r s t group of experiments, the extent of the control (Fig. 11A) and funicular injections into the VMF, VLF (Fig. 11B) , or DLF, DMF (Fig. 11C) were r e s t r i c t e d to the appropriate regions of the cord. Representative i n j e c t i o n and l e s i o n s i t e s are shown in Figures 11D-H. The d i s t r i b u t i o n of l a b e l l e d neurones f o l l o w i n g the f u n i c u l a r i n j e c t i o n and funicular l e s i o n experiments (Figs. 12, 13) are presented i n Tables II and III, respectively. Since the results of the funicular i n j e c t i o n experiments and the subtotal lesion experiments complement each other, the combined data i s presented f o r each b r a i n s t e m - s p i n a l p r o j e c t i o n . F i n a l l y the descending p r o j e c t i o n s w i t h i n each funiculus are summarized and compared to previously published data (Fig. 14) . In the control b i r d (Fig. 11A, G49) the d i s t r i b u t i o n of FB-labelled c e l l s was the same as the d i s t r i b u t i o n of DY-l a b e l l e d c e l l s (Table I I ) . The numbers of FB and DY somata were als o s i m i l a r (-1000), i n d i c a t i n g that both t r a c e r s were r e t r o g r a d e l y t r a n s p o r t e d with equal e f f e c t i v e n e s s from the lumbar cord to the brainstem (approximately 50 cm) . It should be noted, however, that the number of l a b e l l e d neurones afte r a u n i l a t e r a l lumbar i n j e c t i o n would, be at l e a s t twice t h i s 67 TABLE I Summary of Lumbar Injection Sites, Subtotal Spinal Cord lies ions and Subsequent Locomotor Function in Ducks and Geese B i r d Tracer Injection ul/Side Lesion Survival Locomotor Site L R (days) Function G49 FB/DY Control 4 4 19 3 D42 TB VM 5 32 3 D61 TB VL 5 43 3 D 3 8 TB VL,DL 8 31 3 D51 DY DL 5 27 3 G43 FB DM 5 9 3 Intact Region D25 TB IL 10 CL-Hemi 15 2 D26 TB IL 10 CL-Hemi 20 2 G45 TB IL 10 CL-Hemi 30 3 D29 TB CL 10 IL-Hemi 23 2 D24 TB VM, VL 10 BL-DL,DM 23 3 D27 TB VM, VL 5 5 BL-DL,DM 19 2 G89 FDA VM 4 BL-VL 6 0 G91 FDA VM 4 BL-VL 5 0 G95 TB VM 10 BL-VL 21 0 D34 TB VL 5 VM, DL, DM 36 2 D55 TB VL 8 BL-VM 32 3 G93 TB VL 6 6 BL-VM 22 1 G65 FB/DY VL 5 5 L-VM 17 3 G67 FB-L VL 5 L-VM,DM 24 3 G67 DY-R VM, DL 4 R-VL 24 3 D69 FB/DY VL 5 3 BL-VM 23 3 G54 TB DM, DL 7 IL-VL,VM 20 3 G50 FB-L DM, DL 5 L-VM,VL 19 1 G50 DY-R DM, DL 5 R-VM,VL 19 1 Key: Case # p r e f i x denotes duck (D), goose (G) . Tracer: Fast Blue (FB)/ True Blue (TB), Diamidino Yellow (DY), Fluorescein dextran-conjugated amines (FDA). Injection s i t e : ventromedial (VM), v e n t r o l a t e r a l (VL), dorsolateral (DL), dorsomedial (DM). Lesion: c o n t r a l a t e r a l (CL), i p s i l a t e r a l (IL), b i l a t e r a l (BL), rig h t (R),left (L) . The in t a c t region of the spinal cord i s also indicated. Locomotor function: (0) l i t t l e or no active leg movement; (1) standing only; (2) steps with 1 leg (3) walking. 68 SINGLE INJECTION ONLY SINGLE INJECTION + ROSTRAL LESION DOUBLE INJECTION + ROSTRAL LESION Figure 11. Diagrams representing three types of funicular experiments: A: Control, u n i l a t e r a l i n j e c t i o n of 2 d i f f e r e n t retrograde tracers into the lumbar spinal cord (indicated by 2 types of s t i p p l i n g ) ( b i r d G49); L o c a l i z e d i n j e c t i o n of a retrograde t r a c e r i n t o B: the ventromedial (G42) or v e n t r o l a t e r a l (D61) f u n i c u l u s ; C: the v e n t r o l a t e r a l and d o r s o l a t e r a l f u n i c u l i (D38); the dorsomedial (G43), or d o r s o l a t e r a l (D51) f u n i c u l u s ; D-H: S i n g l e i n j e c t i o n plus r o s t r a l l e s i o n ( i n d i c a t e d by c r o s s - h a t c h i n g ) ; I , J : Double i n j e c t i o n with r o s t r a l l e s i o n . € 9 f i g u r e when the i n t e r v a l between each mounted s e c t i o n (30 um x 3) and maximum soma diameters (>30 um, <60 um) i s considered. A f t e r a hemisection c o n t r a l a t e r a l to the i n j e c t i o n , the d i s t r i b u t i o n of l a b e l l e d c e l l s (Table I I I ) was s i m i l a r t o that i n the unlesioned c o n t r o l (G49, Table I I ) . Thus a hemisection ( F i g 11D) appeared t o have a n e g l i g i b l e e f f e c t upon the d i s t r i b u t i o n of retrograde l a b e l l i n g , suggesting that there was no major decussation of descending p r o j e c t i o n s at the high lumbar l e v e l . This f i n d i n g was apparently confirmed i n b i r d D29 (not i l l u s t r a t e d ) , since only one l a b e l l e d neurone was found i n the b r a i n s t e m ( c o n t r a l a t e r a l n u c l e u s c e n t r a l i s m e d u l l a r i s , p a r s d o r s a l i s , Cnd) f o l l o w i n g a h e m i s e c t i o n i p s i l a t e r a l t o the i n j e c t i o n . B rainstem-spinal p r o j e c t i o n s . Dorsomedial medullary-spinal projections: A few r e t r o g r a d e l y l a b e l l e d neurones were observed w i t h i n the v i c i n i t y of the nucleus of the s o l i t a r y t r a c t (nTS) along N X and the region subjacent to X (Ala) predominantly on the c o n t r a l a t e r a l side ( F i g . 12A) . They p r o j e c t v i a the v e n t r a l h a l f of the cord, since they were l a b e l l e d f o l l o w i n g a VLF, or VMF, but not DLF i n j e c t i o n . The p r i n c i p a l pathway appears to be t h e VLF, as l a b e l l i n g was more c o n s i s t e n t f o l l o w i n g a s u b t o t a l l e s i o n of the cord t h a t spared the VLF, than a f t e r a l e s i o n that spared only the VMF ( F i g . 13A,B; Table I I I ) . Medullary reticulospinal projections: Retrogradely l a b e l -l e d c e l l s were found b i l a t e r a l l y w i t h i n the Cnd a f t e r a VMF, or VLF, or DLF i n j e c t i o n , i n d i c a t i n g t h a t t h i s p r o j e c t i o n i s 70 TABLE I I Summary of L a b e l l i n g A f t e r F u n i c u l a r I n j e c t i o n into the Lumbar Spinal Cord T r a c e r : F B / D Y T B / T B TB TB DY I n j e c t i o n L / R V M / B L V L DL+VL DL S i t e : (D49) (G42) (D61) (D38) (D51) L a b e l l e d L R R e g i o n s C L / I L | C L / I L L / R C L / I L C L / I L C L / I L nTS + / " 1 - / ++/ + - / - - / -Cnd + / + + 1 + / + + + / + + + / + + / + - / + Cnv - / - 1 - / - - / - - / - - / -RL + / - 1 + / - - / + - / - + / -Rgc ++/+++| + / + +++/+++ - / + - / + PGL - / - 1 / - - / + + / Rob + + 1 - + + Rp +++ | + + + + + - -Rm + + + | + + + + + + + + + + VeD - / 1 - / - / - / VeL ++/+++| +++/++++ + / + + + / - / -RP ++/+++| ++/+++ + / + / + + / + + C t z + /+ 1 - / + - / - - / + + /'++ RPgc + / + + 1 +++/+++ + + /++ RPO / - 1 - / -LoC + /++ 1 + / + + + / + - / - / + Scd + /++ 1 + + / + + + / + + / - - / - / + Scv ++/+++1 + / + + / + - / - - / + - / -IS + /++ 1 - / + - / -Ru ++++/ | ++/ + / - ++++/ +++ + / PVH / - 1 / - - / - na / -LH / - 1 / - - / - na / -PVM /+ 1 / - - / - na /++++ T r a c e r : F B : F a s t B l u e ; DY: D i a m i d i n o Y e l l o w ; T B : True B l u e . I n j e c t i o n s i t e s : V M : v e n t r o m e d i a l ; V L : v e n t r o l a t e r a l ; D L : d o r s o l a t e r a l f u n i c u l u s . I n j e c t i o n : I L : i p s i l a t e r a l , (L) l e f t , (R) r i g h t s i d e s ; B L : b i l a t e r a l . T o t a l number o f l a b e l l e d c e l l s p e r n u c l e u s / s i d e : (-) < 10; (+) 1 0 - 2 9 ; (++) 3 0 - 5 9 ; (+++) 60 -100 ; (++++) >100. na = no d a t a a v a i l a b l e . A b b r e v i a t i o n s : see page v . 71 TABLEIII Distributions of Retrogradely Labelled Neurones in the Avian Brainstem Following Lumbar Injection and Sub-total Lesion of the Spinal Cord I n t a c t Region of the S p i n a l Cord IL VL, VM VM VL DM, DL n=3 n=2 n=4 n=6 n=3 CL/IL CL/IL CL/IL CL/IL CL/IL nTS + / + ++/ - / - - / Cnd ++/ + + + / + - / - - / - + / + Cnv - / + / - / - / -RL • + + / + + + / + / - + / Rgc ++/+++ + / +++ - / + + /++ - / -PGL + + / - / + -1 - / - / Rob + + • -Rp + + + ++ + + + Rm ++ + + + + . - ++ ++ VeD - / - / - / VeL ++/+++ + / + + /++.++ /-RP - / + + -/++ / + - / -Ctz - / + + - / + - / - / -RPgc / + + /+ /++ RPO / - / -LoC -/++ / + + •/.- 1- - / + Scd -/++ . / + / + -/+ Scv + /•+ / + - / + +/+ IS / + / + / + Ru ++++/ +/ + / ++/ PVH / + / + / - / + LH / - / - /- /-PVM / + / - /+ I n t a c t Region represents the the region remaining a f t e r a sub-t o t a l l e s i o n of the s p i n a l cord r o s t r a l t o an i p s i l a t e r a l i n j e c t i o n at L I . IL: i p s i l a t e r a l h a l f ; VM: ventromedial; VL: v e n t r o l a t e r a l ; DL: d o r s o l a t e r a l ; - DM: dorsomedial f u n i c u l u s . Number of l a b e l l e d c e l l s per nucleus e i t h e r i p s i l a t e r a l (IL) or c o n t r a l a t e r a l (CL) to the i n j e c t i o n & l e s i o n : (-) < 10; (+) 10-29; (++) 30-59; (+++) 60-100; (++++) >100. A b b r e v i a t i o n s : see page v. 72 Figure 12. Photomicrographs of r e t r o g r a d e l y l a b e l l e d neurones f o l l o w i n g an i n j e c t i o n or s p a r i n g o f the v e n t r o l a t e r a l f u n i c u l u s (A-D), ventromedial f u n i c u l u s (E,F) and d o r s o l a t e r a l f u n i c u l u s (G,H). nTS, nucleus of the s o l i t a r y t r a c t ; Cnd, nucleus r e t i c u l a r i s m e d u l l a r i s , pars d o r s a l i s ; Rgc, 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 ; Ctz, corpus trapezoidium; RPgc, nucleus r e t i c u l a r i s p o n t i s c a u d a l i s , pars g i g a n t o c e l l u l a r i s ; VeL, l a t e r a l v e s t i b u l a r nucleus; Ru, red nucleus; PVM, nucleus p a r a v e n t r i c u l a r i s m a g n o c e l l u l a r i s . Scale bars: 50 um. 73 7 4 Figure 13. Diagrams of brainstem sections i n d i c a t i n g the d i s t r i b u t i o n of r e t r o g r a d e l y l a b e l l e d neurones f o l l o w i n g u n i l a t e r a l i n j e c t i o n plus r o s t r a l subtotal s p i n a l cord lesions. Bach s n a i l c i r c l e (o) indicates s neurone which was lab e l l e d when only the i p s i l a t e r a l VLF remained i n t a c t (D34)... Each • • a l l t r i a n g l e (A) indicates a neurone that was l a b e l l e d when only the ventromedial f u n i c u l i were l e f t i n t a c t (693). 75 76 disbursed throughout a l l three f u n i c u l i . S i m i l a r conclusions could be reached from the r e s u l t s i n which the VMF, the VLF or the DLF were spared (Figs. 12B, 13A, B) . Approximately 70% of the l a b e l l i n g was i p s i l a t e r a l when the i n j e c t i o n was centred i n the DLF, but no strong predominance was noted i n the VLF or VMF p r o j e c t i o n s . A few r e t r o g r a d e l y l a b e l l e d neurones were present w i t h i n the nucleus c e n t r a l i s m e d u l l a r i s , pars v e n t r a l i s (Cnv), predominantly on the i p s i l a t e r a l s i d e , f o l l o w i n g i n j e c t i o n s i n t o the VMF or VLF and a l s o when only the VMF or VLF remained i n t a c t a f t e r a s u b t o t a l l e s i o n ( F i g . 13B). The 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) p r o j e c t s v i a the v e n t r a l h a l f of the cord, since l a b e l l e d c e l l s were found w i t h i n the Rgc when e i t h e r the VLF or the VMF was i n j e c t e d or spared i n the l e s i o n experiments. Although the Rgc was a l s o l a b e l l e d when the DLF and DMF was l e f t i n t a c t (Table I I I ) , these l e s i o n s a l s o spared the d o r s a l part of the VLF (e.g. F i g . 11H) . Since the Rgc was not l a b e l l e d a f t e r a DLF i n j e c t i o n ( F i g . 11C) , t h i s suggests t h a t the Rgc does not p r o j e c t v i a the DLF. There was no apparent f u n i c u l a r separation of i p s i l a t e r a l v e r s u s c o n t r a l a t e r a l p r o j e c t i o n s , s i n c e r e t r o g r a d e l y l a b e l l e d neurones were predominantly i p s i l a t e r a l (75%) i n a l l cases (Figs. 12C, 13C,D). Fluorescent somata were l o c a t e d b i l a t e r a l l y w i t h i n the l a t e r a l r e t i c u l a r nucleus (RL) and l a t e r a l p a r a g i g a n t o c e l l u l a r n u c l e u s (PGL) w i t h c o n t r a l a t e r a l predominance. Pontine r e t i c u l o s p i n a l projections: The c a u d a l p o n t i n e r e t i c u l a r formation (RP) and the subjacent corpus trapezoidium 77 (Ctz) region contained l a b e l l e d neurones, predominantly on the i p s i l a t e r a l side, when either the VMF or VLF was injected or l e f t intact (Figs. 12D; 13E,F); however neither the RP nor Ctz were l a b e l l e d when only the medial portion of the VMF was spared (F i g . 11F) . Conversely, the caudal g i g a n t o c e l l u l a r pontine r e t i c u l a r formation (RPgc) was only labelled, on the i p s i l a t e r a l side (20-45 c e l l s ) , when the medial portion of the VMF was s p e c i f i c a l l y injected or l e f t intact (Figs. 12E; 13G) . L a b e l l e d neurones w i t h i n the r o s t r a l pontine r e t i c u l a r formation, s p e c i f i c a l l y the oral d i v i s i o n (RPO), were sparse even i n the control and hemisected birds, but a few i s o l a t e d c e l l s were also found aft e r a VMF i n j e c t i o n or when both the VMF and VLF were l e f t i n t a c t . Raphespinal projections: The raphe p a l l i d u s (Rp) contained many retrogradely l a b e l l e d somata when either the VMF or VLF was s p e c i f i c a l l y injected or l e f t intact (Figs. 13C-F), whereas the raphe obscurus (Rob) was only w e l l l a b e l l e d a f t e r a b i l a t e r a l i n j e c t i o n into the VMF. The raphe magnus (Rm) was primarily l a b e l l e d (30-40 c e l l s ) when either the VLF or DLF was injected or l e f t i n t a c t . Some Rm l a b e l l i n g was present after an in j e c t i o n into the VMF, but only a few is o l a t e d l a b e l l e d c e l l s were found aft e r a subtotal thoracic lesion that preserved the VMF, suggesting that the retrograde l a b e l l i n g following a VMF in j e c t i o n may have resulted from uptake within medial aspects of the VLF or from terminals within the the gray matter. Although the l a b e l l e d c e l l s within the Rm were predominantly i p s i l a t e r a l , a d e f i n i t e l a t e r a l i t y was not always obvious, p a r t i c u l a r l y w i t h i n the Rob, and Rp and consequently no 78 l a t e r a l i t y has been emphasized i n Tables II and I I I . Vestibulospinal projections: Only a few neurones (less than 10) were l a b e l l e d within the contra l a t e r a l descending vestibular nucleus (VeD) following i n j e c t i o n into the VLF or a f t e r a l e s i o n i n which the VLF was l e f t i n t a c t . The i p s i l a t e r a l l a t e r a l v e s t i b u l a r nucleus (VeL) was h e a v i l y l a b e l l e d when the VMF was the sole intact funiculus of the spinal cord for retrograde transport (Table III, Figs. 11F; 12F), but contained only a few fluorescent somata following i n j e c t i o n or sparing of the VLF ( F i g . 11G; 13E) . The contral a t e r a l VeL was sparsely l a b e l l e d (less than 10 neurones) after VLF or VMF i p s i l a t e r a l i n j e c t i o n s . Coeruleospinal projections: Labelled neurones within the locus coeruleus (LoC), were predominantly i p s i l a t e r a l following an i n j e c t i o n into the VMF or DLF. Although no l a b e l l i n g was found aft e r an i n j e c t i o n into the VLF (Fig 11B), l a b e l l i n g was present aft e r a more medial VLF i n j e c t i o n (Fig 11C) and when the VLF was the only region l e f t . Together the data from both series of experiments suggest that the LoC-spinal projections are d i s p e r s e d throughout a l l three f u n i c u l i , but may be greatest within the DLF. The l a b e l l i n g within the dorsal subcoeruleus nucleus (Scd) was most prominent after a discrete i n j e c t i o n of the DLF and when e i t h e r the t h o r a c i c DLF or VLF was unlesioned. Labelled neurones were also evident b i l a t e r a l l y within the ventral subcoeruleus nucleus (Scv), p a r t i c u l a r l y after a DLF in j e c t i o n or when the DLF remained i n t a c t . 7 9 Mesencephalospinal projections: The i n t e r s t i t i a l nucleus (IS) was l a b e l l e d i p s i l a t e r a l l y when the i n j e c t i o n was r e s t r i c t e d to the VMF or the l e s i o n excluded the VMF ( F i g . 131) . The red nucleus (Ru) was l a b e l l e d c o n t r a l a t e r a l l y when the i n j e c t i o n i n c l u d e d the DLF (Figs. 11A,C; 12G). When the DMF and DLF (F i g . HE) were l e s i o n e d a few Ru neurones were s t i l l l a b e l l e d ( F i g . 13H,I), i n d i c a t i n g t h a t some Ru p r o j e c t i o n s t r a v e r s e the v e n t r a l part of the DLF en route to t e r m i n a t i o n . There was no evidence of a Ru p r o j e c t i o n v i a the v e n t r a l f u n i c u l i i n any other experiments. Hypothalamospinal projections: The i p s i l a t e r a l p a r a -v e n t r i c u l a r nucleus (PVM) was l a b e l l e d h e a v i l y a f t e r a DLF i n j e c t i o n ( F i g . 12H), whereas i t was only s p a r s e l y l a b e l l e d when the v e n t r a l h a l f of the cord was i n j e c t e d . No l a b e l l i n g was found when e i t h e r the VMF or VLF were the only i n t a c t regions of the s p i n a l cord. Only i s o l a t e d c e l l s were l a b e l l e d w i t h i n the h y p o t h a l a m i c p a r a v e n t r i c u l a r n u c l e u s (PVH) and l a t e r a l hypothalamus (LH) a f t e r a b i l a t e r a l i n j e c t i o n i n t o the VMF or when the l e s i o n l e f t the VMF i n t a c t ( F i g . 13J) . There were no apparent p r o j e c t i o n s from PVH or LH v i a the DLF or VLF. F u n i c u l a r P r o j e c t i o n Summary The Ventromedial Funiculus. The d i s t r i b u t i o n of l a b e l l e d neurones f o l l o w i n g a d i s c r e t e i n j e c t i o n or l e s i o n of the VMF i n d i c a t e d t h a t the p r i n c i p a l components of the VMF i n c l u d e : i p s i l a t e r a l p r o j e c t i o n s from the RPgc and IS; b i l a t e r a l p r o j e c t i o n s from the Cnd, Cnv, Rgc, RL, PGL, Rob, Rp, RP, VeL, and t o a minor extent the LoC, PVH, and LH. The l o c a t i o n of these pathways at the lumbar l e v e l i s depicted i n F i g . 14, w i t h 80 the t r a j e c t o r y of pathways at the c e r v i c a l l e v e l , as determined by Cabot et a l . (1982), being provided f o r comparison. Following a b i l a t e r a l l e s i o n of the VMF, 2 of 3 birds were capable of self-supported walking (Table I) . Conversely, no b i r d s were capable of s e l f - s u p p o r t e d locomotion when the s u b t o t a l l e s i o n spared only the VMF, b i l a t e r a l l y (n=3). However, the survival time was only f i v e to six days i n two cases (compared to 15 days or more for a l l other cases) . This may not have been sufficent time for recovery of function. The Ventrolateral Funiculus. The components of the VLF in c l u d e d p r o j e c t i o n s from the Cnd on both sides and predominantly i p s i l a t e r a l projections from the Rgc, Rp, Rm, VeL, RP, Ctz, LoC, Scd, and Scv and also c o n t r a l a t e r a l projections from the Ala, nTS, RL, and VeD. After a subtotal l e s i o n of the t h o r a c i c cord where only the b i l a t e r a l VLF remained intact, 2 of 3 birds were able to walk. No birds were capable of walking following a b i l a t e r a l l e s i o n of the VLF (n=3) . When the VLF was lesioned on only one side (n=l; F i g . 11H), walking a b i l i t y was retained. The Dorsolateral Funiculus. The p r i n c i p a l p r o j e c t i o n s contained within the DLF were from the contr a l a t e r a l Ru, the i p s i l a t e r a l PVM, LoC, Scd and b i l a t e r a l l y from the Cnd and Rm. Birds were able to walk i n an apparently normal manner afte r a b i l a t e r a l l e s i o n of the entire dorsal h a l f of the thoracic spinal cord (Fig. HE) . The Dorsomedial Funiculus. No l a b e l l e d c e l l s were found within the brainstem after a DMF i n j e c t i o n (Fig. 11C). 81 CERVICAL (Cabot et al. , 1982) L U M B A R F i g u r e 1 4 . D i a g r a m s i n d i c a t i n g t h e f u n i c u l a r t r a j e c t o r i e s o f d e s c e n d i n g s p i n a l c o r d p r o j e c t i o n s . C e r v i c a l d a t a as d e s c r i b e d fv CaSot et a (1982) f o r t h e l o c a t i o n o f s e v e r a l p a t h w a y s a t t h e b r a c h i a l l e v e l o f t h e c o r d . L u m b a r l e v e l s u m m a r i z e s t h e d a t a f r o m t h e p r e s e n t s t u d y . 82 D I S C U S S I O N T h i s s t u d y has i d e n t i f i e d t h e f u n i c u l a r o r g a n i z a t i o n o f b r a i n s t e m - s p i n a l p r o j e c t i o n s a t t h e t h o r a c o l u m b a r l e v e l i n t h e duck and goose. The d a t a p r o v i d e : 1) c o n f i r m a t i o n and e x t e n s i o n o f t h e f u n c i c u l a r o r g a n i z a t i o n o f d e s c e n d i n g pathways w i t h i n t h e c a u d a l a v i a n s p i n a l c o r d , and 2) f o r t h e f i r s t t i m e , d e l i n e a t e t h e f u n i c u l a r t r a j e c t o r i e s o f o t h e r b r a i n s t e m - s p i n a l pathways (Cnd, Cnv, Rgc, A l a , nTS, RL, LoC) . In t h e f o l l o w i n g d i s c u s s i o n t h e s i m i l a r i t i e s and d i f f e r e n c e s between t h e p r e s e n t f i n d i n g s and t h o s e o f p r e v i o u s s t u d i e s on b i r d s a r e compared t o o t h e r v e r t e b r a t e s . The p r e s e n t s t u d y has shown t h a t p r o j e c t i o n s from d o r s a l m e d u l l a w i t h i n t h e nTS, and v e n t r a l t o X ( A l a ) , p r o j e c t w i t h a s t r o n g c o n t r a l a t e r a l predominance t o t h e lumbar s p i n a l c o r d , v i a t h e VLF and a l s o t h e VMF. A l t h o u g h t h e v e n t r a l group o f a v i a n neurones are d i s t i n c t from t h e nTS i n t h e b i r d , mammalian nTS p r o j e c t i o n s t o t h e lumbar c o r d a l s o t r a v e r s e t h e VLF (Loewy and B u r t o n , 1978) and VMF (Zemlan and P f a f f , 1979) . I n a d d i t i o n , p r o j e c t i o n s from t h e nTS t o t h e lumbar c o r d v i a t h e DMF have been r e p o r t e d f o r t h e monkey ( C a r l t o n e t a l . , 1985), however no neurones were found t o p r o j e c t v i a t h e DMF i n t h e p r e s e n t s t u d y . Descending pathways from Cnd p r o j e c t b i l a t e r a l l y t o t h e l u m b a r c o r d v i a t h e VMF, VLF and DLF i n b i r d s . S p i n a l p r o j e c t i o n s from an e q u i v a l e n t r e g i o n i n mammals, t h e n u c l e u s d o r s a l i s o f t h e 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 , a l s o descend v i a t h e s e f u n i c u l i a t t h e h i g h t h o r a c i c (Kuypers and M a i skey, 1977) and l u m b a r l e v e l s ( M a r t i n e t a l . , 1981a) . The p r e d o m i n a n t 83 i p s i l a t e r a l projection within the DLF, noted i n t h i s study, has also been reported at the lumbar l e v e l for the monkey (Carlton et a l . , 1985) . The Cnv t r a j e c t o r y was not s p e c i f i c a l l y delineated i n a previous study of the descending projections to the c e r v i c a l cord of the pigeon, as the t r i t i a t e d amino acid i n j e c t i o n a l s o i n v o l v e d the Rp (Cabot et a l . , 1982) . The present results demonstrated i p s i l a t e r a l projections v i a the VMF and VLF (but not the DLF) for both the Cnv and Rp i n the duck and goose. Analogous p r o j e c t i o n s (from the i n f e r i o r r e t i c u l a r nucleus i n r e p t i l e s ; nucleus v e n t r a l i s i n mammals) have been reported to descend within the DLF as well as the VMF and VLF i n the l i z a r d (Wolters et a l . , 1982) and monkey (Carlton et a l . , 1985), whereas they apparently traverse the DLF and VLF i n the opossum (Martin et a l . , 1981a). As a n t i c i p a t e d from a previous avian study on the f u n i c u l a r o r g a n i z a t i o n of descending p r o j e c t i o n s at the c e r v i c a l l e v e l (Cabot et a l . , 1982), the Rgc was found to project v i a the VLF and the l a t e r a l portion of the VMF at the lumbar l e v e l . The Rgc was not retrogradely l a b e l l e d from the lumbar cord when only the medial aspects of the caudal thoracic VMF were l e f t i n t a c t . In mammals, however, an a d d i t i o n a l projection v i a the DLF has been reported i n the rat (Zemlan and Pfaff, 1979; Zemlan et a l . , 1984; Martin et a l . , 1985), cat (Holstege and Kuypers, 1982; Mitani et a l . , 1988) and monkey (Carlton et a l . , 1985). Autoradiographic tracing experiments have suggested that the i p s i l a t e r a l and contr a l a t e r a l r e t i c u l o s p i n a l pathways from 84 Rgc p r o j e c t v i a d i f f e r e n t f u n i c u l i i n the cat; the i p s i l a t e r a l p r o j e c t i o n s are conveyed by the VMF, VLF, and DLF, while the c o n t r a l a t e r a l p r o j e c t i o n s r e s i d e w i t h i n the DLF at the lumbar l e v e l (Holstege and Kuypers, 1982) . At the c e r v i c a l l e v e l the Rgc a l s o p r o j e c t s v i a the c o n t r a l a t e r a l VMF i n the c a t (Holstege and Kuypers, 1982; M i t a n i et a l . , 1985) and opossum (Martin et a l . , 1982). In t h i s study, no c l e a r d i s t i n c t i o n was found between the f u n i c u l a r t r a j e c t o r y of the i p s i l a t e r a l versus c o n t r a l a t e r a l p r o j e c t i o n s from the Rgc. The r a t i o of i p s i l a t e r a l to c o n t r a l a t e r a l l a b e l l i n g w i t h i n the Rgc was 3 to 1 when e i t h e r the VMF or VLF were the only f u n i c u l i spared f o r retrograde t r a n s p o r t . This i s s i m i l a r to the r a t i o achieved a f t e r a c o n t r o l i n j e c t i o n of FB and DY on opposite sides of the lumbar cord ( F i g . 11A). Few, i f any f i b r e s , from the Rgc appear to cross at the high lumbar l e v e l of the cord, since no l a b e l l i n g was found i n Rgc when the c o r d was h e m i s e c t e d i p s i l a t e r a l t o the i n j e c t i o n s i t e . However, u s i n g autoradiography, Holstege and Kuypers (1982), have shown tha t some Rgc p r o j e c t i o n s cross at the lumbar l e v e l i n the c a t . Whether t h i s discrepancy i n r e s u l t s i s due to species, t r a c i n g method, or the l e v e l of i n j e c t i o n remains to be determined. In mammals, a c l e a r d i s t i n c t i o n i s made between the g i g a n t o c e l l u l a r (Rgc or NGc, most caudal) and the magnocellular (Rmc, more r o s t r a l and v e n t r a l ) components of the p o n t o 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 on the b a s i s of c y t o a r c h i t e c t u r e ( A n d r e z i k and B e i t z , 1985; Newman, 1985), f u n i c u l a r t r a j e c t o r y and laminar terminations (Martin et a l . , 1979, 1985; Holstege and Kuypers, 1982). For example, axons 85 from t he Rmc, but not the Rgc, p r o j e c t v i a the DLF and terminate w i t h i n laminae I and I I . While no s u b - d i v i s i o n s of the g i g a n t o c e l l u l a r formation have been defined i n the b i r d , neurones w i t h i n the RP adjacent t o Ctz at the l e v e l of VI, are morphologically d i s t i n c t from the d o r s a l RP and caudal Rgc and al s o p r o j e c t v i a the DLF. The RL neurones appeared to p r o j e c t p r i m a r i l y v i a the VLF. Comparable r e s u l t s have been reported i n the cat (Holstege and Kuypers, 1982). In t h i s study, the PGL p r o j e c t e d v i a the VMF and VLF i n the b i r d , whereas i t has been reported t o p r o j e c t a l s o v i a the DLF i n the cat (Holstege and Kuypers, 1982) , and e x c l u s i v e l y v i a the DLF i n the monkey (Carlton et a l . , 1985). The present experiments i n d i c a t e that the o r g a n i z a t i o n of the raphespinal p r o j e c t i o n s i n the b i r d c l o s e l y resemble those found i n mammals. Thus, i n the duck and goose, the Rob pr o j e c t e d p r i m a r i l y v i a the VMF, the Rp predominantly v i a the VLF and the Rm v i a the VLF and DLF, as p r e v i o u s l y documented f o r c e r v i c a l cord p r o j e c t i o n s the pigeon (Cabot et a l . , 1982) and cat (R.F. Mar t i n et a l . , 1978). In the present study, the VeL p r o j e c t i o n s to the lumbar cord were l a r g e l y r e s t r i c t e d t o the VMF. Comparable r e s u l t s have been reported at the lumbar l e v e l i n the cat (Holstege and Kuypers, 1982) and monkey (Carlton et a l , 1985) although there are a l s o some p r o j e c t i o n s v i a the VLF. Only a few c e l l s were l a b e l l e d w i t h i n the VeL, when the VLF was the only f u n i c u l u s l e f t i n t a c t a f t e r a s u b t o t a l s p i n a l cord l e s i o n . The present r e s u l t s on the course of VeD p r o j e c t i o n s are c o n s i s t e n t w i t h 86 data that has demonstrated that the equivalent mammalian nucleus, the i n f e r i o r vestibular nucleus, also projects v i a the VLF (Carlton et a l . , 1985). With regard to pontine r e t i c u l o s p i n a l projections, the present findings are consistent with previous reports i n the cat, that RP and RPO were found to project b i l a t e r a l l y v i a the VMF (Holstege and Kuypers, 1982). In the monkey, however, these projections have been noted to be s o l e l y i p s i l a t e r a l (Carlton et a l . , 1985). In the present study the RPgc did project i p s i l a t e r a l l y v i a the VMF and are therefore similar to the projection at the brachial l e v e l (Cabot et a l . , 1982). The present results indicate that the projections from the LoC and Scd are located within the DLF. These findings are consistent with the trajectory noted at the brachial l e v e l i n the pigeon (Cabot et a l . , 1982). The present results suggest that a small projection i s also contained within the VMF and VLF. The Scv probably corresponds to the ventrolateral pontine region i n mammals. In the cat (Holstege and Kuypers, 1982) and monkey (Carlton et a l . , 1985), as i n the b i r d , t h i s region projects predominantly v i a the c o n t r a l a t e r a l DLF. Projections from the Scv region also course through the VMF and VLF, similar to spinal projections a r i s i n g from the paralemniscal region i n the opossum (Martin et a l . , 1981a); however the paralemniscal region i s more extensive, both i n dorsal and r o s t r a l extent than the Scv i n the b i r d . As suggested by Cabot and colleagues (1982), the IS projections are contained within the dorsomedial portion of the VMF. Similar findings have been reported for mammals at the 87 t h o r a c i c (Tohyama et a l . , 1979a) and lumbar l e v e l of the s p i n a l cord (Martin et a l , 1979). The r u b r o s p i n a l p r o j e c t i o n s were l o c a t e d i n the DLF and adjacent d o r s a l margin of the VLF (extending more v e n t r a l l y than depicted by Cabot et a l . , 1982, F i g . 8; Wild et a l , 1979), since a few c e l l s w i t h i n the Ru were r e t r o g r a d e l y l a b e l l e d d e s p i t e a t r a n s e c t i o n of the e n t i r e d o r s a l h a l f of the cord. These r e s u l t s are c o n s i s t e n t w i t h reports that the Ru terminate w i t h i n lamina V at the base of the d o r s a l horn (Wild et a l . , 197 9) . Although a few 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 were found w i t h i n the Ru f o l l o w i n g an i n j e c t i o n i n t o the VMF t h e s e probably r e s u l t e d from d i f f u s i o n i n t o the gray matter since they were not present when the VMF was the only region that remained i n t a c t . A c c o r d i n g l y, the present r e s u l t s showed no evidence of an i p s i l a t e r a l ventromedial r u b r o s p i n a l p r o j e c t i o n as reported f o r the r a t (Holstege, 1987). The present r e s u l t s show that the o r g a n i z a t i o n of the p r o j e c t i o n s from the avian PVM are c o n s i s t e n t throughout the lower s p i n a l cord since the t r a j e c t o r y through the DLF has been reported by others at the c e r v i c o t h o r a c i c j u n c t i o n (Cabot et a l . , 1982; Berk and F i n k e l s t e i n , 1982); however e n t e r i n g the c e r v i c a l cord t h i s p r o j e c t i o n has been noted to occupy the VLF ( F i n k e l s t e i n and Berk, 1980). The p r e s e n t study f u r t h e r demonstrated th a t the LH and SCE p r o j e c t e d v i a the DLF as w e l l as VMF and VLF. Comparable r e s u l t s have been reported f o r the cat (Basbaum and F i e l d s , 1979). F u n c t i o n a l Considerations 88 The Ventromedial Funiculus. The present results provide confirmation that the major components of the VMF i n the b i r d consist of the i p s i l a t e r a l medial medullary r e t i c u l o s p i n a l , pontine r e t i c u l o s p i n a l , vestibulospinal and i n t e r s t i t i o s p i n a l projections (Cabot et a l . , 1982). In addition, the present study showed that there were small c o n t r i b u t i o n s from the con t r a l a t e r a l Rgc, RP and VeL within the VMF at the lumbar l e v e l . Both b i r d s and mammals are capable of s e l f - s u p p o r t e d walking with only the VMF of the caudal thoracic cord l e f t intact (Eidelberg et a l . , 1981a,b; Sholomenko and Steeves, 1987) . In the present study, none of the birds i n which only the VMF remained intact were capable of walking. In part t h i s may have been due to the short survival time i n two of three cases (5-6 days) aft e r FDA i n j e c t i o n . However one b i r d was unable to walk aft e r 20 days. Since i t i s also true that hindlimb locomotor a b i l i t y i s only t r a n s i e n t l y affected when the VMF i s transected b i l a t e r a l l y (Eidelberg, 1981; Eidelberg et a l . , 1981a,b; Sholomenko and Steeves, 1987), i t i s possible that the projections e s s e n t i a l for locomotion are contained w i t h i n both the VMF and VLF. It i s l i k e l y that lack of locomotion i n birds with only the VMF intact, was due to the fact that lesion was more extensive than i n a previous study where locomotion was preserved (Sholomenko and Steeves, 1987). Thus i n the present study, sparing of projections within the VMF primarily spared those from the VeL, RPgc, and IS, whereas projections descending more l a t e r a l l y within the VMF, such as Rgc, Cnv, RP were apparently lesioned. This provides further 8 9 evidence that the medullary r e t i c u l o s p i n a l projections may be primarily responsible for the preservation of locomotion. The Ventrolateral Funiculus. Consistent c o r r e l a t i o n s between the sp a r i n g of locomotor a c t i v i t y and retrograde l a b e l l i n g of the Rgc, raphe, RL and RP nuclei have been demonstrated following subtotal cord lesions (Eidelberg et a l . , 1981a). The present study confirmed the presence of these pathways within the VLF i n the b i r d at the lumbar l e v e l , and also confirmed f i n d i n g s that s e l f - s u p p o r t e d walking was possible when only the VLF were l e f t intact b i l a t e r a l l y at the low thoracic l e v e l (Sholomenko and Steeves, 1987). In t h i s study, even when only one ventral quarter of the spinal cord was spared a b i r d was capable hopping on the i p s i l a t e r a l l e g , but not b i l a t e r a l s e l f - s u p p o r t e d walking. Similar findings have been reported i n i n the b i r d (Sholomenko and Steeves, 1987) and monkey (Eidelberg et a l . , 1981b) afte r comparable recovery p e r i o d s ; however, a f t e r a few months b i l a t e r a l support i s possible (Eidelberg et a l . , 1981b). While the r o l e of the Rgc has been v e r i f i e d by p h y s i o l o g i c a l s t u d i e s during locomotion (Orlovsky, 1970b; Shefchyk et a l . , 1984; G a r c i a - R i l l et a l . , 1987a), the role of other descending pathways within the VLF, such as the Cnd, Cnv, and RL have not been completely determined. The The Dorsolateral Funiculus. The main descending projections within the DLF are axons from the hypothalamus, the Ru and the coeruleus complex. Additional contributions are provided by f i b r e s from the Cnd, Rm and Ctz. As shown by others 90 (Sholomenko and Steeves, 1987) , a l e s i o n of the DMF and DLF on both sides does not impede walking i n the b i r d . None of these pathways appears to be essential for locomotion, p a r t i c u l a r l y those exclusive to the DLF. Numerous studies have shown that e l e c t r i c a l stimulation of the DLF can evoke locomotion i n a variety of species. In mammals a stimulus a p p l i e d at the high c e r v i c a l l e v e l (Sherrington, 1910) i s presumed to activate fibres emmanating from the TTD or Rpc which provides input to a propriospinal pathway (Kazennikov et a l . , 1983). In f i s h and r e p t i l e s , DLF-stimulation (Lennard and Stein, 1977; Williams et a l . , 1984; S t e i n , 1978) i s thought to a c t i v a t e r e t i c u l o s p i n a l p r o j e c t i o n s , s i n c e they are l o c a t e d w i t h i n the DLF (ten Donkelaar, 1976b; Stein, 1978). In birds, the most e f f e c t i v e s i t e at the c e r v i c a l l e v e l for evoking locomotion included the DLF and VLF (Jacobson and Hollyday, 1982) and therefore may activate r e t i c u l o s p i n a l and DLF. projections. Together these studies indicate alternate locomotor pathways are contained within the DLF although they are not e s s e n t i a l . In summary the funicular organization of avian descending projections at the thoracolumbar l e v e l has been described. Spinal pathways from the Rgc, the Cnd and the RL project v i a the VLF, whereas the VMF conveys projections p r i n c i p a l l y from the VeL, RP, RPgc and IS as well as the Cnd and Rgc. In addition to the Rgc, the RL and Cnd may also contribute to di r e c t descending locomotor drive as suggested by other studies from t h i s laboratory (Steeves et a l . , 1986; Sholomenko and Steeves, i n preparation). V BRAINSTEM LOCOMOTOR SITES 92 INTRODUCTION The supraspinal control of locomotor mechanisms has been studied i n a variety of vertebrates (for reviews see: G r i l l n e r , 1981; Armstrong, 1986; McClellan, 1986). In addition to the les i o n experiments previously discussed (see Ch IV of t h i s t h e s i 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 been used to determine the p r i n c i p l e brainstem regions i n v o l v e d i n the i n i t i a t i o n of locomotion. Shik and associates (1966) were the f i r s t to es t a b l i s h that stimulation i n a region below the i n f e r i o r c o l l i c u l u s , the mesencephalic locomotor region (MLR), l a t e r i d e n t i f i e d as the caudal cuneiform nucleus (Shik et a l . , 1967), could evoke locomotion i n the decerebrate cat. These f i n d i n g s were subsequently confirmed (Steeves et a l . , 1975) and extended to i n c l u d e other n u c l e i i n the same v i c i n i t y ; the l a t e r a l parabrachial nucleus, the periaqueductal gray, and the dorsal pedunculopontine nucleus (PPN) i n the cat (Gar c i a - R i l l et a l . , 1983a,b), and the PPN i n the rat (Skinner and G a r c i a - R i l l , 1984) . In more caudal regions of the cat brainstem, the pontobulbar locomotor s t r i p (PLS) , which extends along a dorsolateral track from the l e v e l of the f a c i a l nucleus to the caudal l e v e l of the medulla (Shik and Yagodnitsyn, 1977; Mori et a l . , 1977), and the ventromedial medullary r e t i c u l a r formation have also been found to be e f f e c t i v e stimulus s i t e s for evoking locomotion (Noga et a l . , 1988). Locomotor stimulation s i t e s have also been demonstrated i n s e v e r a l non-mammalian v e r t e b r a t e species (reviewed by M c C l e l l a n , 1986). In f i s h (Kashin et a l . , 1974, 1981), 93 e f f e c t i v e s i t e s have been located i n the midline of the dorsal midbrain, while i n the t u r t l e locomotion was evoked mainly by stimulation of the l a t e r a l r e t i c u l a r formation (Kazennikov et a l . , 1981) . In birds locomotor s i t e s have been i d e n t i f i e d i n (1) the dorsolateral medulla within the descending nucleus and t r a c t of the t r i g e m i n a l nerve (TTD), nucleus m e d u l l a r i s c e n t r a l i s , pars d o r s a l i s (Cnd), nucleus 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) and (2) i n the ventromedial medullary r e t i c u l a r formation (Steeves et a l . , 1986, 1987). In birds, both the dorsolateral and ventromedial brainstem regions have direc t spinal projections (Cabot et a l . , 1982; Webster and Steeves, 1988). Although these data suggest that medullary locomotor s i t e s are coextensive with areas g i v i n g r i s e to di r e c t spinal projections, i t would be more compelling i f i t could be shown that brainstem neurones, retrogradely l a b e l l e d following a True Blue (TB) i n j e c t i o n into the spinal cord, were within the e f f e c t i v e radius of a focal stimulating electrode capable of evoking locomotion i n the decerebrate duck or goose. METHODS AND MATERIALS Four birds (3 white Pekin ducks, Anas platyrhynchos, and 1 Canada goose, Branta canadensis) were used i n t h i s study. A l l surgical procedures were performed under general and l o c a l anaesthesia. TB was injected into either the low thoracic (3 birds) or c e r v i c a l (C7) spinal cord i n the manner previously described (Webster and Steeves, 1988 and Ch. II of t h i s t h e s i s ) . After 10 to 20 days, each b i r d was reanaesthetised and prepared f o r brainstem s t i m u l a t i o n as p r e v i o u s l y d e s c r i b e d 94 (Steeves et a l . , 1987). In short, a l l pressure points and skin i n c i s i o n s were routinely i n f i l t r a t e d with l o c a l anaesthetic. The l e f t brachial artery and vein were cannulated for monitoring blood pressure and f o r f l u i d i n f u s i o n , r e s p e c t i v e l y , and both i n t e r n a l carotids were li g a t e d . Each b i r d was placed i n a s l i n g over a treadmill belt, the head fixed i n a stereotaxic frame, with the beak clamped i n a horizontal p o s i t i o n . Following a craniotomy, l i g a t i o n and section of the s a g i t t a l sinus, and r e f l e c t i o n of the dura, the telencephalon was removed along a plane extending from the caudal thalamic border to the optic chiasma. Bleeding was c o n t r o l l e d with c a u t e r i z a t i o n and pressure. At the completion of the decerebration, which renders an animal insensient and unable to perceive pain ( K i t c h e l l and Erickson, 1983), anaesthesia was discontinued. The stimulating electrode (Kopf SNE 300) was mounted on the stereotaxic frame at earbar zero. Selected s i t e s were stimulated with a 30-60 sec t r a i n of square-wave pulses of 0.5 msec dur a t i o n at a frequency of 30-50 Hz. In d i f f e r e n t experiments, the threshold current i n t e n s i t y required to evoke locomotion v a r i e d from 25 to 100 uA. A procedure was established whereby the electrode was positioned, inserted, and lowered to the fl o o r of the brainstem. The stimulation t r i a l s were undertaken as the electrode was gradually withdrawn. When locomotion was evoked, the s i t e was c a r e f u l l y explored to determine the point of lowest stimulation threshold. At the end of the t r i a l s for each e f f e c t i v e s i t e , an e l e c t r o l y t i c l e s i o n was made using d i r e c t current of 1-5 mA for a duration 95 of 5-8 sec or a current of 30-50 uA for 30-50 sec. During evoked locomotion, electromyographic (EMG) a c t i v i t y was recorded v i a hook electrodes which had been percutaneously implanted in.the appropriate leg and wing muscles, p r i o r to the commencement of brainstem stimulation. The electrodes were i n s e r t e d i n the i l i o t i b i a l i s c r a n i a l i s (ITC, hip f l e x o r ; mammalian sartorius) muscles, since they have been found to be the most r e l i a b l e markers for demarcating the swing phase of avian walking (Weinstein et a l . , 1984). The in-phase b i l a t e r a l wing a c t i v i t y associated with wing flapping was also recorded by electrodes percutaneously implanted i n the p e c t o r a l i s (wing depressor) muscles (Weinstein, et a l . , 1984). The EMG signals were amplified, f i l t e r e d and fed into an e l e c t r o s t a t i c chart recorder (frequency response range DC - 10,000 Hz). In addition DC potentiometers were attached with a s t r i n g t i e d to the lower leg to record the phase and frequency of stepping. At the end of the stimulation t r i a l s , each b i r d was re-anaesthetized, and perfused t r a n s c a r d i a l l y with saline followed by paraformaldehyde (4%, pH 7.4 i n 0.1 M phosphate b u f f e r ) . The brain and spinal cord injected segments were removed, postfixed for a further 12 hours, then stored i n a cryoprotectant (20% sucrose, 10% glycer o l i n 0.05 M phosphate buffer) for 48 hours before being transversely sectioned at 30 um on a freezing microtome. Every t h i r d section was mounted onto g e l a t i n i z e d s l i d e s and examined with a fluorescent microscope (for d e t a i l s see Ch. II of t h i s t h e s i s ) . Drawings were made of the le s i o n s i t e s and the locations of the retrogradely l a b e l l e d neurones. 96 A few s e c t i o n s c o n t a i n i n g i d e n t i f i e d s t i m u l a t i o n s i t e s were s u b s e q u e n t l y s t a i n e d w i t h c r e s y l v i o l e t or t h i o n i n e . R E S U L T S Low 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 (25-75 uA) o f t h e v e n t r o m e d i a l o r d o r s o l a t e r a l m e d u l l a e v o k e d a l o c o m o t o r s t e p p i n g p a t t e r n ( F i g . 15) i n a l l - b i r d s , . Once t h e t h r e s h o l d s t i m u l u s h a d b e e n e s t a b l i s h e d f o r a p a r t i c u l a r s i t e , t h e p a t t e r n s o f muscle a c t i v i t y , as r e c o r d e d by EMG were o b s e r v e d t o v a r y a c c o r d i n g t o t h e s t r e n g t h o f t h e s t i m u l a t i n g c u r r e n t ( S t e e v e s e t a l . , 1987) . When w a l k i n g movements had been a c t i v a t e d a t l o w i n t e n s i t y (25-50 uA, 30-80 Hz) , f u r t h e r i n c r e a s e i n c u r r e n t s t r e n g t h o r t r e a d m i l l b e l t speed, r e s u l t e d i n i n c r e a s e d f r e q u e n c y o f s t e p p i n g . W i t h a f u r t h e r i n c r e a s e o f t h e s t i m u l a t i n g c u r r e n t s t r e n g t h (to 75-100 uA), wing f l a p p i n g was evoked. Wing-beat f r e q u e n c y v a r i e d from 2-5 Hz and was o n l y p a r t i a l l y dependent upon c u r r e n t s t r e n g t h . The e l e c t r o l y t i c l e s i o n s w hich mark each low t h r e s h o l d s t i m u l a t i o n s i t e were l o c a t e d w i t h i n t h e c a u d a l Cnd ( F i g s . 16A,B), s u b t r i g e m i n a l (ST) and TTD r e g i o n s and t h e c a u d a l n u c l e u s r e t i c u l a r i s m e d u l l a r i s , p a r s v e n t r a l i s (Cnv) ( F i g . 16C) . In t h r e e cases (1 c e r v i c a l , 2 l u m b a r ) , l a b e l l e d neurones were l o c a t e d i n c l o s e p r o x i m i t y t o t h e e l e c t r o l y t i c l e s i o n s m arking t h e e f f e c t i v e l o c o m o t o r s t i m u l a t i o n s i t e s , i n t h e Cnd, Cnv and Rpc ( F i g s . 17A,B) . The c l o s e s t TB c e l l t o t h e c e n t r e o f an e l e c t r o l y t i c l e s i o n was 75um i n t h e Cnd a f t e r a c e r v i c a l i n j e c t i o n ( F i g . 17C). I n o t h e r e x p e r i m e n t s t h e y were w i t h i n 0.5 mm; however i n one b i r d t h e l e s i o n w i t h i n t h e Cnv was c a u d a l t o t h e l e v e l o f TB neurones. 97 Right leg (flexion up) - / W W W W W W W V \ ^ ^ Left leg (flexion up) Right ITC (hip flexor) Left ITC (hip flexor) ' 5 sec off (75 uA, 50 Hz) J Figure 15. Potentiometer and electromyographic records of e l e c t r i c a l l y s t i m u l a t e d evoked walking i n the decerebrate goose. The s t i m u l a t i o n s i t e was w i t h i n the Cnd and the alternate f l e x i o n and extension movements of the hind limbs and a l t e r n a t i n g a c t i v i t y of the i l i o t i b i a l i s c r a n i a l i s ( ITC) muscles was e l i c i t e d at a stimulating current strength of 75 uA. 98 SSP Figure 16. Photomicrographs showing e l e c t r o l y t i c lesions (*) marking evoked locomotion-stimulation s i t e s i n the nucleus r e t i c u l a r i s medullaris, pars dorsalis (Cnd) (A,B) and the nucleus r e t i c u l a r i s m e d u l l a r i s , pars v e n t r a l i s (Cnv) (C). A b b r e v i a t i o n s : X, d o r s a l nucleus of the vagus nerve; SSP supraspinal nucleus; TTD, nucleus and tr a c t of the descending trigeminal nerve. Scale bars: 100 um. 9 9 Figure 17. A: Diagram of the caudal medulla in d i c a t i n g the relationship of locomotor stimulation s i t e s to the d i s t r i b u t i o n of r e t r o g r a d e l y l a b e l l e d neurones f o l l o w i n g a s p i n a l cord i n j e c t i o n of True Blue (TB) . Each large f i l l e d c i r c l e (•) represents a stimulation s i t e from which locomotion could be evoked i n the decerebrate duck or goose. Each small open c i r c l e (o) represents 5 T B - l a b e l l e d neurones a f t e r a c e r v i c a l i n j e c t i o n . Each f i l l e d t r i a n g l e (•) represents 2 TB-labelled neurones after a lumbar i n j e c t i o n . B: Photomicrograph of TB-l a b e l l e d neurones w i t h i n Cnd i n c l o s e proximity to an e l e c t r o l y t i c lesion (*), which marks a locomotor stimulation s i t e within the TTD/Cnd border zone. 100 DISCUSSION The present study has confirmed that discrete e l e c t r i c a l stimulation of the pontomedullary r e t i c u l a r formation can evoke walking and f l y i n g i n decerebrate birds (Steeves et a l . , 1987) . Furthermore the location of retrogradely l a b e l l e d TB somata i n close proximity to the e l e c t r o l y t i c lesions marking the stimulation s i t e s demonstrates that d i r e c t projections to the s p i n a l cord a r i s e from these r e g i o n s . Although only stimulation s i t e s within the caudal medulla were i d e n t i f i e d i n the present small sample of experiments, previous experiments have shown that e f f e c t i v e stimulation s i t e s range from the caudal medulla to the l e v e l of the red nucleus (Steeves et a l . , 1987; Sholomenko, i n preparation). A technical consideration i s whether the l a b e l l e d neurones were within the e f f e c t i v e range of the stimulus at the current i n t e n s i t i e s used i n t h i s study. E f f e c t i v e current spread has been shown to approximate 5.0 um/uA in s i m i l a r studies (Steeves et a l . , 1975), and 0.5 mm i s generally accepted as the radius for i d e n t i f i c a t i o n of putative responsive neurones, using a stimulation strength of less than 100 uA (Ga r c i a - R i l l et a l . , 1983b). The observed distances i n t h i s study indicate that the TB-labelled c e l l s were indeed within e f f e c t i v e range (75-500 um) of e l e c t r i c a l current spread from the t i p of the focal stimulating electrode. Stimulation s i t e s within the medial medullary r e t i c u l a r formation and the region of Cnd bordering the TTD, suggest that both these regions may form d i r e c t descending pathways which i n i t i a t e locomotion by a c t i v a t i n g spinal neuronal networks. 101 These results are similar to those reported for a wide variety of v e r t e b r a t e s i n c l u d i n g lamprey (McClellan and G r i l l n e r , 1984), stingray (Leonard et a l . 1979), t u r t l e (Kazennikov et a l . , 1981), and cat (Mori et a l . , 1978; Shik and Yagodnitsyn, 1977) . Thus there appears to be two d i s t i n c t locomotor regions w i t h i n the caudal r e t i c u l a r formation: the ventromedial gigantocellular and caudal medullary r e t i c u l a r formation and the more d o r s o l a t e r a l TTD/Cnd/Rpc r e g i o n . Both of these regions have been implicated i n the i n i t i a t i o n of locomotion in the cat (Shik et a l . , 1966; Shik and Yagodnitsyn, 1977). The present study has confirmed previous findings that both regions evoke f l y i n g and walking i n the b i r d (Steeves and Weinstein, 1984; Steeves et a l . , 1987). Other studies have shown that f l y i n g can s t i l l be evoked at the same stimulation current strength, af t e r acute thoracic transection i n a decerebrate b i r d (Sholomenko and Steeves, 1987). Therefore, i t i s probable that there i s no d i s t i n c t anatomical separation of the descending pathways that a c t i v a t e the c e r v i c a l versus the lumbar pattern generators. Furthermore, since axons from the medial medulla (Cnv, Rgc) and the dorsolateral Cnd, project to the lumbar cord while those of the Rpc and adjacent TTD do not (Webster and Steeves, 1988), d i r e c t and in d i r e c t descending pathways must be involved. In order to determine the descending inputs to these regions the afferent connections w i l l now be examined. 102 VI DESCENDING AFFERENT PROJECTIONS PONTOMEDULLARY LOCOMOTOR REGIONS 103 INTRODUCTION Both ventromedial and d o r s o l a t e r a l pontomedullary regions have been i d e n t i f i e d as c o n t r i b u t i n g to s p i n a l p r o j e c t i o n s i n v o l v e d i n the i n i t i a t i o n of locomotion i n b i r d s and mammals. In b i r d s , t he v e n t r o m e d i a l r e g i o n c o n s i s t s of the n u c l e u s c e n t r a l i s m e d u l l a r i s , pars v e n t r a l i s (Cnv) and 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 (Rgc). The d o r s o l a t e r a l region i n c l u d e s : l a t e r a l l y , the nucleus and t r a c t of the descending t r i g e m i n a l nerve (TTD); m e d i a l l y , the n u c l e u s c e n t r a l i s m e d u l l a r i s , pars d o r s a l i s (Cnd), and the more r o s t r a l nucleus 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); v e n t r a l l y , the n u c l e u s r e t i c u l a r i s l a t e r a l i s (RL), and subtrigeminal nucleus (ST). A l l of these n u c l e i and t h e i r analogues i n d i f f e r e n t species, have been shown t o p r o j e c t d i r e c t l y to the s p i n a l cord i n r e p t i l e s (ten Donkelaar, et a l . , 1980), b i r d s (Cabot et a l . , 1982; Arends et a l . , 1984; Webster and Steeves, 1988), and mammals (Crutcher et a l . , 1978; Tohyama et a l . , 1979a,b; Zemlan and P f a f f , 1979; Matsushita et a l . , 1981). E l e c t r i c a l s t i m u l a t i o n o f bot h the v e n t r o m e d i a l and d o r s o l a t e r a l p o n t o m e d u l l a r y r e g i o n s e l i c i t s l o c o m o t i o n i n decerebrate t u r t l e s (Kazennikov et a l . , 1981), b i r d s (Steeves et a l . , 1986; 1987; Sholomenko et a l . , i n p r e p a r a t i o n ) , and mammals (Mori et a l . , 1977; G a r c i a - R i l l and Skinner, 1987a; Noga et a l . , 1988). Furthermore, s i n c e 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 n t o b o t h v e n t r o m e d i a l and d o r s o l a t e r a l p o n t o m e d u l l a r y r e g i o n s a l s o evokes locomotion, neuronal somata, and not axons of passage, are probably being a c t i v a t e d by the s t i m u l a t i o n procedures 104 ( G a r c i a - R i l l et a l . , 1983a,b; 1984, 1986; Noga et a l . , 1988; Sholomenko et a l . , i n preparation). Studies i n mammals have shown that projections to the Rgc and magnocellular r e t i c u l a r nucleus (Rmc) are derived from the c e r e b r a l cortex (Keizer and Kuypers, 1984), the s t r i a terminalis, the amygdala (Luppi et a l . , 1988), the hypothalamus (Abols and Basbaum, 1981; Luppi et a l . , 1988), the mesencephalic r e t i c u l a r formation (Gallager and Pert, 1978; Vertes, 1988; Luppi et a l . , 1988), the central gray (PAG) (Gallager and Pert, 1978; Chung et a l . , 1983; Luppi et a l . , 1988), the cuneiform (Edwards, 1975; Chung et a l . , 1983) and subcuneiform regions (Abols and Basbaum, 1981), the dorsal (Luppi et a l . , 1988) and ventral tegmentum (Gallager and Pert, 1978), the superior c o l l i c u l u s (Gallager and Pert, 1978), the pedunculopontine nucleus (PPN) (Moon-Edley and Graybiel, 1983), the vestibular nuclei, the cerebellum, (Abols and Basbaum, 1981; Peterson, 1984), the pontine r e t i c u l a r formation (Brodal, 1958; Gallager and Pert, 1978; Abols and Basbaum, 1981; Vertes, 1988; Luppi et a l . , 1988), the caudal raphe and the caudal medullary r e t i c u l a r formation (nucleus ventralis) (Luppi et a l . , 1988). These projections have been reported for the cat (Abols and Basbaum, 1981; Luppi et a l . , 1988) and rat (Gallager and Pert, 1978; Vertes, 1988). Only midbrain efferents have been described for monkey (Chung et a l . , 1983). In the l i z a r d , projections to the caudal r e t i c u l a r formation (the nucleus r e t i c u l a r i s i n f e r i o r ) from the midbrain arise w i t h i n the d o r s a l thalamus, the p o s t e r i o r entopeduncular 105 nucleus, the posterior commissure, the substantia nigra, the torus semicircularis ( i n f e r i o r c o l l i c u l u s ) , and the nucleus i n t e r c o l l i c u l a r i s (ICo) (ten Donkelaar and De Boer-van Huizen, 1981a). Projections from the p h y s i o l o g i c a l l y defined mesencephalic locomotor region (MLR) to the medullary r e t i c u l a r formation have also been described for the cat (Gar c i a - R i l l et a l . , 1983a; 1987b; Steeves and Jordan, 1984; Bayev et a l . , 1988) and rat (G a r c i a - R i l l et a l . , 1986). Opinions have v a r i e d with regard to the anatomical substrate of the p h y s i o l o g i c a l l y defined pontobulbar locomotor s t r i p (PLS), located within the l a t e r a l medulla and pons (Mori et a l . , 1977). Different studies have suggested that the PLS consists of f i b r e s emmanating from the mesencephalic trigeminal nucleus (Vmes, G a r c i a - R i l l et a l . , 1983b), or Rpc (Selionov and Shik, 1984; Shik, 1985), or TTD (Baev et a l . , 1987; Noga et a l . , 1988). Only recently have sparse projections from the PLS to the medial r e t i c u l a r formation been demonstrated (Bayev et a l . , 1988). In the b i r d , extensive s t u d i e s have d e f i n e d the t r i g e m i n a l subnuclei as w e l l as the a f f e r e n t (Arends and Dubbledam, 1984) and e f f e r e n t connections (Arends et a l . , 1984) . Similar studies with regard to the medial r e t i c u l a r formation are l a c k i n g . The purpose of t h i s study was to i d e n t i f y the a f f e r e n t p r o j e c t i o n s to the ventromedial medullary r e t i c u l a r formation, and the dorsolateral TTD region (including the subjacent Cnd, RL, ST and Rpc), i n birds using a variety of retrograde tracers. 106 MATERIALS AND METHODS Sixteen b i r d s (11 Pekin ducks, Anas platyrhynchos; 1 Canada goose, Branta canadensis; 2 Sulphur-crested cockatoos, Cacatua galerita; and 2 Eastern r o s e l l a s , Platycercus eximius), of both sexes, were used i n t h i s study. The b a s i c procedure i n v o l v e d i n j e c t i o n of small q u a n t i t i e s of f l u o r e s c e n t dye i n t o e i t h e r the ventromedial (n=8) or d o r s o l a t e r a l (n=9) medullary-pontine regions at v a r i o u s r o s t r o c a u d a l l e v e l s . I n j e c t i o n s were made u s i n g s t e r e o t a x i c c o o r d i n a t e s a v a i l a b l e f o r the duck (Zweers, 1971) and goose (Sholomenko, personal communication), w i t h the head and the beak f i x e d i n a h o r i z o n t a l p o s i t i o n i n a s t e r e o t a x i c frame. In t h i s p o s i t i o n ( F i g . 18A), s e c t i o n s cut i n the s t e r e o t a x i c plane are r o t a t e d about a d o r s o r o s t r a l - v e n t r o c a u d a l a x i s (Zweers, 1971) when compared t o the s e c t i o n s d e s c r i b e d i n o t h e r a v i a n a t l a s e s (Karten and Hodos, 1967; van Tienhoven and Juhasz, 1962; Stokes et a l . , 1974); t h e r e f o r e i n t h i s study, as i n previous s t u d i e s (Webster and Steeves, 1988; Chs. II-V of t h i s t h e s i s ) , the b r a i n s were sectioned t r a n v e r s e l y , across the long a x i s ( F i g . 18B). As a consequence most brainstem s e c t i o n s resembled the o u t l i n e s of K a r t e n and Hodos (1967) from medulla t o the hypothalamus but the telencephalon i s cut on a more dorsocaudal plane i n comparison to the pigeon (Karten and Hodos, 1967). In a few cases the i n j e c t i o n needle was i n c l i n e d c a u d a l l y (25-40° to the s t e r e o t a x i c p l a n e ) , so t h a t the s t r u c t u r e s t r a v e r s e d by the i n j e c t i o n t r a c k were v a r i e d . In these cases, the plane of i n j e c t i o n approximated the usual plane of s e c t i o n . In other 1 0 7 cases t h e head was t i l t e d v e n t r a l l y 40° from t h e h o r i z o n t a l , and t h e i n j e c t i o n was made w i t h a v e r t i c a l approach; t h e b r a i n was s e c t i o n e d i n t h e same p l a n e . The b i r d s were a n a e s t h e t i z e d w i t h e i t h e r n i t r o u s o x i d e and Haloth a n e (duck and goose) o r a m i x t u r e o f Ketamine and Rompun ( p a r r o t s ) . A l o c a l a n a e s t h e t i c was i n f i l t r a t e d i n t o p r e s s u r e p o i n t s and s k i n i n c i s i o n edges. The s k i n was i n c i s e d , and a s m a l l r e g i o n o f t h e cran i u m was removed. E i t h e r True B l u e (TB, 5%), F a s t B l u e (FB, 2 % ) , D i a m i d i n o Y e l l o w d i h y d r o c h l o r i d e (DY, 2 % ) , f l u o r e s c e i n - c o n j u g a t e d d e x t r a n amines (FDA, 25%) o r rhodamine-conjugated d e x t r a n amines (RDA, 25%) was i n j e c t e d u n i l a t e r a l l y (0.1 t o 0.2 u l ) t h r o u g h a 1.0 u l H a m i l t o n s y r i n g e . A f t e r a s u r v i v a l p e r i o d o f 3 t o 8 days, each b i r d was a n a e s t h e t i z e d w i t h sodium p e n t o b a r b i t a l ( N e b u t a l , 75 mg/Kg) and p e r f u s e d w i t h one l i t r e o f s a l i n e ( 0 . 9 % ) , f o l l o w e d by one l i t r e o f p a r a f o r m a l d e h y d e i n 0.1 M phosphate b u f f e r (4%, pH 7.4). The b r a i n was d i s s e c t e d , p o s t - f i x e d f o r a f u r t h e r 12 h o u r s , and immersed i n a c r y o p r o t e c t a n t f o r 3 days. The b r a i n s were s e c t i o n e d a t 30 um, e v e r y t h i r d s e c t i o n was mounted o n t o g e l a t i n i z e d s l i d e s and a i r d r i e d i n t h e da r k . Each s e c t i o n was examined w i t h a f l u o r e s c e n t m i c r o s c o p e u s i n g t h e a p p r o p r i a t e f i l t e r s and t h e l o c a t i o n s o f 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 were drawn on o u t l i n e s o f b r a i n s t e m s e c t i o n s . RESULTS The i n j e c t i o n s o f t h e v e n t r o m e d i a l r e t i c u l a r f o r m a t i o n ( F i g s . 19B, 20D, 21A-C) extended from t h e c a u d a l m e d u l l a t o t h e c a u d a l n u c l e u s r e t i c u l a r i s p o n t i s c a u d a l i s (RP) . They were c l e a r l y s e p a r a t e from t h e i n j e c t i o n s i n t o t h e d o r s o l a t e r a l 108 region (Figs. 22B, 23D,) which ranged i n rostrocaudal extent from the obex to the l e v e l of the p r i n c i p a l sensory nucleus (PrV) . The d i s t r i b u t i o n of l a b e l l e d c e l l s was similar i n a l l species studied, but the number of r e t r o g r a d e l y l a b e l l e d neurones appeared greater i n the parrot than the duck. Afferents to the ventromedial r e t i c u l a r formation Injections into the ventromedial r e t i c u l a r formation were l o c a t e d c a u d a l l y w i t h i n the nucleus Cnv (n=5), and more r o s t r a l l y within the Rgc, or the caudal RP (n=3) . The d i s t r i b u t i o n of retrogradely l a b e l l e d neurones was e s s e n t i a l l y the same following a Cnv (Fig. 19) or Rgc (Fig. 20) i n j e c t i o n , but the numbers of l a b e l l e d c e l l s were less a f t e r an i n j e c t i o n into the most caudal Cnv or the caudal RP; however there were a few differences between in d i v i d u a l cases, which may r e f l e c t differences i n the spread of i n j e c t i o n or uptake of dye from fibres traversing the i n j e c t i o n track. For example a 0.2 u l TB in j e c t i o n made with a v e r t i c a l approach encroached s l i g h t l y upon the caudal i n f e r i o r o l i v e (10) i n b i r d D37 (Figs. 19A-J). In b i r d PI (Figs. 20A-M) , a TB i n j e c t i o n (0.2 um) directed into the Rgc, at a dorsocaudal angle, involved the nucleus of the s o l i t a r y t r a c t (nTS), dorsal motor nucleus of the vagus (X), and the hypoglossal nucleus (XII) within the i n j e c t i o n track. Medulla-pons: Retrogradely l a b e l l e d c e l l s (5-20/section) were found b i l a t e r a l l y within the dorsal column nuclei (GC) i n most birds after either Cnv, Rgc or RP injections (Fig. 19A). The external cuneate nucleus (CE) was l a b e l l e d primarily a f t e r 1 0 9 A Figure 18. Schematic diagrams to i l u s t r a t e the orientation of the duck brain: (A) Zweers (1971) stereotaxic p o s i t i o n ; (B) p o s i t i o n for sectioning. A-M indicates approximate l e v e l of the coronal sections depicted i n F i g . 20. 110 Figure 19. The d i s t r i b u t i o n of TB-labelled c e l l s within the duck brainstem following i n j e c t i o n of TB into the Cnv. Diagrams A-J depict coronal sections i n a caudal to r o s t r a l d i r e c t i o n . Each f i l l e d c i r c l e (0) represents 5 c e l l s . Stippled region represents the i n j e c t i o n s i t e . Similar r e s u l t s were found i n the 3 species studied i l l 112 Figure 20. The d i s t r i b u t i o n of TB-labelled c e l l s within the p a r r o t brainstem f o l l o w i n g i n j e c t i o n of TB i n t o the Rgc (indicated by s t i p p l e d region). Diagrams A-M depict coronal sections i n a caudal to r o s t r a l d i r e c t i o n . Each f i l l e d c i r c l e (0) represents 5 c e l l s . 113 114 115 F i g u r e 21. Photomicrographs of Cnv ( A , B ) and Rgc (C) i n j e c t i o n s i t e s and retrogradely l a b e l l e d neurones following Cnv or Rgc i n j e c t i o n (D-G). D: I p s i l a t e r a l nucleus r e t i c u l a r i s c e n t r a l i s , pars v e n t r a l i s (Cnv) and s u b t r i g e m i n a l nucleus (ST); E: I p s i l a t e r a l medial mesencephalic r e t i c u l a r formation, FRM; F: Contralateral 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); G: I p s i l a t e r a l l a t e r a l mesencephalic r e t i c u l a r formation (FRL); Scale bars: 100 um. 116 1 1 7 i n j e c t i o n a t o r r o s t r a l t o t h e obex, p r e d o m i n a n t l y on t h e c o n t r a l a t e r a l s i d e . S i m i l a r l y t h e l a b e l l i n g w i t h i n t h e nTS and v e n t r a l t o X ( t h e n u c l e u s a l a t u s , A l a ) was f o u n d more c o n s i s t e n t l y a f t e r an i n j e c t i o n r o s t r a l t o t h e obex and was a l s o most predominant c o n t r a l a t e r a l l y ( 1 0 - 2 0 / s e c t i o n ) . As n o t e d above f o r P I , some l a b e l l i n g w i t h i n t h e i p s i l a t e r a l nTS appeared t o r e s u l t from damaged f i b r e s w i t h i n t h e i n j e c t i o n t r a c k ( F i g . 20B). R e t r o g r a d e l y l a b e l l e d T B - c e l l s ( 5 - 2 0 / s e c t i o n / s i d e ) were found b i l a t e r a l l y w i t h i n t h e Cnv and Rgc ( F i g s . 19A-C, 20A-F, 21D,F) as w e l l as i n t h e more r o s t r a l corpus t r a p e z o i d i u m (Ctz) and RP ( F i g s . 19D,E, 20G,H). L a b e l l e d c e l l s (5-10/side) were found d o r s o l a t e r a l l y w i t h i n t h e Cnd, p r e d o m i n a n t l y on t h e c o n t r a l a t e r a l s i d e , a f t e r i n j e c t i o n i n t o t h e Cnv o r t h e Rgc; l a b e l l i n g was more p r o m i n e n t a f t e r a r o s t r a l t h a n c a u d a l i n j e c t i o n . B i l a t e r a l l a b e l l i n g was found w i t h i n t h e Rpc a f t e r an Rgc i n j e c t i o n ( F i g s . 20D,E), but i t was s p a r s e a f t e r an i n j e c t i o n i n t o t h e Cnv. L a b e l l e d neurones were p r e s e n t w i t h i n t h e RL and t h e ST b i l a t e r a l l y ( F i g s . 19C, 20C,D). A few l a b e l l e d c e l l s were found i n t h e n u c l e u s g i g a n t o c e l l u l a r i s l a t e r a l i s (PGL) a f t e r r o s t r a l i n j e c t i o n i n t o t h e Rgc, but t h e y were r a r e l y o b s e r v e d f o l l o w i n g i n j e c t i o n i n t o t h e Cnv. R e t r o g r a d e l y l a b e l l e d neurones were found w i t h i n t h e c a u d a l s u b n u c l e u s 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 t r a c t (TTDc), p r e d o m i n a n t l y on t h e c o n t r a l a t e r a l s i d e a f t e r i n j e c t i o n i n t o t h e Rgc or RP; however, t h e TTD was l a b e l l e d i n o n l y t h r e e out o f t h e f i v e Cnv i n j e c t e d b i r d s . Moving r o s t r a l l y , many l a b e l l e d neurones ( 1 0 - 2 0 / s e c t i o n ) 118 were located within the nucleus r e t i c u l a r i s pontis caudalis, pars g i g a n t o c e l l u l a r i s (RPgc), predominantly on the i p s i l a t e r a l side a f t e r an Rgc i n j e c t i o n , but less l a b e l l i n g was evident afte r a Cnv i n j e c t i o n (Figs. 19F, 20J). There were only a few l a b e l l e d c e l l s (< 5/section) i n the nucleus r e t i c u l a r i s pontis o r a l i s (RPO). Retrogradely l a b e l l e d neurones were located within the l a t e r a l vestibular nucleus (VeL) i p s i l a t e r a l l y (10-20/section), and o c c a s i o n a l l y c o n t r a l a t e r a l l y . The c o n t r a l a t e r a l medial vestibular nucleus (VeM) was l a b e l l e d i n three birds, while l a b e l l e d c e l l s within the descending vestibular nucleus (VeD) were only found i n one b i r d (PI). Labelled somata (20-30/section) were present within the cont r a l a t e r a l i n t e r n a l cerebellar nucleus (Cbl) (Figs. 19E, 20F,G). In birds i n which the i n j e c t i o n encroached upon the 10, l a b e l l e d neurones were found within the contralateral 10 and b i l a t e r a l l y within the paramedian nucleus. TB-labelled neurones were found b i l a t e r a l l y within the dorsal subcoeruleus nucleus (Scd) (Figs. 19F, 201), but only a few i n the ventral subcoeruleus (Scv). Labelled c e l l s were present b i l a t e r a l l y within the locus coeruleus (LoC) . These were smaller (15-20 um) than the LoC-spinal neurones and some were located dorsally and r o s t r a l l y (Fig. 19F) . In addition, s e v e r a l l a b e l l e d neurones were found bordering the medial longitudinal fasciculus (MLF) on the contralateral side, i n a region designated the ventral tegmental area of Gudden (Karten and Hodos, 1967), but only i n b i r d P4. A few retrogradely 119 l a b e l l e d neurones were also found dorsal to the f a c i a l nucleus (VII) and i n the pe r i b r a c h i a l region i n close proximity to the fib r e s of the mesencephalic trigeminal nucleus. Mesencephalon-diencephalon: Retrogradely l a b e l l e d neurones were found b i l a t e r a l l y within the ICo (20-30/section) (Figs. 19G,H, 20I-K). TB-labelled neurones (approximately 10/section) were also present within the medial (FRM, Figs. 19H, 20K,L, 21E) and l a t e r a l mesencephalic r e t i c u l a r formation (FRL, Figs. 19G, 20H, 21G) . A few c e l l s (5/section) were l a b e l l e d i n the i p s i l a t e r a l i n t e r s t i t i a l nucleus (IS), while b i l a t e r a l l a b e l l i n g was found i n the nucleus of the posterior commissure (PC, Figs. 19H, 20L). A few l a b e l l e d somata were present within the i p s i l a t e r a l red nucleus (Ru) i n three birds (Fig. 19H) ; however, there were many i n b i r d PI and i n addition the ventral tegmental area (AVT) was also heavily l a b e l l e d . At a s l i g h t l y more r o s t r a l l e v e l of the mesencephalon, a few l a b e l l e d neurones were found l a t e r a l to the occipitomesencephalic t r a c t (OM). These c e l l s formed a narrow column and were orientated i n dorsal to ventral d i r e c t i o n . In D37 these c e l l s were located v e n t r a l l y , l a t e r a l to the ansa l e n t i c u l a r i s (AL, F i g . 191)., but i n other cases they were more dorsal, subjacent to the subrotundus nucleus (Srt). T B - l a b e l l e d neurones were found w i t h i n the l a t e r a l hypothalamic nucleus (LH) , the stratum c e l l u l a r e externum (SCE) (Fig. 191),201) the nucleus p e r i v e n t r i c u l a r i s hypothalmi (PVH), nucleus p a r a v e n t r i c u l a r i s (PVM, F i g s . 19J, 20M), and the suprachiasmatic nucleus (SCN) . In addition, but i n only one b i r d (PI), a few l a b e l l e d neurones were present within the 120 i p s i l a t e r a l p o s t e r i o r d o r s o l a t e r a l n u c l e u s of the thalamus (DPL) . Telencephalon: In t h r e e b i r d s (D83, PI and P4) , 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 ( 2 0 - 30/section) were found b o r d e r i n g the p a l e o s t r i a t u m augmentatum (PA), w i t h i n the intermediate a r c h i s t r i a t u m ( A i ) , (Figs. 19J, 20N) . These were predominantly i p s i l a t e r a l but were found on both sides i n b i r d P I . L a b e l l e d neurones (20-30/section) were a l s o evident w i t h i n the nucleus accumbens (Ac, F i g s . 19J, 20M)), or bed nucleus of the s t r i a t e r m i n a l i s as i t i s termed i n more recent s t u d i e s (Reiner et a l . , 1983). No l a b e l l e d c e l l s were found w i t h i n the d o r s a l telencephalon (the wulst) a f t e r ventromedial i n j e c t i o n s i n t o the pontomedullary r e t i c u l a r formation. A f f e r e n t s to the D o r s o l a t e r a l TTD/Reticular Region Most of the i n j e c t i o n s i n t o the d o r s o l a t e r a l medulla and pons were at the l e v e l s of the i n f e r i o r o l i v e and v e s t i b u l a r n u c l e i . They i n c l u d e d the i n t e r p o l a r i s subnucleus of the TTD (TTDi) and Rpc region ( F i g . 23D) and the more v e n t r a l ST/RL region ( F i g . 23C). Figure 22 d e p i c t s the r e s u l t s from b i r d D36 which r e c e i v e d an extensive i n j e c t i o n i n t o the TTDi/Rpc region ( F i g . 22A). Medulla-pons: 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 were found b i l a t e r a l l y w i t h i n the CE and GC a f t e r i n j e c t i o n i n t o the TTD and Rpc. L a b e l l e d c e l l s were present b i l a t e r a l l y w i t h i n the nTS a f t e r a c a u d a l i n j e c t i o n , but were found o n l y on the i p s i l a t e r a l s i de when the i n j e c t i o n was l o c a t e d i n the r o s t r a l medulla and pons. There was no evidence of a p r o j e c t i o n from 121 Figure 22. The d i s t r i b u t i o n of retrogradely l a b e l l e d neurones i n the duck brainstem f o l l o w i n g i n j e c t i o n of TB i n t o the intermediate t r i g e m i n a l r e g i o n (TTDi). Diagrams A - J d e p i c t tranverse sections i n a caudal to r o s t r a l sequence. Stippled regions indicate i n j e c t i o n s i t e (C,D). Each f i l l e d c i r c l e (•) represents 5 l a b e l l e d soma. 122 123 Figure 23. Photomicrographs of retrogradely l a b e l l e d neurones (A,B, E/F) following i n j e c t i o n into the d orsolateral region of the medulla. A: I p s i l a t e r a l nucleus r e t i c u l a r i s c e n t r a l i s , pars d o r s a l i s (Cnd); B: I p s i l a t e r a l external caudate nucleus (CE); E. I p s i l a t e r a l 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); F. I p s i l a t e r a l nucleus i n t e r c o l l i c u l a r i s (ICo). Ventrolateral (C) and d orsolateral (D) i n j e c t i o n s i t e s i n the subtrigeminal nucleus (ST) and descending trigeminal nucleus (TTD)/ dorsal r e t i c u l a r region (Rpc) region. Scale bars: 100 um. 124 125 the CE, GC, or nTS to the RL region. Injections into the TTDi resulted i n sparse retrograde l a b e l l i n g w i t h i n the caudal TTD nucleus (TTTc) and the c o n t r a l a t e r a l TTDi. S i m i l a r l y an i n j e c t i o n i n t o the TTDo l a b e l l e d only a few c e l l s i n the TTDc. I f there were any p r o j e c t i o n s from TTDo to TTDi, they were masked by the rostrocaudal extent of the i n j e c t i o n s i t e . The Cnd and/or the Rpc were l a b e l l e d d i r e c t l y from an i n j e c t i o n i n t o the d o r s o l a t e r a l r e g i o n . In most cases the c o n t r a l a t e r a l Cnd and Rpc were also l a b e l l e d . There was evidence of r e c i p r o c a l 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 pro-jections between the RL and Rpc. B i l a t e r a l l a b e l l i n g was also found within the PGL a f t e r an i n j e c t i o n into the Rpc or RL. Input to the dorsolateral region from the ventromedial medulla originated from neurones (5-10/side/section) i n the Cnv and the Rgc on both sides. In comparison, there were few p r o j e c t i o n s from the r o s t r a l r e t i c u l a r formation, as only i s o l a t e d l a b e l l e d neurones were found i n the RP, RPgc or RPO. There was b i l a t e r a l l a b e l l i n g w i t h i n the VeM (5-10 cells/section) . The VeD was also l a b e l l e d b i l a t e r a l l y , but usually when the i n j e c t i o n s i t e encroached into the VeD. The 10, paramedian nucleus and Cbl were l a b e l l e d only i n two cases. A few l a b e l l e d neurones (5/section) were present within the i p s i l a t e r a l and/or contr a l a t e r a l LoC, Scd. Labelled c e l l s were also found dorsolateral to the OM i n the pe r i b r a c h i a l region. In b i r d D36, l a b e l l e d c e l l s were also present within the i p s i l a t e r a l ventral nucleus of the l a t e r a l lemniscus (Vlv), and the l a t e r a l lemniscus (LL). 126 Mesencephalon-diencephalon: Many l a b e l l e d neurones were present w i t h i n the i p s i l a t e r a l ICo. The FRL, however was la b e l l e d i p s i l a t e r a l l y i n only one b i r d . The contra l a t e r a l Ru was l a b e l l e d following i n j e c t i o n into the RL region presumably as a consequence of involvement of the rubrospinal t r a c t . In addition, the i p s i l a t e r a l Ru was l a b e l l e d i n one bi r d , which may r e f l e c t t r a c e r uptake by r u b r o - o l i v e r y p r o j e c t i o n s . Retrogradely l a b e l l e d neurones were present w i t h i n the IS b i l a t e r a l l y i n a few cases. B i l a t e r a l l a b e l l i n g was present i n the PC i n only one b i r d . After an i n j e c t i o n into the caudal TTD or RL, there was evidence of a projection from the SCE and PVM, but t h i s was not present i n a l l cases. In addition, l a b e l l e d c e l l s were found l a t e r a l to the OM on the i p s i l a t e r a l side i n three bir d s . Telencephalon: Labelled neurones were located within the Ai b i l a t e r a l l y (10-20/side/section) after i n j e c t i o n into the TTD/Rpc, but not when the i n j e c t i o n was centred i n the RL. There was some l a b e l l i n g i n the Ac but only i n one b i r d . DISCUSSION The major findings of t h i s study were: (1) that i n the avian species examined (Pekin duck, Canada goose, Sulphur-crested cockatoo and Eastern r o s e l l a ) , the medial medullary r e t i c u l a r formation receives the majority of i t s afferent input from other regions of the r e t i c u l a r formation, as p r e v i o u s l y d e s c r i b e d i n other v e r t e b r a t e species (Brodal, 1958); (2) that the l a t e r a l r e t i c u l a r region r e c e i v e d c o n s i d e r a b l y l e s s a f f e r e n t input compared to the medial 127 r e t i c u l a r region; (3) That the TTD projected medially into the Gnd and |\ge; and (4) that the most prominent input from the mesencephalon was from the ICo which projected to both the medial r e t i c u l a r formation and the TTD region; and (5) there were no p r o j e c t i o n s from the r o s t r a l telencephalon to the medulla. There are a number of technical considerations that need to be addressed. F i r s t , the size of the i n j e c t i o n (0.2 ul) was r e l a t i v e l y l a r g e . Nevertheless the medial and l a t e r a l i njections were confined to t h e i r respective regions. Although i t i s p o s s i b l e that f u r t h e r d i f f u s i o n occurred p r i o r to s a c r i f i c e , the results are comparable, i n many respects, to those of mammalian studies i n which smaller injections were used (Gallager and Pert, 1978). Second, there was d i r e c t involvement of dorsal structures (e.g. GC, vestibular) and ventral structures (10) i n the i n j e c t i o n i n some birds . Since the results were sim i l a r even when the angle of i n j e c t i o n was v a r i e d , the i n c l u s i o n of d o r s a l n u c l e i d i d not appear to produce spurious l a b e l l i n g . F i n a l l y , retrograde l a b e l l i n g may be a consequence of the tracer being incorporated by axons traversing the i n j e c t i o n s i t e as well as those terminating therein. The d i s t r i b u t i o n of l a b e l l e d c e l l s was similar, but not i d e n t i c a l , to the d i s t r i b u t i o n after a c e r v i c a l i n j e c t i o n . This indicates that d i f f e r e n t i a l l a b e l l i n g was obtained and that l a b e l l i n g was not solely due to incorporation of the marker by axons en route to the spinal cord. Nevertheless, the present study only represents an i n i t i a l survey and the results require confimation using other techniques. 128 In t h i s discussion the present results are discussed i n r e l a t i o n to findings from previous avian or other vertebrate s t u d i e s and then the f u n c t i o n a l i m p l i c a t i o n s of the connections of these "locomotor" regions are considered. The present results show that the origins of projections to Cnv and Rgc are similar, although those to the Rgc appeared to be more profuse; therefore they w i l l be considered as a whole, since comparable data are available for the mammalian Rgc, but l i t t l e or no data for the analogue of the Cnv, the nucleus v e n t r a l i s . Reticulo-reticulari In the present study the d i s t r i b u t i o n of retrogradely l a b e l l e d neurones following i n j e c t i o n into the Cnv or Rgc showed that both regions r e c e i v e d extensive b i l a t e r a l projections from each other, as well as from the more r o s t r a l pontine and mesencephalic r e t i c u l a r formation (MRF). This pattern of mutual connectivity between components of the medial r e t i c u l a r formation has been demonstrated i n degenerative tracing studies (Brodal, 1958; Petras, 1967) and more recently by retrograde (Gallager and Pert, 1978; Abols and Basbaum, 1981; Vertes, 1988) and anterograde (Jones and Yang, 1985; Vertes, 1988) axonal t r a n s p o r t t r a c i n g techniques. Afferent projections to the Rgc from the nucleus v e n t r a l i s (rat: Vertes, 1988; cat: Petras, 1967) and nucleus dorsalis (Vertes, 1988), the contralateral Rgc (rat: Gallager and Pert, 1978; Jones and Yang, 1985; cat: Abols and Basbaum, 1981), the RP, RPO and MRF b i l a t e r a l l y (rat: Gallager and Pert, 1978; Jones and Yang, 1985; Vertes, 1988; cat: Abols and Basbaum, 1981) have been described previously. The present findings 129 confirm the presence of r e t i c u l o - r e t i c u l a r projections i n the b i r d . Only the ascending connections of the i n f e r i o r r e t i c u l a r region appear to have been studied i n r e p t i l e s (ten Donkelaar and De Boer-van Huizen, 1981b). The present data indicate that the Rgc and Cnv receive a f f e r e n t p r o j e c t i o n s from the RL and the Rpc. Comparable projections to the Rgc have been reported from the RL i n the cat (Petras, 1967) and the Rpc i n the rat (Vertes, 1988) . However, Vertes ( 1 9 8 8 ) , using retrograde and anterograde tra c i n g techniques, described a heavy projection from the Rpc to the medulla and pons, whereas i n t h i s study the data indicate that efferent projections from the Rpc to the Cnv and Rgc are r e l a t i v e l y sparse and predominantly co n t r a l a t e r a l i n the b i r d . The present study a l s o provided evidence f o r a reci p r o c a l projection from the Rgc and Cnv to the Rpc and RL which was b i l a t e r a l . Trigeminal: In t h i s study evidence was found f o r p r o j e c t i o n s from the TTD to the ventromedial r e t i c u l a r formation. The projections (from TTDc) to the Cnv were e n t i r e l y c o n t r a l a t e r a l , whereas the projections to the Rgc (from TTDc and TTDi) and to RP (from TTDo) were b i l a t e r a l but also predominantly c o n t r a l a t e r a l . Comparable r e s u l t s have been reported for the cat i n retrograde HRP studies (Abols and Basbaum, 1981; Baev et a l . , 1988). However, using anterograde tra c i n g Arends et a l . (1984) found no clear evidence of any efferents from any component of the TTD to the medial r e t i c u l a r projection i n the mallard. Therefore the p o s s i b i l i t y that the retrograde l a b e l l i n g found within the TTD i n the present study, 130 may be a consequence of damage to descending trigeminospinal projections traversing the i n j e c t i o n s i t e has to be considered. This i s unlikely to be the sole explanation for my results, however, since the TTD has also been reported to project to other components of the gigantocellular r e t i c u l a r formation (nucleus magnocellularis, Rmc and nucleus g i g a n t o c e l l u l a r i s v e n t r a l i s , Giv), the raphe magnus (Abols and Basbaum, 1981) and the pontine r e t i c u l a r formation (Shammah-Lagnado et a l . , 1987). Vestibular: In the present study, the VeL was found to project b i l a t e r a l l y to the Cnv and Rgc. However, the equivalent projection has been reported to be contralateral i n mammals (cat: Ladpli and Brodal, 1968; Peterson and Abzug, 1975; r a t : Jones and Yang, 1985). The present data also indicated that the ventromedial r e t i c u l a r formation received a projection from the VeM and possibly the VeD; since the l a t t e r was only present i n one b i r d i t may r e f l e c t l a b e l l i n g v i a f i b r e s of passage. However projections from VeM (Ladpli and Brodal, 1968; Gallager and Pert, 1978; Jones and Yang, 1985) and VeD (Ladpli and Brodal, 1968; Peterson and Abzug, 1975; Kunzle, 1985) to the medullary r e r i c u l a r formation have been reported i n other vertebrate species. Cerebellar : A contra l a t e r a l projection from Cbl to the medial r e t i c u l a r formation has been described previously for the pigeon (Karten, 1964) and t u r t l e (Kunzle, 1985) . This concurs with r e p o r t s of a crossed p r o j e c t i o n from the f a s t i g i a l nucleus i n mammals (Batton et a l . , 1977; Ito et a l . , 1970) . In addition, evidence of a sparse projection from the 131 CbL to the Rgc and RP i n the present study i s i n agreement with r e p o r t s of an i p s i l a t e r a l e q u i v a l e n t p r o j e c t i o n , from the dentate nucleus i n mammals (for review see Peterson, 1984) . Although a contra l a t e r a l projection was also apparent i n some birds, t h i s may well r e f l e c t spread of the i n j e c t i o n s i t e v e n t r a l l y into the 10, as a contra l a t e r a l cerebello-olivary projection has been described i n birds (Karten, 1964) and mammals (Martin et a l . , 1976). Pontine: The present results indicate that the LoC and Scd provide input to the medial medullary r e t i c u l a r formation p r i m a r i l y on the c o n t r a l a t e r a l s i d e . This suggests that coeruleoreticular projections form a d i f f e r e n t population from the coeruleospinal projections since the l a t t e r are primarily i p s i l a t e r a l . Sparse b i l a t e r a l or contra l a t e r a l projections from the LoC to Rgc have been previously described for the rat (Gallager and Pert, 1978; Jones and Yang, 1985) and cat (Abols and Basbaum, 1981). More r o s t r a l regions of the pontomedullary r e t i c u l a r formation (Rmc), are also reported to receive only a l i g h t projection from the LoC, but a heavy projection from adjacent regions such as the laterodorsal tegmental nucleus and peri-LoC (Luppi et a l . , 1988). Few projections were noted from the parabrachial region i n the present study. Since the Rmc i s located at the l e v e l of VII, an equivalent projection, i f i t exists i n the bi r d , would require injections into the pons at a more r o s t r a l l e v e l than those of the present study. Mesencephalic: The present data shows that the medial r e t i c u l a r formation receives input from the IS, PC and central gray (Gtc) (PAG, i n mammals) . A sparse projection to the Rgc 132 from the PAG has been noted i n the rat (Gallager and Pert, 1978; Zemlan et a l . , 1984) and cat (Abols and Basbaum, 1981), whereas the projection to the Rmc i s heavy (Gallager and Pert, 1978; Chung et a l . , 1983; Luppi et a l . , 1988). In the present study the i p s i l a t e r a l Ru was l a b e l l e d a f t e r a Cnv and Rgc in j e c t i o n ; however i n a l l such cases the i n j e c t i o n encroached upon the i n f e r i o r o l i v e and therefore r e f l e c t s involvement of rubro-olivary projections as shown for mammals (Martin et a l . , 1983). The contra l a t e r a l Ru, which was retrogradely l a b e l l e d after i n j e c t i o n into the l a t e r a l r e t i c u l a r formation has been shown to provide c o l l a t e r a l s to t h i s region i n previous mammalian studies (Martin and Dom, 1970; Martin et a l . , 1983). This study showed evidence of a b i l a t e r a l but predominantly i p s i l a t e r a l projection from the ICo to the Cnv and Rgc, i n addition to projections from the FRL and FRM. Although t e c t a l neurones were not l a b e l l e d after ventromedial i n j e c t i o n s i n t h i s study, neurones i n the p a r a v e n t r i c u l a r layers of the contra l a t e r a l optic tectum and the ICo and FRL have been l a b e l l e d following ventromedial injections at a more r o s t r a l l e v e l (RP and RPgc) i n the pigeon (Reiner and Karten, 1982). This suggests that c o n t r a l a t e r a l tectobulbar projections terminate i n the pons, as has been described i n mammals. The ICo projections continue t h e i r descent to c e r v i c a l l e v e l s of the cord (Cabot et a l . , 1982; Webster and Steeves, 1988). Both the ICo and the optic tectum projected i p s i l a t e r a l l y to the l a t e r a l r e t i c u l a r formation i n t h i s study. In co n t r a s t to mammals there i s no i p s i l a t e r a l tectospinal pathway i n birds 133 (Cabot et a l . , 1982; Reiner and Karten, 1982). Since the ICo does project to the high c e r v i c a l cord (Cabot et a l . , 1982; Webster and Steeves, 1 9 8 8 ) d i r e c t projections to the RL cannot be distinguished from fibres traversing the i n j e c t i o n s i t e . In view of the widespread connections of the ICo and the subjacent FRL, Reiner and Karten (1982) have suggested that they may be analogous to the deep layers of the mammalian su p e r i o r c o l l i c u l u s and the cuneiform r e g i o n . On cytoarchitectonic and connectional grounds, Edwards ( 1 9 8 0 ) has likened the deep layers of the superior c o l l i c u l u s to the subjacent cuneiform nucleus and the u n d e r l y i n g r e t i c u l a r formation i n the cat. The cuneiform nucleus (Edwards, 1975; Abols and Basbaum, 1981), the subjacent subcuneiform (Abols and Basbaum, 1981), and the a s s o c i a t e d mesencephalic locomotor region (Steeves and Jordan, 1980; G a r c i a - R i l l et a l . , 1983a; Vertes, 1988) have been shown to project to the i p s i l a t e r a l more than the contra l a t e r a l Rgc. These cuneobulbar projections are separate, t h e r e f o r e , from the t e c t o b u l b a r p r o j e c t i o n s , which as already mentioned, descend through the ventromedial pontine r e t i c u l a r formation c o n t r a l a t e r a l l y . Thus the present findings add support to the suggestion of an analogy between the ICo and cuneiform (Reiner and Karten, 1982). Diencephalic: The present findings indicated that several hypothalamic nuclei projected into or through the ventromedial r e t i c u l a r formation, since retrogradely l a b e l l e d neurones were found following i n j e c t i o n into t h i s region. This l a b e l l i n g probably r e f l e c t s damage to hypothalamic efferents from the PVM and the LH to the nTS and X (Berk and Finke l s t e i n , 1983; Berk, 134 1987). This view i s supported by Gallager and Pert (1978), who found retrograde l a b e l l i n g within the hypothalamus only when the Rgc was injected using an oblique approach. Another source of i p s i l a t e r a l projection to the Rgc and Cnv was found l a t e r a l to the AL. However, as t h i s was small and not consistently found i n a l l animals, i t may be due to uptake from damaged fib r e s within the i n j e c t i o n track. Nevertheless, i t i s i n t e r e s t i n g to note that locomotion has been evoked from t h i s region, although u s u a l l y at a s l i g h t l y more l a t e r a l location within the region of the l a t e r a l spiriform nucleus (SpL) (Sholomenko et a l . , i n preparation). Telencephalic: In the present study, retrograde l a b e l l i n g was found i n the latero-caudal aspect of the telencephalon w i t h i n the A i , p r i m a r i l y on the i p s i l a t e r a l s i d e . Since l a b e l l e d c e l l s i n t h i s region were more prevalent i n the parrot than the duck, a species difference i s possible. However, i n preliminary experiments i n the duck (2.0 u l injections, not included i n these r e s u l t s ) , t h i s projection was also present. In studies i n the mallard and pigeon, projections from the A i to the Rpc have been reported (Zeier and Karten, 1971) . I n j e c t i o n s i n t o the d o r s o l a t e r a l medulla confirmed t h i s projection i n the present study. Since none of the ventromedial injections appeared to encroach into the Rpc, the l a b e l l i n g found within the A i after ventromedial injections may be due to c o l l a t e r a l s or damage to fi b r e s of passage within the OM as i t l i e s i n the dorsomedial medulla. No projections were found from the wulst i n t h i s study. 135 This was surprising as f i b r e s have been reported to descend v i a the basal branch of the TSM to the medial and l a t e r a l pontine nuclei (Adamo, 1967) and i n t h e i r descent to the dorsal column nuclei, to send c o l l a t e r a l s to the magnocellular r e t i c u l a r formation (Karten, 1971). Further investigation i s required to c l a r i f y t h i s point. In the present study, the major p r o j e c t i o n s to the dorsolateral region of the brainstem arise from the ST and PGL and, ventromedially, from the r e t i c u l a r formation. Since the i n j e c t i o n s i n v o l v e d the TTD/Rpc and/or the ST/RL, the d i f f e r e n t i a l projections to each nucleus were not determined. Projections to the trigeminal/Rpc region originated from the i p s i l a t e r a l TTDc, Cnd, and Rpc as described previously i n the mallard (Arends and Dubbeldam, 1982). Projections were also found from the Cnv and Rgc, which although r e l a t i v e l y sparse, have not been reported previously. The projection from the A i to the Rpc has been well documented (Zeir and Karten, 1971; Arends and Dubbeldam, 1984). L a b e l l i n g w i t h i n the Ru, and hypothalamus, when present, was heavy and apparently comparable to that found following c e r v i c a l i n j e c t i o n . This indicates that i t was probably due to l a b e l l i n g of the descending spinal t r a c t s . S i m i l a r l y l a b e l l i n g i n the i p s i l a t e r a l ICo and optic tectum may have i n v o l v e d the r e s p e c t i v e t r a c t s . However c o l l a t e r a l s i n t o the Rpc region cannot be r u l e d out, but require further study. The present results suggest that the Cnv projects to and receives input from the Rgc. Therefore these medullary regions from which locomotion can be evoked (Steeves et a l . , 1987), 136 possess r e c i p r o c a l connections. The Cnd, another locomotor s i t e , also projects to the Cnv and Rgc. F i n a l l y both Rgc and Cnv receive dir e c t projections from the ICo. Furthermore, s i t e s within the ICo and FRL have been found to evoke lomomotion i n the decerebrate b i r d , when s t i m u l a t e d e l e c t r i c a l l y , or by i n f u s i o n of neurotransmitter agonists and antagonists (Sholomenko et a l . , i n preparation). Thus, the present study provides evidence for connections between regions that may be associated with the i n i t i a t i o n of locomotion. An analogy with the locomotor connections, previously demonstrated i n mammals, can be made. As discussed previously, the ICo and the FRL may be i n part the avian equivalent of the cuneiform region, which forms the MLR (Shik et a l . , 1967) together with the pedunculopontine nucleus ( P P N ) ( G a r c i a - R i l l et a l . , 1983a,b, 1984, 1986). In addition, both the ICo and MLR project to the Rgc and Cnv, v i a a predominantly i p s i l a t e r a l pathway (present study; Edwards, 1975; G a r c i a - R i l l et a l . , 1983a; Steeves and Jordan, 1984) . Further comparisons are d i f f i c u l t since the projections to the cuneiform nucleus have not been elucidated. However the adjacent P P N , which has been reported to form the anatomical substrate of the MLR i n the rat (Skinner and Garcia-R i l l , 1984; Skinner et a l . , 1985; G a r c i a - R i l l et a l . , 1986) has been shown to project to the Rgc (Ga r c i a - R i l l et a l . , 1983a, 1984, 198 6) and i t receives GABAergic input from the substantia nigra. The ICo receives input from the substantia nigra v i a the tectum (Hunt and Brecha, 1984) . The cytochemistry of the ICo and FRL has not been i n v e s t i g a t e d although a n c i l l a r y 137 studies have i d e n t i f i e d that somatostatin, catecholamine and substance P terminals are present within the ICo (Shiosaka et a l , 1981/ Reiner et a l . , 1983). Now to consider the f u n c t i o n a l connections of the dorsolateral region. In mammals, the dorsolateral locomotor region (the PLS) i s thought to c o n s i s t of descending projections from the mesencephalic trigeminal nucleus (VMes), (G a r c i a - R i l l et a l . , 1983a), to the c e r v i c a l spinal cord i n the cat (Matsushita et al.,1981). In birds, a projection from VMes to the hypoglossal nucleus has been reported i n an elect r o p h y s i o l o g i c a l study (Passetore et a l . , i n Arends and Dubbeldam, 1982)/ however, Arends and Dubbeldam (1982) were unable to confirm t h i s p r o j e c t i o n using a u t o r a d i o g r a p h i c tra c i n g . They did f i n d evidence of a sparse projection from VMes to the i p s i l a t e r a l Cnd (Arends and Dubbeldam, 1982) . In the present study no l a b e l l i n g was found i n VMes after a d o r s o l a t e r a l i n j e c t i o n i n t o the medulla. Although t h i s projection c l e a r l y needs further study, current data indicates that the VMes does not provide substantial input to the PLS i n birds. Descending fibres from the Rpc have also been reported to contribute to the PLS i n mammals (Selionov and Shik, 1984). In birds the Rpc provides input to the Cnd (Arends and Dubbeldam, 1982; present study). Both the Rpc and Cnd are e f f e c t i v e s i t e s for evoked locomotion (Steeves et a l . , 1987). However, the Rpc projects primarily to the c e r v i c a l spinal cord, whereas Cnd projects to the lumbar spinal cord. Therefore, i t i s possible that Rpc may relay v i a Cnd to provide descending locomotor 138 d r i v e to the lumbar s p i n a l cord. Other p r o j e c t i o n s to the d o r s o l a t e r a l region a r i s e w i t h i n the p a r a b r a c h i a l r e g i o n . Although locomotor s i t e s have not been i d e n t i f i e d i n t h i s region i n the b i r d , r o s t r a l p a r a b r a c h i a l locomotor s i t e s have been i d e n t i f i e d i n the cat ( G a r c i a - R i l l , 1983a) . F i n a l l y , p r o j e c t i o n s from the ICo descend i n the l a t e r a l medulla and provide input to the i p s i l a t e r a l l a t e r a l r e t i c u l a r r e g i o n . As discussed p r e v i o u s l y , locomotion can be evoked by s t i m u l a t i o n of the ICo and the n e i g h b o u r i n g mesencephalic r e t i c u l a r formation. In c o n c l u s i o n , p o t e n t i a l sources f o r p r o j e c t i o n s t o the Rgc and Cnv have been i d e n t i f i e d . These have been d i f f e r e n t i a t e d from the regions which p r o j e c t to the d o r s o l a t e r a l TTD/Rpc/Cnd regions. However, the present r e s u l t s need to be confirmed using anterograde t r a c i n g t o f u r t h e r define and confirm the sources of p r o j e c t i o n s t o t h e s e a r e a s . The ICo p r o j e c t s b i l a t e r a l l y to both the Rgc and Cnv and such a connection suggests that i t may be comparable i n part to the cuneiform nucleus and p o s s i b l y , the avian equivalent of the mammalian MLR. Although the PPN i s regarded as a more prominent locus of the MLR, p a r t i c u l a r l y i n the r a t ( G a r c i a - R i l l and Skinner, 198 6) , a homologue of the PPN has not yet been i d e n t i f i e d i n the b i r d . 139 V I I D I S C U S S I O N 140 The objectives of t h i s thesis were to determine: (1) the o r i g i n s of p r o j e c t i o n s descending to the s p i n a l cord i n palmate (web-footed) birds, the duck and the goose; (2) whether there were any novel sources of descending spinal projections i n the dexterous zygodactyl p a r r o t ; (3) the f u n i c u l a r organization of the brainstem-spinal projections within the lumbar spinal cord i n the duck and goose; (4) whether neurones projecting to the spinal cord were within the e f f e c t i v e radius of a f o c a l e l e c t r i c a l s t i m u l a t i n g current that evokes locomotion i n a decerebrate b i r d ; and (5) the sources of afferent input from the r o s t r a l brainstem to the locomotor s i t e s within the caudal medulla. In t h i s discussion, the results are reviewed, compared to other r e l e v a n t s t u d i e s , and d i s c u s s e d i n terms of future i n v e s t i g a t i o n s . F i n a l l y the f u n c t i o n a l s i g n i f i c a n c e of the known connections between "locomotor regions" important for descending locomotor drive i s b r i e f l y summarized. With regard to the f i r s t two objectives, the origins of descending pathways to the high c e r v i c a l s p i n a l cord are si m i l a r i n a l l avian species examined (Canada goose, Pekin duck and Sulphur-crested cockatoo, present study; pigeon Cabot et a l . , 1982; chick Gross and Oppenheim, 1985). These include projections from the CE, GC, nTS, Ala (the region subjacent to X) , the medullary r e t i c u l a r nuclei (Cnv, Cnd, Rgc, RL, PGL, Rpc, ST) , TTD, the raphe nuclei (Rob, Rp, Rm), the vestibular nuclei (VeD, VeL, VeM) , the Cbl, the pontine r e t i c u l a r nuclei (Ctz, RP, RPgc, RPO), the coeruleus complex (LoC, Scd, Scv) , the mesencephalic r e t i c u l a r nuclei (FRL, FRM), the IS, Ru, PC, ICo and the hypothalamic nuclei (PVH, SCE, LH, PVM, SM, SCN) . However there were some differences. No evidence of spinal p r o j e c t i o n s from two hypothalamic n u c l e i (the l a t e r a l mamillary, l a t e r a l tubercle; Gross and Oppenheim, 1985) were found. There were also differences with regard to some of the v e s t i b u l a r n u c l e i . These d i f f e r e n c e s may r e s u l t from differences i n interpretation of the boundaries of s p e c i f i c nuclei, rather than true species differences. In the present study the telencephalon was c a r e f u l l y examined for projections to even the most r o s t r a l levels of the spinal cord, however none were ever found i n the goose, duck or parrot. Since only one parrot was examined for projections to the c e r v i c a l l e v e l , the telencephalospinal projection to the c e r v i c a l cord i n the parrot reported by Zecha (1964), requires further investigation to confirm whether i t i s a true spinal cord projection. Nevertheless, the lack of a telencephalospinal projection was confirmed i n the 5 ducks examined. The origins of projections to the lumbar cord have been described i n a variety of mammalian and non-mammalian species (Kuypers, 1981; Kuypers and Martin, 1982). Recently, they have also been reported for the chick (Gross and Oppenheim, 1985). The TTD, Cbl, and ICo project to the high c e r v i c a l l e v e l . I found no evidence of projections to the lumbar spinal cord from the CE, VeM, SM, and SCN and i n that regard my results d i f f e r from those of Gross and Oppenheim (1985). With regard to the f u n i c u l a r o r g a n i z a t i o n of the descending pathways, t h i s thesis sought to c l a r i f y and expand 142 the findings of Cabot et a l . (1982). As part of an a n c i l l a r y study, they described the pos i t i o n of a few of the descending t r a c t s i n the pigeon and only as f a r as the b r a c h i a l enlargement. My retrograde tracing findings concur with the r e s u l t s of t h e i r a u toradiographic t r a c i n g experiments and lengthen the caudal extent of many of these pathways to the lumbar cord. The f u n i c u l a r t r a j e c t o r i e s are e s s e n t i a l l y unchanged from c e r v i c a l to lumbar enlargements i n the b i r d . In studies of mammals, s h i f t s i n the funicular location of descending pathways have been reported; however these appear to be most obvious when the high c e r v i c a l l e v e l (Petras, 1967) i s compared to the low lumbar l e v e l . In addition to the funicular t r a j e c t o r y , the l a t e r a l i t y of avian lumbar pathways ( i p s i l a t e r a l versus contralateral) has also been c l a r i f i e d i n the present study. F i n a l l y , d i r e c t p r o j e c t i o n s from the telencephalon to the lumbar spinal cord were never observed i n any of the ducks, geese or parrots examined i n the present t h e s i s . Each descending b r a i n s t e m - s p i n a l p r o j e c t i o n i s now reviewed below. As demonstrated previously the nTS projects b i l a t e r a l l y (Cabot et a l . , 1982) to the c e r v i c a l cord. This study has further demonstrated that the projections are primarily v i a the VLF and extend b i l a t e r a l l y to the lumbar l e v e l . These findings are consistent with studies i n both r e p t i l e s and mammals and i l l u s t r a t e the constancy of t h i s spinal pathway. However, the present study has not c l e a r l y i d e n t i f i e d which of the sub-nuclei of the nTS project as far as the lumbar l e v e l . According to the c y t o l o g i c a l d e s c r i p t i o n given by Katz and Karten (1983a), the retrogradely l a b e l l e d neurones i n the nTS, i n the present study were i n the v i c i n i t y of the ventromedial and vent r o l a t e r a l subnuclei. In the cat, these regions project to thoracic and lumbar levels and are known to be involved i n cardiac and respiratory control (Loewy and Burton, 1978). As shown by others, a medullary region, ventral to X and d i s t i n c t from nTS, projects to c e r v i c a l (Cabot et a l . , 1982) and lumbar levels of the spinal cord (nucleus alatus; Gross and Oppenheim, 1985) . Furthermore, t h i s study has shown that the lumbar p r o j e c t i o n i s greater than that from the nTS and descends, b i l a t e r a l l y with a strong contralateral predominance, within the VLF, the DLF and, to a lesser extent, the VMF. In b i r d s , more som a t o s t a t i n - c o n t a i n i n g neurones than catecholaminergic neurones have been i d e n t i f i e d w i t h i n the region ventral to X (Guglielmone and Panzica, 1983). The r e t i c u l o s p i n a l pathways from the caudal medulla originate from the Cnd and Cnv. In birds (Cabot et a l . , 1982; Gross and Oppenheim, 1985) and mammals, the Cnv (or the equ i v a l e n t nucleus v e n t r a l i s ) (Zemlan and P f a f f , 1979), projection i s heavy to the c e r v i c a l cord, but r e l a t i v e l y sparse at lumbar levels and courses v i a the VLF and VMF (Martin et a l . , 1981a; C a r l t o n et a l . , 1985; present study). Stimulation of the avian Cnv has been shown to evoke locomotion (Steeves et a l . , 1987). The nucleus v e n t r a l i s has been c i t e d as an e f f e c t i v e locomotor s i t e i n the cat ( G a r c i a - R i l l and Skinner, 1987a). Locomotor s i t e s at s i m i l a r l e v e l s of the brainstem as part of the magnocellular tegmental f i e l d (FTM, 144 a c c o r d i n g t o Berman, 1968) (Noga e t a l . , 1988). A l l a v a i l a b l e e v i d e n c e i n d i c a t e s t h a t l o c o m o t i o n c a n be e v o k e d f r o m e q u i v a l e n t s t i m u l a t i o n s i t e s w i t h i n t h e a v i a n and mammalian 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 ( S t e e v e s e t a l . , 1987; Sholomenko, i n p r e p a r a t i o n ) . The Cnd p r o j e c t s b i l a t e r a l l y t o t h e c e r v i c a l and lumbar l e v e l s o f t h e c o r d (Cabot e t a l . , 1982; Gross and Oppenheim e t a l . , 1985). As shown i n t h i s s t u d y , t h e d e s c e n d i n g Cnd f i b r e s appear t o have some c o m p a r t m e n t a l i z a t i o n w i t h a predominance o f i p s i l a t e r a l f i b r e s i n t h e DLF, and c o n t r a l a t e r a l f i b r e s w i t h i n t h e VMF and VLF. In mammals such as t h e r a t , t h e Cnd p e r i k a r y a a re r e p o r t e d t o be u n i f o r m l y s m a l l ( A n d r e i k and B e i t z , 1985), whereas l a r g e d i a m e t e r neurones are p r e s e n t w i t h i n t h e a v i a n c a u d a l Cnd; t h e s e p r o j e c t p r i m a r i l y t o t h e c o n t r a l a t e r a l s p i n a l c o r d . I t i s p o s s i b l e t h a t some o f t h e p r o j e c t i o n s from t h e s e l a r g e d i a m e t e r c e l l s may be p a r t o f an a v i a n e q u i v a l e n t t o t h e mammalian n u c l e u s r e t r o a m b i g u u s , s i n c e t h e y a re c a u d a l t o t h e n u c l e u s ambiguus i d e n t i f i e d by K a t z and K a r t e n (1983) . A l t h o u g h t h e y are s i t u a t e d i n t h e v i c i n i t y o f t h e l a t e r a l c e r v i c a l n u c l e u s , i t i s u n l i k e l y t h e y c o n s t i t u t e p a r t o f t h a t n u c l e u s s i n c e t h e y p r o j e c t t o t h e lumbar s p i n a l c o r d . F u r t h e r e x p e r i m e n t s a r e needed t o c l a r i f y t h e n a t u r e and e x t e n t o f t h e s e c o n n e c t i o n s . W h i l e r e s p i r a t o r y as w e l l as g u s t a t o r y f u n c t i o n s a re a s s o c i a t e d w i t h t h i s r e g i o n , t h e Cnd as d e f i n e d i n t h i s s t u d y (an o b l i q u e band o f c e l l s , d o r s o l a t e r a l t o Cnv and m e d i a l t o TTD i n t h e c a u d a l m e d u l l a ) a p p e a r s t o be i n v o l v e d i n l o c o m o t i o n . Cnd p r o j e c t i o n s were found c o i n c i d e n t w i t h t h e Rgc and Cnv w i t h i n the VLF, which has been shown i n t h i s and previous studies (Steeves and Jordan, 1980; Sholomenko and Steeves, 1987) to be an e s s e n t i a l pathway f o r descending locomotor d r i v e . Furthermore the Cnd has been shown to be an e f f e c t i v e s i t e for brainstem stimulated locomotion i n the b i r d (Steeves et a l . , 1987; Sholomenko, i n preparation). The Rgc projects primarily i p s i l a t e r a l l y to the c e r v i c a l and lumbar levels of the cord, as shown i n t h i s and other studies i n birds and mammals (Cabot et a l . , 1982; Martin et a l . , 1981a). This study has confirmed that i n the bird, as i n mammals, the Rgc projects v i a the VLF and VMF. In addition, a topographic organization was i d e n t i f i e d i n the bi r d , i n that the dorsal Rgc projects to the c e r v i c a l but not to the lumbar l e v e l . This finding i s similar to that documented i n r e p t i l e s (Newman et a l . , 1983) and mammals (Martin et a l . , 1981a; Zemlan et a l . , 1984). Apart from the d i s t i n c t i o n between the ventral and dorsal regions of the Rgc, no other topographical organization was i d e n t i f i e d i n the present study. In mammals, subdivisions of the Rgc have been i d e n t i f i e d on the basis of cytoarchitecture (Andreik and Beitz, 1985; Newman, 1985) and connections (Martin et a l . , 1981a, 1985). Further s t u d i e s are r e q u i r e d to define the morphological c h a r a c t e r i s t i c s , cytochemistry and d i f f e r e n t i a l connections of the avian Rgc. Functionally, stimulation of the Rgc-spinal pathway i n i t i a t e s locomotion i n birds (Steeves, et a l . , 1987) and mammals (Ga r c i a - R i l l and Skinner, 1987a). In addition to acti v a t i n g locomotion, there appears to be ongoing modification 146 of locomotion by Rgc stimulation (for review see Armstrong, 1986) . The Ctz-spinal projections i n the b i r d have been described previously (Cabot et a l . , 1982) and project as far as the lumbar l e v e l of the spinal cord (Gross and Oppenheim, 1985). The present study has shown that the spinal projections from the region of the Ctz descend primarily i p s i l a t e r a l l y v i a the VLF and the DLF. Although the Ctz consists of an auditory pathway, neurones within t h i s region (ventral pons) have a d i s t i n c t morphology compared to those i n more d o r s a l RP regions. The function of the Ctz-spinal pathway i s not known. The l a b e l l e d neurones i n Ctz may form a l a t e r a l extension of the adjacent raphe magnus, since the Rm also projects v i a the DLF and VLF. A l t e r n a t i v e l y these Ctz-spinal projections may be considered as component of the r e t i c u l a r formation, since they are at the same l e v e l as Rmc; however data i s i n s u f f i c i e n t to make further comparisons. The RL and ST project to the c e r v i c a l (Cabot et a l . , 1982) and the lumbar l e v e l (Gross and Oppenheim, 1985) . This study has c l a r i f i e d t hat, l i k e the r e t i c u l o s p i n a l pathway, the projections are primarily v i a the VLF. A functional role of the RL and ST i n avian locomotion cannot be excluded because aft e r a subtotal thoracic spinal cord lesion, except for the VLF, animals are s t i l l capable of locomotion (Sholomenko and Steeves, 1987) . The raphespinal. projections course within the DLF, VLF and VMF as described by Cabot and colleagues (1982). Functionally they appear to be associated with autonomic regulations (Cabot 147 et a l . , 1982), nociception and regulation of postural tone (Mori et a l . , 1982). Although the raphe has not been implicated in the i n i t i a t i o n of locomotion i n the bird , the raphe and the adjacent medial r e t i c u l a r formation have been found to be e f f e c t i v e locomotor s i t e s i n the cat (Noga et a l . , 1988). The vestibulospinal projections arise from the VeLv, VeLd, VeD and VeM i n the duck and goose as reported for the pigeon. The present study confirms e a r l i e r retrograde degeneration studies that the VeL-spinal projections descend b i l a t e r a l l y within the ventral funiculus (Wallenberg, 1904, c i t e d by Wold, 1978); however, the VeLd i s e n t i r e l y i p s i l a t e r a l , while only the VeLv has both i p s i l a t e r a l and contralateral projections. Although the vestibulospinal t r a c t s appear to be non-essential for the i n i t i a t i o n of locomotion (Yu and Eidelberg, 1981; J e l l et a l . , 1985), they appear to have a s i g n i f i c a n t role i n the regulation of extensor muscle a c t i v i t y (Orlovsky, 1972) . With regard to the coeruleospinal pathways, the present study extends previous findings reported for the pigeon (Cabot et a l . , 1982) and chicken (Gross and Oppenheim, 1985). In a d d i t i o n to confirming the f u n i c u l a r o r g a n i z a t i o n of the cervic a l • p r o j e c t i o n , a widespread trajectory for coeruleospinal pathways was observed at lumbar lev e l s , within the DLF, VLF and possibly the VMF. Although the functional role has not been sp e c i f i e d for birds, the LoC and Scd are reported to play a role i n i n h i b i t i o n of flexor r e f l e x afferents, modulation of sensory input and f a c i l i t a t i o n of both extensor and flexor motoneurone e x c i t a b i l i t y i n mammals (for review see Fung and 148 Barnes, 1984). The pontine r e t i c u l a r formation (RP, RPgc) was found i n th i s study to project to the lumbar cord almost exclusively v i a the VMF. This finding i s consistent with those of previous studies i n birds (Cabot et a l . , 1982) and mammals (Martin et a l . , 1979, 1981a). In mammals t h i s region has been implicated i n s e v e r a l f u n c t i o n a l domains i n c l u d i n g i n i t i a t i o n of locomotion (Steeves et a l . , 1987) and regulation of the neck and trunk (Mori et a l . , 1977, 1978; Peterson, 1979). The rubrospinal pathway has been shown to project v i a the contral a t e r a l DLF i n birds (Wild et a l . , 1979) as well as mammals. It has an active role during locomotion and primarily i n f l u e n c e s both f l e x o r interneurones (Orlovsky, 1972) and fusimotor neurones (Armstrong, 198 6). There are direc t rubral motoneuronal connections i n mammals, but only at the c e r v i c a l l e v e l (Robinson et a l . , 1987; Holstege, 1987) and these have been shown to play a role i n reaching and grasp (Houk et a l . , 1988) . Although the rubrospinal t r a c t i s not es s e n t i a l for self-supported locomotion, i t s role i n hindlimb function i n s k i l l e d locomotion such as rung walking has yet to be determined. The IS-spinal projections descend v i a the VMF and are exclusively i p s i l a t e r a l i n a l l birds and mammals investigated. The IS-spinal pathway at c e r v i c a l l e v e l s i s associated with coordination of eye and neck movements. The function of the lumbar projections are not clear. The i n t e r s t i t i a l nucleus i n f i s h and t u r t l e has been reported to be an e f f e c t i v e locomotor stimulation s i t e ; t h i s i s not true of the b i r d and as has been 149 shown by Sholomenko and Steeves (1987) and the present study, locomotion i s not preserved when t h i s projection i s spared and the r e t i c u l o s p i n a l t r a c t s are lesioned. The hypothalamic pathways were, as i n previous studies, (Cabot et a l . , 1982) found to project v i a the DLF. In addition, a small contribution was apparent within the dorsal aspects of the VLF. These projections have been implicated i n descending hormonal control. Although no involvement i n direc t locomotor control has been i d e n t i f i e d for t h i s region, i t may play a role in motivated behaviours (Kuypers, 1982). In addition to the points mentioned above, other aspects concerning avian descending s p i n a l pathways need to be addressed. For instance, how extensive i s the c o l l a t e r a l i z a t i o n of descending spinal systems at di f f e r e n t levels of the avian spinal cord? An attempt was made to determine t h i s i n the present study, using the retrograde tracers SITS and DY at the c e r v i c a l and lumbar lev e l s , respectively. SITS has the supposed advantage that i t i s not transported by fib r e s of passage (Schmued and Swanson, 1982), but the disadvantage that SITS has been reported to produce toxic effects i n rats (Lawes et a l . , 1984). This unfortunately was also the case i n the b i r d and for t h i s reason other combinations of tracers w i l l have to be used in future experiments (eg. WGA-HRP or cholera toxin; Hayes and Rustioni, 1981; Luppi et a l . , 1988). I was unable to determine whether neurones that project to both sides of the spinal cord decussate at the spinal l e v e l . Although a hemisection p r i o r to an i p s i l a t e r a l i n j e c t i o n at a 150 more caudal l e v e l provided negative results i n t h i s study, t h i s can not be regarded as c o n c l u s i v e , since i t was only demonstrated i n one b i r d . Nevertheless, similar methods have been report e d to y i e l d negative r e s u l t s i n other s t u d i e s , p a r t i c u l a r l y at high c e r v i c a l levels i n mammals (Kuypers and Maisky, 1977). It i s p o s s i b l e t h e r e f o r e that decussation across the midline of the avian cord may be more prevalent at the l e v e l of the c e r v i c a l or lumbar enlargements. Positive r e s u l t s of f i b r e s c r o s s i n g the mammalian cord have been reported using anterograde tracing techniques at the lumbar l e v e l (Holstege and Kuypers, 1982) . Confirmation with retrograde techniques w i l l require discrete injections, since in the present study the double l a b e l l i n g technique provided ambiguous r e s u l t s due to s l i g h t d i f f u s i o n of one or both tracers across the midline of the cord. Another question that needs to be addressed i s the histochemical nature of these descending neuronal projections. The histochemistry of the avian brainstem has been described (Shiosaka et a l . , 1981; Guglielmone and Panzica, 1984; Reiner et a l . , 1983; Steeves et a l . , 1986b), but the simultaneous determination of connections and the putative neurotransmitters has been accomplished i n only a few studies (eg. Smolen et a l . , 1979). Therefore double l a b e l l i n g s t u d i e s using immuno-histochemical agents and retrograde tracers could help c l a r i f y the functional role attributed to s p e c i f i c brainstem regions. With regard to the fourth objective, t h i s study v e r i f i e d that s i t e s within the Cnv and Cnd, from which locomotion could be evoked by focal e l e c t r i c a l stimulation i n a decerebrate b i r d , were i n c l o s e proximity to neurones with d i r e c t projections to the spinal cord. The e f f e c t i v e spread of the stimulating current has been shown to be i n the range of 500 um at current strengths of 100 uA or less (Steeves et a l . , 1975) and retrogradely l a b e l l e d c e l l s were found well within t h i s distance. The coincident location of an e f f e c t i v e locomotor stimulation s i t e and the source of a spinal projection may suggest a causal relationship, but i t does not prove such a l i a i s o n . Furthermore, 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 i s non-s p e c i f i c and may a c t i v a t e somata, dendrites of d i s t a n t neurones, or axons traversing the stimulation s i t e . Recent findings have also demonstrated that infusion of s p e c i f i c neurotransmitter agonists or antagonists (eg. carbachol, NMDA) into the Cnd or Cnv also evokes locomotion i n a decerebrate b i r d (Sholomenko, i n preparation). This suggests that neuronal somata and dendrites, or immediate presynaptic terminals, rather than axons of passage, were being activated by the focal e l e c t r i c a l stimulation used i n t h i s study. This adds cons i d e r a b l e strength to the argument f o r the r e t i c u l o s p i n a l projections being a f i n a l common pathway for i n i t i a t i n g locomotion. However, f u r t h e r d e f i n i t i o n of the precise a c t i v i t y of r e t i c u l o s p i n a l neurones before, during and af t e r locomotion i s required for i d e n t i f i c a t i o n of t h e i r exact role i n locomotion. As to the f i n a l objective of t h i s thesis, t h i s study has o u t l i n e d the descending a f f e r e n t p r o j e c t i o n s to the ventromedial and dorsolateral regions of the avian medulla. 152 With regard to the pathways projecting to the ventromedial region, there were many s i m i l a r i t i e s to those p r e v i o u s l y described i n mammals. There were, however, differences and ambiguities, which may r e f l e c t differences i n technique or species. This i s also true of afferent projections to the d o r s o l a t e r a l region, which have i n part been d e s c r i b e d previously for the b i r d (Arends et a l . , 1984). Since the afferents to the medial medullary r e t i c u l a r formation have only been examined i n a few mammalian species and never been investigated previously i n the bird, comparative data i s sparse. The afferents to the avian Cnv and Rgc included dense b i l a t e r a l r e c i p r o c a l p r o j e c t i o n s from medial pontomedullary r e t i c u l a r regions (Cnv, Rgc, RP) . In agreement with a recent report for the rat (Vertes, 1988), there were r e l a t i v e l y fewer a f f e r e n t s from more r o s t r a l r e t i c u l a r formation (RPgc, RPO, FRL, FRM). Furthermore, there were only sparse a f f e r e n t inputs to the medullary Cnv and Rgc from ventr o l a t e r a l r e t i c u l a r regions (RL, PGL, ST). A projection from the TTD and adjacent Rpc was also noted to the ventromedial r e t i c u l a r formation (especially Rgc). This projection was predominantly from the contralateral brainstem. Although comparable results have been reported for the cat i n retrograde HRP studies (Abols and Basbaum, 1981; Bayev et a l . , 1988) , t h i s TTD/Rpc to Rgc connection was not seen i n the mal l a r d duck (Arends et a l . , 1984) and needs f u r t h e r investigation. Projections from more r o s t r a l regions of the brainstem to the Cnv and Rgc are r e l a t i v e l y sparse i n comparison to 153 neighbouring inputs from the caudal brainstem. Nevertheless both Cnv and Rgc r e c e i v e p r o j e c t i o n s from the LoC, Scd (Gallager and Pert, 1978/ Jones and Yang, 1985/ Abols and Basbaum, 1981), IS, PC, Gtc, FRL and FRM (Gallager and Pert, 1978/ Zemlan et a l . , 1984/ Abols and Basbaum, 1981). In addition, the PVM and LH may provide afferents to the Rgc and Cnv. The only substantial projections from the midbrain to the medullary r e t i c u l a r formation emmanated from the ICo, FRM, and FRL. These p r o j e c t i o n s were b i l a t e r a l , but predominantly i p s i l a t e r a l , and have been previously demonstrated to descend to the pontomedullary r e t i c u l a r formation (Reiner and Karten (1982) and as far as the c e r v i c a l spinal cord (Cabot et a l . , 1982/ Webster and Steeves, 1988). The afferents to the dorsolateral region (TTD/Rpc) of the medulla included intratrigeminal connections, and b i l a t e r a l projections from the medial r e t i c u l a r formation (Cnv, Cnd, Rgc) and c o n t r a l a t e r a l Rpc. Further s t u d i e s using anterograde t r a c e r s such as PHA-L are r e q u i r e d to d e l i n e a t e the d i f f e r e n t i a l inputs to TTD versus Rpc. 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 w i t h i n the r o s t r a l avian brainstem have recently been i d e n t i f i e d where focal stimulation ( e l e c t r i c a l or chemical) w i l l evoke locomotion (Sholomenko, i n preparation). These include the FRM, FRL, ICo, r o s t r a l TTD, RP. The p r e c i s e anatomical and cytochemical nature of the connections to and from these regions s t i l l need to be elucidated. 154 The functional implications of t h i s study, with regard to locomotion, are that the origins of pathways that project to the lumbar cord have been i d e n t i f i e d which appear to be i n v o l v e d i n the i n i t i a t i o n of locomotion (Sholomenko and Steeves, 1987; Steeves et a l . , 1987). Since locomotion i s only blocked by sectioning the ventral f u n i c u l i of the spinal cord (Sholomenko and Steeves, 1987), i t suggests that the brainstem-spinal pathways necessary for locomotion must project v i a the ventral f u n i c u l i . Surveying the data from my thesis suggests that descending pathways or i g i n a t i n g within the Rgc, Cnv, and Cnd are the best candidates since stimulation in and around a l l of these regions evokes avian locomotion (Steeves et a l . , 1987) . This study has shown i n the b i r d that the Rgc projects to the cord v i a the v e n t r o l a t e r a l f u n i c u l u s and ventromedial funiculus. The Cnv was found to have a li m i t e d projection to the lumbar cord v i a the ventral f u n i c u l i , whereas Cnd has a p r o j e c t i o n to the lumbar cord, v i a the d o r s o l a t e r a l and v e n t r o l a t e r a l f u n i c u l i , that i s intermediate i n d e n s i t y compared to Rgc and Cnv. The Rgc i s of major interest as r e t i c u l o s p i n a l projections from t h i s region have been consistently recognized as necessary for spontaneous (Eidelberg et a l . , 1981a,b), as well as evoked (Steeves and Jordan, 1980; Shekchyk et a l . , 1984) mammalian locomotion. In mammals, r e t i c u l o s p i n a l projections from Rgc are located i n the medial as well as the l a t e r a l f u n i c u l i (Zemlan et a l . , 1984; Martin et a l . , 1981a; Carlton et a l . , 1985; Mitani et a l . , 1988) . 155 Stimulation of the l a t e r a l aspects of the cord has also been shown to evoke locomotion i n f i s h , t u r t l e , b i r d and mammals (Lennard and Stein, 1977; Stein, 1978; Jacobson and Hollyday, 1982; Kazennikov et a l . , 1983a). Reports have varied as to the most e f f e c t i v e s i t e of stimulation, but generally i t involves the dorsolateral column at the c e r v i c a l l e v e l of the cord. From my data, projections from the Rm, LoC, Scv, PVM, Ru, and Cnd traverse the dorsolateral funiculus. Since complete sectioning of the dorsal h a l f of the cord does not prevent avian locomotion (Sholomenko and Steeves, 1987), projections such as the Cnd, which also project v i a the VLF, would appear to be of more fundamental importance to locomotion than PVM, Ru, and Rm. Although LoC and Scv projections are course through the VLF they have no known role i n locomotion. The question concerning the Cnv i s how locomotion i s induced from stimulation of t h i s s i t e when i t has only sparse projections to the lumbar cord? My data has demonstrated that Cnv p r o j e c t s to and r e c e i v e s input from the Rgc. It i s possible, therefore, that Cnv stimulation i n d i r e c t l y activates spinal locomotor networks v i a Rgc. A l t e r n a t i v e l y , locomotion may be i n t i t i a t e d v i a c e r v i c a l cord propriospinal neurones that are f i r s t a c t i v a t e d v i a the dense Cnv to c e r v i c a l cord projection. Although axons from the TTD and the Rpc do not project to the lumbar l e v e l , both regions have been implicated i n the i n i t i a t i o n of locomotion since t h e i r s t i m u l a t i o n evokes locomotion i n the decerebrate b i r d (Steeves et a l . , 1987). An 156 alternative TTD/Rpc pathway has been i d e n t i f i e d to the c e r v i c a l l e v e l v i a the d o r s o l a t e r a l f u n i c u l u s i n a l l v e r t e b r a t e s examined (stingray, Livingstone and Leonard, i n preparation; duck, Arends et a l , 1984; cat, Kazennikov et a l . , 1983). Therefore t h e i r influence at more caudal levels of the cord may be v i a the ac t i v a t i o n of propriospinal neurones at c e r v i c a l levels that descend throughout the cord. As suggested i n t h i s study, TTD and Rpc may also project into the medial r e t i c u l a r formation and RL and therefore, these projections may also form a f i n a l common path from l a t e r a l locomotor regions to the lumbar l e v e l . The descending input from r o s t r a l brainstem locomotor regions i s less clear. Major input to both the medial and l a t e r a l regions of the r e t i c u l a r formation emanates from the ICo, a locomotor region i d e n t i f i e d within the mesencephalon (Sholomenko, i n p r e p a r a t i o n ) . Another i d e n t i f i e d locomotor region i s the mesencephalic r e t i c u l a r formation (FRM and FRL d i v i s i o n s ) which has axonal p r o j e c t i o n s to the medial pontomedullary r e t i c u l a r formation. Reiner and Karten (1982) have suggested that the ICo i s homologus to the cuneiform nucleus i n mammals and the present results have also shown some s i m i l a r i t y between the descending projections from the ICo and those previously described for the cuneiform nucleus (Edwards, 1975). Although Edwards (1975) examined p r o j e c t i o n s from the r o s t r a l cuneiform nucleus, subsequent tracing studies from the p h y s i o l o g i c a l l y defined MLR have also i d e n t i f i e d projections from the caudal cuneiform to the medullary r e t i c u l a r formation (Steeves and Jordan, 1984). 157 While i t i s tempting to speculate that the ICo projection may be the avian equivalent to the mammalian MLR, some caution i s necessary u n t i l f u r t h e r s t u d i e s have determined the locomotor r o l e of the mesencephalic r e t i c u l a r formation, including the p o s s i b i l i t y that i t i s the avian MLR. Exact p a r a l l e l s with the mammalian MLR cannot be drawn since the MLR does not project to the spinal cord (Steeves and Jordan, 1984), whereas the ICo does innervate the r o s t r a l c e r v i c a l cord (Cabot et a l . , 1982) . Both the avian ICo and mammalian MLR regions receive major input from the basal ganglia and are recognized as general sensory-motor integration centres. For example, the ICo has been implicated i n the generation of other motor patterns; namely v o c a l i z a t i o n . ICo stimulation evokes, and conversely ICo lesions abolish v o c a l i z a t i o n i n the b i r d ( P h i l l i p s , 1983). The next question i s how these brainstem locomotor c i r c u i t s are influenced from more r o s t r a l regions of the brain. Lesion studies have demonstrated that the b i r d can locomote normally following extirpation of the cortex (ten Cate, 1975). The only apparent d e f i c i t s due to removal of the telencephalon are l o s s of goal d i r e c t e d behaviours and d i s c r i m i n a t i v e learning. The major projections from the telencephalon to the brainstem are from the archistriatum v i a the OM. The OM sends c o l l a t e r a l s to the ICo during i t s descent to the brainstem (primary t a r g e t Rpc) . It i s a l s o p o s s i b l e that t h i s OM projection i s involved with the control of TTD/Rpc locomotor regions. 158 Other projections from the telencephalon relay v i a the basal ganglia to the SpL and substantia nigra and then to the tectum. In order to gain insight into the functional aspects of these connections, locomotion needs to be examined p h y s i o l o g i c a l l y i n intact birds. In conclusion, the present studies have i d e n t i f i e d the origins of brainstem-spinal projections and the t r a j e c t o r i e s of these pathways at the low thoracic l e v e l i n ducks and geese. No evidence was found for a telencephalospinal projection even i n the p r e h e n s i l e p a r r o t . Apart from t h i s d i s t i c t i o n , the desending spinal pathways i n birds are similar to those that have been d e s c r i b e d f o r mammals. P r o j e c t i o n s from the ventromedial medullary r e t i c u l a r formation course through the v e n t r o l a t e r a l f u n i c u l u s and hindlimb locomotion has been correlated with t h e i r preservation at thoracic le v e l s of the cord. Regions which p r o j e c t to the medullary r e t i c u l a r formation 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 and the mesencephalic r e t i c u l a r formation, which may be equivalent to the mammalian mesencephalic locomotor region. 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