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The distribution of somatostatin immunoreactivity in the subcortical auditory system of three mammalian… Von Krosigk, Marcus 1986

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THE DISTRIBUTION OF SOMATOSTATIN IMMUNOREACTIVITY IN THE SUBCORTICAL AUDITORY SYSTEM OF THREE MAMMALIAN SPECIES: THE CAT; GUINEA PIG; AND RAT. By MARCUS VON KROSIGK B . S c , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES NEUROLOGICAL SCIENCES PROGRAMME We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A u g u s t 1986 (c) Marcus von Krosigk, 1986 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f \^^yct\i^."f f y The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 D a t e / l ^ o s l 2 0 } 3-6 (3/81) i i Abstract Using current immunohistochemical methods the d i s t r i b u t i o n of somatostatin was examined i n the s u b c o r t i c a l auditory pathways of three mammalian species. The d i s t r i b u t i o n s of immunoreactivity were si m i l a r i n some respects and quite varied i n others. The differences and s i m i l a r i t i e s could be considered i n the context of three categories of previously described projections: ascending auditory pathways; ascending auditory and somatosensory pathways; and descending auditory pathways. The greatest heterogeneity was associated with immunostaining i n the ascending auditory n u c l e i ; t h i s was only found i n rodents. Immunolabelled c e l l s were seen i n the rat spherical c e l l region of the anterior ventral cochlear nucleus (SC-AVCN) and i n the dorsal region of the ventral nucleus of the l a t e r a l lemniscus (VLL), while immunoreactive puncta were prominent i n the MNTB (medial nucleus of the trapezoid body). In the guinea pig a density of SOM-IR c e l l s , much lower than that of the r a t , was seen i n the SC-AVCN while immunoreactive neuropil was only seen apposing the p r i n c i p a l c e l l s of the medial superior o l i v e . In a l l species somatostatin immunoreactivity was associated with the ascending projection from immunolabelled c e l l s i n the i n t e r c o l l i c u l a r area (ICA), and possibly the external cortex of the i n f e r i o r c o l l i c u l u s (EC), which project to the midbrain r e t i c u l a r formation (MRF), a s i t e of i i i common imraunolabelled neuropil. These regions are associated with both ascending auditory and somatosensory information. In the descending auditory regions somatostatin immunoreactivity was prominent i n a l l species examined. A l l species showed immunolabelled c e l l s i n the dorsal and external c o r t i c e s of the i n f e r i o r c o l l i c u l u s . C e l l s seen i n the medial VLL region do not appear to form ascending pathways and the VLL i s known to provide descending pathways. A l l species showed immunoreactive neuropil i n the medial VLL, the dorsal p e r i o l i v a r y regions of the superior o l i v a r y complex and i n the ventral nucleus of the trapezoid body. Comparison of the immunoreactivities, both s i m i l a r i t i e s and differences, to known projections, i s used to describe the possible projections of somatostatin immunoreactive c e l l s . The discussion includes some speculation on the possible coexistence of somatostatin with other peptides and i t s possible functional s i g n i f i c a n c e . The p o s s i b i l i t y of descending mechanisms of c o r t i c a l and subcortical auditory control of the cochlear nucleus and cochlea i s derived from a comparison of peptide and c l a s s i c a l neurotransmitter d i s t r i b u t i o n s with physiology and pharmacology. i v T a b l e o f C o n t e n t s INTRODUCTION. .• 1 The B r a i n s t e m A u d i t o r y S y s t e m 7 METHODS AND MATERIALS 12 RESULTS 15 The C o c h l e a r N u c l e u s 15 V e n t r a l C o c h l e a r N u c l e u s 15 D o r s a l C o c h l e a r N u c l e u s 31 The S u p e r i o r O l i v a r y Complex 34 P e r i o l i v a r y R e g i o n s 34 D o r s o m e d i a l P e r i o l i v a r y N u c l e u s 34 D o r s a l P e r i o l i v a r y N u c l e u s 43 D o r s o l a t e r a l P e r i o l i v a r y N u c l e u s 43 L a t e r a l P e r i o l i v a r y R e g i o n 56 V e n t r a l P e r i o l i v a r y N u c l e u s 56 S u p e r i o r P a r a o l i v a r y N u c l e u s 57 M e d i a l N u c l e u s o f t h e T r a p e z o i d Body 57 V e n t r a l N u c l e u s o f t h e T r a p e z o i d Body 64 S u p e r i o r O l i v a r y N u c l e i 67 The N u c l e i o f t h e L a t e r a l L e m n i s c u s 67 N u c l e u s o f t h e V e n t r o l a t e r a l L e m n i s c u s 67 N u c l e u s o f t h e D o r s o l a t e r a l L e m n i s c u s 89 V The Sagulum 89 The D o r s a l Perimeter of the DLL 90 The I n f e r i o r C o l l i c u l u s 90 DISCUSSION 131 The D i s t r i b u t i o n of SOM-IR Across Species 131 Comparison t o P r e v i o u s S t u d i e s 135 P o s s i b l e Somatostatin Immunoreactive Pathways 136 Ascending A u d i t o r y Pathways 137 Ascending Multimodal Pathways 141 Descending A u d i t o r y Pathways 142 Chemical Neuroanatomy 149 P h y s i o l o g y and Pharmacology 156 Concluding Remarks 163 QUESTIONS RAISED 169 General Questions 169 S p e c i f i c Questions 169 REFERENCES 171 v i L i s t of Tables Table 1. Summary of the d i s t r i b u t i o n of somatostatin immunoreactivity i n the su b c o r t i c a l auditory nuclei 133 v i i L i s t of F i g u r e s F i g u r e 1. Simple summary of the a u d i t o r y pathways 3 F i g u r e 2. Somatostatin immunoreactivity i n the guinea p i g v e n t r a l c o c h l e a r nucleus; low m a g n i f i c a t i o n 17,18 F i g u r e 3. Somatostatin immunoreactivity seen throughout the guinea p i g v e n t r a l c o c h l e a r n u c l e u s ; medium and h i g h m a g n i f i c a t i o n s 21,23 F i g u r e 4. Somatostatin immunoreactivity i n the r a t v e n t r a l c o c h l e a r n u c l e u s ; low m a g n i f i c a t i o n 26,27 F i g u r e 5. Somatostatin immunoreactivity i n the r a t v e n t r a l c o c h l e a r nucleus; medium and h i g h m a g n i f i c a t i o n s 29 F i g u r e 6. Somatostatin immunoreactivty seen i n the c a t and guinea p i g d o r s a l c o c h l e a r nucleus and the c a t a n t e r i o r v e n t r a l c o c h l e a r nucleus; medium m a g n i f i c a t i o n 33 F i g u r e 7. Somatostatin immunoreactivity i n the c a t s u p e r i o r o l i v a r y complex; low m a g n i f i c a t i o n . . . . . 36,37 v i i i F i g u r e 8. Somatostatin immunoreactivity seen i n the c a t v e n t r a l nucleus of the t r a p e z o i d body; medium and h i g h m a g n i f i c a t i o n s 39 F i g u r e 9. Somatostatin immunoreactivity seen i n the c a t p e r i o l i v a r y r e g i o n s ; medium and h i g h m a g n i f i c a t i o n s 42 F i g u r e 10. Somatostatin immunoreactivity found i n the guinea p i g s u p e r i o r o l i v a r y complex; low m a g n i f i c a t i o n 46 F i g u r e 11. Somatostatin immunoreactivity a s s o c i a t e d w i t h the guinea p i g medial s u p e r i o r o l i v e and medial nucleus of the t r a p e z o i d body; medium and h i g h m a g n i f i c a t i o n s 48 F i g u r e 12. Somatostatin immunoreactivity seen i n s u p e r i o r p a r a o l i v a r y and d o r s a l p e r i o l i v a r y r e g i o n s ; medium and h i g h m a g n i f i c a t i o n s 50 F i g u r e 13. Somatostatin immunoreactivity seen i n the v a r i o u s r e g i o n s of the guinea p i g s u p e r i o r o l i v a r y complex; medium and h i g h m a g n i f i c a t i o n s 52 F i g u r e 14. Somatostatin immunoreactivity a s s o c i a t e d w i t h the guinea p i g v e n t r a l nucleus of the t r a p e z o i d body; medium and hi g h m a g n i f i c a t i o n s 54,55 i x F i g u r e 15. S o m a t o s t a t i n i m m u n o r e a c t i v i t y s e e n i n t h e r a t s u p e r i o r o l i v a r y c o mplex; low m a g n i f i c a t i o n 59 F i g u r e 16. S o m a t o s t a t i n i m m u n o r e a c t i v i t y a s s o c i a t e d w i t h t h e r a t m e d i a l a n d v e n t r a l n u c l e i o f t h e t r a p e z o i d body; medium and h i g h m a g n i f i c a t i o n s 61 F i g u r e 17. S o m a t o s t a t i n i m m u n o r e a c t i v i t y s e e n i n t h e r a t p e r i o l i v a r y r e g i o n s ; medium a n d h i g h m a g n i f i c a t i o n s 63 F i g u r e 18. S o m a t o s t a t i n i m m u n o r e a c t i v i t y f o u n d i n t h e c a t v e n t r o l a t e r a l tegmentum; low m a g n i f i c a t i o n 69 F i g u r e 19. S o m a t o s t a t i n i m m u n o r e a c t i v i t y a s s o c i a t e d w i t h t h e c a t n u c l e i o f t h e l a t e r a l l e m n i s c u s ; medium and h i g h m a g n i f i c a t i o n s 71 F i g u r e 20. S o m a t o s t a t i n i m m u n o r e a c t i v i t y s e e n i n t h e g u i n e a p i g l a t e r a l l e m n i s c u s r e g i o n s ; low m a g n i f i c a t i o n 74 F i g u r e 21. S o m a t o s t a t i n i m m u n o r e a c t i v i t y f r o m s e l e c t e d r e g i o n s o f t h e g u i n e a p i g v e n t r a l n u c l e u s o f t h e l a t e r a l l e m n i s c u s ; medium and h i g h m a g n i f i c a t i o n s 76 F i g u r e 22. S o m a t o s t a t i n i m m u n o r e a c t i v i t y s e e n a t t h e d o r s a l p e r i m e t e r o f t h e g u i n e a p i g d o r s a l n u c l e u s o f t h e l a t e r a l l e m n i s c u s ; medium and h i g h m a g n i f i c a t i o n s 78 X F i g u r e 23. Somatostatin immunoreactivity a s s o c i a t e d w i t h the r a t l a t e r a l tegmental r e g i o n s ; low, medium and h i g h m a g n i f i c a t i o n s 82 F i g u r e 24. Somatostatin immunoreactivity a s s o c i a t e d w i t h the r a t v e n t r a l nucleus of the l a t e r a l lemniscus; medium m a g n i f i c a t i o n 84 F i g u r e 25. Somatostatin immunoreactivity a s s o c i a t e d w i t h the r a t d o r s a l , v e n t r a l nucleus of the l a t e r a l lemniscus; medium and h i g h m a g n i f i c a t i o n s 86 F i g u r e 26. Somatostatin immunoreactivity seen embedded i n f i b r e s of the r a t r o s t r a l , l a t e r a l lemniscus; medium and hi g h m a g n i f i c a t i o n s 88 F i g u r e 27. Somatostatin immunoreactivity seen i n the c a t i n f e r i o r c o l l i c u l u s ; low m a g n i f i c a t i o n 92 F i g u r e 28. Somatostatin immunoreactivity from s e l e c t e d r e g i o n s of the c a t i n f e r i o r c o l l i c u l u s ; medium and h i g h m a g n i f i c a t i o n s 94 F i g u r e 29. Somatostatin immunoreactivity from the r o s t r a l r e g i o n of the c a t i n f e r i o r c o l l i c u l u s ; medium and h i g h m a g n i f i c a t i o n s 96 x i F i g u r e 30. Somatostatin immunoreactivity found i n the r o s t r a l r e g i o n s of the c a t i n f e r i o r c o l l i c u l u s ; high-magnif i c a t i o n 98 F i g u r e 31. Somatostatin immunoreactivity a s s o c i a t e d w i t h the guinea p i g i n f e r i o r c o l l i c u l u s ; low m a g n i f i c a t i o n 103-105, 107 F i g u r e 32. So m a t o s t a t i n immunoreactivity seen i n the c a u d a l to mid r e g i o n s of the guinea p i g i n f e r i o r c o l l i c u l u s ; medium m a g n i f i c a t i o n 110,112,114 F i g u r e 33. Somatostatin immunoreactivity from s e l e c t e d r e g i o n s of the guinea p i g i n f e r i o r c o l l i c u l u s ; h i g h m a g n i f i c a t i o n 116 F i g u r e 34. Somatostatin immunoreactivity seen i n the r a t i n f e r i o r c o l l i c u l u s ; low m a g n i f i c a t i o n 120,122 F i g u r e 35. Somatostatin immunoreactivity from s e l e c t e d r e g i o n s of the r a t i n f e r i o r c o l l i c u l u s ; medium m a g n i f i c a t i o n 124,126 F i g u r e 36. Somatostatin immunoreactivity from s e l e c t e d r e g i o n s of the r a t i n f e r i o r c o l l i c u l u s ; h i g h m a g n i f i c a t i o n 128 x i i F i g u r e 37. A summary diagram of the ascending a u d i t o r y n u c l e i which c o n t a i n s o m a t o s t a t i n immunoreactive c e l l s . . . 1 3 9 F i g u r e 38. A summary diagram of the descending a u d i t o r y n u c l e i and t h e i r p o s s i b l e p r o j e c t i o n s 144 F i g u r e 39. P o s s i b l e s i t e s of neurochemical c o e x i s t e n c e or p a r a l l e l p r o j e c t i o n s 151 F i g u r e 40. P o s s i b l e s i t e s and f u n c t i o n s of the descending a u d i t o r y pathways 165 Acknowledgement I would l i k e t o begin by t h a n k i n g Dr. J.C. Brown (Dept. P h y s i o l o g y , U n i v e r s i t y of B r i t i s h Columbia) f o r p r o v i d i n g the s o m a t o s t a t i n monoclonal antibody. Without Dr. Brown's g e n e r o s i t y t h i s t h e s i s would have been c o n s i d e r a b l y s h o r t e r . I would a l s o l i k e t o express my a p p r e c i a t i o n t o the people of the Kinsmen L a b o r a t o r y f o r t h e i r camaraderie, a s s i s t a n c e and a d v i c e . I am p a r t i c u l a r l y i n d e b t e d to Dr.s E.G. McGeer and S.R. V i n c e n t (Steve and Edie) f o r t h e i r p a t i e n c e , support and guidance over the p a s t y e a r s . To them I owe a s p e c i a l thanks and wish them "more f u n " f o r the f u t u r e . 1 INTRODUCTION ( R e i c h l e n , '83; Ror s t a d e t a l . , '80) The d i s t r i b u t i o n of s o m a t o s t a t i n i n the brainstem a u d i t o r y system of th r e e s p e c i e s : c a t , r a t and guinea p i g was examined u s i n g c u r r e n t immunohistochemical methods. The p r o c e s s of h e a r i n g begins w i t h sound s t r i k i n g the tympanic membrane of the middle e a r . The st i m u l u s then, v i a the o s s i c l e s and o v a l window, produces compression of the f l u i d of the c o c h l e a . T h i s compression s e t s up a wave motion along the b a s i l a r membrane. The peak amplitude of t h i s wave i s a f u n c t i o n of the st i m u l u s frequency. The frequency which produces t h i s peak i s r e f e r r e d t o as the c h a r a c t e r i s t i c frequency (and v a r i e s w i t h the p o s i t i o n along the b a s i l a r membrane). S i t u a t e d on the b a s i l a r membrane i s the organ of C o r t i . The organ of C o r t i c o n t a i n s both i n n e r and out e r h a i r c e l l s . I t i s the i n n e r h a i r c e l l s which appear p r i m a r i l y i n v o l v e d i n the ascending a u d i t o r y a f f e r e n t pathway. P u t a t i v e l y these c e l l s d e p o l a r i z e i n response t o the wave motion of the b a s i l a r membrane which r e s u l t s i n the r e l e a s e of n e u r o t r a n s m i t t e r onto apposing IHC a f f e r e n t s . These a f f e r e n t s o r i g i n a t e from the b i p o l a r c e l l s whose p e r i k a r y a ( F i g 1) are found w i t h i n the s p i r a l g a n g l i o n . These s p i r a l g a n g l i o n c e l l s p r o j e c t v i a the e i g h t h nerve t o terminate i n the c o c h l e a r n u c l e u s . T h i s nucleus i n t u r n p r o j e c t s ( F i g 1) t o the s u p e r i o r o l i v a r y complex (SOC), the d o r s a l (DLL) and 2 F i g u r e 1. A simple summary diagram of the a u d i t o r y pathways. B o l d l i n e s show the ascending a u d i t o r y pathways. The dashed l i n e s i n d i c a t e the descending a u d i t o r y pathways. The t h i n s o l i d l i n e (1) i n d i c a t e s an ascending somatosensory pathway t o the i n f e r i o r c o l l i c u l u s . The d o t t e d l i n e (2) i n d i c a t e s a p r o j e c t i o n from the IC t o the c o n t r a l a t e r a l IC. The d o t t e d l i n e (3) i n d i c a t e s an ascending multimodal sensory pathway. A p o s s i b l e p r o j e c t i o n (?) may e x i s t from the IC t o the VLL; t h i s i s shown i n the i l l u s t r a t i o n s of Burne e t a l . ('81) and Van Noort ('69), but not d e s c r i b e d i n t h e i r t e x t s . Symbols:A , c o n t r a l a t e r a l . A b b r e v i a t i o n s : ANC, a u d i t o r y neocortex; BCG, b i p o l a r c e l l g a n g l i o n ; CN, c o c h l e a r nucleus; DCN , d o r s a l column n u c l e i ( i n a l l o t h e r f i g u r e s DCN r e f e r s t o the d o r s a l c o c h l e a r n u c l e u s ) ; IC, i n f e r i o r c o l l i c u l u s ; IHC, in n e r h a i r c e l l s ; LCN, l a t e r a l c e r v i c a l nucleus; LPN, l a t e r a l p o n t i n e nucleus; MGB, medial g e n i c u l a t e body; MRF, mid b r a i n r e t i c u l a r f o r mation; NLL, n u c l e i of the l a t e r a l lemniscus; OHC, out e r h a i r c e l l s ; SOC, s u p e r i o r o l i v a r y complex. 3 D C N * C N L C N 4 v e n t r a l (VLL) n u c l e i of the l a t e r a l lemniscus ( F i g 1) and the c e n t r a l nucleus of the i n f e r i o r c o l l i c u l u s (ICC). The s u p e r i o r o l i v a r y complex, l o c a t e d i n the v e n t r a l ponto-me d u l l a r y brainstem, p r o j e c t s t o the VLL, DLL and the ICC ( F i g 1). Both the DLL and VLL a l s o p r o j e c t t o the ICC. T h i s core r e g i o n o f the i n f e r i o r c o l l i c u l u s p r o j e c t s i n t u r n t o the v e n t r a l d i v i s i o n of the medial g e n i c u l a t e body (MGBv). The MGBv p r o j e c t s t o the primary a u d i t o r y neocortex where, presumably, c o n s c i o u s r e c o g n i t i o n of sound o c c u r s . Although not w e l l s t u d i e d , a descending a u d i t o r y path (dashed l i n e s F i g 1) a l s o e x i s t s . A f f e r e n t s from the a u d i t o r y c o r t e x i n n e r v a t e the p e r i c e n t r a l ( p e r i l C C ) r e g i o n s of the i n f e r i o r c o l l i c u l u s and the l a t e r a l r e g i o n s of the pont i n e n u c l e i . P e r i c e n t r a l r e g i o n s of the i n f e r i o r c o l l i c u l u s p r o v i d e descending e f f e r e n t s t o the p e r i o l i v a r y r e g i o n s (periSOC). These p e r i o l i v a r y r e g i o n s i n t u r n p r o v i d e descending e f f e r e n t s t o the c o c h l e a r nucleus and to the i n n e r h a i r c e l l a f f e r e n t s and the out e r h a i r c e l l s of the c o c h l e a . C e l l s of the a u d i t o r y system, l i k e the b a s i l a r membrane, have a frequency t o which they w i l l show maximal response. T h i s i s c a l l e d the c h a r a c t e r i s t i c frequency (CF). W i t h i n most a u d i t o r y n u c l e i these CFs change on a continuum as one moves through a nuc l e u s . T h i s p a t t e r n i s r e f e r r e d t o as t o n o t o p i c o r , when compared t o the c o c h l e a , c o c h l e o t o p i c . 5 Most n e u r o t r a n s m i t t e r s and p e p t i d e s have been r e p o r t e d i n a t l e a s t one r e g i o n of the a u d i t o r y system. T h i s l i t e r a t u r e w i l l be examined f u r t h e r i n the d i s c u s s i o n . The i n v e s t i g a t i o n i n t o s o m a t o s t a t i n immunoreactivity, SOM-IR, began w i t h a study of the l a r g e SOM-IR t e r m i n a l s i n the r a t medial nucleus of the t r a p e z o i d body (MNTB), which forms p a r t of the s u p e r i o r o l i v a r y complex (SOC). The guinea p i g was subsequently examined f o r s i m i l a r t e r m i n a l s . The p a t t e r n of SOM-IR t e r m i n a l s seen i n the r a t MNTB was not found i n the guinea p i g . The d i s t r i b u t i o n s of SOM-IR throughout the a u d i t o r y systems of these rodents, showed both s i m i l a r i t i e s and marked d i f f e r e n c e s . The next q u e s t i o n was how d i d the d i s t r i b u t i o n of SOM-IR var y between rodents and other s p e c i e s ? The c a t seemed t o be the bes t c h o i c e as t h i s animal i s a common s u b j e c t i n a u d i t o r y r e s e a r c h . While p r e v i o u s s t u d i e s have shown the d i s t r i b u t i o n of SOM-IR i n the r a t and guinea p i g b r a i n s , they p r o v i d e l i t t l e d e s c r i p t i o n of c e l l type or comparison t o p r e v i o u s work on a u d i t o r y a f f e r e n t and e f f e r e n t pathways. With t h i s i n mind I sought t o d e s c r i b e , compare and c o n t r a s t the d i s t r i b u t i o n of SOM-IR i n the s u b c o r t i c a l a u d i t o r y pathways of the c a t , guinea p i g and r a t . Somatostatin, SOM, was f i r s t c h a r a c t e r i z e d as a t e t r a d e c a p e p t i d e , SOM-14. I t s name so m a t o s t a t i n , or somatotropin r e l e a s e i n h i b i t i n g f a c t o r , r e f e r s t o i t s i n h i b i t i o n of growth hormone r e l e a s e from the a n t e r i o r p i t u i t a r y . Subsequently a 14 amino a c i d N - t e r m i n a l e x t e n s i o n 6 of SOM-14, SOM-28, and l a r g e r forms of so m a t o s t a t i n have been d i s c o v e r e d . These l a r g e r forms were b i o l o g i c a l l y a c t i v e on t h e i r own and as such r e p r e s e n t more than j u s t p r e c u r s o r s . Somatostatin t h e r e f o r e i s not a s i n g l e p e p t i d e but r e f e r s t o a f a m i l y of p e p t i d e s whose mol e c u l a r weight v a r i e s from around 1600 to 3100 d a l t o n s . The d i s t r i b u t i o n of s o m a t o s t a t i n i s not r e s t r i c t e d t o the hypothalamus. Somatostatin i s found i n the pancreas, i n t e s t i n e and nervous system of v e r t e b r a t e s and as such i s found both i n neurons and endocrine c e l l s . C e n t r a l l y , s o m a t o s t a t i n i s r e l e a s e d as a hormone from the median eminence and i s a l s o found i n p e r i k a r y a and p r e s y n a p t i c t e r m i n a l s throughout the CNS. Somatostatin has been found i n a l l c l a s s e s of v e r t e b r a t e s and i n i n v e r t e b r a t e s i n c l u d i n g i n s e c t s and pro t o z o a . Of the d i f f e r e n t forms of so m a t o s t a t i n , SOM-14 appears the b e s t conserved. In a comparison between a r a t medullary carcinoma l i n e and a n g l e r f i s h t h e r e was an 83% c o n s e r v a t i o n of n u c l e o t i d e s c o d i n g f o r SOM-14. The N - t e r m i n a l extended SOM-28, however, was l e s s w e l l conserved. U s i n g the same sources c i t e d above, t h e r e was o n l y a 69% c o n s e r v a t i o n of n u c l e o t i d e s , w i t h most c o n s e r v a t i o n b e i n g i n the 1-14 amino a c i d sequence. As a n e u r o t r a n s m i t t e r c a n d i d a t e , s o m a t o s t a t i n , l i k e o t h e r neuropeptides, d i f f e r s from the c l a s s i c n e u r o t r a n s m i t t e r s i n t h a t i t i s d e r i v e d from a l a r g e p o l y p e p t i d e s y n t h e s i z e d , l i k e a l l p r o t e i n s , under the 7 guidance of a s p e c i f i c mRNA. Post t r a n s l a t i o n a l m o d i f i c a t i o n of the preprohormone or prohomone takes p l a c e w i t h i n the endoplasmic r e t i c u l u m or the l a m e l l a e of the G o l g i apparatus. Subsequently the p e p t i d e , s o m a t o s t a t i n , i s bound i n v e s i c l e s and t r a n s p o r t e d a x o n a l l y t o p r e s y n a p t i c t e r m i n a l s . Other n e u r o t r a n s m i t t e r c r i t e r i a , such as the presence of b i n d i n g s i t e s , potassium evoked r e l e a s e , changes i n spontaneous c e l l u l a r a c t i v i t y f o l l o w i n g i o n t o p h o r e t i c a p p l i c a t i o n and b e h a v i o u r a l e f f e c t s , have a l l been demonstrated f o r s o m a t o s t a t i n . P r e v i o u s s t u d i e s have shown s o m a t o s t a t i n i n the brainstem of the r a t (Johansson e t a l . , '84; V i n c e n t e t a l . , '85) and g u i n e a - p i g ( T a b e r - P i e r c e e t a l . , '85). The pr e s e n t study i n c l u d e s the c a t and focuses on the ponto-medullary, tegmental and t e c t a l a u d i t o r y n u c l e i . The Brainstem A u d i t o r y System The b r a i n s t e m a u d i t o r y r e g i o n s i n c l u d e d were the c o c h l e a r n u c l e i , the s u p e r i o r o l i v a r y complex, the l e m n i s c a l n u c l e i , the sagulum, and the i n f e r i o r c o l l i c u l u s . F u r t h e r s u b d i v i s i o n of these n u c l e i has taken many forms. V a r i a t i o n s r e s u l t from the use of d i f f e r e n t h i s t o l o g i c a l methods or from the d i f f e r e n t c r i t e r i a , i . e . , c e l l s i z e , c e l l morphology, d e n d r i t i c a r b o r i z a t i o n and/or d e n d r i t i c o r i e n t a t i o n . These v a r i o u s n u c l e i have been i m p l i c a t e d i n both the ascending and descending a u d i t o r y pathways ( H a r r i s o n and Howe, '74a; '74b; H a r r i s o n and Feldman, '70). 8 A more d e t a i l e d summary of brainstem a u d i t o r y anatomy than t h a t o u t l i n e d i n F i g u r e 1 i s f i r s t r e q u i r e d . Primary a f f e r e n t s a r i s i n g i n the c o c h l e a r s p i r a l g a n g l i o n terminate on the second order a u d i t o r y neurons found p r i m a r i l y i n the c o c h l e a r nucleus (CN). The c o c h l e a r nucleus i s d i v i d e d i n t o a v e n t r a l c o c h l e a r nucleus (VCN) and a d o r s a l c o c h l e a r nucleus (DCN). The b i f u r c a t i o n of the e i g h t h nerve i n t o ascending and descending branches d i v i d e s the VCN i n t o an a n t e r i o r (AVCN) and p o s t e r i o r (PVCN) d i v i s i o n r e s p e c t i v e l y . The DCN i s d o r s a l to and contiguous w i t h the PVCN. There are many d i v i s i o n s of the c o c h l e a r n u c l e i , although a r e l a t i v e l y common d i v i s i o n of the VCN f o r c a t (Osen, '69), g u i n e a - p i g ( P r i z i g , '68) and r a t ( H a r r i s o n and I r v i n g , '66a; 66b) e x i s t s . S l i g h t d i f f e r e n c e s e x i s t between s p e c i e s and methods. Osen's method uses a d e s c r i p t i o n of the p r i n c i p a l c e l l s f o r a r e g i o n w h i l e the rodent nomenclatures use Roman numerals t o c l a s s i f y s i m i l a r r e g i o n s . Region I I I , the r o s t r a l p o l e of the AVCN, has predominantly medium-large t o l a r g e s p h e r i c a l c e l l s . Region I l i e s immediately c a u d a l t o r e g i o n I I I and i s r e f e r r e d t o as the s m a l l s p h e r i c a l c e l l r e g i o n . Regions I and I I I were to g e t h e r r e f e r r e d t o as the s p h e r i c a l c e l l r e g i o n of the AVCN (SC-AVCN) (Warr, '82). Region I I i s l o c a t e d v e n t r a l l y through both AVCN/PVCN. Region I I i s c h a r a c t e r i z e d by medium-large c e l l s w i t h e c c e n t r i c n u c l e i which have been c a l l e d g l o b u l a r c e l l s . Regions IV and V l i e w i t h i n the PVCN. Region IV i s c h a r a c t e r i z e d by l a r g e m u l t i p o l a r s r e f e r r e d to as octopus 9 c e l l s . Region V forms the ca u d a l e x t e n t of the PVCN. Regions II-V can be c o l l e c t i v e l y r e f e r r e d t o as the m u l t i p o l a r and g l o b u l a r - c e l l r e g i o n (Warr, '82). A cap of v a r i o u s types of s m a l l c e l l s l i e s a long the d o r s a l perimeter of the ca u d a l AVCN and r o s t r a l PVCN. At the l a t e r a l and d o r s a l perimeter l i e s a granule c e l l l a y e r . The ex t e n t of the granule c e l l l a y e r v a r i e s somewhat between s p e c i e s . The granule c e l l l a y e r forms the s u p e r f i c i a l s u r f a c e o f the l a t e r a l AVCN and the border between PVCN and DCN (Mugnaini e t a l . , '80). The s u p e r i o r o l i v a r y complex (SOC) has been s u b d i v i d e d i n v a r i o u s ways and, a c c o r d i n g l y , c e r t a i n r e g i o n s have been g i v e n a v a r i e t y of names. While the nomenclature v a r i e s , the d i v i s i o n s and d e s c r i p t i o n s of the v a r i o u s r e g i o n s remain r e l a t i v e l y c o n s t a n t . A review of a u d i t o r y nomenclature i s gi v e n by H a r r i s o n and Howe ('74a; '74b). For s i m p l i c i t y and c l a r i t y , p r e v a i l i n g nomenclature, which p r o v i d e s an ac c u r a t e r e p r e s e n t a t i o n of the SOM-IR d i s t r i b u t i o n , w i l l be used. The SOC i s s u b d i v i d e d i n t o the LSO ( l a t e r a l s u p e r i o r o l i v e ) , MSO (medial s u p e r i o r o l i v e ) , MNTB (medial nucleus of the t r a p e z o i d body), VNTB ( v e n t r a l nucleus of the t r a p e z o i d body), SPO ( s u p e r i o r p a r a o l i v a r y n u c l e u s ) , DMPO (dorsomedial p e r i o l i v a r y nucleus) and the DLPO ( d o r s o l a t e r a l p e r i o l i v a r y r e g i o n ) . A n o t a b l e d i f f e r e n c e between the c a t and rodents i s the SPO. T h i s nucleus i s seen between the MNTB and the MSO and i s found i n rodents, m a r s u p i a l s and ce t a c e a but i s not found i n the c a t . I t c o n t a i n s both s m a l l and l a r g e m u l t i p o l a r c e l l s , i n c l u d i n g the l a r g e s t c e l l s i n the 10 s u p e r i o r o l i v a r y complex ( H a r r i s o n and Feldman, '70). T h i s nucleus i s d i f f e r e n t from the DMPO. The DMPO i n c a t s i s composed of s m a l l e l o n g a t e d c e l l s and s m a l l t o medium s i z e m u l t i p o l a r c e l l s ( H a r r i s o n and Feldman, '70; Morest '69). The l a t t e r send d e n d r i t e s i n t o the r e t i c u l a r f o r m a t i o n o r i n t o the MNTB ( i n the r a t these c e l l s a l s o d i r e c t p r o c e s s e s i n t o the MNTB). The DPO l i e s between the d o r s a l aspect of MSO and LSO. The DLPO i s found a l o n g the d o r s o l a t e r a l t o l a t e r a l margin of the SPO. The VNTB c o n s i s t s of i s l a n d s of c e l l s seen i n the v e n t r a l f i b r e s of the t r a p e z o i d body. The MNTB i s composed p r i n c i p a l l y of round or o v a l c e l l s and l i e w i t h i n the medial f i b r e s of the t r a p e z o i d body. The VPO c o n s i s t s of round, f u s i f o r m and a few m u l t i p o l a r c e l l s and l i e s a l o n g the v e n t r a l aspect of the LSO. The LSO i s a sigmoid shaped nucleus composed p r i m a r i l y of f u s i f o r m c e l l s which, i n c o r o n a l s e c t i o n , l i e p e r p e n d i c u l a r t o the s u r f a c e of the nu c l e u s . The MSO c o n s i s t s of a column of f u s i f o r m c e l l s which o r i e n t t h e i r long axes i n a m e d i o l a t e r a l d i r e c t i o n . The r o s t r a l extreme of the SOC i s contiguous, but not continuous, w i t h the ca u d a l p o l e of the nucleus of the v e n t r o l a t e r a l lemniscus (VLL). The l e m n i s c a l n u c l e i l i e r o u g h l y between the medial and l a t e r a l bands of the l a t e r a l l emniscus. The n u c l e i of the l a t e r a l lemniscus can be d i v i d e d i n t o a d o r s a l and v e n t r a l groups. The v e n t r a l nucleus has been f u r t h e r s u b d i v i d e d i n t o a d o r s a l , m edial and v e n t r a l r e g i o n . T h i s d o r s a l r e g i o n of the VLL i s 11 a l t e r n a t i v e l y r e f e r r e d t o as the i n t e r s t i t i a l nucleus of the l a t e r a l lemniscus (ILL) (Adams, '79). The sagulum i s a s m a l l r e g i o n which l i e s l a t e r a l " t o the l a t e r a l l e m n i s c a l band and i n the v e n t r a l l a t e r a l border of the i n f e r i o r c o l l i c u l u s ( I C ) . The nomenclature of the IC d i v i s i o n s v a r i e s but s i m i l a r r e g i o n s have been g e n e r a l l y r e c o g n i z e d r e g a r d l e s s of the nomenclature. One e x c e p t i o n i s the d i v i s i o n of the IC suggested by Berman ('69), which f a i l s t o d i f f e r e n t i a t e a dorsomedial s u b d i v i s i o n of the IC. As was r e c e n t l y suggested (Morest and O l i v e r , '84), t h i s d i s c r e p a n c y was p r o b a b l y due to the use of N i s s l s t a i n t o d i f f e r e n t i a t e the r e g i o n s of the IC. To d e s c r i b e the d i s t r i b u t i o n of SOM-IR w i t h i n the IC, we can c o n s i d e r a simple d i v i s i o n i n t o a c e n t r a l nucleus (ICC) and d o r s a l (DC) and e x t e r n a l (EC) c o r t i c e s . A s s o c i a t e d IC s t r u c t u r e s i n c l u d e the brachium of the i n f e r i o r c o l l i c u l u s (BIC), the r o s t r a l p o l e (RP), the i n t e r c o l l i c u l a r area (ICA) and the commissure of the i n f e r i o r c o l l i c u l u s (COIC). The a f f e r e n t s and e f f e r e n t s of these a u d i t o r y n u c l e i w i l l be d e t a i l e d when d i s c u s s i n g the p o s s i b l e o r i g i n of SOM-IR a u d i t o r y e f f e r e n t s . MATERIALS AND METHODS Three a d u l t male c a t s , 3-4 kg, s i x a d u l t male guinea p i g s , 150-250 grams, and f i v e a d u l t male Sprauge Dawley r a t s , 200-250 grams, were used. The animals were a l l g i v e n l e t h a l i n j e c t i o n s of sodium p e n t o b a r b i t a l . The c a t s were then g i v e n a b o l u s of h e p a r i n (10,000 u n i t s , i n t r a c a r d i a c ) w h i l e the rodents were p e r f u s e d w i t h 50 mis of h e p e r i n i z e d normal s a l i n e (20 IU/ml). Cats and rodents were p e r f u s e d w i t h e i t h e r 4% paraformaldehyde i n phosphate b u f f e r or a 2% paraformaldehyde Zamboni's f i x a t i v e (Zamboni and Demartino, '67), pH 7.4. A l l s p e c i e s were p e r f u s e d v i a a o r t i c c a n n u l a t i o n . F o l l o w i n g p e r f u s i o n , the b r a i n s were removed, p o s t f i x e d i n the same f i x a t i v e o v e r n i g h t a t 4° C and then p l a c e d i n c r y o p r o t e c t a n t (25% sucrose, 10% g l y c e r o l i n 0.05 M phosphate b u f f e r ) a t 4° C and l e f t u n t i l sunk. The r e s u l t s were the same r e g a r d l e s s of the f i x a t i v e . B r a i n s were c u t c o r o n a l l y a t 30 microns on a R e i c h e r t Jung f r e e z i n g microtome. S e c t i o n s were incubated i n 0.3% H 2 0 2 i n t r i s - b u f f e r e d s a l i n e (TBS), 50 mM pH 7.4, f o r 3 X 20 min. w i t h 2 X 5 min. i n TBS alone between each i n c u b a t i o n i n H2O2. S e c t i o n s were next i n c u b a t e d i n primary antibody s o l u t i o n c o n t a i n i n g mouse a n t i - s o m a t o s t a t i n IgG monoclonal, 2-4% normal goat serum (NGS) and 0.3% T r i t o n X-100 (TX) i n TBS, pH 7.4. S e c t i o n s were in c u b a t e d i n primary antibody s o l u t i o n f o r 48 hours a t 4 U C w i t h c o n s t a n t a g i t a t i o n on a r o t a r y mixer and then washed f o r 3 x 20 min. i n TBS a t room temperature. The secondary a n t i b o d y s o l u t i o n c o n s i s t e d of b i o t i n y l a t e d horse anti-mouse (Vector S t a i n ) , 4% NGS and 0.3% TX i n TBS. S e c t i o n s p l a c e d i n the secondary antibody s o l u t i o n f o r one hour a t room temperature w i t h c o n s t a n t a g i t a t i o n . S e c t i o n s were then washed 3 x 20 min. i n TBS and p l a c e d i n t e r t i a r y s o l u t i o n . T h i s was made by mixing 88 u,l of a v i d i n and 88 u.1 of b i o t i n y l a t e d p e r oxidase i n 10 ml of TBS, a t l e a s t 20 min. b e f o r e use. NGS and TX were added t o t h i s t e r t i a r y s o l u t i o n t o a f i n a l c o n c e n t r a t i o n of 4% and 0.3% r e s p e c t i v e l y . I n c u b a t i o n was f o r 1 hour a t room temperature w i t h continuous a g i t a t i o n . S e c t i o n s were a g a i n washed 3 x 20 min. i n TBS. S e c t i o n s were then i n c u b a t e d i n 50 mM t r i s b u f f e r , pH 7.4, c o n t a i n i n g e i t h e r 0.02% diaminobenzidine (DAB) and 0.001 M i m i d a z o l e or 0.02% DAB and 0.6% n i c k e l ammonium s u l f a t e f o r 10-15 min. p r i o r t o the a d d i t i o n of H2O2/ 0.0075% or 0.0015%, r e s p e c t i v e l y . S e c t i o n s were i n c u b a t e d f o r a f u r t h e r 10-15 or l e s s than 5 min., r e s p e c t i v e l y . S e c t i o n s were washed i n a t l e a s t 3 changes of TBS f o r no l e s s than 10 min., mounted on chrome-alum coated g l a s s s l i d e s and a i r d r i e d . The mounted s e c t i o n s were taken through an a l c o h o l g r a d i e n t s e r i e s , passed through x y l e n e and then mounted w i t h Permount and c o v e r s l i p p e d . S e c t i o n s were observed and photographed under the l i g h t microscope. 14 The primary antibody, d i r e c t e d a g a i n s t s y n t h e t i c c y c l i c SOM 1-14, has been c h a r a c t e r i z e d and d e s c r i b e d elsewhere (V i n c e n t e t a l . , '85). C o n t r o l experiments i n v o l v i n g p r e a b s o r p t i o n of the primary antibody w i t h s y n t h e t i c c y c l i c SOM 1-14 e l i m i n a t e d immunohistochemical s t a i n i n g . 15 RESULTS The r e s u l t s w i l l f o l l o w the ascending a u d i t o r y pathway, b e g i n n i n g w i t h the c o c h l e a r n u c l e i and ending w i t h the i n f e r i o r c o l l i c u l u s ( I C ) . W i t h i n each a u d i t o r y nucleus or s u b d i v i s i o n s t h e r e o f , the d i s t r i b u t i o n of SOM-IR, i n both ascending and descending a u d i t o r y pathways, w i l l be c o n s i d e r e d . The p o s s i b l e o r i g i n s of the SOM-IR puncta w i l l be d e a l t w i t h i n the d i s c u s s i o n . Cochlear Nucleus V e n t r a l Cochlear Nucleus, VCN The c a t showed a v i r t u a l absence of SOM-IR soma throughout the VCN. The few c e l l s seen were medium s i z e d , m u l t i p o l a r c e l l s and were u s u a l l y l o c a t e d a t the p e r i p h e r y of the nucleus ( F i g 6C). A low t o moderate d e n s i t y of SOM-IR puncta, were seen a s s o c i a t e d w i t h the granule c e l l l a y e r of the c a t VCN ( F i g 6C). The guinea p i g VCN c o n t a i n e d a moderate t o h i g h d e n s i t y of SOM-IR puncta, and a moderate t o low d e n s i t y of immunoreactive c e l l s i n the AVCN. In the r o s t r a l p o l e of the AVCN few c e l l s were seen ( F i g 2A). Moving c a u d a l l y s m a l l t o medium s i z e d , m u l t i p o l a r or b i p o l a r c e l l s w i t h o v a l , t r i a n g u l a r or s p i n d l e shaped somas ( F i g 2B,C, 3A,B,E,F) were seen. I n i t i a l l y these c e l l s appeared a t the 16 F i g u r e 2. R o s t r a l guinea p i g a n t e r i o r v e n t r a l c o c h l e a r nucleus (AVCN) t p c a u d a l guinea p i g p o s t e r i o r v e n t r a l c o c h l e a r nucleus (PVCN). Immunoreactive puncta show e x t e n s i v e p e r i c e l l u l a r a p p o s i t i o n throughout the v e n t r a l c o c h l e a r nucleus (VCN). Increa s e d immunoreactivity along the l a t e r a l s u r f a c e of the VCN was a s s o c i a t e d w i t h the gr a n u l e c e l l l a y e r of the PVCN. (A) A moderate t o h i g h d e n s i t y of som a t o s t a t i n immunoreactive puncta form p e r i c e l l u l a r a p p o s i t i o n i n the r o s t r a l p o l e of the guinea p i g AVCN. The granule c e l l l a y e r (apposing arrows), l o c a t e d l a t e r a l l y , shows a h i g h e r l e v e l of s o m a t o s t a t i n immunoreactivity than does the adj a c e n t r e g i o n s of the AVCN. (B) C a u d a l l y t h e r e was an i n c r e a s e i n immunoreactive c e l l s seen p e r i p h e r a l l y . Medium t o l a r g e m u l t i p o l a r c e l l s tend t o be found c e n t r a l l y w i t h i n the AVCN. (C) Medium t o l a r g e m u l t i p o l a r c e l l s were seen p r i m a r i l y i n the d o r s a l t w o - t h i r d s of the AVCN (arrowheads). M e d i a l l y a s i m i l a r p a t t e r n of immunoreactive puncta and s m a l l t o medium c e l l s were seen c l u s t e r e d among the f i b r e s of the v e s t i b u l a r nerve (arrow). (D) The d o r s a l aspect of the PVCN c o n t a i n s both s m a l l and l a r g e s o m a t o s t a t i n immunoreactive c e l l s . (E) Caudal PVCN a t the border w i t h the d o r s a l c o c h l e a r nucleus (DCN). Rare, l a r g e m u l t i p o l a r immunoreactive c e l l s were seen w i t h i n the PVCN (arrow). The m a j o r i t y of l a r g e m u l t i p o l a r c e l l s were covered by e x t e n s i v e p e r i s o m a t i c and p e r i d e n d r i t i c immunoreactive puncta (see F i g 2M-0). The c a l i b r a t i o n bar i n d i c a t e s 80 microns f o r A,B and 500 microns f o r C,D,E. 17 DCN '4 pypN !-•• '.V . \ " "V -19 d o r s a l , l a t e r a l and medial p e r i m e t e r s of the r o s t r a l AVCN ( F i g 2B). L a t e r a l l y these c e l l s were u s u a l l y o r i e n t e d w i t h the l o n g axes of t h e i r soma p a r a l l e l t o , and embedded w i t h i n , the t r a p e z o i d body ( F i g 2B). In the pe r i m e t e r , these c e l l s have o v a l , s p h e r i c a l o r t r i a n g u l a r c e l l b odies w i t h a b i p o l a r or m u l t i p o l a r d e n d r i t i c appearance. The s m a l l c e l l s found d o r s a l l y ( F i g 3A) were somewhat s m a l l e r than t h e i r v e n t r a l c o u n t e r p a r t s ( F i g 3B). Moving c a u d a l l y t h e r e was an i n c r e a s e i n medium and l a r g e m u l t i p o l a r c e l l s i n the d o r s a l t w o - t h i r d s of the AVCN ( F i g 2B,C). These c e l l s have e i t h e r t r i a n g u l a r or s p i n d l e shaped somas ( F i g 3B,C,G,H). The s m a l l t o medium immunoreactive c e l l s of the SC-AVCN ( F i g 3A,F) were s t i l l seen i n the perimeter of the nucleus w i t h the medium and l a r g e s i z e d m u l t i p o l a r c e l l s u s u a l l y occupying a more c e n t r a l p o s i t i o n . In the c a u d a l guinea p i g AVCN and r o s t r a l PVCN, a t the l e v e l of the nerve r o o t , ( F i g 2D) t h e r e were medium t o l a r g e m u l t i p o l a r c e l l s i n the d o r s a l t w o - t h i r d s of the nucleus as w e l l as medium s i z e d m u l t i p o l a r c e l l s w i t h o v a l and s p h e r i c a l c e l l b odies ( F i g 3G,H,J). The l a t t e r were found i n both d o r s a l and v e n t r a l PVCN. At t h i s l e v e l ( F i g 2D) s m a l l t o medium c e l l s were s t i l l seen a t the p e r i p h e r y of the VCN ( F i g 3J,K). Small o v a l and s p i n d l e shaped c e l l s were s t i l l found a t the d o r s a l and dorsomedial borders VCN ( F i g 3I,J,K) . Moving t o the ca u d a l PVCN, the number of l a r g e and medium, m u l t i p o l a r c e l l s d e c l i n e d and, a t the caud a l PVCN, were i n f r e q u e n t ( F i g 2E). 20 F i g u r e 3. I l l u s t r a t e s s o m a t o s t a t i n immunoreactivity seen through the a n t e r i o r v e n t r a l c o c h l e a r nucleus (AVCN) and the p o s t e r i o r v e n t r a l c o c h l e a r nucleus (PVCN). P e r i c e l l u l a r immunoreactive puncta can be seen i n most p a r t s of F i g u r e 3. (A) High m a g n i f i c a t i o n of s m a l l t o medium s i z e d c e l l s seen a t d o r s a l p e r i m e t e r of the r o s t r a l AVCN ( F i g 3B). (B) Medium to medium-large c e l l ( s h o r t arrow) and o c c a s i o n a l s m a l l t o medium c e l l s (arrow) seen c e n t r a l l y w i t h i n AVCN. (C) Large c e l l s seen d o r s a l l y throughout the AVCN ( F i g 2C). (D) High d e n s i t y of immunoreactive puncta making p e r i c e l l u l a r (arrow) and p e r i d e n d r i t i c (arrowhead) a p p o s i t i o n t o medium to medium-large c e l l s of the s p h e r i c a l c e l l r e g i o n of the AVCN (SC-AVCN). (E) A h i g h d e n s i t y of immunoreactive puncta can be seen l a t e r a l l y i n the granule c e l l l a y e r (GCL) (see F i g 3D). Small t o medium c e l l s were l o c a t e d (arrows) medial t o the GCL. Immunoreactive puncta were seen making p e r i c e l l u l a r a p p o s i t i o n . (F) High m a g n i f i c a t i o n of the s m a l l t o medium s i z e c e l l s shown i n (E) which predominate i n the p e r i p h e r y of the VCN. Immunolabelled p e r i c e l l u l a r puncta can be seen forming p e r i c e l l u l a r a p p o s i t i o n . (G,H) Large immunoreactive m u l t i p o l a r c e l l s were seen along the d o r s a l aspect of the ca u d a l AVCN border. 2 1 22 F i g u r e 3 ( c o n t . ) . (I) The PVCN/DCN border (see F i g 3E). Small, medium and l a r g e c e l l s were seen. (J) High m a g n i f i c a t i o n of l a r g e m u l t i p o l a r and s m a l l t o medium c e l l s seen a t the c a u d a l VCN border (from F i g 31,2D). (K) Small t o medium c e l l s seen d o r s a l l y a t the PVCN/DCN border ( F i g 31,2D). (L) High m a g n i f i c a t i o n of immunoreactive puncta seen a l o n g mid l a t e r a l l e v e l of the PVCN ( F i g 3D). Large t o medium m u l t i p o l a r c e l l s were o u t l i n e d by p e r i s o m a t i c (arrows) and p e r i d e n d r i t i c immunoreactive puncta (arrowheads). (M-0) Immunoreactivity seen i n the c a u d a l PVCN a t the border w i t h the,DCN ( F i g 3F). (M) Immunoreactive puncta form both p e r i c e l l u l a r (arrows) and p e r i d e n d r i t i c a p p o s i t i o n (arrowheads). (N) Large immunoreactive c e l l ( s h o r t arrow) seen next t o l a r g e c e l l apposed by p e r i c e l l u l a r immunoreactive puncta ( l o n g e r arrows). (0) Medium m a g n i f i c a t i o n of the PVCN ( F i g 3M,N). Note immunoreactive puncta forming p e r i c e l l u l a r ( s h o r t arrows) and p e r i d e n d r i t i c a p p o s i t i o n (arrowheads). C a l i b r a t i o n bars i n f i g u r e s E,I,0 i n d i c a t e 80 microns and i n the remaining f i g u r e s i n d i c a t e 30 microns. 23 24 Immunoreactive puncta were seen throughout the AVCN and PVCN ( F i g 2A, 3 ) . In the AVCN these puncta formed p r i m a r i l y p e r i c e l l u l a r and d e n d r i t i c a p p o s i t i o n s ( F i g 3A,D-F) which o u t l i n e d the s p h e r i c a l or o v a l somas. In the PVCN these puncta form dense p e r i c e l l u l a r and p e r i d e n d r i t i c a p p o s i t i o n ( F i g 3L-0). P e r i d e n d r i t i c a p p o s i t i o n was most prominent i n the d o r s a l t w o - t h i r d s of the PVCN. T h i s p e r i c e l l u l a r and p e r i d e n d r i t i c a p p o s i t i o n o u t l i n e d l a r g e and medium s i z e d , m u l t i p o l a r neurons of the PVCN. In the r a t , a h i g h d e n s i t y of s o m a t o s t a t i n immunoreactive soma were seen throughout the AVCN ( F i g 4A-D). Except i n the v e n t r a l g l o b u l a r c e l l r e g i o n which showed a markedly lower d e n s i t y of immunoreactive soma ( F i g 4E). In c o n t r a s t t o the AVCN, the PVCN showed a low d e n s i t y of immunoreactive c e l l s . T h i s d i f f e r e n c e was c l e a r l y seen a t the AVCN/PVCN border ( F i g 4E). Most immunoreactive c e l l s w i t h i n the PVCN were u s u a l l y l o c a t e d d o r s a l l y . Immunoreactive c e l l bodies were found i n h i g h d e n s i t y throughout the s p h e r i c a l c e l l r e g i o n of the AVCN, SC-AVCN. The r o s t r a l p o l e of the AVCN c o n t a i n s SOM-IR s p h e r i c a l t o o v a l c e l l s ( F i g 5A). Immunoreactive c e l l s appeared t o correspond t o the s p h e r i c a l c e l l s ( F i g 5A). The c e l l s of the r o s t r a l p o l e tend t o be l a r g e r than those found c a u d a l l y . T h i s change i n s i z e has been noted i n most p r e v i o u s r e p o r t s ( H a r r i s o n and I r v i n g , '66a; '66b; Osen, '69). Proceeding c a u d a l l y , SOM-IR was continuous throughout 25 F i g u r e 4. Somatostatin immunoreactivity from r o s t r a l t o c a u d a l VCN i n the r a t . (A) Medium s p h e r o i d s o m a t o s t a t i n immunoreactive c e l l s throughout the r o s t r a l p o l e of the r a t AVCN. (B) R o s t r a l l y , immunolabelling of s m a l l t o medium c e l l s was found throughout the AVCN. A s l i g h t decrease i n d e n s i t y was found v e n t r a l l y . (C) Small , medium, and o c c a s i o n a l , l a r g e m u l t i p o l a r c e l l s (arrows) were seen a t t h i s l e v e l of the SC-AVCN. (D) The c a u d a l AVCN shows a marked decrease i n the d e n s i t y o f immunolabelled s m a l l t o medium c e l l s v e n t r a l l y i n the g l o b u l a r c e l l r e g i o n . Large m u l t i p o l a r c e l l s were seen i n low d e n s i t y throughout, w h i l e s m a l l e r c e l l s were common i n the d o r s a l AVCN. (E) The remnant of s m a l l t o medium c e l l s seen r o s t r a l l y were s t i l l found a t t h i s l e v e l . Large m u l t i p o l a r s were seen s c a t t e r e d throughout t h i s c a u d a l AVCN r e g i o n (arrowhead). (F) PVCN shows remnant of s m a l l t o medium c e l l s . Large m u l t i p o l a r s were s c a t t e r e d throughout t h i s r e g i o n . (G) Caudal PVCN s m a l l c e l l s were s c a t t e r e d throughout the d o r s a l PVCN. O c c a s i o n a l l a r g e , m u l t i p o l a r c e l l s were seen v e n t r a l l y (arrowhead). (H) W i t h i n the DCN a low d e n s i t y of s m a l l t o medium c e l l s c o n t i n u e s i n t o the a c o u s t i c s t r i a . C a l i b r a t i o n bar f o r A i n d i c a t e s 200 microns and f o r B-H i n d i c a t e s 200 microns. 26 27 28 F i g u r e 5. Medium and h i g h m a g n i f i c a t i o n photomicrographs of so m a t o s t a t i n immunoreactivity p r i m a r i l y w i t h i n the r a t VCN. (A) Medium and c e l l s seen a t the r o s t r a l p o l e of the AVCN. (B) High m a g n i f i c a t i o n photomicrographs of v e n t r a l one t h i r d of the r a t AVCN a t the l e v e l of F i g 4B,C. Immunoreactive s m a l l t o medium s i z e , s p h e r i c a l and o v a l c e l l s (arrow) and a l s o a l a r g e m u l t i p o l a r c e lKshort arrow). (C) Immunoreactive s m a l l t o medium c e l l s o f t e n appear t o form c i r c u l a r c l u s t e r s i n the AVCN. (D) Immunoreactive l a r g e , m u l t i p o l a r ( s h o r t arrow) and s m a l l t o medium c e l l s ( l o n g arrows) of the r a t SC-AVCN. (E) Small t o medium c e l l s seen a t the dorsomedial border of the SC-AVCN (approximately a t the l e v e l F i g 4C) Small t o medium s i z e c e l l s w i t h i n the cauda l AVCN. These c e l l s were seen d o r s o m e d i a l l y a t the l e v e l of F i g 4D. (G) O c c a s i o n a l l a r g e , m u l t i p o l a r ( s h o r t arrow) and b i p o l a r c e l l s ( l o n g arrow) were seen i n the r a t PVCN. A mixture of s m a l l t o medium c e l l s were a l s o found w i t h i n t h i s r e g i o n ( F i g 4F,G). C a l i b r a t i o n bars i n d i c a t e 30 microns f o r A-F and 80 microns f o r G. 29 30 a l l r e g i o n s of the SC-AVCN ( F i g 4B,C). Immunoreactivity appeared p r i m a r i l y i n s m a l l t o medium c e l l s w i t h s p h e r i c a l or o v a l shaped c e l l bodies ( F i g 5B-F). While SOM-IR c e l l s i n the r o s t r a l SC-AVCN appeared l a r g e r , they were continuous w i t h s m a l l e r s p h e r o i d c e l l s c a u d a l l y ( F i g 5B-D). D o r s a l l y SOM-IR c e l l s were s i m i l a r t o those seen v e n t r a l l y ( F i g 5E,F). Large m u l t i p o l a r c e l l s were a l s o p o s i t i v e f o r so m a t o s t a t i n ( F i g 4C, 5B,E). These two c e l l types appeared throughout the VCN ( F i g 4C-E, 5B,C,G) and account f o r the m a j o r i t y of l a b e l l e d c e l l s . Moving c a u d a l l y , immunoreactivity d i m i n i s h e d v e n t r a l l y . T h i s decrease corresponded t o the i n c r e a s e i n the s i z e of the g l o b u l a r -c e l l and m u l t i p o l a r c e l l r e g i o n which c o n t a i n e d l i t t l e SOM-IR. Few i f any of the g l o b u l a r c e l l s show SOM-IR ( F i g 4D,E). W i t h i n the r a t PVCN s t a i n i n g was p r i m a r i l y r e s t r i c t e d t o the d o r s a l r e g i o n ( F i g 4F,G). SOM-IR c e l l s here i n c l u d e d medium and l a r g e m u l t i p o l a r s , as w e l l as the s m a l l e r s p h e r i c a l c e l l s ( F i g 4G, 5G). The l a r g e m u l t i p o l a r c e l l s decreased i n number as the c a u d a l r a t PVCN was approached, l e a v i n g o n l y the s m a l l e r c e l l s ( F i g 4G,H). A few of the s m a l l e r s p h e r i c a l and o v a l c e l l s c o n t i n u e d i n t o the a c o u s t i c s t r i a ( F i g 4H). Here these c e l l s o r i e n t e d t h e i r p r o x i m a l p r o c e s s e s d o r s a l l y , p a r a l l e l t o the s t r i a . 31 D o r s a l C ochlear Nucleus, DCN In the c a t , the DCN c o n t a i n e d few immunoreactive c e l l s . Those found were i n the deep polymorphic l a y e r o f the DCN. Immunoreactive c e l l s were el o n g a t e f u s i f o r m , whose p r o x i m a l processes and soma ran p a r a l l e l t o the a c o u s t i c s t r i a and j u s t s u p e r f i c i a l t o or w i t h i n t h a t s t r i a . These c e l l s a l s o appeared w i t h i n the DAS, as the a c o u s t i c s t r i a l e f t the v e n t r a l s u r f a c e of the DCN and coursed toward the m i d l i n e of the brainstem. Puncta were seen s c a t t e r e d throughout a l l l a y e r s of DCN ( F i g 6A). The guinea p i g DCN showed a p a t t e r n of immunoreactivity s i m i l a r t o t h a t o f the c a t . Small t o medium s i z e d , b i p o l a r c e l l s were seen l y i n g j u s t s u p e r f i c i a l t o , or running w i t h i n , and p a r a l l e l t o the a c o u s t i c s t r i a . O c c a s i o n a l c e l l s would s t a i n i n l a y e r s 3-5 but these were r a r e . Puncta were seen throughout the DCN ( F i g 6B). The r a t showed s i m i l a r p a t t e r n s of s c a t t e r e d SOM-IR soma. The m a j o r i t y of c e l l s s t a i n e d were i n or j u s t s u p e r f i c i a l t o the a c o u s t i c s t r i a . Here, as i n the other s p e c i e s , the p r o x i m a l processes were seen running d o r s a l l y i n the body of the a c o u s t i c s t r i a . The SOM-IR puncta were seen s c a t t e r e d throughout the r a t DCN and were i n much lower d e n s i t y than e i t h e r the c a t or guinea p i g . F i g u r e 6. (A) and (B) demonstrate the f i n e immunoreactive puncta w i t h i n the DCN of the c a t and g u i n e a - p i g . r e s p e c t i v e l y . (C) Immunoreactive puncta and o c c a s i o n a l m u l t i p o l a r c e l l s were seen w i t h i n the c a t a n t e r i o r v e n t r a l c o c h l e a r nucleus (AVCN). C a l i b r a t i o n bar i n d i c a t e s 80 microns f o r A-C. A b b r e v i a t i o n s : GCL, g r anule c e l l l a y e r . 33 34 S u p e r i o r O l i v a r y Complex P e r i o l i v a r y C e l l s In the c a t , r a t and guinea p i g , medium s i z e d , e l o n g a t e SOM-IR c e l l s w i t h s p i n d l e shaped c e l l b odies were seen s c a t t e r e d i n low d e n s i t y through a l l d o r s a l p e r i o l i v a r y r e g i o n s . Small c e l l s w i t h o v a l and t r i a n g u l a r o r s p i n d l e shaped c e l l b o d i es were seen i n most p e r i o l i v a r y r e g i o n s . In a l l s p e c i e s , f i n e puncta and v a r i c o s i t i e s were seen i n a l l p e r i o l i v a r y r e g i o n s . Dorsomedial P e r i o l i v a r y Nucleus, DMPO In the c a t , t h e r e were r a r e SOM-IR medium s i z e d , m u l t i p o l a r c e l l s ( t r i a n g u l a r c e l l bodies) and s m a l l c e l l s w i t h o v a l and round soma. The l a t t e r were o c c a s i o n a l l y seen as b i p o l a r . C a u d a l l y , l a r g e , m u l t i p o l a r , e l o n g a t e c e l l s were seen. At the r o s t r a l e x t e n t of the nucl e u s , medium m u l t i p o l a r and s m a l l , o v a l or round c e l l s were embedded i n a dense plexus of f i n e beaded v a r i c o s i t i e s . These c e l l s were s i m i l a r t o those seen a l o n g the medial perimeter of the MNTB. O c c a s i o n a l medium s i z e d , f u s i f o r m c e l l s were seen w i t h i n the MNTB but these were r a r e . Small o v a l and s p i n d l e shaped c e l l s were found w i t h i n the guinea p i g DMPO ( F i g 10B,D, 13C). C a u d a l l y these were l o c a l i z e d d o r s a l t o the MNTB ( F i g IOA). Moving r o s t r a l l y , these c e l l s appeared d i s p l a c e d 35 F i g u r e 7. A s e r i e s of low m a g n i f i c a t i o n c o r o n a l s e c t i o n s through the c a t s u p e r i o r o l i v a r y complex (SOC). Most immunoreactivity l i e s o u t s i d e the l a t e r a l and medial-, s u p e r i o r o l i v e , LSO and MSO, r e s p e c t i v e l y . (A) Mid l e v e l of the SOC. Note immunolabelled p e r i k a r y a were found w i t h i n the v e n t r a l p e r i o l i v a r y nucleus (VPO) (arrowhead). Small c l u s t e r s of c e l l s were seen i n the d o r s a l p e r i o l i v a r y r e g i o n , between the LSO and MSO (arrow). (B) At t h i s r o s t r a l l e v e l of the SOC immunoreactive p e r i k a r y a were seen i n the v e n t r a l nucleus of the t r a p e z o i d body (VNTB) and medial t o the medial nucleus of the t r a p e z o i d body (MNTB). Note the s m a l l c l u s t e r of immunolabelled c e l l s l a t e r a l t o the MSO seen a t t h i s l e v e l (arrow). A mixture of s m a l l and medium s i z e d c e l l s was seen i n a l l p e r i o l i v a r y r e g i o n s . (C) The r o s t r a l extreme of the SOC a t the border w i t h the v e n t r a l nucleus of the l a t e r a l lemniscus (VLL). Note t h a t the immunolabelled c e l l s seen w i t h i n VNTB area were s t i l l seen a t t h i s l e v e l (arrowhead). S c a t t e r e d s m a l l t o medium c e l l s occupy the space between the r e c e d i n g SOC and the c a u d a l p o l e of the VNTB. Note the band of immunoreactive n e u r o p i l seen i n the v e n t r a l tegmentum (arrow). C a l i b r a t i o n bar i n d i c a t e s 500 microns f o r a l l f i g u r e s . A b b r e v i a t i o n s : DMPO, dorsomedial p e r i o l i v a r y nucleus; DPO, d o r s a l p e r i o l i v a r y n ucleus; LSO, l a t e r a l s u p e r i o r o l i v a r y nucleus; MSO, medial s u p e r i o r o l i v a r y nucleus." LSO DMPO MSO MNTB VPO ' ^ VNTB 37 38 F i g u r e 8. Somatostatin immunoreactivity i n the c a t v e n t r a l nucleus of the t r a p e z o i d body (VNTB). (A) Immunoreactive p e r i k a r y a seen a t r o s t r a l to mid l e v e l SOC. A mixture o f s m a l l and medium c e l l s can be seen. There was a mixture of b i p o l a r (arrow) and m u l t i p o l a r ( s h o r t arrow) c e l l s w i t h i n the v e n t r a l r e g i o n . Many c e l l s send processes t a n g e n t i a l t o the p a s s i n g t r a p e z o i d f i b r e s (arrowhead). (B) Small c e l l s were found w i t h i n VNTB. (C) F i n e immunolabelled puncta (arrow) seen throughout VNTB, as w e l l as immunoreactive l a b e l l i n g o f a c e l l body (arrowheads). C a l i b r a t i o n bars i n d i c a t e s 80 microns f o r A and 30 microns f o r B, C. 40 F i g u r e 9. Somatostatin immunoreactivity i n c a t p e r i o l i v a r y r e g i o n s . (A) V e n t r a l p e r i o l i v a r y r e g i o n (VPO). Note t h a t a v a r i e t y of s m a l l t o medium s i z e c e l l s were seen. Round ( s h o r t arrow), o v a l ( l o n g arrow), and t r i a n g u l a r (arrowhead) p e r i k a r y a were seen throughout the VPO. (B) High m a g n i f i c a t i o n i n s e t from F i g 9A showing the c e l l s of the VPO. A medium s i z e , t r i a n g u l a r m u l t i p o l a r c e l l and two s m a l l e r , curved o v a l c e l l s were seen. (C) Immunoreactivity seen w i t h i n the d o r s a l p e r i o l i v a r y r e g i o n . A c l u s t e r of immunolabelled, s m a l l t o medium c e l l s were found j u s t d o r s a l t o and between the LSO and MSO ( s i m i l a r t o the s m a l l c l u s t e r of c e l l s shown i n F i g 7A). O c c a s i o n a l l a r g e m u l t i p o l a r c e l l s were seen between the d o r s a l aspect of the LSO/MSO and j u s t v e n t r a l t o the s m a l l c l u s t e r of c e l l s . D o r s a l and l a t e r a l p e r i o l i v a r y r e g i o n s of have low t o moderate d e n s i t i e s of immunoreactive puncta and v a r i c o s i t i e s . (D) Medium m a g n i f i c a t i o n photomicrographs of l a t e r a l p e r i o l i v a r y immunoreactivity. Note moderate d e n s i t y of immunoreactive puncta and f i b r e s seen l a t e r a l l y . Immunoreactive c e l l s of s m a l l t o medium s i z e were seen throughout the l a t e r a l p e r i o l i v a r y r e g i o n . P e r i k a r y a were u s u a l l y o v a l (arrow) t o t r i a n g u l a r (arrowhead) i n shape. Note most immunolabelled c e l l s l i e p a r a l l e l t o s u r f a c e of the adjacent o l i v a r y n u cleus. 41 F i g u r e 9 ( c o n t . ) , (E) High m a g n i f i c a t i o n photomicrographs of c a t medial nucleus of the t r a p e z o i d body (MNTB). Note f i n e s o m a t o s t a t i n immunoreactive puncta form p e r i c e l l u l a r -a p p o s i t i o n s (arrowheads). O c c a s i o n a l f u s i f o r m c e l l s were seen w i t h i n the MNTB. C a l i b r a t i o n bars f o r A,C,D i n d i c a t e 80 microns and 30 microns f o r B,E. 42 43 d o r s o m e d i a l l y by the SPO ( F i g 10B,C). A low d e n s i t y o f p u n c t a , s i m i l a r t o those seen w i t h i n other r e g i o n s of the SOC ( F i g 13C) were a l s o found i n the DMPO. W i t h i n the r a t DMPO, a moderate t o hi g h d e n s i t y of s m a l l m u l t i p o l a r c e l l s were found ( F i g 15,D, 17A,B). In s a g i t t a l s e c t i o n s , these c e l l s r an the l e n g t h of the medial r e g i o n of the MNTB. A moderate d e n s i t y of f i n e immunoreactive puncta and v a r i c o s i t i e s were a l s o seen i n the DMPO ( F i g 17B). D o r s a l P e r i o l i v a r y Nucleus, DPO The c a t DPO, l o c a t e d between and j u s t d o r s a l t o the LSO/MSO border, c o n t a i n e d a c l u s t e r of s m a l l t o medium s i z e d c e l l s ( F i g 7A). These c e l l s l a y alo n g the d o r s a l perimeter of the ca u d a l DPO. O c c a s i o n a l l a r g e , m u l t i p o l a r neurons were j u s t v e n t r a l t o t h i s c l u s t e r of c e l l s ( F i g 7A, 9C). In the guinea p i g ( F i g 13E) and r a t ( F i g 17E) t h i s c l u s t e r of c e l l s was not seen. O c c a s i o n a l c e l l s were seen i n t h i s r e g i o n but these c e l l s appeared as s m a l l t o l a r g e c e l l s w i t h s p i n d l e shaped or f u s i f o r m p e r i k a r y a and are s i m i l a r t o c e l l s seen throughout most p e r i o l i v a r y r e g i o n s . D o r s o l a t e r a l P e r i o l i v a r y Nucleus, DLPO In the r a t , o c c a s i o n a l immunoreactive c e l l s were seen i n the c a u d a l DLPO. These c e l l s were not e v i d e n t i n e i t h e r the ca t or guinea p i g . 44 F i g u r e 10. A s e r i e s of low m a g n i f i c a t i o n photomicrographs of s e r i a l c o r o n a l s e c t i o n s through the guinea p i g s u p e r i o r o l i v a r y complex (SOC) proc e e d i n g c a u d a l - r o s t r a l l y . Medium and s m a l l c e l l s w i t h o v a l and t r i a n g u l a r p e r i k a r y a were seen throughout the p e r i o l i v a r y r e g i o n s . (A) Caudal guinea p i g SOC. Note i n t e n s e l y immunoreactive c e l l s w i t h i n the VNTB (arrow). Small immunoreactive c e l l s were seen w i t h i n the DMPO. (B) At t h i s l e v e l , DMPO c e l l s were d i s p l a c e d l a t e r a l l y by the SPO ( s u p e r i o r p a r a o l i v a r y n u c l e u s ) . The VNTB shows a hi g h d e n s i t y of immunoreactivity. A l a r g e m u l t i p o l a r c e l l was seen w i t h i n the SPO (arrow). S c a t t e r e d immunoreactive c e l l s were seen throughout the d o r s a l p e r i o l i v a r y r e g i o n . (C) R o s t r a l guinea p i g SOC. Immunoreactive c e l l s i n the VNTB have spread l a t e r a l l y as a t h i n sheet. Large m u l t i p o l a r s were seen w i t h i n the SPO (arrow). (D) Immunolabelled VNTB c e l l s were s t i l l p r e s e n t v e n t r a l l y . There was a tendency f o r VNTB c e l l s t o change t h e i r o r i e n t a t i o n from b e i n g p a r a l l e l t o the t r a p e z o i d f i b r e s t o be i n g p a r a l l e l t o ascending immunoreactive f i b r e s c o u r s i n g d o r s o l a t e r a l l y . S c a t t e r e d immunoreactive c e l l s were seen a l o n g the d o r s a l p e r i m e t e r , embedded w i t h i n the t r a p e z o i d f i b r e s (arrowheads). 45 F i g u r e 10 (cont.) (E) At the l e v e l of the c a u d a l VLL, the d e n s i t y of l a r g e and medium s t a i n e d p e r i k a r y a i n the SOC decreases. Immunostained c e l l s , c o u r s i n g d o r s o l a t e r a l l y , can be seen v e n t r a l l y . Immunoreactive l a r g e m u l t i p o l a r c e l l s (arrowhead) appear s i m i l a r t o and continuous w i t h c e l l s of the SPO. (F) While immunoreactive c e l l s were s t i l l seen, the l a r g e r c e l l s seen c a u d a l l y were no longer e v i d e n t i n the v e n t r a l immunoreactive band. The c a l i b r a t i o n bar i n d i c a t e s 500 microns. A b b r e v i a t i o n s : LSO, l a t e r a l s u p e r i o r o l i v a r y nucleus; MSO, medial s u p e r i o r o l i v a r y nucleus; VLL, nucleus of the v e n t r a l l a t e r a l n u c l e u s . 46 47 F i g u r e 11. (A) Medium m a g n i f i c a t i o n photomicrographs of immunoreactive puncta which form p e r i d e n d r i t i c and p e r i c e l l u l a r a p p o s i t i o n throughout the guinea p i g m e d i a l s u p e r i o r o l i v e (MSO). (B) Immunoreactive puncta which form p e r i s o m a t i c and p e r i d e n d r i t i c a p p o s i t i o n w i t h i n the MSO. (C) A low d e n s i t y of f i n e immunoreactive puncta was seen i n the guinea p i g LSO. (D) Immunoreactive puncta forming p e r i c e l l u l a r a p p o s i t i o n w i t h c e l l s i n the medial nucleus of the t r a p e z o i d body (MNTB). F a i n t immunoreactive v a r i c o s i t i e s can a l s o be seen w i t h i n the MNTB. C a l i b r a t i o n bars i n d i c a t e 80 microns f o r A and 30 microns f o r B-D. 48 F i g u r e 12. (A) shows two l a r g e m u l t i p o l a r s seen w i t h i n the guinea p i g s u p e r i o r p a r a o l i v a r y nucleus (SPO) a t the c a u d a l mid l e v e l of the SOC. Small t o medium c e l l s (arrowheads) were seen a l o n g the d o r s a l p e r i m e t e r of the SPO. Note low d e n s i t y o f immunolabelled puncta seen w i t h i n the SPO. (B) High m a g n i f i c a t i o n photomicrographs of r o s t r a l SPO c e l l s . Some of the m u l t i p o l a r c e l l s seen a t t h i s l e v e l appear s i m i l a r t o those seen i n r o s t r a l d o r s a l p e r i o l i v a r y r e g i o n s (C-D) The r o s t r a l d o r s a l p e r i o l i v a r y c e l l s . A mixture of medium t o l a r g e c e l l s were seen here (see F i g 10F). C a l i b r a t i o n b a r s f o r A i n d i c a t e s 80 microns and 30 microns f o r B, C, D. 50 51 F i g u r e 13. (A) O c c a s i o n a l s m a l l c e l l s l o c a t e d between the d o r s a l aspect of the guinea p i g l a t e r a l s u p e r i o r o l i v e (LSO) and medial s u p e r i o r o l i v e (MSO). (B) Small c e l l s found medial t o the MNTB a t i t s r o s t r a l e x t e n t . (C) Caudal guinea p i g DMPO. Small c e l l s w i t h o v a l t o t r i a n g u l a r p e r i k a r y a . (D) Immunoreactive puncta seen i n the cau d a l VLL ( l e v e l of F i g 10D,F). (E) Small c e l l s seen along the d o r s a l p e r i m e t e r of the LSO. Small c e l l s have o v a l t o f u s i f o r m p e r i k a r y a . Note the d i f f e r e n c e i n the d e n s i t i e s of immunolabelled puncta seen between the p e r i o l i v a r y and LSO r e g i o n s . C a l i b r a t i o n bar i n d i c a t e s 30 microns f o r A-D. 52 53 F i g u r e 14. The guinea p i g v e n t r a l nucleus of the t r a p e z o i d body (VNTB). (A,B) Immunoreactive c e l l s and n e u r o p i l of the VNTB. There was a tendency f o r the l a r g e s t c e l l s t o be found m e d i a l l y . Dense immunoreactive n e u r o p i l and puncta appear between l a t e r a l t r a p e z o i d f i b r e s . Small t o l a r g e immunoreactive c e l l s were seen w i t h i n the VNTB. (B) Mid t o r o s t r a l l e v e l of the VNTB from a c o l c h i c i n e - p r e t r e a t e d animal. Note most c e l l s d i r e c t a p i c a l p rocesses (arrowheads) d o r s o l a t e r a l l y . O c c a s i o n a l c e l l s d i r e c t p rocesses m e d i a l l y , p a r a l l e l t o the t r a p e z o i d body (arrow). (C) The r o s t r a l e x t e n t of the VNTB. Large c e l l s of the VNTB were decreased r o s t r a l l y (see F i g 10E). C e l l s a t t h i s l e v e l appear d o r s o l a t e r a l l y . Dense immunoreactive n e u r o p i l was s t i l l seen v e n t r a l l y . (D) S l i g h t l y r o s t r a l t o F i g 13C the l a r g e c e l l s seen c a u d a l l y i n the VNTB were no longer p r e s e n t . The dense v e n t r a l band of immunoreactive n e u r o p i l c o n t i n u e s along the v e n t r a l s u r f a c e of the v e n t r a l nucleus of the l a t e r a l lemniscus (VLL) (throughout i t s whole l e n g t h ) . The immunoreactive puncta and v a r i c o s i t i e s were a l s o seen throughout the VLL. C a l i b r a t i o n bars i n d i c a t e 80 microns f o r A and 30 microns f o r B,C,D. 54 56 L a t e r a l P e r i o l i v a r y Region, LPR The l a t e r a l p e r i o l i v a r y r e g i o n , which apposes the l a t e r a l s u r f a c e of the LSO, showed a s m a l l number of-SOM-IR c e l l s i n the c a t ( F i g 9D). These c e l l s were s i m i l a r t o those s c a t t e r e d i n d o r s a l p e r i o l i v a r y r e g i o n s . The c e l l s were s m a l l t o medium i n s i z e w i t h o v a l , s p i n d l e or t r i a n g u l a r shaped p e r i k a r y a . F i n e puncta and v a r i c o s i t i e s seen i n t h i s r e g i o n were s i m i l a r t o those seen i n other p e r i o l i v a r y r e g i o n s ( F i g 6D). At r o s t r a l l e v e l s a s l i g h t i n c r e a s e i n c e l l d e n s i t y was seen i n the LPR. The r a t and guinea p i g showed s c a t t e r e d s m a l l , o v a l and f u s i f o r m c e l l s ( F i g 13A, 17D) throughout the LPR, s i m i l a r t o those seen i n o t h e r p e r i o l i v a r y r e g i o n s . The s l i g h t i n c r e a s e i n immunoreactive c e l l s , seen i n the c a t , was not d e t e c t e d i n the r o s t r a l LPR of rodents. The f i n e puncta and v a r i c o s i t i e s , seen i n the d o r s a l p e r i o l i v a r y r e g i o n s , were a l s o seen i n LPR. V e n t r a l P e r i o l i v a r y Nucleus, VPO The c a t VPO c o n t a i n s a low d e n s i t y of medium t o s m a l l , SOM-IR c e l l s ( F i g 7A). The c e l l s appeared as m u l t i p o l a r or b i p o l a r w i t h o v a l , s p i n d l e - s h a p e d , round or t r i a n g u l a r soma ( F i g 9A,B). At r o s t r a l l e v e l s , few c e l l s were seen. The s m a l l , o v a l or spi n d l e - s h a p e d c e l l s seen c a u d a l l y were s i m i l a r t o those seen i n a s m a l l c l u s t e r of c e l l s seen i n the r o s t r a l LPR. O c c a s i o n a l f i n e puncta and v a r i c o s i t i e s c o u l d be seen i n the VPO r e g i o n ( F i g 9A,B). 57 Immunoreactive soma i n the VPO of the guinea p i g and r a t were r a r e . In the r a t , o c c a s i o n a l s m a l l o v a l c e l l s , s i m i l a r t o those s c a t t e r e d throughout the v e n t r a l t r a p e z o i d r e g i o n were seen. A low d e n s i t y of f i n e beaded v a r i c o s i t i e s and puncta s i m i l a r t o those seen i n the c a t c o u l d be seen i n the v e n t r a l t r a p e z o i d r e g i o n i n both the r a t and guinea p i g . S u p e r i o r P a r a o l i v a r y Nucleus, SPO In the guinea p i g , a low d e n s i t y of l a r g e and s m a l l m u l t i p o l a r immunoreactive c e l l s were seen. C e l l s had o v a l t o t r i a n g u l a r p e r i k a r y a ( F i g 10A-E, 11A-D). There was a l s o a low d e n s i t y of puncta throughout t h i s r e g i o n ( F i g 11A-D). In the r a t t h e r e was a low d e n s i t y of f i n e puncta and v a r i c o s i t i e s w i t h i n the SPO but immunoreactive c e l l s were not seen. T h i s r e g i o n i s not found i n c a t s ( H a r r i s o n and Feldman, '70). Med i a l Nucleus of the T r a p e z o i d Body, MNTB Only o c c a s i o n a l f u s i f o r m c e l l s were seen w i t h i n the c a t MNTB ( F i g 9E). Most c e l l s i n t h i s r e g i o n l a y a l o n g the d o r s a l , v e n t r a l , o r medial p e r i m e t e r of the MNTB and appeared t o be a s s o c i a t e d w i t h o t h e r r e g i o n s of the SOC. Only i n the r o s t r a l extreme d i d o c c a s i o n a l c e l l s appear i n the medial t r a p e z o i d body. These immunoreactive c e l l s appeared t o be a s s o c i a t e d w i t h more r o s t r a l p e r i o l i v a r y c e l l groups. A low 58 F i g u r e 15. (A-C) Low m a g n i f i c a t i o n photomicrographs of c o r o n a l s e c t i o n s through the r a t b r a i n , c a u d a l t o r o s t r a l . Note c e l l and n e u r o p i l immunoreactivity was seen i n most p e r i o l i v a r y r e g i o n s . (A) Rare s o m a t o s t a t i n immunoreactive c e l l s were seen i n the caud a l l a t e r a l s u p e r i o r o l i v e (LSO). (B) O c c a s i o n a l SOM-IR c e l l s were seen i n the ca u d a l d o r s o l a t e r a l p e r i o l i v a r y r e g i o n (DLPO) (arrowhead). Note arrows i n d i c a t i n g the l o c a t i o n of the n u c l e i . (D) Low m a g n i f i c a t i o n photomicrograph of a s a g i t t a l s e c t i o n o f medial r e g i o n s of the s u p e r i o r o l i v a r y complex (SOC). Small c e l l s were seen w i t h i n the dorsomedial p e r i o l i v a r y r e g i o n (DMPO). Note the s l i g h t i n c r e a s e i n immunoreactive n e u r o p i l seen i n r o s t r a l m e dial nucleus of the t r a p e z o i d body (MNTB). T r i a n g l e s o u t l i n e the border of the MNTB. C i r c l e s o u t l i n e the d o r s a l p e r i m e t e r of the DMPO, which l i e s d o r s a l t o the medial r e g i o n s o f the MNTB. The arrows i n d i c a t e the l o c a t i o n of the DMPO. (E) Coronal s e c t i o n of the MNTB. The d e n s i t y of immunoreactive puncta forming p e r i c e l l u l a r a p p o s i t i o n s (arrowhead) appears h i g h e r d o r s o m e d i a l l y than v e n t r o l a t e r a l l y . Small c e l l s seen m e d i a l l y (arrow) appear s i m i l a r t o those seen d o r s a l l y i n the DMPO. C a l i b r a t i o n bars i n d i c a t e 500 microns f o r A-D and 80 microns f o r E. A b b r e v i a t i o n s VNTB, v e n t r a l nucleus of the t r a p e z o i d body. 59 60 F i g u r e 16. (A,B) Photomicrographs of the r a t MNTB i n s a g i t t a l plane taken i n two d i f f e r e n t f o c a l planes from a s i n g l e s e c t i o n . Note the change i n the d i s t r i b u t i o n of immunoreactive puncta on the soma s u r f a c e (arrowheads). (C) Medium m a g n i f i c a t i o n photomicrograph of the r a t VNTB. Immunoreactive puncta were seen, i n low d e n s i t y , v e n t r a l l y and v e n t r o m e d i a l l y (arrowheads). (D) Immunoreactive puncta (arrowheads) and o c c a s i o n a l s m a l l c e l l s were found i n the r a t VNTB. C a l i b r a t i o n bars i n d i c a t e 80 f o r (D) and 30 microns f o r A, B, D. 6 1 62 F i g u r e 17. (A,B) I l l u s t r a t e the immunoreactive c e l l s and n e u r o p i l seen i n the r a t dorsomedial p e r i o l i v a r y nucleus (DMPO). C e l l s w i t h i n the DMPO appear o v a l t o t r i a n g u l a r . (C) O c c a s i o n a l immunoreactive c e l l s seen i n the c a u d a l l a t e r a l s u p e r i o r o l i v e (LSO) (see F i g 15A). (D) A low d e n s i t y of s m a l l c e l l s l i e s a d jacent t o the LSO (arrowheads). (E) Immunoreactive c e l l s seen i n the d o r s a l p e r i o l i v a r y r e g i o n s . (F) Small t o medium s i z e d , m u l t i p o l a r c e l l s were found i n the d o r s o l a t e r a l p e r i o l i v a r y r e g i o n . C a l i b r a t i o n bars i n d i c a t e 80 microns f o r A and 30 microns f o r B-F. A b b r e v i a t i o n s , SPO s u p e r i o r p a r a o l i v a r y n u cleus. 63 64 d e n s i t y of f i n e beaded v a r i c o s i t i e s formed p e r i c e l l u l a r a p p o s i t i o n w i t h c e l l s of the c a t MNTB ( F i g 9E). As i n the c a t , the guinea p i g showed c e l l s a t the per i m e t e r of the nucleus which appeared s i m i l a r t o c e l l s of ne i g h b o r i n g r e g i o n s of the SOC. In the guinea p i g , a low d e n s i t y of s m a l l puncta made p e r i c e l l u l a r a p p o s i t i o n w i t h most c e l l s of the MNTB ( F i g 15E). As i n the c a t , immunoreactive c e l l s appeared o n l y i n the r o s t r a l extreme of the m e dial t r a p e z o i d body ( F i g 13B). In r a t , as i n oth e r s p e c i e s , immunoreactive c e l l s were seen a t the pe r i m e t e r of the MNTB ( F i g 15E). In the r a t , however, a h i g h d e n s i t y o f l a r g e SOM-IR puncta apposed the soma and proxi m a l d e n d r i t e s of c e l l s i n the MNTB ( F i g 15A, 16A,B). The d e n s i t y of the p e r i c e l l u l a r a p p o s i t i o n s can be seen i n F i g 16A,B. Somatostatin immunoelectron microscopy (unpublished o b s e r v a t i o n s ) r e v e a l e d these synapses as d i s t i n c t from the c a l y c e s of He l d and they appeared s i m i l a r t o the l a r g e t e r m i n a l s d e s c r i b e d by Casey and Feldman ('85). The d e n s i t i e s o f immunoreactive puncta were much h i g h e r and t h e i r s i z e l a r g e r i n the r a t than i n e i t h e r the c a t or the guinea p i g . While these immunolabelled puncta appeared throughout the r a t MNTB, the d e n s i t y of immunoreactive t e r m i n a l s were h i g h e r a t the dorsomedial l e v e l and r o s t r a l l e v e l s of the r a t MNTB ( F i g 15E). V e n t r a l Nucleus of the T r a p e z o i d Body, VNTB The c a t VNTB showed a low d e n s i t y of SOM-IR c e l l s ( F i g 65 7A,B). Immunoreactive c e l l s v a r i e d from s m a l l t o medium ( F i g 8A-C). P e r i k a r y a of s m a l l c e l l s tended t o be o v a l or round. La r g e r , m u l t i p o l a r c e l l s had o v a l or t r i a n g u l a r p e r i k a r y a . B i p o l a r c e l l s of v a r i o u s s i z e s were a l s o seen throughout the VNTB. M u l t i p o l a r c e l l s o f t e n l a y p a r a l l e l t o p a s s i n g t r a p e z o i d f i b r e s and would send t h e i r p r o x i m a l p r o c e s s e s p e r p e n d i c u l a r or s l i g h t l y o b l i q u e t o the p a s s i n g t r a p e z o i d f i b r e s ( F i g 8A). Other c e l l s send t h e i r p roximal processes i n t o the p a s s i n g f i b r e s ( F i g 7B). R o s t r a l l y c e l l s u s u a l l y l a y p a r a l l e l t o ascending t r a p e z o i d f i b r e s ( F i g 7C). A moderate d e n s i t y of f i n e puncta and v a r i c o s i t i e s were seen throughout the VNTB ( F i g 8C). The guinea p i g VNTB c o n t a i n e d a h i g h d e n s i t y of i n t e n s e l y l a b e l l e d c e l l s . C a u d a l l y , these c e l l s were r e s t r i c t e d t o the medial VNTB ( F i g 10A). Moving r o s t r a l l y , however, the c e l l s spread i n t o a t h i n band of c e l l s embedded w i t h i n the laminae of the v e n t r a l t r a p e z o i d body ( F i g 10D,E). F u r t h e r r o s t r a l l y , t h i s t h i n sheet of l a t e r a l c e l l s d i s appeared, l e a v i n g the medial group of c e l l s ( F i g 10D,E). T h i s medial group of c e l l s moved s l i g h t l y d o r s o l a t e r a l f u r t h e r r o s t r a l l y ( F i g 10D,E). These c e l l s , a t t h e i r most r o s t r a l e x t e n t , formed a group which l a y v e n t r a l to the c a u d a l border of the VLL. A v a r i e t y of c e l l s were seen i n the guinea p i g VNTB. These c e l l s appeared as medium and s m a l l , o v a l and round c e l l s , as w e l l as medium and l a r g e , m u l t i p o l a r c e l l s w i t h t r i a n g u l a r and o v a l p e r i k a r y a ( F i g 14). C a u d a l l y , the v a r i o u s m o r p h o l o g i c a l types were found 66 t o g e t h e r . Moving r o s t r a l l y , t h e r e was a tendency f o r the s m a l l and medium s i z e d c e l l s t o be found v e n t r o l a t e r a l l y w h i l e l a r g e r c e l l s were found i n more medial r e g i o n s o f the VNTB ( F i g 14A,B). C o l c h i c i n e treatment r e v e a l e d t h a t many c e l l s d i r e c t t h e i r p r ocesses d o r s o l a t e r a l l y ( F i g 14B). C a u d a l l y the long axes of the p e r i k a r y a tended t o be p a r a l l e l t o the p a s s i n g t r a p e z o i d f i b r e s ( F i g 14A, 10A-C). R o s t r a l l y , however, these c e l l s changed t h e i r o r i e n t a t i o n so t h a t they were roughl y p a r a l l e l t o the ascending f i b r e s seen a t the SOC/VLL border ( F i g 14C, 10D,E). In the guinea p i g , t h e r e was a moderate t o h i g h d e n s i t y of puncta w i t h i n the VNTB ( F i g 10, 14). M u l t i p o l a r c e l l s i n the guinea p i g o r i e n t e d t h e i r d e n d r i t e s d o r s a l l y . R o s t r a l l y , the medium and l a r g e m u l t i p o l a r c e l l s moved l a t e r a l l y and s l i g h t l y d o r s a l as the pontine n u c l e i occupy the v e n t r o m e d i a l tegmentum ( F i g 10D,E, 14C). These c e l l s end v e n t r a l t o the l a t e r a l lemniscus and were mixed w i t h s m a l l e r , o v a l and f u s i f o r m c e l l s which apposed the base of the VLL. The m u l t i p o l a r c e l l s of the VNTB d i d not appear to c o n t i n u e r o s t r a l l y a t the base of the VLL ( F i g 14D). In the r a t , r a r e s m a l l , o v a l c e l l s were seen i n the VNTB and throughout the v e n t r a l t r a p e z o i d r e g i o n ( F i g 16D). Immunoreactive c e l l s i n the r a t VNTB were i n much lower d e n s i t y and showed a l e s s v a r i e d morphology than those seen i n the c a t or guinea p i g . There was a moderate d e n s i t y of 67 s m a l l / f i n e immunoreactive puncta and v a r i c o s i t i e s ( F i g 160,0), s i m i l a r t o those seen i n the c a t and guinea p i g . O l i v a r y N u c l e i In both the c a t and the guinea p i g , SOM-IR soma were not seen i n e i t h e r the LSO or MSO. In the r a t , immunoreactive c e l l s were seen o n l y a t cau d a l l e v e l s a t the per i m e t e r of the LSO. These c e l l s were s m a l l t o medium w i t h o v a l and round p e r i k a r y a ( F i g 17C). W i t h i n the guinea p i g LSO a low d e n s i t y of f i n e immunoreactive puncta was seen ( F i g 11C). Small SOM-IR puncta were seen making p e r i c e l l u l a r and p e r i d e n d r i t i c a p p o s i t i o n t o c e l l s of MSO ( F i g 11A,B). In the c a t and r a t , immunoreactive puncta were v i r t u a l l y n i l i n both the LSO and MSO. N u c l e i of the L a t e r a l Lemniscal Nucleus of the V e n t r o l a t e r a l Lemniscus, VLL In a l l t h r e e s p e c i e s examined, c e l l s were o c c a s i o n a l l y seen w i t h i n the c e n t r a l and l a t e r a l aspects of the lemniscus, w h i l e SOM-IR occurs i n low to moderate d e n s i t y o n l y a l o n g the medial p e r i m e t e r of the VLL. Through the c a u d a l and mid l e v e l s of the c a t VLL th e r e was a low d e n s i t y of immunostaining, predominantly along the perim e t e r of t h i s r e g i o n ( F i g 18). At the r o s t r a l e x t e n t of 68 F i g u r e 18. (A-C) Coronal s e c t i o n s , p roceeding c a u d a l -r o s t r a l l y through the c a t l a t e r a l tegmentum. (A) S c a t t e r e d immunoreactive c e l l s seen a t pe r i m e t e r of the nucleus of the v e n t r o l a t e r a l lemniscus (VLL). Immunolabelled c e l l s w i t h i n the nucleus of the d o r s o l a t e r a l lemniscus (DLL) were r a r e . Note the band of immunolabelled p e r i k a r y a w i t h i n the v e n t r a l sagulum (arrowhead). (B) R o s t r a l c a t VLL. Note i n c r e a s e i n the immunolabelled c e l l s v e n t r a l l y w i t h i n the f i b r e s of the l a t e r a l lemniscus (arrows). Note the band of immunolabelled p e r i k a r y a w i t h i n the v e n t r a l sagulum (SAG) (arrowheads). (C) The r o s t r a l extreme of the l a t e r a l lemniscus. Note t h a t an i n c r e a s e i n immunolabelled c e l l s o ccurs among the v e n t r a l l a t e r a l l e m n i s c a l f i b r e s (arrows) and c l u s t e r s of c e l l s w i t h i n the r o s t r a l l a t e r a l lemniscus (arrowheads) were p r e s e n t . The c a l i b r a t i o n bar i n d i c a t e s 500 microns. 69 70 F i g u r e 19. (A) The c a t r o s t r a l and v e n t r a l l a t e r a l l emniscus. Small t o medium c e l l s l o c a t e d l a t e r a l l y were seen throughout the r o s t r a l l e v e l . The medial c e l l s were seen throughout the nucleus of the v e n t r o l a t e r a l lemniscus (VLL) alo n g the medial perimeter(arrowheads). The l a t e r a l c e l l group shows a mixture of s m a l l t o medium s i z e d c e l l s . (B) Immunoreactive c e l l s and n e u r o p i l seen i n F i g 19A. Most c e l l s were seen t o have o v a l t o round p e r i k a r y a and l i e p a r a l l e l t o the l e m n i s c a l laminae of the l a t e r a l lemniscus. (C) Medium s i z e d , m u l t i p o l a r c e l l s l o c a t e d along the medial border of the l a t e r a l lemniscus ( F i g 18C). Immunoreactive puncta and v a r i c o s i t i e s were s c a t t e r e d throughout t h i s r e g i o n . (D) Immunolabelled c e l l s l o c a t e d on the medial p e r i m e t e r of the VLL. C e l l s v a r y from s m a l l t o medium w i t h t r i a n g u l a r (arrowhead), round ( l o n g arrow) or o v a l ( s h o r t arrow) p e r i k a r y a . F i n e immunoreactive puncta and v a r i c o s i t i e s were seen w i t h i n t h i s r e g i o n . (E) O c c a s i o n a l s m a l l c e l l s seen i n the nucleus of the d o r s o l a t e r a l lemniscus (DLL). Note low d e n s i t y of f i n e immunoreactive puncta and v a r i c o s i t i e s . (F) Oval t o round, s m a l l t o medium c e l l s were seen along the ve n t r o m e d i a l border of the c a t sagulum (from F i g 18A). C a l i b r a t i o n bars i n d i c a t e 80 microns f o r (A) and 30 microns f o r (B-F). 7 1 72 the l a t e r a l lemniscus, t h e r e was an i n c r e a s e i n immunoreactive c e l l s i n the v e n t r o l a t e r a l l e m n i s c a l r e g i o n ( F i g 18). C e l l s which appeared i n low d e n s i t y a t the v e n t r a l and medial p e r i m e t e r of the VLL were s m a l l t o medium w i t h oval,round or t r i a n g u l a r p e r i k a r y a . R o s t r a l l y , t h e r e was an i n c r e a s e l a t e r a l l y i n s m a l l t o medium s i z e d c e l l s ( F i g 18B,C). The m a j o r i t y of these c e l l s were s m a l l , o v a l c e l l s o r i e n t e d p a r a l l e l t o the ascending l e m n i s c a l f i b r e s , a lthough a few t r i a n g u l a r c e l l s were a l s o seen ( F i g 19A,B). These r o s t r a l c e l l s appeared d i f f e r e n t from those seen along the medial p e r i m e t e r ( F i g 19A,D). The l a t t e r v a r i e d from s m a l l t o l a r g e w i t h o v a l , t r i a n g u l a r and m u l t i p o l a r p e r i k a r y a . There was an i n c r e a s e i n immunoreactive n e u r o p i l a s s o c i a t e d w i t h these r o s t r a l c e l l s . At more d o r s a l and r o s t r a l l e v e l s of the VLL, t h e r e was a low d e n s i t y of medium, m u l t i p o l a r c e l l s ( F i g 19C). These l a r g e c e l l s u s u a l l y o r i e n t e d t h e i r p e r i k a r y a and p r o x i m a l processes t r a n s v e r s e t o the ascending l e m n i s c a l f i b r e s . Immunoreactive puncta and v a r i c o s i t i e s can be seen i n low d e n s i t y throughout the VLL. In the guinea p i g VLL, a t the c a u d a l v e n t r a l border, SOM-IR c e l l s which were continuous w i t h the VNTB appeared to be d i s p l a c e d l a t e r a l l y by the p o n t i n e n u c l e i ( F i g 10D,E). These c e l l s appeared i n r o s t r a l c o n t i g u i t y w i t h the VNTB/LNTB of the SOC. The l a r g e r VNTB c e l l d i d not appear t o c o n t i n u e i n t o the r o s t r a l v e n t r a l p e r i m e t e r of the VLL. In the guinea p i g , c e l l s appeared p r i m a r i l y along the medial 73 F i g u r e 20. (A-C) Coronal s e c t i o n s through the guinea p i g l a t e r a l lemniscus p r o c e e d i n g c a u d a l l y t o r o s t r a l l y . The m a j o r i t y of c e l l s were seen a l o n g the medial p e r i m e t e r of the nucleus of the v e n t r o l a t e r a l lemniscus (VLL). Note the n e u r o p i l immunoreactive s t a i n i n g a long the v e n t r a l p erimeter of VLL ( s h o r t arrows) i n A,B. The medial border of the VLL a l s o shows a band of immunoreactive n e u r o p i l . (A) Mid l e v e l of the guinea p i g VLL and nucleus of the d o r s o l a t e r a l lemniscus (DLL). Note the dense band of immunoreactive s t a i n i n g d o r s a l l y , c o u r s i n g l a t e r a l l y a c r o s s the d o r s a l DLL (arrowheads). (B) The guinea p i g r o s t r a l l a t e r a l lemniscus. Note the i n c r e a s e d d e n s i t y of immunoreactive p e r i k a r y a l o c a t e d v e n t r a l l y (arrows). Immunoreactivity was s t i l l seen a t the d o r s o l a t e r a l and dorsomedial borders of the DLL (arrowheads). (C) At the r o s t r a l extreme of the l a t e r a l lemniscus. Immunolabelled c e l l s (arrows) were l o c a t e d i n the f i b r e s of the v e n t r a l l a t e r a l lemniscus. Immunolabelling can s t i l l be seen immediately d o r s a l t o the DLL (arrowheads). C a l i b r a t i o n bar i n d i c a t e s 500 microns. A b b r e v i a t i o n s : SAG, sagulum n u c l e u s . 74 75 F i g u r e 21. (A) The v e n t r a l immunoreactive band forming the base of the guinea p i g nucleus of the v e n t r o l a t e r a l lemniscus (VLL) ( F i g 20A,B). The s m a l l to medium c e l l s were seen w i t h i n and d o r s a l t o the v e n t r a l band. Immunoreactive puncta and v a r i c o s i t i e s can be seen w i t h i n t h i s v e n t r a l p e r i m e t e r and, a t a lower d e n s i t y , w i t h i n the VLL. (B) Immunoreactivity a t the medial p e r i m e t e r of the VLL. The immunolabelled v a r i c o s i t i e s can be seen running p e r p e n d i c u l a r and t a n g e n t i a l t o ascending l e m n i s c a l f i b r e s . C e l l s seen were p r i m a r i l y s m a l l and o v a l (arrowhead), round ( s h o r t arrow) or t r i a n g u l a r ( l o n g arrow) soma. (C) C e l l s seen a t the c a u d a l v e n t r a l p e r i m e t e r of the VLL. P r i m a r i l y s m a l l , o v a l c e l l s and o c c a s i o n a l s m a l l , e l o n g a t e t r i a n g u l a r c e l l s were seen c a u d a l l y . ( D ) A h i g h m a g n i f i c a t i o n i n s e t from F i g 21B. Small to medium c e l l s can be seen as w e l l as immunolabelled f i b r e s (arrow) and puncta (arrowhead). C a l i b r a t i o n bars i n d i c a t e 80 microns f o r A,B and 30 microns f o r C,D. 77 F i g u r e 22. (A) Immunolabelling i n the guinea p i g seen immediately d o r s a l t o the d o r s a l nucleus of the l a t e r a l lemniscus. Small s p h e r i c a l c e l l s (arrow) were s c a t t e r e d through areas which c o n t a i n immunolabelled puncta and v a r i c o s i t i e s . Most immunolabelled v a r i c o s i t i e s run t a n g e n t i a l t o the l e m n i s c a l f i b r e s . (B) Immunolabelling seen r o s t r a l l y i n the v e n t r a l l a t e r a l lemniscus (from F i g 20C). C e l l s l i e r o u g h l y p a r a l l e l t o the laminae of the l a t e r a l lemniscus. Processes however tend t o l i e t a n g e n t i a l t o the ascending laminae (double arrow). C a l i b r a t i o n bar i n d i c a t e s 30 microns f o r A and 80 microns f o r B. 7 8 79 and l a t e r a l edge of the l a t e r a l l e m n i s c a l f i b r e s . V e n t r a l l y , immunolabelled c e l l s and puncta formed a d e n s e l y s t a i n e d band along the v e n t r a l border of the l a t e r a l lemniscus ( F i g 10F, 14D, 20). The d e n s i t y of immunoreactive n e u r o p i l decreased s l i g h t l y a long the medial border of the VLL ( F i g 20, 21A,B). C a u d a l l y a p o p u l a t i o n of s m a l l e l o n g a t e c e l l s w i t h o v a l and t r i a n g u l a r p e r i k a r y a ( F i g 14D, 21A,C) were l o c a t e d a l o n g the medial perimeter of the VLL. While r o s t r a l l y o v a l and round, s m a l l t o medium c e l l s , predominated i n t h i s r e g i o n ( F i g 21B,D). Most medial o v a l c e l l s ran r o u g h l y p a r a l l e l t o the ascending l e m n i s c a l f i b r e s . More l a t e r a l c e l l s , w i t h t r i a n g u l a r p e r i k a r y a , showed mixed o r i e n t a t i o n ( F i g 21B). O c c a s i o n a l medium m u l t i p o l a r s were seen i n the dorsomedial aspect of VLL. At the r o s t r a l extreme of the l a t e r a l lemniscus t h e r e was a l a r g e i n c r e a s e l a t e r a l l y i n s m a l l t o medium s i z e d , o v a l t o round c e l l s ( F i g 20C, 22B). These c e l l s appeared e i t h e r p a r a l l e l o r s l i g h t l y o b l i q u e t o the ascending f i b r e s of the l a t e r a l lemniscus. A moderate t o low d e n s i t y of s m a l l immunoreactive puncta and v a r i c o s i t i e s can be seen throughout the guinea p i g VLL ( F i g 21). Along the perimeter of the VLL t h e r e was a h i g h d e n s i t y of immunoreactive puncta and v a r i c o s i t i e s ( F i g 21). R o s t r a l l y ( F i g 20C), immunolabelled v a r i c o s i t i e s appeared t a n g e n t i a l t o the f i b r e s of the l a t e r a l lemniscus ( F i g 22B). In the r a t the dorsomedial e x t e n t of VLL showed the g r e a t e s t p r o l i f e r a t i o n of c e l l s ( F i g 23A,B, 24A,C). C e l l s c o u l d a l s o 80 be seen, however, along the medial and v e n t r a l border of the l a t e r a l lemniscus (LL) ( F i g 23A,B, 24B). R o s t r a l l y , the d e n s i t y of c e l l s embedded i n the v e n t r o l a t e r a l LL i n c r e a s e s ( F i g 23C). The SOM-IR c e l l s a long the medial edge of the LL were medium and s m a l l c e l l s w i t h o v a l and round p e r i k a r y a ( F i g 24B). The s m a l l c e l l s were prominent a l o n g the ven t r o m e d i a l border of the VLL ( F i g 24D). W i t h i n the ven t r o m e d i a l r e g i o n most c e l l s were o r i e n t e d w i t h t h e i r long axes p a r a l l e l t o the ascending l e m n i s c a l f i b r e s ( F i g 23D, 24D). At the dorsomedial border of the VLL th e r e was a sharp i n c r e a s e i n immunolabelled c e l l s . These were s m a l l and medium c e l l s w i t h o v a l , round and t r i a n g u l a r p e r i k a r y a ( F i g 24A). These c e l l s were o r i e n t e d w i t h t h e i r soma and pro x i m a l d e n d r i t e s p e r p e n d i c u l a r or s l i g h t l y o b l i q u e t o the l a t e r a l lemniscus ( F i g 24A). At t h e i r d o r s a l extreme, some immunoreactive c e l l s coursed l a t e r a l l y , v e n t r a l t o the DLL, ac r o s s the d o r s a l VLL ( F i g 24C, 25). These c e l l s were s i m i l a r t o those seen d o r s o m e d i a l l y but a l s o i n c l u d e d medium and s m a l l f u s i f o r m c e l l s ( F i g 25). These l a t e r a l l y c o u r s i n g c e l l s were u s u a l l y p e r p e n d i c u l a r t o l e m n i s c a l f i b r e s . At the r o s t r a l extreme of the LL, s m a l l o v a l c e l l s w i t h i n the v e n t r o l a t e r a l r e g i o n of the LL ( F i g 23C, 26B) were seen. These c e l l s l a y p a r a l l e l or s l i g h t l y o b l i q u e t o the f i b r e s of the l a t e r a l lemniscus. F i n e puncta and v a r i c o s i t i e s were seen along the v e n t r a l and l a t e r a l p e r i m e t e r of the VLL ( F i g 23D, 24B,D). The d e n s i t y of immunoreactive puncta seen along the v e n t r a l 81 F i g u r e 23. (A-C) A c a u d a l - r o s t r a l s e r i e s of c o r o n a l s e c t i o n s through the r a t l a t e r a l tegmentum. (A) Immunolabelling seen through most l e v e l s of the v e n t r o l a t e r a l (VLL) and d o r s o l a t e r a l (DLL) n u c l e i of the l a t e r a l lemniscus. The p r o l i f e r a t i o n of immunolabelled c e l l s (arrows) along the dorsomedial perimeter was seen a t t h i s l o c a t i o n through most of the VLL. A number of these dorsomedial c e l l s course l a t e r a l l y a t t h e i r d o r s a l e x t e n t ( s h o r t arrow). V e n t r a l l y , c e l l s were found a l o n g the medial p e r i m e t e r of the VLL. Note the s m a l l band of immunoreactive n e u r o p i l seen a t the d o r s a l border of the DLL (arrowheads). T h i s immunoreactive band appears d i f f e r e n t from P r o b s t ' s commissure. (B) R o s t r a l VLL and DLL. There was an i n c r e a s e i n immunolabelled c e l l s w i t h i n the f i b r e s of the v e n t r o l a t e r a l lemniscus ( s h o r t arrows). The f a i n t immunostained band was s t i l l p r e s e n t d o r s a l t o the DLL (arrowhead). An immunolabelled band a t the v e n t r a l p e r i m e t e r was seen throughout the VLL a t t h i s l o c a t i o n ( l o n g arrow). (C) The r o s t r a l extreme of the l a t e r a l l e m n i s c a l f i b r e s . There was a dense band of c e l l s w i t h i n the l e m n i s c a l f i b r e s v e n t r o l a t e r a l s (arrow). (D) Immunolabelling seen a t the v e n t r a l p e r i m e t e r of VLL. O c c a s i o n a l s m a l l to medium c e l l s were seen a l o n g the v e n t r a l p e r i m e t e r . Note immunolabelled puncta w i t h i n the v e n t r a l band and d o r s a l l y w i t h i n the VLL (arrowheads). Immunoreactive v a r i c o s i t i e s were seen w i t h i n the VLL and the v e n t r a l band. C a l i b r a t i o n bars i n d i c a t e 500 microns f o r A-C and 80 microns D. 82 83 F i g u r e 24. (A) Immunoreactive s m a l l t o medium c e l l s of the r a t which course l a t e r a l l y from the medial p e r i m e t e r of the d o r s a l e x t e n t of the v e n t r a l nucleus of the l a t e r a l lemniscus (VLL) ( F i g 23A). Immunoreactive c e l l s i n t h i s r e g i o n l i e t a n g e n t i a l t o the ascending l e m n i s c a l f i b r e s . Immunoreactive v a r i c o s i t i e s a l s o course l a t e r a l l y w i t h the c e l l s . ( B ) C e l l s a l o n g the ventromedial p e r i m e t e r of VLL. A low d e n s i t y of s m a l l t o medium c e l l s decrease l a t e r a l l y . (C.D) Immunolabelled c e l l s seen a t the r o s t r a l l a t e r a l lemniscus. L a t e r a l - m e d i a l band of immunoreactive c e l l s , puncta and v a r i c o s i t i e s seen a t the d o r s a l aspect of VLL. There was a g e n e r a l i n c r e a s e i n immunolabelled p e r i k a r y a seen c e n t r a l l y w i t h i n the VLL. Medium and s m a l l c e l l s were seen throughout the d o r s a l VLL r e g i o n . (D) There was an i n c r e a s e v e n t r a l l y i n immunolabelled p e r i k a r y a i n the r o s t r a l VLL. The v e n t r a l perimeter shows a decrease i n immunoreactivity. Small t o medium c e l l s were found s c a t t e r e d through the v e n t r a l lemniscus. C a l i b r a t i o n bar i n d i c a t e s 80 microns f o r A-D. 84 85 F i g u r e 25. (A) Immunoreactive c e l l s , puncta and v a r i c o s i t i e s (arrowhead) course l a t e r a l l y over the d o r s a l aspect of the r a t v e n t r a l nucleus of the l a t e r a l lemniscus (VLL). -(B,C) Small t o medium c e l l s seen w i t h i n t h i s l a t e r a l l y c o u r s i n g band. C e l l s tend t o have s p h e r o i d (arrowheads) t o t r i a n g u l a r (arrow) p e r i k a r y a . (C) Immunoreactive f u s i f o r m c e l l s (arrowhead) a l s o occur w i t h i n t h i s band. C a l i b r a t i o n bars i n d i c a t e 80 microns f o r A and 30 microns f o r B,C. 8 6 87 F i g u r e 26. Immunoreactive c e l l s , puncta and v a r i c o s i t i e s found i n the r o s t r a l extreme of the r a t l a t e r a l l e m n i s c a l f i b r e s (from F i g 20C). (A) Immunolabelled s m a l l c e l l s i n the r o s t r a l l e m n i s c a l f i b r e s (at the l e v e l of F i g 23C). C e l l s and f a s c i c l e s appear t o be o r i e n t e d t a n g e n t i a l l y t o the f i b r e s of the l a t e r a l lemniscus. Immunoreactive c e l l s and n e u r o p i l o f t e n appear c l u s t e r e d w i t h i n the f i b r e s of the r o s t r a l lemniscus (arrow). (B) Small c e l l s predominate w i t h i n the r o s t r a l l e m n i s c a l f i b r e s . Dense immunoreactive n e u r o p i l was a s s o c i a t e d w i t h these c l u s t e r s of s m a l l c e l l s s i m i l a r t o t h a t i n d i c a t e d i n (A) (arrow). C a l i b r a t i o n bars i n d i c a t e 80 microns f o r A and 30 microns f o r B. 8 8 89 p e r i m e t e r of the VLL was s l i g h t l y h i g h e r than t h a t seen l a t e r a l l y ( F i g 23A,B). R o s t r a l l y the v e n t r a l immunoreactive band was not seen ( F i g 23C). F i n e puncta and v a r i c o s i t i e s c o u l d a l s o be seen w i t h i n the c e l l group t h a t courses l a t e r a l l y a l o n g the d o r s a l and dorsomedial aspect of VLL. Immunoreactive puncta and v a r i c o s i t i e s were seen throughout the VLL i n low d e n s i t y ( F i g 24, 25). These s m a l l c e l l s i n the r o s t r a l LL were seen i n a l l s p e c i e s . Whether these c e l l s were p a r t of the a u d i t o r y system remains t o be demonstrated. D o r s a l Nucleus of the L a t e r a l Lemniscus, DLL In a l l t h r e e s p e c i e s , c a t , g u i n e a p i g and r a t , t h e r e were o n l y o c c a s i o n a l immunoreactive soma seen i n the DLL. These c e l l s were s m a l l t o medium i n s i z e w i t h o v a l , round or t r i a n g u l a r p e r i k a r y a ( F i g 19C). S c a t t e r e d immunoreactive puncta and v a r i c o s i t i e s occur i n the DLL of a l l s p e c i e s ( F i g 19E). Sagulum In the c a t , a low to moderate d e n s i t y of c e l l s was seen w i t h i n the sagulum. C e l l s were seen along the v e n t r a l sagulum where i t borders DLL ( F i g 18A,B). The c e l l s of the sagulum were s m a l l , w i t h o v a l and t r i a n g u l a r p e r i k a r y a ( F i g 19C). Immunoreactive c e l l s were r a r e i n the sagulum of the r a t and guinea p i g . Immunoreactive puncta and v a r i c o s i t i e s s i m i l a r t o those seen i n the VLL were seen i n the sagulum of a l l t h r e e s p e c i e s . D o r s a l Perimeter of the DLL In a l l t h r e e s p e c i e s , the SOM-IR c e l l s and puncta were seen j u s t d o r s a l t o the DLL ( F i g 20, 23). There was a low d e n s i t y of immunoreactive s m a l l round t o o v a l c e l l s found w i t h i n t h i s r e g i o n . These c e l l s were o f t e n seen embedded w i t h i n immunostained f a s c i c l e s and puncta ( F i g 22A). Puncta and f a s c i c l e s s t a i n e d i n t h i s r e g i o n ran t a n g e n t i a l l y t o the ascending l e m n i s c a l f i b r e s ( F i g 22A). At v a r i o u s l e v e l s of the VLL, t h i s immunoreactive n e u r o p i l formed a complete band a c r o s s the DLL/IC border. T h i s p a t t e r n of s t a i n i n g was most prominent i n the guinea p i g and r a t (moderate d e n s i t y ) and was a l s o seen, but t o a l e s s e r degree, i n the c a t . I n f e r i o r c o l l i c u l u s , IC In a l l t h r e e s p e c i e s , the immunostaining was predominantly found throughout the p e r i c e n t r a l r e g i o n s of the i n f e r i o r c o l l i c u l u s (IC) the e x t e r n a l c o r t e x (EC) d o r s a l c o r t e x (DC) and the i n t e r c o l l i c u l a r area (ICA). The d e n s i t y of immunolabelling of c e l l s and puncta w i t h i n the c e n t r a l nucleus showed a marked decrease as the c e n t r a l nucleus of 91 F i g u r e 27. A s e r i e s of low m a g n i f i c a t i o n photomicrographs c o r o n a l s e c t i o n s through the c a t i n f e r i o r c o l l i c u l u s . S e c t i o n s proceed i n a c a u d a l t o r o s t r a l o r d e r . Note i n F i g u r e 27A-C the m a j o r i t y of immunolabelled p e r i k a r y a were found p e r i c e n t r a l l y . (A) The c a u d a l l e v e l of the c a t IC. Note a t t h i s l e v e l a few c e l l s are l o c a t e d w i t h i n the c e n t r a l r e g i o n of the IC. However the m a j o r i t y of immunolabelled c e l l s were s t i l l p e r i c e n t r a l . (B) Mid l e v e l c a t IC. Here the c e n t r a l nucleus (ICC) c o n t a i n s o n l y a few s c a t t e r e d immunolabelled c e l l s . The d e n s i t y of the c e l l s seen v e n t r a l l y has a l s o d i m i n i s h e d from t h a t i n p r e v i o u s c a u d a l l e v e l s . A rough o u t l i n e of the ICC was i n d i c a t e d by the d o t t e d o u t l i n e . (C) R o s t r a l c a t IC. F i b r e s from the commissure of the i n f e r i o r c o l l i c u l u s course t o the l a t e r a l p e rimeter of the IC. The frequency of the immunolabelled c e l l s begins t o i n c r e a s e c e n t r a l l y . Note immunolabelled band which marks the medial border o f the IC (arrowheads). (D) Shows the r o s t r a l p o l e of the IC. Immunolabelled c e l l s were seen throughout the f i b r e s of the commissure of the i n f e r i o r c o l l i c u l u s . Note t h a t the c e n t r a l r e g i o n of the r o s t r a l p o l e (RP) shows a p a u c i t y of immunolabelled p e r i k a r y a . The immunoreactive band of n e u r o p i l has s h i f t e d w i t h the r e c e d i n g IC t o d e l i n e a t e the d o r s a l border of the r o s t r a l p o l e of the IC (arrowheads). C a l i b r a t i o n bar i n d i c a t e s 1 mm. A b b r e v i a t i o n s : COIC, commissure of the i n f e r i o r c o l l i c u l u s ; DC, d o r s a l c o r t e x ; e x t e r n a l c o r t e x ; ICA, i n t e r c o l l i c u l a r a rea; ICC, c e n t r a l nucleus of the i n f e r i o r c o l l i c u l u s . 92 93 F i g u r e 28. S e l e c t e d r e g i o n s of the c a t IC. (A) Shows s m a l l immunolabelled c e l l s seen a l o n g the d o r s a l t o medial perimeter of the ICC. Note t h a t c e l l s appear t o l i e p a r a l l e l t o ascending laminae. (B) I l l u s t r a t e s the immunolabelling the d o r s o l a t e r a l edge of the c a u d a l and mid l e v e l s of the IC ( F i g 28A-B). Note t h a t s m a l l c e l l s predominate. I n c r e a s e d immunoreactive puncta and n e u r o p i l along the d o r s o l a t e r a l tectum were a s s o c i a t e d w i t h the s u p e r f i c i a l f i b r o c e l l u l a r l a y e r s of the e x t e r n a l and d o r s a l c o r t i c e s . (C) The v e n t r o l a t e r a l r e g i o n of the IC. A mixture of immunoreactive c e l l s w i t h s m a l l t o medium m u l t i p o l a r , and s m a l l o v a l c e l l s . Note t h a t the o v a l c e l l s are l o c a t e d more c e n t r a l l y than the m u l t i p o l a r c e l l s and run p a r a l l e l t o the ascending laminae. M u l t i p o l a r c e l l s appear t a n g e n t i a l t o the ascending l e m n i s c a l laminae. (D) The c a t commissure of the i n f e r i o r c o l l i c u l u s (COIC). Immunoreactive c e l l s ( l o n g arrow) occur a t a low d e n s i t y w i t h i n the commissure of the i n f e r i o r c o l l i c u l u s . Immunoreactive v a r i c o s i t i e s run p a r a l l e l (arrowhead) and t a n g e n t i a l ( s h o r t arrow) to the commissural f i b r e s . (E) I n s e t from F i g 27A. Small c e l l s seen along the dorsomedial p e r i m e t e r of the c e n t r a l nucleus of the i n f e r i o r c o l l i c u l u s (ICC) and i n h i g h e r d e n s i t y i n the a d j a c e n t d o r s a l c o r t e x (DC). (F) I l l u s t r a t e s dorsomedial DC immunoreactivity. Medium m u l t i p o l a r and s m a l l e r immunolabelled c e l l s were seen w i t h i n t h i s r e g i o n . C a l i b r a t i o n bars i n d i c a t e 80 microns f o r A-D and 30 microns f o r E-F. 94 95 F i g u r e 29. S e l e c t e d r e g i o n s of the c a t r o s t r a l i n f e r i o r c o l l i c u l u s ( I C ) . (A) Immunolabelled c e l l s seen a t the r o s t r a l IC, the d o r s o l a t e r a l border ( l e v e l 28C) w i t h i n the i n t e r c o l l i c u l a r a rea (ICA). There was an i n c r e a s e i n medium s i z e d c e l l s w i t h t r i a n g u l a r p e r i k a r y a . Small o v a l c e l l s seen c a u d a l l y were s t i l l p r e s e n t . (B) The r o s t r a l IC taken a t the l e v e l of F i g 27C. At t h i s r o s t r a l l e v e l a mixture of s m a l l t o medium c e l l s were seen throughout the commissural f i b r e s . O c c a s i o n a l s m a l l c e l l s were seen i n the d o r s a l m o l e c u l a r l a y e r (arrowhead). Note the s m a l l pocket of medium t o s m a l l , o v a l t o t r i a n g u l a r c e l l s seen apposing the molecular l a y e r (arrow). (C) Immunoreactive c e l l s seen a t the r o s t r a l p o l e of the c a t ICA. A mixture of s m a l l , medium and l a r g e c e l l s were seen i n t h i s r e g i o n . L a r g e r c e l l s seen l a t e r a l l y were found throughout commissural f i b r e s . M e d i a l l y t h e r e was a predominance of s m a l l c e l l s . (D) Immunoreactivity seen a t r o s t r a l IC, w i t h i n the ICA. These c e l l s l i e w i t h i n commissural f i b r e s . Note t h a t most c e l l s and pr o c e s s e s were o r i e n t e d t a n g e n t i a l l y t o p a s s i n g f i b r e s . A low d e n s i t y of f i n e puncta can be seen i n both F i g 29C,E. (E) The l a t e r a l boundary of the r o s t r a l p o l e and as such was v e n t r a l t o C and D. The c e l l s i n t h i s r e g i o n form a more heterogeneous group than t h a t seen d o r s a l l y . There was a n o t a b l e decrease i n the t r i a n g u l a r c e l l s seen d o r s a l l y ( F i g C,E). C a l i b r a t i o n bar i n d i c a t e s 160 microns f o r B and 80 microns f o r A-F. 97 F i g u r e 30. High m a g n i f i c a t i o n photomicrographs from the r o s t r a l p o l e of the c a t IC. (A) Immunoreactivity from the area of r o s t r a l IC immediately v e n t r a l t o the mo l e c u l a r l a y e r ( F i g 29B). Medium and s m a l l c e l l s were seen w i t h i n t h i s r e g i o n . (B) Immunoreactivity w i t h i n the f i b r e s of the commissure of IC a t the r o s t r a l and d o r s o l a t e r a l border of IC. A mixture of s m a l l t o medium c e l l s were seen i n t h i s r e g i o n (from F i g 29E). (C-D) Immunoreactivity seen i n the ICA. Small and medium c e l l s were most common w i t h i n the r e g i o n s a d j a c e n t t o the r o s t r a l p o l e . C a l i b r a t i o n bars i n d i c a t e 30 microns. 98 A f 3 g 4 u \ C ^ D » * tj t w 4 • p 99 the i n f e r i o r c o l l i c u l u s (ICC) was approached from the p e r i c e n t r a l a r e a s . The major p o p u l a t i o n of immunoreactive c e l l s w i t h i n the c a t IC was s m a l l c e l l s w i t h oval,round and t r i a n g u l a r soma ( F i g 27, 28A-C,E,F). These immunoreactive c e l l s i n c r e a s e d a t the dorsomedial perimeter of the c e n t r a l n u c l e u s . These s m a l l c e l l s and t h e i r p r o x i m a l p r o c e s s e s u s u a l l y ran p a r a l l e l t o the ascending f i b r e s ( F i g 28A,E). Immunoreactive v a r i c o s i t i e s and puncta were r a r e l y found w i t h i n the c a t ICC. C a u d a l l y , immunoreactive c e l l s were seen throughout a l l p e r i c e n t r a l r e g i o n s of the c a t IC ( F i g 27A). R o s t r a l l y , t h e r e was a decrease i n the d e n s i t y of c e l l s seen i n v e n t r a l p e r i c e n t r a l r e g i o n s ( F i g 27B,C). F u r t h e r r o s t r a l l y , c e l l s were seen w i t h i n most r e g i o n s of the c a t IC and commissural f i b r e s ( F i g 27D). Small c e l l s were seen throughout the p e r i c e n t r a l c a t IC ( F i g 27). These s m a l l c e l l s were, however, most prominent a t caud a l and mid l e v e l p e r i c e n t r a l r e g i o n s . As a group, these c e l l s were s m a l l t o medium w i t h o v a l , round and t r i a n g u l a r p e r i k a r y a ( F i g 27). Other c e l l s immunolabelled f o r s o m a t o s t a t i n were medium s i z e d m u l t i p o l a r s w i t h oval,round and t r i a n g u l a r p e r i k a r y a . The DC c o n t a i n e d these c e l l s and a l s o s l i g h t l y l a r g e r c e l l s w i t h t r i a n g u l a r p e r i k a r y a ( F i g 28F). Most p e r i c e n t r a l c e l l s were o r i e n t e d p a r a l l e l t o the ascending laminae formed by l e m n i s c a l a f f e r e n t s . Puncta and 100 v a r i c o s i t i e s were predominant a l o n g the d o r s a l and l a t e r a l s u p e r f i c i a l f i b r o c e l l u l a r l a y e r s of the DC and EC ( F i g 28B, 29B). R o s t r a l l y t h e r e was an i n c r e a s e i n the s i z e and d e n s i t y of immunoreactive p e r i k a r y a . T h i s p a r a l l e l e d the r o s t r a l r e d u c t i o n i n the s i z e of the ICC and the COIC f i b r e s c o u r s i n g l a t e r a l l y t o the brachium of the i n f e r i o r c o l l i c u l u s (BIC). Medium c e l l s became more prominent a t t h i s l e v e l ( F i g 29B) and i n c r e a s e d r o s t r a l l y w i t h i n the ICA ( F i g 29, 30). These c e l l s were t r i a n g u l a r t o o v a l m u l t i p o l a r , o r o v a l b i p o l a r c e l l s ( F i g 30). O r i e n t a t i o n of c e l l s w i t h i n the r o s t r a l EC, p e r i ICC, was both p a r a l l e l and p e r p e n d i c u l a r t o the p a s s i n g commissural f i b r e s . These SOM-IR c e l l s were seen c a u d a l l y w i t h i n the p e r i c e n t r a l IC r e g i o n s and r o s t r a l l y w i t h i n the c a t ICA. O c c a s i o n a l s m a l l o v a l c e l l s were seen w i t h i n the most s u p e r f i c i a l l a y e r s of IC ( F i g 29B, 30A,B). At the r o s t r a l c a t IC, medium s i z e d c e l l s w i t h t r i a n g u l a r p e r i k a r y a predominated w i t h i n the ICA. Other c e l l s seen i n t h i s r e g i o n were medium t o s m a l l c e l l s w i t h o v a l or round p e r i k a r y a . Most c e l l s i n t h i s r e g i o n , and t h e i r p r o x i m a l d e n d r i t e s , were o r i e n t e d t a n g e n t i a l l y t o the p a s s i n g commissural f i b r e s ( F i g 29C,D, 30C). W i t h i n the l a t e r a l p e r i m e t e r of the c a t r o s t r a l p o l e , c e l l s tended t o show mixed o r i e n t a t i o n . C e l l s appeared m u l t i p o l a r and b i p o l a r , w i t h t r i a n g u l a r t o o v a l p e r i k a r y a . At r o s t r a l l e v e l s of the c a t IC, a f a i n t band of immunoreactive n e u r o p i l d e l i m i t e d the medial border of IC 101 ( F i g 27C). T h i s border moved l a t e r a l l y w i t h the r e c e d i n g IC/ICA r e g i o n . Most immunoreactive puncta were seen along the d o r s a l and d o r s o l a t e r a l tectum. Immunoreactive puncta, v a r i c o s i t i e s and o c c a s i o n a l s m a l l c e l l s were seen w i t h i n the medial c a t COIC ( F i g 28B). Immunoreactive v a r i c o s i t i e s r an both p a r a l l e l and p e r p e n d i c u l a r t o commissural f i b r e s ( F i g 28B) In the guinea p i g , the d e n s i t y of SOM-IR decreased as the ICC was approached from the p e r i c e n t r a l r e g i o n , EC, DC ( F i g 31). C a u d a l l y , the EC c o n t a i n e d immunoreactive p e r i k a r y a and puncta ( F i g 31A). C e l l s seen along the dorsomedial p e r i m e t e r of the c e n t r a l nucleus were u s u a l l y s m a l l c e l l s w i t h oval,round and t r i a n g u l a r p e r i k a r y a ( F i g 32D). These c e l l s l a y p a r a l l e l t o the ascending l e m n i s c a l laminae. Medium s i z e d t r i a n g u l a r c e l l s , as w e l l as s m a l l e r o v a l c e l l s , were seen along the v e n t r o l a t e r a l and ven t r o m e d i a l borders of the guinea p i g ICC ( F i g 32B,E). In g e n e r a l , c e l l s w i t h i n the c e n t r a l nucleus were found i n hi g h e r d e n s i t y i n areas immediately a d j a c e n t t o the p e r i c e n t r a l r e g i o n s ( F i g 32D vs F i g 32E). A low d e n s i t y of f i n e puncta and v a r i c o s i t i e s was seen w i t h i n the guinea p i g ICC. The m a j o r i t y of immunoreactivity i n the guinea p i g was found i n the EC, DC and ICA. C a u d a l l y , c e l l s were l o c a t e d 102 F i g u r e 31. A s e r i e s of c o r o n a l s e c t i o n s through the guinea p i g i n f e r i o r c o l l i c u l u s ( I C ) . F i g u r e s were pre s e n t e d i n a c a u d a l t o r o s t r a l f a s h i o n . Note the decrease of immunoreactivity w i t h i n the c e n t r a l nucleus of the IC between F i g A and B. (A) Caudal guinea p i g IC. Note a t t h i s l e v e l immunoreactive c e l l s were seen throughout the IC. Patches of low immunoreactivity can be seen d o r s a l l y . (B) The guinea p i g IC a t the l e v e l of the cau d a l completion of the commissure of the i n f e r i o r c o l l i c u l u s (COIC). Note the decrease i n immunoreactive p e r i k a r y a as the c e n t r a l nucleus of the i n f e r i o r c o l l i c u s (ICC) was approached from the p e r i c e n t r a l r e g i o n s . Along the l a t e r a l p e r i m e t e r t h e r e was an i n c r e a s e i n immunoreactivity a s s o c i a t e d w i t h the s u p e r f i c i a l f i b r o c e l l u l a r l a y e r s of the e x t e r n a l (EC) and d o r s a l (DC) c o r t i c e s . (C) The guinea p i g IC, r o s t r a l t o B. Note t h a t the p a t t e r n immunoreactivity was s i m i l a r t o t h a t seen i n B. Again the d e n s i t y of immunoreactive c e l l s decreases as the ICC was approached from p e r i c e n t r a l r e g i o n s , DC and EC. The d o r s a l and l a t e r a l t e c t a l s u r f a c e l a y e r s show h i g h e r l e v e l s of immunoreactivity than the ad j a c e n t IC a r e a s . (D) The s t a r t of the r o s t r a l IC of the guinea p i g . At t h i s l e v e l , f i b r e s of the of COIC course t o the l a t e r a l p e r i m e t e r of the tectum. The brachium of the IC shows a h i g h l e v e l of immunoreactive n e u r o p i l (arrow). M e d i a l l y , an immunoreactive band forms the medial border of the IC (arrowheads). Note t h a t t h i s immunoreactive band moves l a t e r a l l y i n more r o s t r a l l e v e l s of guinea p i g IC. 103 104 105 106 F i g u r e 31 ( c o n t . ) . (E,F) The r o s t r a l extreme of the guinea-p i g IC. Note i n both f i g u r e s the medial band of immunoreactivity seen i n C s h i f t s l a t e r a l l y t o form the d o r s a l border of the EC/DC/ICA (arrowheads). In both E and F i n c r e a s e d immunoreactivity was a s s o c i a t e d w i t h the dorsomedial aspect of the brachium of the i n f e r i o r c o l l i c u l u s (BIC) (arrow). (E) R o s t r a l IC. At t h i s l e v e l of the IC the immunolabelling w i t h i n the commissure has s h i f t e d l a t e r a l l y . C e l l s w i t h i n the commissural f i b r e s were o r i e n t e d t a n g e n t i a l l y t o p a s s i n g commissural f i b r e s . C e l l s w i t h i n the c e n t r a l r o s t r a l p o l e , l o c a t e d v e n t r o m e d i a l l y , show a mixed o r i e n t a t i o n . The BIC shows immunolabelled n e u r o p i l a t i t s d o r s a l p e r i m e t e r . (F) The r o s t r a l p o l e of the IC. V e n t r a l and v e n t r o m e d i a l c e l l s show mixed o r i e n t a t i o n s , w h i l e d o r s a l and d o r s o l a t e r a l c e l l s have a t a n g e n t i a l o r i e n t a t i o n . The l a t t e r , a l o n g w i t h the d o r s a l band of n e u r o p i l , form a continuous band w i t h the d o r s a l border of the BIC. C a l i b r a t i o n bar i n d i c a t e s 500 microns. A b b r e v i a t i o n s : PAG, p e r i a q u e d u c t a l grey; SC, s u p e r i o r c o l l i c u l u s . 107 108 F i g u r e 32. Photomicrographs of the guinea p i g i n f e r i o r c o l l i c u l u s ( I C ) . (A-K) c a u d a l t o mid l e v e l s of the guinea p i g IC. Note i n a l l f i g u r e s immunoreactive c e l l s and v a r i c o s i t i e s tend t o run p a r a l l e l t o ascending laminae. (A) The d o r s o l a t e r a l border of the guinea p i g IC. The m a j o r i t y o f immunoreactive c e l l s were s m a l l w i t h o v a l , round or t r i a n g u l a r p e r i k a r y a . Medium s i z e d c e l l s were a l s o seen i n t h i s r e g i o n . Note the dense immunoreactive f a s c i c l e s a s s o c i a t e d w i t h the brachium of the i n f e r i o r c o l l i c u l u s . (B) The l a t e r a l border of the guinea p i g IC c o n t a i n s immunolabelled s m a l l c e l l s and l a r g e r medium s i z e d , m u l t i p o l a r c e l l s (arrowhead). Note the i n c r e a s e i n d e n s i t y of f a s c i c l e s and puncta a s s o c i a t e d w i t h the BIC and the v e r y l a r g e p e r i c e n t r a l c e l l s h i g h l i g h t e d by the a p p o s i t i o n of f i n e immunoreactive puncta (arrow). (C) P e r i c e n t r a l r e g i o n s of the d o r s a l and medial IC . The d e n s i t y of s m a l l c e l l s has i n c r e a s e d i n the more d o r s a l r e g i o n s . As i n D, s m a l l c e l l s and t h e i r a p i c a l p r o c e s s e s l i e p a r a l l e l t o ascending laminae. (D) The immunoreactivity seen a t the dorsomedial perimeter of the c e n t r a l nucleus and i n the dorsomedial p e r i c e n t r a l a r e a s . Note c e l l s and a p i c a l p r o c e s s e s run p a r a l l e l t o ascending laminae (arrowheads). These c e l l s v ary from o v a l and round t o t r i a n g u l a r . (E) The v e n t r o l a t e r a l border of the g u i n e a - p i g IC. V e n t r o l a t e r a l l y , medium s i z e d m u l t i p o l a r c e l l s (arrowheads) were prominent. Small c e l l s however were s t i l l seen here. (F) The immunoreactivity seen w i t h i n the c a u d a l COIC. Note the dense s t a i n i n g n e u r o p i l 109 seen l a t e r a l l y c o n t a i n s s m a l l t o medium s i z e d , o v a l c e l l s . S i m i l a r c e l l s were a l s o seen s c a t t e r e d throughout the c e n t r a l commissural r e g i o n . Immunoreactive v a r i c o s i t i e s seen c e n t r a l l y both t r a v e l t r a n s v e r s e (arrowhead) and p a r a l l e l (arrow) t o the commissure of the IC. 110 I l l F i g u r e 32 ( c o n t . ) . (G,H) Immunostaining from w i t h i n the commissural f i b r e s of the r o s t r a l l e v e l s of the IC ( F i g 30D-E ) . At more r o s t r a l l e v e l s t h e r e an i n c r e a s e i n the s i z e of immunoreactive c e l l s compared t o those seen c a u d a l l y . C e l l s i z e v a r i e s r o s t r a l l y from s m a l l t o l a r g e . (G) Immunoreactivity seen among the d o r s a l commissural f i b r e s a t the l e v e l of F i g 31D-E. Note the v a r i e t y of c e l l s and o r i e n t a t i o n s . There was an i n c r e a s e i n medium t o l a r g e c e l l s seen a t t h i s l e v e l (arrowheads). (H) C e l l s seen w i t h i n the dorsomedial commissural f i b r e s a t the l e v e l of F i g 31E. Small t o medium c e l l s were seen throughout the commissural f i b r e s . C e l l s tend t o d i r e c t t h e i r p r ocesses t a n g e n t i a l l y , (arrowheads) t o the commissural f i b r e s . Immunoreactive v a r i c o s i t i e s a l s o appear t a n g e n t i a l t o the commissural f i b r e s and p a r a l l e l t o c e l l p r o c e s s e s . (I) I l l u s t r a t e s the immunostaining seen w i t h i n the r o s t r a l ICA. (J) The c e n t r a l r e g i o n o f the r o s t r a l p o l e of the guinea p i g IC. Ventromedial c e l l s d i d not show the t a n g e n t i a l o r i e n t a t i o n seen d o r s a l l y or l a t e r a l l y i n the ICA. Note t h a t the v e r y l a r g e c e l l s found c a u d a l l y were s t i l l seen, h i g h l i g h t e d by f i n e puncta (arrow). As i n the l a t e r a l r e g i o n , immunoreactive c e l l s here were a heterogeneous p o p u l a t i o n . 112 113 F i g u r e 32 ( c o n t . ) . (K) Immunoreactivity i n the d o r s o l a t e r a l ICA. R o s t r a l l y , s o m a t o s t a t i n immunoreactive c e l l s were predominantly medium s i z e d . Small c e l l s were, however, s t i l l p r e s e n t . Note the o r i e n t a t i o n of immunoreactive c e l l s , p r o c e s s e s , and v a r i c o s i t i e s t a n g e n t i a l t o the commissural f i b r e . Small and medium c e l l s were seen i n the ICA. Most medium c e l l s were m u l t i p o l a r , w i t h t r i a n g u l a r t o o v a l p e r i k a r y a . O c c a s i o n a l , e l o n g a t e , b i p o l a r c e l l s were seen throughout the r o s t r a l p o l e . C a l i b r a t i o n bars i n d i c a t e 80 microns f o r A-K. 114 115 F i g u r e 33. I l l u s t r a t e s s e l e c t e d r e g i o n s of the guinea p i g IC. (A) Very l a r g e m u l t i p o l a r c e l l s seen throughout a l l p e r i c e n t r a l r e g i o n s ( r o s t r a l t o caudal) of the IC. These p a r t i c u l a r c e l l s were seen i n the v e n t r o l a t e r a l t o l a t e r a l r e g i o n of IC. Note t h a t these c e l l s were not s t a i n e d but were h i g h l i g h t e d by v e r y f i v e immunoreactive puncta (arrowhead) and v a r i c o s i t i e s . The l a t t e r form p e r i d e n d r i t i c and p e r i s o m a t i c a p p o s i t i o n . Dense p e r i s o m a t i c immunoreactive f i b r e s o f t e n h i g h l i g h t the c e l l s (arrow). (B) Small c e l l s were seen a t the dorsomedial p e r i m e t e r of the c e n t r a l nucleus and w i t h i n dorsomedial p e r i c e n t r a l r e g i o n s . (C) The c e l l s seen i n the r o s t r a l ICA. The mixture of c e l l s seen here tend t o o r i e n t t h e i r p e r i k a r y a and/or processes t a n g e n t i a l t o the p a s s i n g f i b r e s . (D) Immunoreactivity seen i n the brachium of the i n f e r i o r c o l l i c u l u s (BIC). Note t h a t the dense immunoreactive v a r i c o s i t i e s course d o r s o l a t e r a l l y i n the BIC ( F i g 31E-F). O c c a s i o n a l s m a l l t o medium o v a l c e l l s were seen w i t h i n the BIC (arrowhead). (E) Medium s i z e d m u l t i p o l a r s seen i n the v e n t r a l c a u d a l IC ( F i g 32C). C a l i b r a t i o n bar i n d i c a t e s 30 microns. 117 throughout the e x t e r n a l c o r t e x . Medium SOM-IR, m u l t i p o l a r c e l l s were found v e n t r a l l y ( F i g 32E) and l a t e r a l l y ( F i g 32B) i n the ca u d a l EC. These m u l t i p o l a r c e l l s had o v a l and t r i a n g u l a r p e r i k a r y a ( F i g 33E). Small t o medium s i z e d c e l l s w i t h o v a l t o t r i a n g u l a r p e r i k a r y a were seen throughout a l l p e r i c e n t r a l r e g i o n s ( F i g 32). Most c e l l s and prox i m a l processes r a n p a r a l l e l t o the ascending laminae. In the dorsomedial r e g i o n of the ca u d a l t o mid l e v e l s of the guinea p i g IC and DC, ( F i g 31A,B,C) the m a j o r i t y of c e l l s were s m a l l c e l l s w i t h o v a l , round or t r i a n g u l a r p e r i k a r y a ( F i g 33B). As w i t h most c e l l s , these c e l l s p a r a l l e l the ascending laminae ( F i g 32). O c c a s i o n a l , v e r y l a r g e m u l t i p o l a r c e l l s were a l s o seen a l o n g the v e n t r o l a t e r a l guinea p i g EC. These c e l l s were u s u a l l y h i g h l i g h t e d by p e r i c e l l u l a r f i n e puncta and o c c a s i o n a l immunoreactive bands ( F i g 33A). There was a moderate t o h i g h d e n s i t y of immunoreactive puncta throughout the p e r i c e n t r a l IC. The h i g h e s t immunoreactivity o c c u r r e d along the l a t e r a l and d o r s a l p e r i m e t e r s of the tectum. Some r e f e r t o t h i s as the BIC wh i l e o t h e r s r e f e r t o i t as the s u p e r f i c i a l f i b r o c e l l u l a r l a y e r s of the EC and DC. Immunoreactive v a r i c o s i t i e s appeared t o run p a r a l l e l t o the t e c t a l s u r f a c e ( F i g 31B,C). C a u d a l l y , immunoreactive patches were seen d o r s o m e d i a l l y i n c a u d a l IC, EC and DC. These patches p a r a l l e l e d the d i s t r i b u t i o n of s m a l l c e l l s w i t h i n the c a u d a l dorsomedial r e g i o n ( F i g 31A,B,C). In the r o s t r a l p e r i c e n t r a l guinea p i g IC, where f i b r e s o f the COIC course l a t e r a l l y ( F i g 31C), t h e r e was an 118 i n c r e a s e i n l a r g e and medium s i z e d c e l l s . Small c e l l s were s t i l l found throughout t h i s r e g i o n of the IC. The l a r g e r c e l l s u s u a l l y were o r i e n t e d t a n g e n t i a l l y t o the f i b r e s of the commissure IC ( F i g 31D, 32H). T h i s t r e n d c o n t i n u e d i n t o the guinea p i g ICA ( F i g 31E,F). At r o s t r a l l e v e l s of the IC, the t a n g e n t i a l o r i e n t a t i o n of c e l l s was more pronounced ( F i g 32G,H,I,K). There was a l a r g e h e t e r o g e n e i t y of c e l l types a t these r o s t r a l l e v e l s . W i t h i n the guinea p i g ICA, s m a l l t o l a r g e SOM-IR c e l l s were found. C e l l s appeared as both b i p o l a r and m u l t i p o l a r ( F i g 32G,H,I,K). The r o s t r a l p o l e of the IC showed s i m i l a r c e l l s t o those seen l a t e r a l l y ( F i g 31E,F). The o r i e n t a t i o n of the r o s t r a l p o l e c e l l s , however, was mixed ( F i g 32). These c e l l s were i n lower d e n s i t y a t the r o s t r a l l i m i t o f the r o s t r a l p o l e . There was a low t o moderate d e n s i t y of immunoreactive puncta and v a r i c o s i t i e s throughout the r o s t r a l guinea p i g IC. A band of moderately l a b e l l e d n e u r o p i l d e l i n e a t e d the medial l i m i t of the r o s t r a l IC ( F i g 31D). Moving r o s t r a l l y , t h i s band moved l a t e r a l l y w i t h the r e c e d i n g IC. At the r o s t r a l ICA r e g i o n , t h i s immunoreactive band marked the d o r s a l border ( F i g 31E,F). The dorsomedial border of the guinea p i g BIC showed a h i g h d e n s i t y of immunoreactive puncta and v a r i c o s i t i e s ( F i g 31E,F). O c c a s i o n a l immunoreactive, medium s i z e d , o v a l c e l l s were seen w i t h i n the BIC ( F i g 33D). 119 F i g u r e 34. Coronal s e c t i o n s of the r a t i n f e r i o r c o l l i c u l u s . S e c t i o n s are p r e s e n t e d i n a c a u d a l t o r o s t r a l f a s h i o n . Note t h a t the decrease i n the d e n s i t y of immunolabelled c e l l s w i t h i n the c e n t r a l nucleus of the i n f e r i o r c o l l i c u l u s (ICC). Note t h a t the i n c r e a s e i n the immunoreactive n e u r o p i l seen at the d o r s o l a t e r a l perimeter was a s s o c i a t e d w i t h the BIC. (A) The c a u d a l r a t the IC. Immunolabelled c e l l s were l o c a t e d throughout IC. The s l i g h t i n c r e a s e of immunoreactive n e u r o p i l a l o n g the d o r s o l a t e r a l border was a s s o c i a t e d w i t h the brachium of the i n f e r i o r c o l l i c u l u s (BIC). (B) Mid l e v e l IC. A decrease i n the c e l l d e n s i t y was seen c e n t r a l l y . Again i n c r e a s e d immunoreactive n e u r o p i l was a s s o c i a t e d w i t h the BIC. (C) The r o s t r a l IC. Note t h a t the commissural f i b r e s of the IC course t o the l a t e r a l extreme of the IC. C e n t r a l r e g i o n s of the IC a g a i n show a low d e n s i t y of immunoreactivity as compared w i t h p e r i c e n t r a l r e g i o n s . At t h i s l e v e l of the IC, a band of immunoreactive n e u r o p i l (arrowhead) marks the medial l i m i t of the IC. J u s t l a t e r a l t o t h i s band was another band of immunoreactivity ( s i m i l a r t o the guinea p i g , F i g 31D). (D) The r o s t r a l p o l e , RP, of the r a t IC. Note the m a j o r i t y of c e l l s were w i t h i n the ICA. In both E and F an immunoreactive band (arrowheads) can be seen t o form the d o r s a l s u r f a c e of the r o s t r a l IC. Along the d o r s o l a t e r a l p e r i m e t e r an i n c r e a s e i n immunoreactivity was s t i l l a s s o c i a t e d w i t h the BIC (arrows). Note t h a t the c e n t r a l t o v e n t r o m e d i a l r e g i o n shows decreased immunoreactivity. 120 121 F i g u r e 34 ( c o n t . ) . (E) R o s t r a l IC. Immunoreactive c e l l s were p r i m a r i l y a s s o c i a t e d w i t h the commissural f i b r e s of the IC. Note t h a t c e l l s i n the d o r s o l a t e r a l r e g i o n run t a n g e n t i a l t o the commissural f i b r e s and p a r a l l e l t o the d o r s a l band of immunoreactive n e u r o p i l (arrowhead). (F) The extreme r o s t r a l p o l e of the r a t IC. Note the decrease i n immunoreactive c e l l s which were seen, d o r s o l a t e r a l l y , i n E. C a l i b r a t i o n bar i n d i c a t e s 500 microns. A b b r e v i a t i o n s : COIC, commissure of the i n f e r i o r c o l l i c u l u s ; DC, d o r s a l c o r t e x ; ICA, i n t e r c o l l i c u l a r area; ICC, c e n t r a l nucleus of the i n f e r i o r c o l l i c l u s ; RP, r o s t r a l p o l e . 122 123 F i g u r e 35. Immunoreactivity seen i n the r a t IC. Small c e l l s were seen throughout the p e r i c e n t r a l IC. Immunoreactive v a r i c o s i t i e s were seen p r i m a r i l y a l o n g the d o r s o l a t e r a l and dorsomedial t e c t a l s u r f a c e s . (A) The d o r s o l a t e r a l r a t IC. Immunoreactive c e l l s have o v a l , round and t r i a n g u l a r p e r i k a r y a . Most c e l l s l i e p a r a l l e l t o the ascending laminae. Note t h a t immunolabelled v a r i c o s i t i e s and f a s c i c l e s were t a n g e n t i a l t o the laminae, which were immediately v e n t r a l . (B) Immunoreactivity seen a l o n g the dorsomedial p e r i m e t e r of the c e n t r a l nucleus of the i n f e r i o r c o l l i c u l u s (ICC). The i n c r e a s e m e d i a l l y i n c e l l s was a s s o c i a t e d w i t h the p e r i c e n t r a l r e g i o n s of the IC. (C) The dorsomedial s u r f a c e of the r a t IC and DC. A mixture of s m a l l t o medium c e l l s showed v a r i a b l e s i z e and o r i e n t a t i o n . Immunoreactive v a r i c o s i t i e s seen d o r s a l l y run p a r a l l e l t o the d o r s a l s u r f a c e . (D) The v e n t r o l a t e r a l IC. Small t o medium c e l l s predominate i n t h i s r e g i o n . 124 125 F i g u r e 35 ( c o n t . ) . (E-H) The r o s t r a l r a t IC. (E) The d o r s o l a t e r a l immunoreactivity seen i n the r o s t r a l p o l e of the IC. C e l l s were predominantly s m a l l t o medium i n s i z e w i t h o v a l , round or t r i a n g u l a r p e r i k a r y a . The l a t t e r were o f t e n seen as m u l t i p o l a r . Note most c e l l s i n t h i s r e g i o n run t a n g e n t i a l t o the commissural f i b r e s , as d i d the f a i n t v a r i c o s i t i e s seen w i t h i n t h i s r e g i o n . (F) The l a t e r a l a spect of the r o s t r a l p o l e of the IC. Small c e l l s predominate i n t h i s r e g i o n . C e l l s here d i d not show the t a n g e n t i a l o r i e n t a t i o n which was seen d o r s a l l y and l a t e r a l l y i n the i n t e r c o l l i c u l a r area (ICA). (G) Immunoreactivity i n the d o r s o l a t e r a l ICA. A mixture of s m a l l t o medium c e l l s was found i n t h i s a r e a . Most c e l l s were o r i e n t e d t a n g e n t i a l l y t o the commissural f i b r e s . C e l l s appear as b i p o l a r or m u l t i p o l a r , w i t h o v a l or round p e r i k a r y a . (H) Immunoreactivity w i t h i n the medial r e g i o n of the commissure of the i n f e r i o r c o l l i c u l u s (COIC). Small c e l l s were seen i n low d e n s i t y i n t h i s r e g i o n . Immunolabelled v a r i c o s i t i e s (arrowheads) were seen t r a v e l l i n g both p a r a l l e l and o b l i q u e t o the main body of commissural f i b r e s . C a l i b r a t i o n bar i n d i c a t e s 80 microns. 126 F i g u r e 36. Immunoreactivity i n the r a t i n f e r i o r c o l l i c u l u s ( I C ) . Note most c e l l s v a r y from s m a l l t o medium i n s i z e . P e r i k a r y a were o v a l , round or t r i a n g u l a r . (A) Small c e l l s l o c a t e d a t the d o r s o l a t e r a l border of the IC. (B) Small c e l l s seen i n the d o r s a l IC. Most c e l l s were o r i e n t e d p a r a l l e l t o the ascending laminae. (C) Immunoreactive c e l l s seen w i t h i n the r o s t r a l p o l e of the IC. Medium s i z e d , t r i a n g u l a r c e l l s (arrowheads) predominate i n t h i s r e g i o n but s c a t t e r e d s m a l l c e l l s were a l s o seen. (D) Immunoreactive s m a l l b i p o l a r c e l l s and v a r i o u s m u l t i p o l a r c e l l s were seen w i t h i n the ICA. C a l i b r a t i o n bar i n d i c a t e s 30 microns. 128 Throughout the guinea p i g IC, v e r y l a r g e m u l t i p o l a r c e l l s were seen i n a l l p e r i c e n t r a l r e g i o n s ( F i g 32B,E,G,J, 33A,E). These c e l l s were o f t e n h i g h l i g h t e d by f i n e immunoreactive puncta forming p e r i c e l l u l a r and p e r i d e n d r i t i c a p p o s i t i o n . The r a t showed a p e r i c e n t r a l d i s t r i b u t i o n of SOM-IR s i m i l a r t o t h a t seen i n other s p e c i e s , but the d e n s i t y of the s c a t t e r e d SOM-IR c e l l s w i t h i n the ICC was g r e a t e r than i n the other s p e c i e s . Small t o medium s i z e d c e l l s were seen throughout a l l p e r i c e n t r a l r e g i o n s ( F i g 34A-D). These c e l l s tended t o have o v a l , round or t r i a n g u l a r p e r i k a r y a ( F i g 35, 36A,B). These c e l l s decreased as they approached the ICC ( F i g 34 E,F,G). R o s t r a l l y , t h e r e was an i n c r e a s e i n medium s i z e d c e l l s w i t h t r i a n g u l a r p e r i k a r y a ( F i g 35A,G). Many r o s t r a l c e l l s show a t a n g e n t i a l o r i e n t a t i o n t o the f i b r e s of the COIC ( F i g 35G). A l s o seen w i t h i n these r o s t r a l commissural f i b r e s , along the d o r s a l and d o r s o l a t e r a l border of the r o s t r a l ICA, were s m a l l t o medium s i z e d , o v a l c e l l s which show a s i m i l a r t a n g e n t i a l o r i e n t a t i o n ( F i g 35G,H). V a r i o u s s m a l l and medium s i z e d , o v a l c e l l s , seen a t the perimeter of the r o s t r a l c e l l s a d jacent t o the r o s t r a l p o l e , tended t o show mixed o r i e n t a t i o n ( F i g 35F). Low t o moderate d e n s i t i e s of immunoreactive puncta and v a r i c o s i t i e s were seen throughout the p e r i c e n t r a l r a t IC. The h i g h e s t d e n s i t y of immunoreactivity was seen a l o n g the d o r s a l and d o r s o l a t e r a l margins of the tectum. R o s t r a l l y , immunoreactive puncta were s t i l l seen, but a t low t o 130 moderate d e n s i t y . At r o s t r a l l e v e l s ( F i g 34C-F), t h e r e was a l i g h t immunoreactive band of n e u r o p i l . T h i s band d e l i n e a t e d the m edial border of the i n f e r i o r c o l l i c u l u s and. a t i t s r o s t r a l e x t e n t , marked the d o r s a l border of the r o s t r a l IC and the ICA ( F i g 34C-F). T h i s immunoreactive band ran from the v e n t r o m e d i a l edge of the r a t IC t o the d o r s a l r e g i o n s of the brachium of the i n f e r i o r c o l l i c u l u s , BIC. The h i g h e s t d e n s i t y of immunostained puncta and v a r i c o s i t i e s w i t h i n the r o s t r a l BIC occurs a t i t s d o r s o l a t e r a l border ( F i g 35E,F). A low d e n s i t y of immunoreactive medium-small c e l l s , w i t h t r i a n g u l a r p e r i k a r y a , were seen throughout the r a t COIC ( F i g 35H). Immunoreactive puncta and v a r i c o s i t i e s were a l s o l o c a t e d w i t h i n the r a t COIC. The v a r i c o s i t i e s ran both t a n g e n t i a l l y and p a r a l l e l t o the p a s s i n g commissural f i b r e s . In a l l t h r e e s p e c i e s , the p a t t e r n of som a t o s t a t i n s t a i n i n g was s i m i l a r throughout the IC and the r e were re g i o n s where SOM-IR was absent. T h i s r e g i o n corresponded t o the c e n t r a l r e g i o n . In the c a t , t h i s r e g i o n appeared t o be p a r t o r a l l of the ICC. In the roden t s , the absence of s t a i n was s i m i l a r but not i d e n t i c a l t o the c a t or t o each o t h e r . S t a i n i n g i n the ICC decreased as one moved c e n t r a l l y from the p e r i c e n t r a l ICC. The c o r t i c a l l a y e r s and commissural n u c l e i of a l l s p e c i e s showed SOM-IR. The guinea p i g IC c o n t a i n e d a h i g h e r d e n s i t y of SOM-IR puncta and c e l l s than e i t h e r the c a t or the r a t IC. The SOM-IR was s i m i l a r i n these l a t t e r two s p e c i e s . 131 DISCUSSION The d i s c u s s i o n w i l l address s e v e r a l p o i n t s ; the d i f f e r e n c e s and s i m i l a r i t i e s between s p e c i e s i n the d i s t r i b u t i o n of SOM-IR; comparison t o p r e v i o u s s t u d i e s on SOM-IR d i s t r i b u t i o n ; the p o s s i b l e p r o j e c t i o n s of the SOM-IR c e l l s ; o t h e r a u d i t o r y neurochemicals and t h e i r p o s s i b l e r e l a t i o n s h i p t o the SOM-IR; and the p h y s i o l o g y and pharmacology of SOM-IR r e g i o n s and the p o s s i b l e a c t i o n s of SOM-IR and oth e r neurochemicals which have shown s i m i l a r d i s t r i b u t i o n s t o SOM-IR. The D i s t r i b u t i o n of SOM-IR Across S p e c i e s The r e p o r t e d d i s t r i b u t i o n s of SOM-IR are summarized i n Table 1. Note t h a t a c r o s s the t h r e e s p e c i e s s t u d i e d many re g i o n s showed s i m i l a r d i s t r i b u t i o n s of SOM-IR. In a l l s p e c i e s , s t r i k i n g s i m i l a r i t i e s were found i n the d o r s a l p e r i o l i v a r y r e g i o n s of the SOC, VNTB, the medial and r o s t r a l p e r imeter of the VLL, the p e r i c e n t r a l d i s t r i b u t i o n of SOM-IR i n the DC, EC and ICA and the presence of f i n e SOM-IR puncta i n the MRF. The most s t r i k i n g s p e c i e s d i f f e r e n c e s i n SOM-IR were seen i n the VCN. In the r a t a h i g h d e n s i t y of SOM-IR c e l l s were seen i n the SC-AVCN. In the guinea p i g , immunoreactive c e l l s were i n lower d e n s i t y than i n the r a t AVCN w i t h h i g h e s t d e n s i t y of c e l l s b e i n g found i n the caud a l AVCN. 132 Tab l e 1. A summary of the d i s t r i b u t i o n of SOM-IR i n the c a t , guinea p i g and r a t . Symbols i n d i c a t e the f o l l o w i n g d e n s i t y of s o m a t o s t a t i n immunoreactivity: -, n i l ; ±, r a r e ; +,-. low d e n s i t y ; ++, moderate d e n s i t y ; +++, h i g h d e n s i t y . A b b r e v i a t i o n s : AVCN, a n t e r i o r v e n t r a l c o c h l e a r nucleus; SC-AVCN, s p h e r i c a l c e l l r e g i o n of the a n t e r i o r v e n t r a l c o c h l e a r nucleus; CN, c o c h l e a r nucleus; COIC, commissure of the i n f e r i o r c o l l i c u l u s ; DC, d o r s a l c o r t e x of the i n f e r i o r c o l l i c u l u s ; DCN, d o r s a l c o c h l e a r nucleus; DLL, d o r s a l nucleus of the l a t e r a l lemniscus; DLPO, d o r s o l a t e r a l p e r i o l i v a r y n u c l eus; DMPO, dorsomedial p e r i o l i v a r y n u c l eus; EC, e x t e r n a l c o r t e x of the l a t e r a l lemniscus; IC, i n f e r i o r c o l l i c u l u s ; ICA, i n t e r c o l l i c u l a r area; ICC, c e n t r a l nucleus of the i n f e r i o r c o l l i c u l u s ; NLL, n u c l e i of the l a t e r a l lemniscus; LSO, l a t e r a l s u p e r i o r o l i v e ; M/G-AVCN, m u l t i p o l a r and g l o b u l a r c e l l r e g i o n of the a n t e r i o r v e n t r a l c o c h l e a r nucleus; MNTB, medial nucleus of the t r a p e z o i d body; MRF, midbrain r e t i c u l a r f o r mation; MSO, medial s u p e r i o r o l i v e ; PVCN, p o s t e r i o r v e n t r a l c o c h l e a r nucleus; SPO, s u p e r i o r p a r a o l i v a r y n u c leus; VLL, v e n t r a l nucleus o f the l a t e r a l lemniscus; VLL^, d o r s a l p e r i m e t e r o f the v e n t r a l nucleus of the l a t e r a l lemniscus; V L L m p , medial perimeter of the v e n t r a l nucleus o f the l a t e r a l lemniscus; V L L r , r o s t r a l p e r i m e t e r of the nucleus of the v e n t r a l l a t e r a l lemniscus; V L L v p , v e n t r a l p erimeter of the nucleus of the v e n t r a l l a t e r a l lemniscus; VNTB, v e n t r a l nucleus of the t r a p e z o i d body; VPO, v e n t r a l p e r i o l i v a r y n u c l e u s . 133 NUCLEUS CAT GUINEA PIG RAT C e l l s N e u r o p i l C e l l s N e u r o p i l C e l l s N e u r o p i l Cochlear Nucleus AVCN SC-AVCN M/G-AVCN + + + + + + ++ ++ +++ + + + PVCN + + + ++ + + GCL + + ++ + + + DCN + + + + + + S u p e r i o r O l i v a r y Complex DMPO DPO DLPO LPR VPO SPO MNTB VNTB LSO MSO + ++ + + + + + + + + + + + ++ + + + + + + + +++ + + + + + + + ++ + ++ ++ + + + + + + + + + + + + + ++ ++ N u c l e i of the L a t e r a l Lemniscus V L L v p V L L m p V L L d VLL V L L r DLL + + + + ++ + ++ + + + ++ + + + + + ++ + ++ + + + ++ + • + + ++ + ++ + ++ + + + ++ + d o r s a l t o DLL + ++ + ++ + ++ Sagulum + + + + + + I n f e r i o r C o l l i c u l u s ICC DC EC ICA COIC + ++ ++ ++ + + + ++ + + +++ +++ ++ + + ++ + ++ + + ++ ++ ++ + + + ++ + MRF + ++ + ++ + ++ 134 Unique t o the guinea p i g , were the SOM-IR puncta which formed e x t e n s i v e p e r i c e l l u l a r a p p o s i t i o n throughout the VCN. The c a t VCN, i n c o n t r a s t t o the r a t and guinea p i g , showed l i t t l e i n the way of SOM-IR throughout the main body of the VCN. While i n h i g h e r d e n s i t y i n the guinea p i g , the gr a n u l e c e l l l a y e r of both the c a t and guinea p i g VCN showed immunoreactive puncta. These two s p e c i e s a l s o showed s i m i l a r d e n s i t i e s and d i s t r i b u t i o n s of SOM-IR puncta i n the DCN. In c o n t r a s t t o these two s p e c i e s , SOM-IR puncta i n the r a t DCN and granule c e l l l a y e r were c o m p a r a t i v e l y r a r e . SOM-IR puncta i n the MSO were unique t o the guinea p i g . A l s o p e c u l i a r t o t h i s s p e c i e s were s c a t t e r e d immunoreactive puncta i n the LSO. S c a t t e r e d SOM-IR puncta were seen i n s i m i l a r d i s t r i b u t i o n and d e n s i t i e s i n the MNTB of the c a t and guinea p i g . In c o n t r a s t t o these two s p e c i e s , the r a t MNTB c o n t a i n e d immunoreactive puncta which were both l a r g e r and i n h i g h e r d e n s i t y . These immunoreactive puncta were most prominent i n the r o s t r a l and dorsomedial r e g i o n s of the r a t MNTB. Somatostatin immunoelectron microscopy of the r a t MNTB (unpublished o b s e r v a t i o n s ) r e v e a l e d l a r g e t e r m i n a l s s i m i l a r t o those d e s c r i b e d by Casey and Feldman ('85), which were d i s t i n c t from the c a l y c e s of Held. Immunoreactive VNTB c e l l s were most numerous i n the guinea p i g , i n lower d e n s i t y i n the c a t and were r a r e i n the r a t . Immunoreactive puncta were found i n the VNTB of a l l s p e c i e s . A s m a l l group o f SOM-IR c e l l s i n the DMPO were seen i n the r a t , s i m i l a r c e l l s were i n lower d e n s i t y i n the guinea p i g , and found o n l y 135 o c c a s i o n a l l y i n the c a t . A s m a l l group of immunoreactive c e l l s were unique t o the c a t VPO and ca u d a l DPO. O c c a s i o n a l c e l l s were a l s o seen i n the ca u d a l r a t LSO and DLPO. A low d e n s i t y of l a r g e c e l l s was seen i n the guinea p i g SPO but not the r a t SPO. The SPO i s not p r e s e n t i n the c a t . The r a t d o r s a l VLL c o n t a i n e d a unique p o p u l a t i o n of immunoreactive c e l l s which coursed l a t e r a l l y a c r o s s d o r s a l VLL. While a s i m i l a r p e r i c e n t r a l d i s t r i b u t i o n was seen i n the IC of a l l s p e c i e s , the DC of the guinea p i g c o n t a i n e d a h i g h e r d e n s i t y of SOM-IR c e l l s and n e u r o p i l than e i t h e r the c a t or the r a t . Comparison t o P r e v i o u s S t u d i e s T h i s study agrees w e l l w i t h an e a r l i e r study of SOM-IR d i s t r i b u t i o n i n the guinea p i g brainstem ( T a b e r - P i e r c e e t a l . , '85). In g e n e r a l we r e p o r t a h i g h e r d e n s i t y of SOM-IR i n the VCN, SOC and IC. We have a l s o added the presence of SOM-IR puncta apposing the d e n d r i t e s of the MSO and a t the perimeter of the VLL. The d e n s i t y and d i s t r i b u t i o n of SOM-IR seen i n the guinea p i g MGB r e g i o n i n t h i s study i s i n agreement w i t h T a b e r - P i e r c e e t a l . ('85). Reports of SOM-IR (Johansson e t a l . , '84; Shiosaka e t a l . , ' 81,-Takatsuki e t a l . , '81; V i n c e n t e t a l . , '85) i n the r a t have been q u i t e v a r i a b l e . Our d i s t r i b u t i o n of SOM-IR i n the a d u l t i s s i m i l a r t o t h a t d e s c r i b e d f o r the 12 day o l d r a t (Shiosaka e t a l . , '81; T a k a t s u k i e t a l . , '81). These authors, however, r e p o r t e d an absence of SOM-IR i n the brains t e m a u d i t o r y system of the a d u l t r a t . T h i s d i s c r e p a n c y 136 would a r i s e i f the antibody used were t o r e c o g n i z e o n l y one form of SOM, e i t h e r SOM-14, SOM-25, SOM-28 o r , a l a r g e r form. The antibody used i n t h i s study r e c o g n i z e s SOM-14, SOM-25, SOM-28, and l a r g e r forms of SOM. I t i s p o s s i b l e t h a t d u r i n g l a t e r stages of p o s t n a t a l development an enzyme, which c l e a v e s l a r g e r v e r s i o n s o f SOM, i s expressed o r , a l t e r n a t i v e l y , t u r n e d o f f . T a k a t s u k i e t a l . ('81) used c y c l i c SOM-14 as an a n t i g e n but d i d not c h a r a c t e r i z e the antibody f u r t h e r . The r e p o r t of Johansson e t a l . ('84) i s i n ge n e r a l agreement w i t h the f i n d i n g s p r e s e n t e d here. T h e i r r e p o r t noted t h a t the d i s t r i b u t i o n o f SOM-IR puncta i n the r o s t r a l MNTB was g r e a t e r than the ca u d a l MNTB. T h i s study a l s o noted t h a t these puncta predominated i n the dorsomedial r e g i o n s of the r o s t r a l MNTB. P o s s i b l e Somatostatin Immunoreactive Pathways The p o s s i b l e p r o j e c t i o n s of SOM-IR c e l l s are shown i n F i g u r e s 37,38. The SOM-IR pathways are suggested on the b a s i s of SOM-IR p e r i k a r y a i n an a u d i t o r y nucleus and of SOM-IR puncta i n the i n d i c a t e d t e r m i n a l f i e l d . When immunoreactive c e l l s were seen i n a s i m i l a r l o c a t i o n i n a l l s p e c i e s , the co r r e s p o n d i n g t e r m i n a l f i e l d or f i e l d s were a l s o immunoreactive i n a l l s p e c i e s . S i m i l a r l y s p e c i e s showing unique p o p u l a t i o n s of immunoreactive c e l l s u s u a l l y d i s p l a y e d unique immunoreactivity i n c o r r e s p o n d i n g t e r m i n a l f i e l d s . The p o s s i b l e SOM-IR p r o j e c t i o n s w i l l be d i v i d e d i n t o : ( F i g 37) ascending a u d i t o r y pathways; and ( F i g 37) 137 multimodal sensory pathways and descending a u d i t o r y pathways. Ascending A u d i t o r y Pathways SOM-IR was found i n the ascending a u d i t o r y pathways o n l y i n rodents ( F i g 37). The d i s t r i b u t i o n was, moreover, l i m i t e d and d i f f e r e n t between the r a t and guinea p i g . In g e n e r a l , SOM-IR was l o c a t e d i n the descending a u d i t o r y systems of a l l s p e c i e s . The SOM-IR c e l l s which may form the rodent ascending a u d i t o r y system were l o c a t e d i n the SC-AVCN, and the d o r s a l VLL of the r a t and the c a u d a l AVCN of the guinea p i g . The n u c l e i which form the m a j o r i t y of the ascending a u d i t o r y pathways are the CN, LSO, MSO, MNTB, VLL and DLL ( F i g 38). These n u c l e i converge upon the c a t ICC ( A i t k i n and P h i l l i p s , '84a). While the VNTB p r o j e c t s t o the IC (Adams, '83), the p r e c i s e t e r m i n a t i o n o f the VNTB i n p u t has y e t to be determined. The o r i g i n of SOM-IR puncta i n the r a t MNTB may be e i t h e r from the unique p o p u l a t i o n s of r a t SOM-IR c e l l s seen i n the SC-AVCN or the d o r s a l VLL r e g i o n . The p a t t e r n of f i b r e d e g e n e r a t i o n seen i n the c a t MNTB, f o l l o w i n g l e s i o n s i n the c a t SC-AVCN (Warr, '82), p a r a l l e l s the d i s t r i b u t i o n o f r a t SOM-IR puncta seen i n the MNTB. Thus i n the r a t there may be a s p e c i f i c p r o j e c t i o n from the SC-AVCN t o the MNTB which c o n t a i n s SOM-IR. Another p o s s i b l e source of the r a t MNTB puncta may be the SOM-IR c e l l s of the d o r s a l VLL which 138 F i g u r e 37. Probable s o m a t o s t a t i n c o n t a i n i n g t r a c t s ( b o l d l i n e s ) i n the ascending a u d i t o r y system of the guinea p i g (GP) and r a t (R). T h i n l i n e s i n d i c a t e t r a c t s which do. not appear t o c o n t a i n s o m a t o s t a t i n immunoreactivity. A p o s s i b l e p r o j e c t i o n (?) may e x i s t from the r a t SC-AVCN ( s p h e r i c a l c e l l r e g i o n o f the a n t e r i o r v e n t r a l c o c h l e a r nucleus) t o the MNTB (medial nucleus of the t r a p e z o i d body) (see t e x t f o r d e t a i l s ) . Not shown are n u c l e a r s u b d i v i s i o n s not s t r i c t l y p e r t i n e n t t o the argument. Note the p r o j e c t i o n from the d o r s a l r e g i o n of the VLL t o the r a t MNTB may form a descending s o m a t o s t a t i n e r g i c pathway and was i n c l u d e d t o i n d i c a t e the two p o s s i b l e sources of SOM-IR i n n e r v a t i o n of the r a t MNTB. A p o s s i b l e SOM-IR p r o j e c t i o n from the i n f e r i o r c o l l i c u l u s to the midbrain r e t i c u l a r f o r m a t i o n i s not i n c l u d e d s i n c e i t i s prob a b l y multimodal r a t h e r than s t r i c t l y a u d i t o r y (see t e x t ) . Symbols: • , ca u d a l ; A, c o n t r a l a t e r a l . A b b r e v i a t i o n s : ANC, a u d i t o r y neocortex; AVCN, a n t e r i o r v e n t r a l c o c h l e a r nucleus; BCG, b i p o l a r c e l l g a n g l i o n ; IHC, i n n e r h a i r c e l l s ; LSO, l a t e r a l s u p e r i o r o l i v e ; MGB me d i a l g e n i c u l a t e body; MSO, medial s u p e r i o r o l i v e ; NLL, n u c l e i of the l a t e r a l lemniscus; VCN, v e n t r a l c o c h l e a r n u c l e u s ; VLL, v e n t r a l nucleus of the l a t e r a l l emniscus. 139 A N C M G B S O C L S O M S O M N T B S O C A M N T B A M S 0 A V C N A V C N 1 S C -A V C N B C G 140 were a l s o seen u n i q u e l y i n the r a t . T h i s p r o j e c t i o n has been demonstrated i n the c a t but t e r m i n a t i o n appeared i n the l a t e r a l r e g i o n s of the c a t MNTB (Kudo, '81). I t was not t h i s l a t e r a l r e g i o n , but r a t h e r the medial r e g i o n , which c o n t a i n e d the h i g h e s t d e n s i t y o f SOM-IR puncta i n the r a t . The SOM-IR c e l l s of the guinea p i g cau d a l AVCN pr o b a b l y p r o j e c t t o the MSO ( F i g 37). T h i s pathway has been d e s c r i b e d i n the r a t ( H a r r i s o n and Feldman, '70; H a r r i s o n and I r v i n g , '66a; '66b) and i n more d e t a i l i n the c a t (Cant and Casseday, '86; H a r r i s o n and Howe, '74; Warr, '72). The m a j o r i t y of the MSO i n n e r v a t i o n i n the c a t has been r e p o r t e d t o come from more r o s t r a l r e g i o n s of the AVCN; however, i n p u t from the mid t o caud a l AVCN was a l s o demonstrated (Cant and Casseday, '86; Shneiderman and Henkel, '85; Warr, '82). In the c a t , c e l l s i n the SC-AVCN r e g i o n p r o j e c t b i l a t e r a l l y t o the MSO. The p a t t e r n of the MSO i n n e r v a t i o n i s worth mentioning. In t r a n s v e r s e s e c t i o n s the MSO c e l l s form a v e r t i c a l s t a c k . The d e n d r i t e s of the p r i n c i p a l , f u s i f o r m , c e l l s are o r i e n t e d l a t e r a l - m e d i a l l y and are p e r p e n d i c u l a r t o the long a x i s of the MSO. The s p h e r i c a l cell-AVCN c e l l s p r o j e c t t o the l a t e r a l d e n d r i t e s i p s i l a t e r a l l y and medial d e n d r i t e s c o n t r a l a t e r a l l y of the MSO f u s i f o r m c e l l s of the c a t ( K i s s and Majorossy, '83; Schwartz, '84; Shneiderman and Henkel, '85; Warr, '82). Two d i f f e r e n t types of AVCN b i l a t e r a l p r o j e c t i o n s t o the p a r t s of the c a t SOC have been suggested (Shneiderman and Henkel, '85). T h i s would be expected p h y s i o l o g i c a l l y , as the LSO was 141 c h a r a c t e r i z e d by a g r e a t e r p r o p o r t i o n of h i g h b e s t f r e q u e n c i e s w h i l e the MSO was c h a r a c t e r i z e d by a g r e a t e r p r o p o r t i o n of low b e s t f r e q u e n c i e s (Guinan e t a l . , '72). Shneiderman and Henkel ('85) have suggested t h a t one p r o j e c t i o n p r o v i d e s p r e c i s e t e r m i n a t i o n t o the i p s i l a t e r a l LSO and MSO and a d i f f u s e t e r m i n a t i o n t o the c o n t r a l a t e r a l MSO. The second p r o j e c t i o n p r o v i d e s the p r e c i s e b i l a t e r a l t e r m i n a t i o n i n the MSO w i t h an ind e t e r m i n a t e p r o j e c t i o n t o the c o n t r a l a t e r a l LSO. As few SOM-IR puncta were seen i n the guinea p i g LSO, i t i s prob a b l y t h i s second pathway which c o n t a i n s SOM-IR i n the guinea p i g . Multimodal Ascending Pathways In a l l s p e c i e s , the SOM-IR c e l l s of the ICA, and p o s s i b l y the EC, may c o n t r i b u t e t o multimodal ascending somatosensory and a u d i t o r y p r o j e c t i o n s t o the midbrain r e t i c u l a r f o r m a t i o n (MRF) which l i e s a d j a c e n t t o the v e n t r a l d i v i s i o n of the medial g e n i c u l a t e body (MGBv). T h i s pathway was d e s c r i b e d i n the c a t (Andersen e t a l . , '84b; Henkel, '83; Kudo and N i i m i , 80; Kudo e t a l . , '83) and r a t (LeDoux e t a l . , '85) and appeared t o be multimodal sensory r a t h e r than s t r i c t l y a u d i t o r y . The MRF does not form p a r t of the ascending a u d i t o r y pathway. I t i s p o s s i b l e t h a t these SOM-IR f i b r e s of the ICA and EC form the immunoreactive band which was seen t o d e l i n e a t e the dorsomedial border of the r o s t r a l IC and t o be continuous w i t h the d o r s a l o n e - t h i r d of the BIC. In a l l s p e c i e s a moderate d e n s i t y of f i n e SOM-IR puncta 142 was seen i n t h a t r e g i o n of the MRF which was l o c a t e d m edial t o the MGBv. Descending A u d i t o r y Pathways In c o n t r a s t t o the ascending a u d i t o r y system ( F i g 37), the descending a u d i t o r y pathways ( F i g 38) c o n t a i n e d more SOM-IR w i t h g r e a t e r s i m i l a r i t i e s i n SOM-IR d i s t r i b u t i o n between s p e c i e s . A more d e t a i l e d d e s c r i p t i o n of the descending a u d i t o r y pathways than g i v e n i n F i g u r e 1 w i l l f i r s t be p r o v i d e d . The descending a u d i t o r y pathways o r i g i n a t e from the a u d i t o r y neocortex, the DC, the l e m n i s c a l n u c l e i , the d o r s a l p e r i o l i v a r y r e g i o n s and the VNTB. Whether the EC a l s o forms a descending pathway has been a p o i n t of some debate. As d e s c r i b e d f o r the c a t (Andersen e t a l . , '80a; Rockel and Jones, '73), the r a t ( B e y e r l , '78; Druga and Skyka, '84a; Faye-Lund, '85) and the monkey ( F i t z P a t r i c k and Imig, '73), the descending a u d i t o r y path from the a u d i t o r y neocortex p r o j e c t s t o d i s c r e t e r e g i o n s of the DC and l a t e r a l p o n t i n e nucleus (LPN). D i s c r e t e i n j e c t i o n s of anterograde l a b e l r e v e a l e d t h a t the c e n t r i f u g a l i n n e r v a t i o n of the c a t DC terminated i n two d i s c r e t e r o s t r o c a u d a l bands. While n e o c o r t i c a l p r o j e c t i o n s t o the EC have a l s o been r e p o r t e d (Druga and Skyka, '84a; Faye-Lund, '85; Rockel and Jones, '73; Van Noort, '69), th e r e was some debate as t o whether these o r i g i n a t e from the a u d i t o r y or the adjacent somatosensory neocortex (Andersen e t a l . , 80a). 143 F i g u r e 38. A summary diagram showing the descending a u d i t o r y pathways which may c o n t a i n s o m a t o s t a t i n immunoreactivity. For s i m p l i c i t y , non-commissural p r o j e c t i o n s are shown f o r o n l y one hemisphere and many p r o j e c t i o n s are shown as branched which may be independent. Probable s o m a t o s t a t i n c o n t a i n i n g t r a c t s ( b o l d l i n e s ) of the c a t (C), guinea p i g ( ^  ) or c a t and guinea p i g ( ) or c a t , guinea p i g and r a t ( 5fc ). L i g h t l i n e s i n d i c a t e t r a c t s which do not appear t o c o n t a i n SOM-IR i n any of these s p e c i e s . Not shown i s the c o n t r o v e r s i a l p r o j e c t i o n from a u d i t o r y c o r t e x t o the EC of the i n f e r i o r c o l l i c u l u s or n u c l e a r s u b d i v i s i o n s not s t r i c t l y p e r t i n e n t t o the d i s c u s s i o n (see t e x t f o r d e t a i l s ) . The p e r i h i l a r r e g i o n of the l a t e r a l s u p e r i o r o l i v e (PLSO) p r o j e c t s t o the IHC ( i n n e r h a i r c e l l s ) i n the c a t w h i l e i n rodents the same p r o j e c t i o n o r i g i n a t e s from the LSO proper. Symbols: A, c o n t r a l a t e r a l . A b b r e v i a t i o n s : AVCN, a n t e r i o r v e n t r a l c o c h l e a r nucleus; ANC, a u d i t o r y neocortex; CN, c o c h l e a r n u c l e u s ; DCN, d o r s a l c o c h l e a r nucleus; DLPO, d o r s o l a t e r a l p e r i o l i v a r y nucleus; DMPO, dorsomedial p e r i o l i v a r y n u c l eus; DPO, d o r s a l p e r i o l i v a r y nucleus; IC, i n f e r i o r c o l l i c u l u s ; IHC, i n n e r h a i r c e l l s ; GCL, granule c e l l l a y e r ; LSO, l a t e r a l s u p e r i o r o l i v e ; NLL, n u c l e i of the l a t e r a l lemniscus; OHC, outer h a i r c e l l s ; PVCN, p o s t e r i o r v e n t r a l c o c h l e a r n u c leus; SOC, s u p e r i o r o l i v a r y complex; VLL, v e n t r a l nucleus of the l a t e r a l lemniscus; VNTB, v e n t r a l nucleus of t r a p e z o i d body; VPO, v e n t r a l p e r i o l i v a r y n ucleus. 144 o I C ? * N L L C V L L ) s o c D M P O V N T B D L P O , L S O V P O D P O ( P L S O ) 145 The DC has been r e p o r t e d t o p r o j e c t t o the c o n t r a l a t e r a l DC i n the mouse (Hernandez e t a l . , '86) and the c a t (Andersen e t a l . , '80b; Kudo and N i i m i , '80), although t h i s p r o j e c t i o n has been q u e s t i o n e d i n the c a t ( P h i l l i p s and A i t k i n , '84b). The use of a d i f f e r e n t t r a c i n g technique i n t h i s l a s t study suggests t h a t f u r t h e r r e s e a r c h i s r e q u i r e d on t h i s p o s s i b l e p r o j e c t i o n . In the mouse t h i s DC p r o j e c t i o n t o the c o n t r a l a t e r a l DC, l i k e the c e n t r i f u g a l p r o j e c t i o n t o DC, terminated i n two bands (Hernandez e t a l . , '86). In the r a t , t h i s DC p r o j e c t i o n t erminated i n the c o n t r a l a t e r a l DC i n r e g i o n s a d j a c e n t t o the c e l l s of o r i g i n . The DC of the c a t p r o v i d e s descending p r o j e c t i o n s t o the medial and v e n t r a l VLL r e g i o n s as w e l l as the d o r s a l p e r i o l i v a r y r e g i o n s and VNTB of the SOC (Burne e t a l . , '81; H a r r i s o n and Howe, '74b; Hashikawa, '83; Van Noort, '69). Anterograde t r a c i n g (Burne e t a l . , '81) r e v e a l e d l a b e l l i n g a t the medial and v e n t r a l p e r i m e t e r s of the VLL. The anterograde l a b e l l i n g a t the v e n t r a l p erimeter of the VLL was p a r t i c u l a r l y dense and appeared continuous w i t h t h a t r e p o r t e d i n the adj a c e n t l a t e r a l p o n t i n e nucleus (LPN). The LPN i n t u r n p r o j e c t e d t o the c e r e b e l l u m (path not shown). Presumably t h i s i s the pathway which p r o v i d e s c e r e b e l l a r c e l l s w i t h a u d i t o r y i n p u t ( A z i z i e t a l . , '85). In the c a t , the same d o r s a l p e r i o l i v a r y and the VNTB r e g i o n s which r e c e i v e e f f e r e n t s from the DC a l s o r e c e i v e them from the VLL (Kudo, '81; W h i t e l y and Henkel, '84). What these s t u d i e s do not show i s whether the descending VLL p r o j e c t i o n o r i g i n a t e d 146 from the medial p e r i m e t e r or from the more c e n t r a l r e g i o n s of the VLL. The d o r s a l and l a t e r a l p e r i o l i v a r y r e g i o n s and the VNTB were shown to p r o j e c t t o the c o c h l e a r nucleus i n the c a t (Adams, '83; E l v e r l a n d , '77) and the t r e e shrew (Covey e t a l . , '84). In the c a t , these p r o j e c t i o n s t o the CN o r i g i n a t e from c e l l s which terminate e x c l u s i v e l y i n the CN and from c o l l a t e r a l s of SOC c e l l s which p r o j e c t t o the c o c h l e a (Adams, '83). The p r o j e c t i o n from p e r i o l i v a r y r e g i o n s and VNTB t o the organ of C o r t i can be d i v i d e d i n t o a l a t e r a l and a medial o l i v o c o c h l e a r system. The d i v i s i o n can be approximated by an imaginary l i n e which s a g i t t a l l y b i s e c t s the MSO. The medial and l a t e r a l o l i v o c o c h l e a r c e l l s p r o v i d e d descending e f f e r e n t s t o the o u t e r h a i r c e l l s and i n n e r h a i r c e l l s , r e s p e c t i v e l y . The p r o j e c t i o n i n a l l s p e c i e s s t u d i e d can be d i v i d e d i n t o a l a t e r a l and medial d i v i s i o n . Both the l a t e r a l and medial o l i v o c o c h l e a r systems were r e p o r t e d t o p r o j e c t back t o the organ of C o r t i i n a t o n o t o p i c f a s h i o n i n the c a t (Guinan, '84) and the guinea p i g (Robertson, '84; Robertson and Gummer, '85). The medial system p r o j e c t e d p r i m a r i l y (80-90%) t o the c o n t r a l a t e r a l c o c h l e a w i t h the remainder of c e l l s p r o j e c t i n g i p s i l a t e r a l l y . The c o n t r a l a t e r a l l y p r o j e c t i n g c e l l s do so v i a medium s i z e d m y e l i nated f i b r e s which run d o r s a l l y and then course l a t e r a l l y beneath the f l o o r of the f o u r t h v e n t r i c l e . I n the c a t (Warr, '75; '78) and guinea p i g (Robertson, '85) these medial o l i v o c o c h l e a r c e l l s were found 147 i n the VNTB, MNTB, and DMPO. In the r a t , however, the medial o l i v o c o c h l e a r c e l l s appeared o n l y i n the VNTB (White and Warr, 1 8 3 ) . The l a t e r a l o l i v o c o c h l e a r system of the c a t was found p r i m a r i l y i n the p e r i h i l a r r e g i o n of the LSO although the l a t e r a l o l i v o c o c h l e a r c e l l s were found i n other p e r i o l i v a r y r e g i o n s l a t e r a l t o the MSO (Adams, '83; Warr, 75; 78; Guinan e t a l . , '83; '84). In rodents, l a t e r a l o l i v o c o c h l e a r c e l l s , s i m i l a r t o those seen i n the c a t p e r i h i l a r r e g i o n , were l o c a t e d w i t h i n the body of the LSO (Robertson, '85; White and Warr, '83). While the l o c a t i o n of the medial o l i v o c o c h l e a r system was s i m i l a r i n the c a t and guinea p i g , the l o c a t i o n of the l a t e r a l o l i v o c o c h l e a r system appeared s i m i l a r i n the r a t and guinea p i g . The l a t e r a l o l i v o c o c h l e a r e f f e r e n t s were e s s e n t i a l l y i p s i l a t e r a l i n rodents w h i l e i n the c a t up t o 10-20% of the c e l l s p r o j e c t e d c o n t r a l a t e r a l l y . The medial o l i v o c o c h l e a r e f f e r e n t s terminate on the p e r i k a r y a of ou t e r h a i r c e l l s . In c o n t r a s t the l a t e r a l o l i v o c o c h l e a r e f f e r e n t s terminate on the a f f e r e n t ends of the b i p o l a r s p i r a l g a n g l i o n c e l l s which appose the i n n e r h a i r c e l l s (Bodian, '83; Guinan e t a l . , '83; '84). While some s p e c i e s d i f f e r e n c e s e x i s t between the l o c a t i o n of the l a t e r a l and medial o l i v o c o c h l e a r c e l l s , the o l i v o c o c h l e a r systems of a l l s p e c i e s showed s i m i l a r c e l l morphologies and s i m i l a r anatomical p r o j e c t i o n s . In a l l s p e c i e s both a l a t e r a l and medial o l i v o c o c h l e a r system 148 e x i s t . The VNTB made up p a r t of the medial o l i v o c o c h l e a r system i n the c a t , guinea p i g and r a t . The p o s s i b l e descending p r o j e c t i o n s which c o n t a i n SOM-IR are summarized i n F i g u r e 38. In a l l s p e c i e s the SOM-IR c e l l s of the DC, and perhaps the EC, may form the descending p r o j e c t i o n s t o the medial and l a t e r a l p e rimeter of the VLL, the p e r i o l i v a r y r e g i o n s of the SOC and the VNTB. The hi g h e r d e n s i t y of immunoreactive c e l l s i n the guinea p i g DC may r e p r e s e n t a s u b p o p u l a t i o n of c e l l s which were not immunoreactive i n the c a t or r a t . T h i s s u b p o p u l a t i o n p r o b a b l y p r o j e c t s t o the c o n t r a l a t e r a l DC and would account f o r the much h i g h e r d e n s i t y of SOM-IR n e u r o p i l i n the guinea p i g DC. In a l l s p e c i e s the SOM-IR c e l l s of the medial VLL may p r o j e c t t o the d o r s a l p e r i o l i v a r y r e g i o n s and to the VNTB of the SOC. In both the c a t and guinea p i g , but not the r a t , SOM-IR c e l l s were seen i n the VNTB. Corresponding immunolabelled t e r m i n a l f i e l d s were l o c a t e d i n the DCN and the granule c e l l l a y e r of the AVCN. The d e n s i t i e s of the SOM-IR puncta i n the DCN of both the c a t and guinea p i g were s i m i l a r , s u g g e s t i n g a s i m i l a r s u b p o p u l a t i o n of SOM-IR VNTB c e l l s p r o j e c t i n g t o the DCN. In c o n t r a s t , the guinea p i g showed a h i g h e r d e n s i t y of SOM-IR puncta i n the gr a n u l e c e l l l a y e r than d i d the c a t . T h i s may r e f l e c t the h i g h e r d e n s i t y of SOM-IR c e l l s i n the guinea p i g VNTB. 149 The SOM-IR puncta, seen throughout the guinea p i g VCN, pro b a b l y o r i g i n a t e d from the SOM-IR c e l l s of the VNTB which s p e c i f i c a l l y p r o j e c t t o the CN. The s c a t t e r e d SOM-IR puncta seen i n the c a t VCN may have o r i g i n a t e d from the r a r e SOM-IR in t e r n e u r o n s or from c o l l a t e r a l s of o l i v o c o c h l e a r f i b r e s . While the c o c h l e a was not examined immunohistochemically, i t i s p o s s i b l e t h a t the SOM-IR c e l l s of the c a t and guinea p i g VNTB p r o j e c t t o the out e r h a i r c e l l s of the c o c h l e a and t h a t the SOM-IR c e l l s which were seen i n the c a t VPO and ca u d a l DPO, c o n t r i b u t e t o the l a t e r a l o l i v o c o c h l e a r system. Chemical Neuroanatomy The p o s s i b l e p a t t e r n s of c o e x i s t e n c e or p a r a l l e l p r o j e c t i o n s are i l l u s t r a t e d i n F i g u r e 39. The d i s t r i b u t i o n of e n k e p h a l i n immunoreactivity (ENK-IR) and neuropeptide-Y immunoreactivity (NPY-IR) (Yamazoe e t a l . , '85) i n the r a t a u d i t o r y system, was l i m i t e d t o the descending a u d i t o r y pathways. Both p e p t i d e s had immunoreactive p e r i k a r y a r e s t r i c t e d t o the EC and DC r e g i o n s of the IC. Both p e p t i d e s \ a l s o showed o v e r l a p p i n g d i s t r i b u t i o n s of immunoreactive n e u r o p i l a t the medial perimeter of the VLL and i n the d o r s a l p e r i o l i v a r y r e g i o n s of the SOC. In a d d i t i o n , ENK-IR c e l l were a l s o r e p o r t e d i n the r a t medial VLL and VNTB r e g i o n s . In the guinea p i g , ENK-IR c e l l s were r e p o r t e d i n the LSO and the periLSO r e g i o n s ( A l t s h u l e r e t a l . , '83; '84). ENK-IR t e r m i n a l s have a l s o been shown apposing the 150 F i g u r e 39. P o s s i b l e occurrence s o m a t o s t a t i n w i t h GABA, ENK, SOM, ACh i n the same (c o e x i s t e n c e ) or p a r a l l e l pathways. T h i s i s based on the p r e v i o u s l y r e p o r t e d p r o j e c t i o n s and d i s t r i b u t i o n s of these neurochemicals. The evidence f o r c o e x i s t e n c e o r p a r a l l e l pathways, s p e c i e s d i f f e r e n c e s and othe r d e t a i l s are presented i n t e x t . Not shown i s a p o s s i b l e gabaergic p r o j e c t i o n from the IC to the DCN. PLSO (see t e x t or F i g u r e 38 f o r d e t a i l s ) . Symbols: 5^ , p r o b a b l y found i n a l l s p e c i e s ; gaba o n l y i n the c a t and r a t ; r a t does not c o n t a i n s o m a t o s t a t i n ; s o m a t o s t a t i n found o n l y i n the c a t and not the guinea p i g or r a t ; • , c o n t r a l a t e r a l . Neurochemical a b b r e v i a t i o n s : A, a c e t y l c h o l i n e ; E, enk e p h a l i n ; G, GABA; N, neuropeptide-Y; S, s o m a t o s t a t i n . Anatomical a b b r e v i a t i o n s : AVCN, a n t e r i o r v e n t r a l c o c h l e a r nucleus; CN, c o c h l e a r nucleus; DC, d o r s a l c o r t e x of the i n f e r i o r c o l l i c u l u s ; DCN, d o r s a l c o c h l e a r nucleus; DLPO, d o r s o l a t e r a l p e r i o l i v a r y nucleus; DMPO, dorsomedial p e r i o l i v a r y n u c l eus; DPO, d o r s a l p e r i o l i v a r y n u c l eus; IC, i n f e r i o r c o l l i c u l u s ; EC, e x t e r n a l c o r t e x of the i n f e r i o r c o l l i c u l u s ; IHC, i n n e r h a i r c e l l s ; GCL, granule c e l l l a y e r ; LSO, l a t e r a l s u p e r i o r o l i v e ; NLL, n u c l e i of the l a t e r a l lemniscus; OHC, outer h a i r c e l l s ; PLSO, p e r i h i l a r r e g i o n of the l a t e r a l s u p e r i o r o l i v a r y n u c l eus; PVCN, p o s t e r i o r v e n t r a l c o c h l e a r nucleus; SOC, s u p e r i o r o l i v a r y complex; VLL,. v e n t r a l nucleus of the l a t e r a l lemniscus; VNTB, v e n t r a l nucleus of t r a p e z o i d body; VPO, v e n t r a l p e r i o l i v a r y n u cleus. E G i N S * ? O H C A I H C 152 guinea p i g ou t e r h a i r c e l l somas and on the i n n e r h a i r c e l l a f f e r e n t s (Fex and A l t s c h u l e r , '85). The presence of the NPY-IR n e u r o p i l i n the medial VLL and d o r s a l p e r i o l i v a r y r e g i o n s , w i t h the NPY-IR c e l l s o n l y i n the DC and EC supports the n o t i o n t h a t these immunoreactive c e l l s supply the descending p r o j e c t i o n s . I t has a l s o been suggested t h a t , i n the c a t , the ENK-IR c e l l s of the VNTB i n n e r v a t e the granule c e l l l a y e r of the VCN (Adams, '84). I t i s a l s o presumably the ENK-IR c e l l s of the VNTB which p r o v i d e the ou t e r h a i r c e l l s w i t h ENK-IR t e r m i n a l s and the l a t e r a l o l i v o c o c h l e a r system whose ENK-IR e f f e r e n t s appose the i n n e r h a i r c e l l a f f e r e n t s . There have been s t u d i e s of GABA- or GAD-IR i n the a u d i t o r y system i n the c a t (Adams and Mugnaini, '84a; '84b), the guinea p i g (Thompson e t a l . , '85), the r a t (Moore and Moore, '84; S h i r a i s h i e t a l . , '85; O t t e r s e n and Storm-Mathisen, '84) and g e r b i l . While these s t u d i e s v a r y somewhat they a l l had s i m i l a r d e s c r i p t i o n s of immunoreactive c e l l s i n a l l p a r t s of the IC (EC, DC, and ICC), i n the VNTB, DLL, medial VLL r e g i o n and DCN. An e x t e n s i v e network of p e r i c e l l u l a r puncta has been g e n e r a l l y r e p o r t e d throughout the VCN. In the guinea p i g , both GABA- and GAD-IR puncta were seen apposing p r i m a r i l y the a p i c a l outer h a i r c e l l soma but were a l s o seen apposing the i n n e r h a i r c e l l a f f e r e n t s (Fex and A l t s c h u l e r , '84; Fex e t a l . , '86). Compared t o the d i s t r i b u t i o n , of the GABA- and GAD-IR, GABA transaminase a c t i v i t y was d e t e c t e d o n l y i n c e l l s i n the 153 DLL, ICC, and s c a t t e r e d c e l l s i n the medial VLL r e g i o n . There has been no e x p l a n a t i o n f o r t h i s d i s c r e p a n c y . The DLL and ICC are r e g i o n s which c o n t a i n few p e p t i d e r g i c neurons; whether t h i s i s s i g n i f i c a n t or merely c o i n c i d e n c e remains t o be determined. The GABA- or GAD-IR puncta seen throughout the VCN may o r i g i n a t e from the IC or the VNTB o r , a l t e r n a t i v e l y , may be i n t e r n e u r o n s of the CN. Support f o r CN i n t e r n e u r o n s comes from work i n the r a t which f a i l e d t o show a change i n the p a t t e r n of GAD-IR puncta f o l l o w i n g s u r g i c a l i s o l a t i o n of the CN from the r e s t of the brainstem ( S h i r a i s h i e t a l . , '85). Other authors, however, have r e p o r t e d a 30% decrease i n GAD-a c t i v i t y i n the VCN, measured b i o c h e m i c a l l y (Davies, '77; F i s c h e r and Davies, '76), f o l l o w i n g l e s i o n s of the d o r s a l a c o u s t i c s t r i a . T h i s would support the n o t i o n t h a t some of the gabaergic i n p u t t o the VCN i s from the IC. A problem w i t h a p u t a t i v e VCN gabaergic i n t e r n e u r o n i s t h a t , d e s p i t e the e x t e n s i v e network of p e r i c e l l u l a r GABA- and GAD-IR puncta seen throughout the VCN, the GAD a c t i v i t y of the VCN i s amongst the lowest i n the a u d i t o r y system. Even i n the r a t , where the GAD a c t i v i t y i s twice t h a t of the guinea p i g or c a t (Davies, '77; F i s h e r and Davies, '76), GABA- or GAD-IR c e l l s have r a r e l y been r e p o r t e d . I n the IC, where the GAD a c t i v i t y i s much hi g h e r than the VCN, c e l l s have been r e p o r t e d . Whether t h i s d i f f e r e n c e i n GAD a c t i v i t y r e f l e c t s the d i f f e r e n c e between GAD l o c a t e d i n c e l l s i n the IC versus t e r m i n a l s i n the VCN remains t o be demonstrated. The 154 p o s s i b i l i t y t h a t the GABA- and GAD-IR c e l l s of the VNTB, form the e x t e n s i v e p e r i c e l l u l a r a p p o s i t i o n seen throughout the VCN cannot be ig n o r e d . A r e l a t i v e l y d i f f u s e p r o j e c t i o n from the VNTB and v e n t r a l t r a p e z o i d r e g i o n has been demonstrated i n two q u i t e d i f f e r e n t s p e c i e s , the c a t (Adams, '83; E l v e r l a n d , '77) and the t r e e shrew (Covey e t a l . , '84). GABA- or GAD-IR i s seen i n the v e n t r a l t r a p e z o i d r e g i o n of a l l s p e c i e s s t u d i e d . Evidence t o the c o n t r a r y i s t h a t l e s i o n s of the t r a p e z o i d body, the r e p o r t e d e f f e r e n t bundle of the VNTB, f a i l s t o reduce G A D - a c t i v i t y (Davies, '77; Fex and Davies, '76) or change the appearance of GAD-IR ( S h i r a i s h i e t a l . , '85) puncta i n the VCN. These s t u d i e s however had s i g n i f i c a n t d i f f e r e n c e s i n other aspects of t h e i r l e s i o n r e s u l t s , s u g g e s t i n g t h a t f u r t h e r work may be necessary. The p o s s i b i l i t y t h a t the descending GABA- and GAD-IR o r i g i n a t e s i n the primary a f f e r e n t s appears u n l i k e l y (Canzek and Reubi, '80; K l i n k e and O e r t e l , '77). While a l l these p r o j e c t i o n s are p o s s i b l e , f u r t h e r work i s r e q u i r e d t o e l u c i d a t e the nature of the gabaergic i n n e r v a t i o n of the VCN. C h o l i n e r g i c c e l l s were r e p o r t e d i n the LSO i n rodents, the p e r i h i l a r r e g i o n s of the c a t LSO and i n the periLSO r e g i o n s i n both s p e c i e s . In these s p e c i e s c h o l i n e r g i c c e l l s were a l s o r e p o r t e d f o r the VNTB. These two d i f f e r e n t c h o l i n e r g i c p o p u l a t i o n s p r o b a b l y c o n t r i b u t e t o the l a t e r a l and m e dial o l i v o c o c h l e a r system t o form the c h o l i n e r g i c t e r m i n a l s r e p o r t e d apposing the outer h a i r c e l l s and the 155 a f f e r e n t s of the i n n e r h a i r c e l l s ( A l t s c h u l e r e t a l . , '85). In the guinea p i g , the l a t e r a l o l i v o c o c h l e a r c e l l s c o n t a i n both ENK-IR and a c e t y l c h o l i n e s t e r a s e , and ENK-IR and c h o l i n e a c e t y l t r a n s f e r a s e immunoreactivity (ChAT-IR) ( A l t s h u l e r e t a l . , '83; A l t s h u l e r e t a l . , '84). T h i s p a t t e r n of c o e x i s t e n c e was not seen i n the medial o l i v o c o c h l e a r system. While CHAT-IR and ENK-IR c o e x i s t i n the l a t e r a l o l i v o c o c h l e a r system, i t would appear l i k e l y t h a t GABA- and ENK-IR may c o e x i s t i n the medial o l i v o c o c h l e a r system i n the c a t , r a t and guinea p i g . GABA-, SOM-, and ENK-IR may c o e x i s t i n these same c e l l s i n the c a t and guinea p i g which p r o j e c t to the o u t e r h a i r c e l l s . In the c a t and guinea p i g , the p r o j e c t i o n t o the granule c e l l l a y e r from the VNTB may a l s o c o n t a i n GABA-, SOM-, and ENK-IR, w h i l e i n the r a t t h i s same path may c o n t a i n o n l y GABA and ENK-IR. Whether t h i s p a t t e r n of GABA-, SOM-, ENK-IR i s seen i n the descending p r o j e c t i o n s from the DC and medial VLL remains t o be demonstrated. NPY-IR may c o e x i s t w i t h the a f o r e mentioned neurochemicals or may r e p r e s e n t a p a r a l l e l pathway from the DC. In the guinea p i g , GABA and SOM-IR may c o e x i s t i n VCN puncta which may o r i g i n a t e from l o c a l neurons or a l t e r n a t i v e l y from the VNTB. The IC i s a l e s s l i k e l y source of these GABA/SOM-IR puncta as t h i s p r o j e c t i o n has been shown t o o r i g i n a t e from the ICC (Hashikawa, '83), where no SOM-IR was seen i n the guinea p i g . In the c a t and r a t the same p r o j e c t i o n p r o b a b l y c o n t a i n s o n l y GABA. 156 In the c a t , the VNTB p r o j e c t i o n t o the DCN d i d not c o n t a i n a c e t y l c h o l i n e s t e r a s e and was presumably non-c h o l i n e r g i c ( F a r l e y and Warr, '81). T h i s VNTB i n p u t t o the DCN may be gabaergic i n rodent s p e c i e s , as i s suggested by the s i m i l a r d i s t r i b u t i o n s of both GABA-IR and GAD-IR i n the a u d i t o r y brainstem. T h i s may a l s o be the case i n the c a t where l e v e l s of GAD a c t i v i t y , s i m i l a r t o those of the guinea p i g , have been i d e n t i f i e d i n the DCN (Davies, '77; F i s h e r and Davies, '76). The c e l l s which p r o j e c t from the VNTB to the DCN may c o n t a i n both SOM and GABA i n the c a t and guinea p i g and o n l y GABA i n the r a t . Few r e p o r t s are a v a i l a b l e on the d i s t r i b u t i o n of c e l l s which use glutamate as a p u t a t i v e n e u r o t r a n s m i t t e r . The r e p o r t of O t t e r s e n and Storm-Mathisen ('84), however, shows glutamate c o n t a i n i n g c e l l s i n those r e g i o n s of the IC and SOC which form descending a u d i t o r y pathways. P h y s i o l o g y and Pharmacology With the e x c e p t i o n of the o l i v o c o c h l e a r system, the m a j o r i t y o f a u d i t o r y p h y s i o l o g y has fo c u s s e d on the ascending a u d i t o r y pathways. Comparison of the a u d i t o r y p h y s i o l o g y between s p e c i e s has been, a t b e s t , piecemeal. From the s c a t t e r e d s t u d i e s i n d i f f e r e n t s p e c i e s i t would appear t h a t the t o n o t o p i c order of the a u d i t o r y n u c l e i , and the c e l l response p a t t e r n s of those n u c l e i , appear t o be pr e s e r v e d a c r o s s s p e c i e s . 157 The p h y s i o l o g y of the o l i v o c o c h l e a r p r o j e c t i o n s , i n p a r t i c u l a r the medial o l i v o c o c h l e a r system, has been s t u d i e d i n s e v e r a l s p e c i e s . More r e c e n t work has focused on the m e dial o l i v o c o c h l e a r system of the guinea p i g . S t i m u l a t i o n of the m edial o l i v o c o c h l e a r f i b r e bundle r e s u l t s i n the a t t e n u a t i o n of response t o a u d i t o r y s t i m u l i (Brown and N u t t a l , '85). T h i s e f f e c t was mimicked i n the c a t i f medial o l i v o c o c h l e a r c e l l s were s t i m u l a t e d d i r e c t l y ( G i l f o r d and Guinan, '83). S i m i l a r a t t e n u a t i o n of a u d i t o r y responses o c c u r r e d w i t h u n i l a t e r a l e x t r a c o c h l e a r s t i m u l a t i o n (Dodson, '86; Rajan and Johnstone, '83). The absence of d i r e c t e f f e r e n t t e r m i n a l s t o the i n n e r h a i r c e l l ' s soma, the absence of o u t e r h a i r c e l l s e f f e r e n t s to the i n n e r h a i r c e l l s , and the l a c k of an i n n e r h a i r c e l l response f o l l o w i n g COCB s t i m u l a t i o n , p r e c l u d e r e l e a s e of a d i r e c t c h e m i c a l e f f e c t o r on the i n n e r h a i r c e l l s . The p u t a t i v e t h e o r i e s propose a mechanical mechanism f o r the m o d i f i c a t i o n , by the o u t e r h a i r c e l l s , of the i n n e r h a i r c e l l response to t o n a l s t i m u l i (Kim, '85; S i e g e l and Kim, '82). By changing t h e i r l e n g t h , the o u t e r h a i r c e l l s c o u l d a f f e c t the r i g i d i t y of the t e c t o r i a l membrane and thus, presumably, the response to the frequency of t o n a l s t i m u l i . T h i s has been shown t o be the case w i t h s e l e c t i v e l e s i o n i n g of the outer h a i r c e l l s , r e s u l t i n g i n an a t t e n u a t i o n of responses t o a u d i t o r y s t i m u l i as measured i n the a u d i t o r y nerve ( e a r l i e r and P u j o l , '82). D e s t r u c t i o n of the outer h a i r c e l l s , however, d i d not change the response a u d i t o r y s t i m u l u s i n t e n s i t y (Prosen, '81), nor 158 does i t a f f e c t the membrane p o t e n t i a l of the i n n e r h a i r c e l l s (Brown and N u t t a l , '85). I s o l a t e d guinea p i g out e r h a i r c e l l s shortened and lengthened when c u r r e n t was passed through the b a t h i n g medium. The d i r e c t i o n of change was a f u n c t i o n of the p o l a r i t y of the c u r r e n t (Brownell, '85). T h i s study a l s o demonstrated t h a t the guinea p i g outer h a i r c e l l s s h o r t e n i n response t o ACh i n t r o d u c e d i n t o the b a t h i n g medium. Ac c o r d i n g t o the model proposed by Brownell ('85), t h i s s h o r t e n i n g a c t i o n induced by ACh would make the t e c t o r i a l membrane l e s s r i g i d and f a c i l i t a t e a u d i t o r y responses t o the c h a r a c t e r i s t i c frequency f o r t h a t p o s i t i o n of the b a s i l a r membrane. T h i s suggests a p o s s i b l e f u n c t i o n f o r the GABA/ENK/SOM ( i n the c a t and guinea p i g) or SOM alone medial o l i v o c o c h l e a r e f f e r e n t s to the out e r h a i r c e l l s . In a number of r e g i o n s , SOM has an i n h i b i t o r y a c t i o n on c o n s t r i c t i o n . In the r a t vas d e f e r e n s , and the r a b b i t ear a r t e r y , f o r example, SOM reduced the c o n t r a c t i l e response t o a d r e n a l i n e by a p o s t s y n a p t i c mechanism, a p p a r e n t l y by a d i r e c t a c t i o n . In the guinea p i g ileum, SOM decreased the amplitude of c o n t r a c t i o n s i n response t o e l e c t r i c a l f i e l d s t i m u l a t i o n but f a i l e d t o i n h i b i t the c o n t r a c t i l e response t o exogenous a p p l i c a t i o n of ACh. T h i s l e d t o s p e c u l a t i o n t h a t the i n h i b i t o r y a c t i o n of SOM was due to p r e s y n a p t i c i n h i b i t i o n of ACh r e l e a s e and not t o a d i r e c t a c t i o n on the pos t s y n a p t i c membrane ( R e i c h l e n , '83; Ror s t a d e t a l . , '80). 159 I n the medial o l i v o c o c h l e a r system both i n h i b i t o r y and e x c i t a t o r y mechanisms may e x i s t . The l a t t e r , as suggested by Brownell ('85), would be mediated by ACh w i t h the former b e i n g mediated by GABA and SOM. As mentioned e a r l i e r , s h o r t e n i n g of the outer h a i r c e l l s by ACh may i n c r e a s e the compliance of the mechanical u n i t of the c o c h l e a and thus the response t o a u d i t o r y s t i m u l i . Conversely, l e n g t h e n i n g of the o u t e r h a i r c e l l s would make t h i s u n i t more r i g i d and atte n u a t e the response t o a u d i t o r y s t i m u l i (Brownell, '85). I t i s p o s s i b l e t h a t t h i s l e n g t h e n i n g i s through an i n h i b i t o r y mechanism mediated by GABA and t h a t SOM may a c t to f a c i l i t a t e the i n h i b i t o r y a c t i o n of GABA by b l o c k i n g p r e s y n a p t i c a l l y the r e l e a s e of ACh, as was the case f o r the guinea p i g ileum. A l t e r n a t i v e l y the a c t i o n of SOM may be a d i r e c t one on the post s y n a p t i c membrane of the out e r h a i r c e l l s . From the proposed model (Brownell, '85), i t would appear t h a t i t was the l e n g t h e n i n g , i n h i b i t o r y a c t i o n which predominated f o l l o w i n g s t i m u l a t i o n of the medial o l i v o c o c h l e a r neurons or of the c r o s s e d o l i v o c o c h l e a r bundle. T h i s a c t i o n would not be mediated by ACh a c c o r d i n g t o B r o w n e l l ('85). Regardless of the p r e c i s e e f f e c t , the presence o f gab a e r g i c and c h o l i n e r g i c o u t e r h a i r c e l l e f f e r e n t s suggests a bimodal response, w i t h SOM p o s s i b l y a n t a g o n i z i n g the c h o l i n e r g i c e f f e c t . M i c r o i o n t o p h o r e t i c a p p l i c a t i o n of GABA or ACh i n t o the c a t m a c c u l i s a c c u l i , presumably a f f e c t i n g o n l y the i n n e r h a i r c e l l a f f e r e n t s , r e s u l t e d i n f a c i l i t a t i o n and 160 i n h i b i t i o n , r e s p e c t i v e l y , of spontaneous a c t i v i t y measured i n the a f f e r e n t f i b r e s t o the i n n e r h a i r c e l l s ( F e l i x and Ehrnberger, '77). In c o n t r a s t , spontaneous a c t i v i t y of the i n n e r h a i r c e l l s was not a f f e c t e d by COCB s t i m u l a t i o n (Brown and N u t t a l , '84). T h i s suggests t h a t the GABA- and GAD-IR e f f e r e n t s which appose the out e r h a i r c e l l a f f e r e n t s are not c o l l a t e r a l s o f the medial o l i v o c o c h l e a r f i b r e s , but r a t h e r o r i g i n a t e from the l a t e r a l o l i v o c o c h l e a r neurons. I t i s p o s s i b l e t h a t i n n e r h a i r c e l l s can be b i m o d a l l y i n f l u e n c e d i n a manner which i s d i s t i n c t from the outer h a i r c e l l s . I t i s i n t e r e s t i n g t h a t a common denominator f o r h a i r c e l l i n h i b i t i o n may be the presence of ENK-IR. In the l a t e r a l o l i v o c o c h l e a r system, ACh and ENK appear t o c o e x i s t . The a c t i o n of ACh on the spontaneous f i r i n g r a t e of the i n n e r h a i r c e l l s was r e p o r t e d as i n h i b i t o r y . In c o n t r a s t , c h o l i n e r g i c c e l l s i n the medial o l i v o c o c h l e a r system do not appear t o c o n t a i n ENK. The a c t i o n of ACh on outer h a i r c e l l s , from the model proposed by Brownell ('85), appears f a c i l i t a t o r y . In the medial o l i v o c o c h l e a r system GABA and ENK may c o e x i s t , and GABA may cause a t t e n u a t i o n of the response t o a u d i t o r y s t i m u l i . Whether the presence of ENK i s f u n c t i o n a l l y important t o h a i r c e l l i n h i b i t i o n remains t o be demonstrated. As an a s i d e , the out e r h a i r c e l l s found i n the base of the c o c h l e a seem t o be p a r t i c u l a r l y s u s c e p t i b l e t o damage f o l l o w i n g exposure t o aminoglycoside a n t i b i o t i c s , loop d i u r e t i c s , or prolonged round window s t i m u l a t i o n , w h i l e the 161 o u t e r h a i r c e l l s i n the a p i c a l r e g i o n s had more r e s i s t a n c e t o these damaging e f f e c t s . Outer h a i r c e l l s i n the a p i c a l r e g i o n s , u n l i k e t h e i r b a s a l c o u n t e r p a r t s , r e c e i v e GABA- and GAD-IR puncta,. presumably o r i g i n a t i n g from medial o l i v o c o c h l e a r c e l l s l o c a t e d i n the VNTB. Does the presence of GABA prevent the damage t o these outer h a i r c e l l s ? Aminoglycoside a n t i b i o t i c s are b e l i e v e d t o i n h i b i t the carbohydrate metabolism of the out e r h a i r c e l l s (Jobe and Brown, '80). One p o s s i b i l i t y i s t h a t the GABA-shunt (McGeer e t a l . , '83) may p r o v i d e an a l t e r n a t e source of energy, perhaps enough t o att e n u a t e the t o x i c i t y of aminoglycoside a n t i b i o t i c s . As mentioned e a r l i e r , s t i m u l a t i o n of the c a t o l i v o c o c h l e a r bundle produced a v a r i e t y of responses: e x c i t a t o r y , i n h i b i t o r y and complex ( S t a r r and Wernick, '68). I o n t o p h o r e t i c a p p l i c a t i o n of GABA i n the c h i n c h i l l a DCN produced i n h i b i t i o n and complex responses t o both spontaneous and tone evoked a c t i v i t y (Caspary e t a l . , '79). While most of the p r o j e c t i o n s t o the DCN from the VNTB were shown t o be i p s i l a t e r a l (Covey e t a l . , '84; E l v e r l a n d , '77), some of the f i b r e s from the VNTB must course t o the DCN v i a the c r o s s e d o l i v o c o c h l e a r bundle, as s t i m u l a t i o n of the c a t ' s c r o s s e d o l i v o c o c h l e a r bundle r e s u l t s i n a v a r i e t y of responses i n the DCN: e x c i t a t o r y , i n h i b i t o r y , and complex ( S t a r r and Wernick, '68). C e l l s of the DCN were s a i d t o be i n h i b i t e d by the p r e s e n t a t i o n of sound t o the c o n t r a l a t e r a l ear, w h i l e most c e l l s of the c a t VNTB were b i n a u r a l l y 162 e x c i t e d . These VNTB c e l l s would t h e r e f o r e not be good c a n d i d a t e s f o r the DCN i n h i b i t i o n . There were, however, a low d e n s i t y of c e l l s , l o c a t e d m e d i a l l y w i t h i n the c a t VNTB, which were i p s i l a t e r a l l y e x c i t e d (Guinan, '72) and these c e l l s might be a source of i n h i b i t o r y e f f e r e n t s t o the c o n t r a l a t e r a l DCN. A l t e r n a t e l y , s i n c e both i o n t o p h o r e t i c a p p l i c a t i o n of GABA and s t i m u l a t i o n of the c r o s s e d o l i v o c o c h l e a r bundle a l s o r e s u l t s i n complex responses i n the DCN (Caspary, '79), the l a t t e r may be due t o the gabaergic ( p o s s i b l y GABA/SOM-IR) c e l l s l o c a t e d m e d i a l l y i n the VNTB. The p h y s i o l o g y of the DC i s not always c l e a r . An e a r l y r e p o r t i n t h a t the c a t suggested the DC does not respond t o t o n a l s t i m u l i (Merzenich and Re i d , '74). Another study i n the c a t r e p o r t e d r a t h e r broad t u n i n g curves f o r the d o r s a l d i v i s i o n s of the DC ( A i t k i n e t a l . , '75). U n f o r t u n a t e l y , t h i s study used a c l a s s i f i c a t i o n scheme (Berman, '68) which d i d not r e c o g n i z e the m a j o r i t y o f the DC, making comparison d i f f i c u l t . More r e c e n t l y , a r e p o r t i n the mouse showed the DC was t o n o t o p i c i n a manner which was c o e x t e n s i v e w i t h the ICC ( S t i e b l e r and Ehret, '85). F u r t h e r c l a r i f i c a t i o n of these d i s c r e p a n c i e s i s r e q u i r e d . Although l i t t l e i s understood of the f u n c t i o n of the DC, i t s connections suggest t h a t the DC may p r o v i d e a focus f o r descending feedback mechanisms from the a u d i t o r y neocortex and oth e r a u d i t o r y a f f e r e n t s t o the DC. 163 The EC and ICA r e c e i v e both a u d i t o r y and somatosensory i n p u t . The c e l l s of the EC have been shown to have broad t u n i n g c u r v e s . In a s i m i l a r f a s h i o n , the c e l l s of the EC and ICA a l s o have l a r g e somesthetic r e c e p t i v e f i e l d s i n the c a t ( A i t k i n e t a l . , '75; '; '81), opossum (Robards, '79; Robards e t a l . , '79), and mouse ( W i l l a r d e t a l . , '79). In the mouse, the p h y s i o l o g i c a l p r o p e r t i e s of c e l l s i n the EC make them p a r t i c u l a r l y good c a n d i d a t e s f o r mediators of a c o u s t i c and somatosensory s t a r t l e r e f l e x e s ( W i l l a r d e t a l . , '79). The p r o j e c t i o n of the ICA t o r e g i o n s i n the m i dbrain r e t i c u l a r f o r m a t i o n p r o v i d e s a p o s s i b l e motor path out of the CNS. Concluding Remarks The assumption used throughout t h i s d i s c u s s i o n has been t h a t the a n a t omical s t r u c t u r e , a f f e r e n t s and e f f e r e n t s are r e asonably w e l l conserved between s p e c i e s even i f the d i s t r i b u t i o n s of t h e i r n e u r o t r a n s m i t t e r s may d i f f e r . The p h y s i o l o g y of these brainstem a u d i t o r y r e g i o n s a l s o appears t o be conserved between s p e c i e s although a v a i l a b l e comparative d a t a are scanty. D i f f e r e n c e s have been r e p o r t e d f o r the l o c a t i o n of c e l l s which form the descending o l i v o c o c h l e a r bundle but the c e l l morphologies were v e r y s i m i l a r between s p e c i e s . Although the e x t e n t of b i l a t e r a l p r o j e c t i o n s v a r i e s s l i g h t l y , the predominant p a t t e r n s of t e r m i n a t i o n a l s o appear to be conserved between s p e c i e s (Robertson, '84; '85; White and Warr, '83). In a l l t h e r e i s , i n the f i n a l a n a l y s i s , both a 164 F i g u r e 40. The p o s s i b l e a c t i o n o f the descending pathways. Arrows p o i n t i n g up ( f ) i n d i c a t e e x c i t a t o r y a c t i o n s , w h i l e arrows which p o i n t down ( \ ) i n d i c a t e i n h i b i t o r y a c t i o n s . While based on c i r c u m s t a n t i a l evidence, the e x i s t e n c e o f ) and a c e t y l c h o l i n e , and i n h i b i t o r y agents, such as GABA i n the descending a u d i t o r y pathways suggests a descending bimodal i n f l u e n c e . PLSO (see t e x t or F i g u r e 38 f o r d e t a i l s ) . Symbol: A, c o n t r a l a t e r a l . A b b r e v i a t i o n s : ANC, a u d i t o r y neocortex; AVCN, a n t e r i o r v e n t r a l c o c h l e a r nucleus; CN, c o c h l e a r n u c l eus; DC, d o r s a l c o r t e x of the i n f e r i o r c o l l i c u l u s ; DCN, d o r s a l c o c h l e a r nucleus; DLPO, d o r s o l a t e r a l p e r i o l i v a r y n u c l eus; DMPO, dorsomedial p e r i o l i v a r y nucleus; DPO, d o r s a l p e r i o l i v a r y n u c l eus; IC, i n f e r i o r c o l l i c u l u s ; IHC, i n n e r h a i r c e l l s ; GCL, gr a n u l e c e l l l a y e r ; LSO, l a t e r a l s u p e r i o r o l i v e ; NLL, n u c l e i of the l a t e r a l lemniscus; OHC, outer h a i r c e l l s ; PLSO, p e r i h i l a r r e g i o n s of the l a t e r a l s u p e r i o r o l i v e ; PVCN, p o s t e r i o r v e n t r a l c o c h l e a r nucleus; SOC, s u p e r i o r o l i v a r y complex; VLL, v e n t r a l nucleus of the l a t e r a l lemniscus; VNTB, v e n t r a l nucleus of t r a p e z o i d body; VPO, v e n t r a l p e r i o l i v a r y n u c l e u s . 165 D M P O VNTB D L P O , L S O V P O D P O ( P L S O ) 166 l a t e r a l and medial o l i v o c o c h l e a r systems. The l a t t e r p r o j e c t s t o the out e r h a i r c e l l s w h i l e the former p r o j e c t s t o the i n n e r h a i r c e l l s . The anatomy and immunohistochemistry a l l p o i n t t o a descending mechanism of a u d i t o r y c o n t r o l ( F i g 40). The n e o c o r t i c a l i n p u t and other t e c t a l i n p u t t o DC, can, v i a the descending DC pathways t o the d o r s a l p e r i o l i v a r y r e g i o n s and the VNTB, i n f l u e n c e the c o c h l e a r nucleus, the outer h a i r c e l l s and the i n n e r h a i r c e l l a f f e r e n t s of the organ of C o r t i . S u b c o r t i c a l l y , o t h e r r e g i o n s of the IC, such as the ICC, can i n f l u e n c e these r e g i o n s v i a i n p u t t o the DC. In a s i m i l a r f a s h i o n , the CN, the outer h a i r c e l l s and i n n e r h a i r c e l l a f f e r e n t s c o u l d be i n f l u e n c e d by the VLL v i a i t s i n p u t t o the d o r s a l p e r i o l i v a r y r e g i o n s and the VNTB. The e f f e r e n t pathways t o the out e r h a i r c e l l s and i n n e r h a i r c e l l a f f e r e n t s are ga b a e r g i c , c h o l i n e r g i c and p e p t i d e r g i c . The presence o f GABA and ACh suggests a bimodal i n f l u e n c e on the h a i r c e l l s o f the organ of C o r t i . Indeed the i n n e r h a i r c e l l a f f e r e n t s ( F e l i x and Ehrnberger, '77) and the outer h a i r c e l l s (Brownell, '85) have been shown to be capable of bimodal responses. Taken t o g e t h e r , t h i s suggests both c o r t i c a l and s u b c o r t i c a l descending pathways which u l t i m a t e l y i n f l u e n c e the a u d i t o r y r e c e p t o r . These descending a u d i t o r y pathways may thus p r o v i d e a p o s s i b l e mechanism of h a b i t u a t i o n or s e n s i t i z a t i o n t o s p e c i f i c t o n a l f r e q u e n c i e s . The descending a u d i t o r y pathways from the neocortex t o the DC and from the p e r i o l i v a r y r e g i o n s and VNTB to both the CN 167 and the c o c h l e a have been documented. What remains t o be demonstrated i s the nature of the descending a u d i t o r y pathways from DC t o the medial VLL r e g i o n s and t o the d o r s a l p e r i o l i v a r y r e g i o n s and VNTB. Andersen e t a l . ('80a) b r i e f l y mentioned a r e c i p r o c a l c o n n e c t i o n from the IC t o the VNTB, which would suggest t h a t d i s c r e t e p r o j e c t i o n s e x i s t between these two r e g i o n s . U n f o r t u n a t e l y the c l a s s i f i c a t i o n scheme used i n t h i s study f a i l e d t o d i f f e r e n t i a t e a DC r e g i o n from the ICC. The f u n c t i o n of SOM i n the ascending multimodal pathways from ICA and p o s s i b l y EC t o the MRF remains t o be demonstrated. Whatever i t s f u n c t i o n , SOM-IR was a s s o c i a t e d w i t h the descending a u d i t o r y pathways of a l l s p e c i e s . When SOM-IR was seen i n the ascending a u d i t o r y n u c l e i , i t was s p e c i e s s p e c i f i c . S i m i l a r p a t t e r n s of p e p t i d e immunoreactivity i n the descending pathways have been d e s c r i b e d f o r ENK and NPY-IR i n the r a t . Whether these p e p t i d e s i n the descending a u d i t o r y pathways a r e , l i k e SOM-IR, conserved between s p e c i e s remains t o be demonstrated. Of the c l a s s i c a l n e u r o t r a n s m i t t e r s , GABA appears to show a s i m i l a r d i s t r i b u t i o n t o both SOM-IR and ENK-IR. Whether SOM-IR c o e x i s t s w i t h these p e p t i d e s or w i t h GABA, or whether these neurochemicals are found i n p a r a l l e l pathways, remains t o be demonstrated. As a t o o l comparative neuroanatomy can p r o v i d e i n s i g h t s i n t o the o r g a n i z a t i o n of CNS pathways. T h i s i s e s p e c i a l l y 168 t r u e f o r o v e r l a p p i n g subpopulations of c e l l s w i t h o v e r l a p p i n g t e r m i n a l f i e l d s , which are d i f f i c u l t t o r e s o l v e w i t h c u r r e n t t r a c t t r a c i n g methods. Comparative immunohistochemistry alone, or i n combination w i t h t r a c t t r a c i n g , can p r o v i d e v a l u a b l e i n f o r m a t i o n on the d i s c r e t e o r g a n i z a t i o n of the CNS. 169 QUESTIONS RAISED General Questions A. Are anatomical p r o j e c t i o n s the same i n a l l s p e c i e s ? B. Are the p h y s i o l o g i c a l responses of a g i v e n r e g i o n conserved i n a l l s p e c i e s ? C. I f the answer t o the above i s "yes", then i s a s i m i l a r response due t o the a c t i o n upon the same i o n c h a n n e l ( s ) . D. I f the answer t o the above i s "yes", then do d i f f e r e n t neurochemicals u l t i m a t e l y a c t upon the same i o n channel(s) t o produce the same p h y s i o l o g i c a l response. S p e c i f i c Questions 1. What i s the s p e c i f i c nature of the descending a u d i t o r y pathways o r i g i n a t i n g i n the i n f e r i o r c o l l i c u l u s . 2. Do GABA and SOM c o e x i s t i n the a u d i t o r y system? Do SOM and NPY c o e x i s t i n the descending a u d i t o r y pathways o r i g i n a t i n g i n the EC and DC? 3. I f the GABA shunt were b l o c k e d , w i t h GABAculine f o r example, would the a p i c a l outer h a i r c e l l s show the same s u s c e p t i b l e as b a s a l outer h a i r c e l l s t o kanamycin t o x i c i t y ? 4. Do the c e l l s which p r o j e c t t o the granule c e l l l a y e r c o n t a i n GABA/SOM/enkephalin? 5. Do SOM c e l l s i n the IC p a r t i c i p a t e i n descending a u d i t o r y p r o j e c t i o n s t o the VLL and SOC. 170 6. What i s the nature of the p r o j e c t i o n of the SOM-IR c e l l s seen a t the medial border of the VLL and i n the r o s t r a l l e m n i s c a l f i b r e s ? 7. Are SOM-IR c e l l s i n the ICA the source of puncta i n the midbrain r e t i c u l a r formation? 10. Are t h e r e s p e c i e s d i f f e r e n c e s i n the descending a u d i t o r y pathways? I s t h i s the case f o r the IC i n p u t t o the DC? 11. 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