UBC Theses and Dissertations

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UBC Theses and Dissertations

Studies on characterized nucleus gigantocellularis neurones Harris, David Platt 1980

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STUDIES ON CHARACTERIZED NUCLEUS GIGANTOCELLULABIS NEURONES by DAVID PLAIT HASBIS B . S c , the U n i v e r s i t y of V i c t o r i a A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOB THE DEGBEE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( D i v i s i o n o f Pharmacology and T o x i c o l o g y i n the F a c u l t y cf Pharmaceutical Sciences) Be accept t h i s t h e s i s as conforming to the r e g u i r e d standards THE UNIVERSITY OF BRITISH COLUMBIA HAY 1980 © D A V I D PL ATT HARRIS, 1980 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis f o r s c h olarly purposes may be granted by the Head of my Department or by h i s representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. D i v i s i o n of Pharmacology and Toxicology Faculty of Pharmaceutical Sciences The Univesity of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6H). 1W5 A p r i l 23, 1980. ABSTBACT P e r i a q u e d u c t a l gray (PAG) s t i m u l a t i o n produced a n t i n o c i c e p t i o n may be mediated by i n h i b i t i o n of an ascending n o c i c e p t i v e pathway at the brainstem l e v e l and/or the a c t i v a t i o n of descending i n h i b i t i o n on s p i n a l n o c i c e p t o r - d r i v e n neurones. Experiments were performed on urethane a n a e s t h e t i z e d r a t s to e s t a b l i s h i f t h e r e i s a monosynaptic pathway from the PAG to the nucleus g i g a n t o c e l l u l a r i s (nGC). / S i n g l e u n i t s i n the PAG were a b l e to be a n t i d r o m i c a l l y a c t i v a t e d by s t i m u l a t i o n of the nGC, i n d i c a t i n g t h a t a d i r e c t pathway does e x i s t . To f u r t h e r c l a r i f y nGC involvement i n the two p o s s i b i l i t i e s s t a t e d above, s i n g l e u n i t s i n the nGC were c h a r a c t e r i z e d a c c o r d i n g t o t h e i r responses t o n a t u r a l s t i m u l i . . About o n e - h a l f of the neurones t e s t e d sere e x c i t e d by noxious s t i m u l i . S i m i l a r l y , about one-half of those t e s t e d with PAG s t i m u l a t i o n were i n h i b i t e d . Of those neurones a f f e c t e d by both noxious p e r i p h e r a l s t i m u l i and PAG s t i m u l a t i o n , the l a r g e s t group (3755) were e x c i t e d by the noxious s t i m u l i and i n h i b i t e d by the PAG s t i m u l a t i o n . Intravenous and i o n t o p h o r e t i c f l u o x e t i n e , a s p e c i f i c s e r o t o n i n uptake b l o c k e r , enhanced the p e r i o d of i n h i b i t i o n and reduced the e x c i t a t i o n of nGC neurones due t o PAG s t i m u l a t i o n . Furthermore, i n h i b i t i o n of nGC neurones by i o n t o p h o r e t i c s e r o t o n i n or f l u o x e t i n e was c o r r e l a t e d with the PAG s t i m u l a t i o n produced i n h i b i t i o n of these neurones, su g g e s t i n g t h a t t h i s i n h i b i t i o n was mediated by s e r o t o n i n . These d a t a suggest t h a t many nGC neurones are i n an a s c e n d i n n o c i c e p t i v e pathway and under i n h i b i t o r y c o n t r o l o f t h e PAG. John G. S i n c l a i r , Ph. D., S u p e r v i s o r . i v TABLE OF COMMENTS A B S T R A C * T * * • • • * * • • * « * • # * • * • • # • •.».• * • * • *.:« * * • * • • « • • • X X LIST OF T ABLES ....................................... .... . , v i i laXST O f F XG OR £S • • < * . « < • • « * • « • »"•,:-». * * * * * * • =• • •* *-* « * * * • • • » • • • vxxx ACKNOWLEDGEMENT ........................................... X X N T BODOCT XO N • * * ,» * • » » • * » . • * • • » • • • * • • • • *.»- • • • • • • » * * * * • 1 BACKGROUND , 4 A. N o c i c e p t o r s and t h e i r A f f e r e n t F i b r e s .............. 4 E. S p i n a l Cord S u b s t r a t e s o f N o c i c e p t i o n .............. 5 Laminae of the S p i n a l Cord Gray Matter .......... 5 Lamina I — the Marginal Layer ................ 5 Laminae I I and I I I -the Su b s t a n t i a G e l a t i n o s a 6 Laminae IV, V, and VI - the Nucleus P r o p r i u s 8 Ascending N o c i c e p t i v e Pathways .................. 9 The Spinothalamic T r a c t ...................... 10 The S p i n o r e t i c u l a r T r a c t ..................... 12 The S p i n o c e r v i c o t h a l a m i c T r a c t ............... 13 The S p i n o t e c t a l T r a c t ........................ 14 C. Mesencephalic N u c l e i ............................... 15 The P e r i a q u e d u c t a l Gray Matter .................. 15 The D o r s a l and Medial Raphe ..................... 23 D. Medullary N u c l e i ................................... 25 The Nucleus R e t i c u l a r i s G i g a n t o c e l l u l a r i s ....... 25 The Caudal Raphe ................................, 31 MATERIALS AND METHODS .................................... . 38 A. S u r g i c a l P r e p a r a t i o n • 38 E. E l e c t r o d e P r e p a r a t i o n and P o s i t i o n i n g .............. 39 P r e p a r a t i o n ... . . . .. . *,'• ...... .... .... .... ... * ...... *. < 39 P o s i t i o n i n g ..................................... 41 C. Equipment I n t e r c o n n e c t i o n .......................... 42 P e r i a q u e d u c t a l Gray Neurones - Antidromic A c t i v a t i o n . . . . . . . . . . . . . . . . . ... .... .... 43 Nucleus G i g a n t o c e l l u l a r i s Neurones - E f f e c t of PAG S t i m u l a t i o n ...... . . ...... . . ..,. ...... ...... ... 49 D. P r o t o c o l ............ ....................... ........ ., 49 P e r i a q u e d u c t a l Gray Neurones - Antidromic A c t i v a t i o n . ... » . ... ............ .............. 49 Nucleus G i g a n t o c e l l u l a r i s Neurones - E f f e c t of PAG •> S t i m u l a t i o n ..... ............. ... 53 E. H i s t o l o g y .......................................... 54 RESULTS 56 A P e r i a q u e d u c t a l Gray Neurones ........................ 56 Responses to Noxious P e r i p h e r a l S t i m u l i .......... 56 Responses to S t i m u l a t i o n o f the nGC ............. 59 B. Nucleus R e t i c u l a r i s G i g a n t o c e l l u l a r i s Neurones ..... 65 E f f e c t o f Drugs on U n i t A c t i v i t y ................ 72 P e r i p h e r a l S t i m u l i .............................. 72 S t i m u l a t i o n of the P e r i a q u e d u c t a l Gray .......... , 81 DISCUSSION- , 93 A, P e r i a q u e d u c t a l Gray Neurones ........................ 93 Noxious P e r i p h e r a l S t i m u l i ...................... 93 S t i m u l a t i o n of the Nucleus G i g a n t o c e l l u l a r i s .... 94 B» Nucleus G i g a n t o c e l l u l a r i s Neurones .................100 E f f e c t s of Drugs ..................... ....... . ...... 100 Responses to P e r i p h e r a l S t i m u l i .................. 102 Responses t o PAG S t i m u l a t i o n .................... ,104 v i C. C o n c l u s i o n ....... ........ 106 SUMMARY . ,, ............ ...... ...... ......... ,110 APPENDIX . . , . . ... ............. ...... . ... .... ..... ......... 111 BIBLIOGRAPHY 122 v i i LIS5 OF TABLES I . C l a s s i f i c a t i o n of PAG area u n i t s by t h e i r f u l f i l m e n t of a n t i d r c m i c i t y c r i t e r i a ......................... 62 I I . Responses of nGC area neurones t o v a r i o u s t e s t s .... 73 I I I . Comparison of nGC u n i t responses to noxious s t i m u l i and iontophoresed drugs ............................ 82 IV. E f f e c t of i n t r a v e n o u s f l u o x e t i n e on the responses of nGC neurones t o PAG or s c i a t i c nerve s t i m u l a t i o n . . 8 3 V. Comparison of nGC neuronal responses to noxious p e r i p h e r a l s t i m u l i and PAG s t i m u l a t i o n ............ 87 VI. Comparison of nGC u n i t responses to PAG s t i m u l a t i o n and i o n t o p h o r e t i c s e r o t o n i n or f l u o x e t i n e ......... 88 v i i i LIST OF FIG OSES 1 . Schematic of equipment setup f o r s t u d y i n g p e r i a q u e d u c t a l gray neurones ........................ 45 2. Schematic of equipment setup f o r the p r o c e s s i n g of taped responses ..................................... 48 3. Schematic of equipment setup f o r s t u d y i n g nucleus g i g a n t o c e l l u l a r i s neurones .......................... 51 4. L o c a t i o n of PAG area neurones responding t o noxious p e r i p h e r a l s t i m u l i .................................. 58 5. L o c a t i o n of nGC s t i m u l a t i o n s i t e s .....................61 6. L o c a t i o n o f PAG area u n i t s a c t i v a t e d o r t h o d r o m i c a l l y from the nGC 64 7. L o c a t i o n of PAG area u n i t s suspected of being a n t i d r o m i c a l l y a c t i v a t e d from the nGC 67 8. Example of c o l l i s i o n between spontaneous s p i k e s and a n t i d r o m i c s p i k e s evoked by nGC s t i m u l a t i o n ......... 69 9. C o l l i s i o n of spontaneous a c t i o n p o t e n t i a l s s i t h t r i g g e r e d a c t i o n p o t e n t i a l s .........................71 10. Examples o f nGC u n i t responses t o i o n t o p h o r e s i s and p e r p h e r a l s t i m u l i ................................... 75 1 1 . L o c a t i o n of nGC area u n i t s responding to noxious p e r i p h e r a l s t i m u l i ...... ............................. 78 12. Examples of nGC u n i t responses to PAG and s c i a t i c nerve s t i m u l a t i o n ............ ......... .......... .......... 80 13. L o c a t i o n of PAG s t i m u l a t i o n s i t e s ..................... 85 14. Examples of f l u o x e t i n e enhancement of nGC neuronal i n h i b i t i o n by PAG s t i m u l a t i o n ....................... 90 15. Example of f l u o x e t i n e r e d u c t i o n of nGC e x c i t a t i o n by i x PAG s t i m u l a t i o n ..................................... 92 16. Schemes of nGC mediation of PAG SPA 108 17. Power supply module- schematic .......................113 18. Mindow d i s c r i m i n a t o r module - schematic ...............115 19. Spike i n t e g r a t o r module- schematic ...................117 20. Delay l i n e module - schematic .119 21. Raster scan module - schematic ........................121 X ACKNOWLEDGEMENT Si n c e r e thanks are extended t o Dr. John G. S i n c l a i r f o r h i s support, e x p e r t i s e , and encouragement, and t o Amy and George T i e n f o r t h e i r g e n e r o s i t y . This study was funded by a Medical Research C o u n c i l of Canada r e s e a r c h grant held by Dr. S i n c l a i r . To my w i f e , L i z a , and my mother. B o t h . 1 INTRODUCTION In 1969 Reynolds reported t h a t e l e c t r i c a l s t i m u l a t i o n o f the p e r i a q u e d u c t a l gray matter produced s u f f i c i e n t a n a l g e s i a t o allow laparotomies to be performed on unanaesthetized r a t s . T h i s paper caused an abundance of r e s e a r c h on s t i m u l a t i o n produced a n t i n o c i c e p t i o n from d i f f e r e n t b r a i n r e g i o n s , i n c l u d i n g the p e r i a q u e d u c t a l gray {Reynolds, 1969; Mayer et a l . , 1971; Oleson et a l . , 1S78) and the nucleus r e t i c u l a r i s g i g a n t o c e l l u l a r i s (nGC) (Takagi e t a l . , 1975; flcCreery and B l o e d e l , 1S75). In p a r t i c u l a r , s t i m u l a t i o n of the p e r i a q u e d u c t a l gray produces both a r e d u c t i o n i n b e h a v i o u r a l responses (Oleson e t a l . , 1978) and r e f l e x responses (Mayer et a l . # 1971; A k i l e t a l . , 1976; Yaksh et a l . , 1976c; Yeung e t a l . , 1977; Oleson e t a l . , 1S78; Rhodes and L i e b e s k i n d , 1978) to noxious s t i m u l i . In a d d i t i o n , t h i s r e d u c t i o n was repor t e d t o l a s t up t o f i v e minutes beyond the p e r i o d of s t i m u l a t i o n (Mayer et a l . , 1971; Rhodes and l i e b e s k i n d , 1978)., However, the mechanism of s t i m u l a t i o n produced a n t i n o c i c e p t i o n i n g e n e r a l , and s p e c i f i c a l l y t h a t of the p e r i a q u e d u c t a l gray, i s s t i l l not known i n any d e t a i l . P e r i a q u e d u c t a l gray s t i m u l a t i o n produced a n t i n o c i c e p t i o n may be mediated by the i n h i b i t i o n o f an ascending n o c i c e p t i v e pathway a t any l e v e l of the nervous system, i n c l u d i n g the s p i n a l c o r d or brainstem l e v e l s . Noxious p e r i p h e r a l i n f o r m a t i o n t r a v e l s through the d o r s a l horn and e v e n t u a l l y reaches the thalamus, e i t h e r d i r e c t l y v i a the s p i n o t h a l a m i c t r a c t , or i n d i r e c t l y v i a the s p i n o r e t i c u l a r t r a c t , the s p i n o c e r v i c a l t r a c t , or p r o p r i o s p i n a l l y . The s p i n o r e t i c u l a r t r a c t , 2 propriospinal f i b r e s , and probably c o l l a t e r a l s of the spinothalamic tract are also known to terminate i n the nucleus r e t i c u l a r i s g i g a n t o c e l l u l a r i s (Hehler et a l . , i960; McCreery and Bloedel, 1975). This thesis started with the premise that periaqueductal gray stimulation produced antinociception may be mediated by the nucleus r e t i c u l a r i s g i g a n t o c e l l u l a r i s . This nucleus may, on one hand, be involved i n t h i s antinociception by i n h i b i t i o n of an ascending nociceptive pathway at the spin a l cord l e v e l . E l e c t r i c a l stimulation of the periaqueductal gray has been . reported to i n h i b i t s p i n a l cord nociceptor-driven neurones (pliveras et a l . , 1974a). However, the periaqueductal gray has few d i r e c t axonal projections to the spin a l cord, therefore, these i n h i b i t o r y e f f e c t s must be mediated by other brainstem n u c l e i . The most often suggested nucleus for t h i s role i s the nucleus raphe magnus {cf. Mayer and Price, 1976), but the nGC i s also a p o s s i b i l i t y . The nucleus g i g a n t o c e l l u l a r i s i s known to project to the sp i n a l cord (Wolstencroft, 1964) and e l e c t r i c a l stimulation of the nGC i n h i b i t s spinal cord nociceptor-driven neurones (Takagi et a l . , 1975). Therefore, the nucleus g i g a n t o c e l l u l a r i s may mediate periaqueductal gray stimulation produced antinociception by i n h i b i t i n g an ascending nociceptive pathway at the spinal l e v e l . Conversely, the nucleus g i g a n t o c e l l u l a r i s may mediate the antinociception by i n h i b i t i o n of the ascending pathway at the brainstem l e v e l . The nGC i s known to receive noxious input from the s p i n a l cord (Schiebel and Schiebel, 1968; Wolstencroft, 1964; Casey, 1969, 1971a). The nucleus g i g a n t o c e l l u l a r i s i s 3 a l s o known to have axonal p r o j e c t i o n s to the thalamus (Bowsher et a l . , 1968) , suggesting that nGC neurones may be i n an ascending n o c i c e p t i v e pathway. A l s o , p e r i a q u e d u c t a l gray s t i m u l a t i o n has been repo r t e d to i n h i b i t nucleus g i g a n t o c e l l u l a r i s neurones (Morrow and Casey, 1 9 7 6 ) . T h e r e f o r e , p e r i a q u e d u c t a l gray s t i m u l a t i o n produced a n t i n o c i c e p t i o n may be mediated by the nucleus g i g a n t o c e l l u l a r i s by i n h i b i t i o n of ascending n o c i c e p t i v e i n f o r m a t i o n a t the brainstem l e v e l . T h i s t h e s i s i n v o l v e d experiments i n which neurones i n the v i c i n i t y of the p e r i a g u e d u c t a l gray were a n t i d r o m i c a l l y a c t i v a t e d by s t i m u l a t i o n of the nucleus g i g a n t o c e l l u l a r i s and experiments c o r r e l a t i n g the responses of nGC neurones to v a r i o u s t e s t s . These t e s t s i n c l u d e d noxious and non-noxious p e r i p h e r a l s t i m u l i , s t i m u l a t i o n o f the PAG, and i n t r a v e n o u s l y and i o n t o p h o r e t i c a l l y administered s e r o t o n i n and f l u o x e t i n e , a s p e c i f i c s e r o t o n i n uptake b l o c k e r . As w e l l , the e f f e c t s o f int r a v e n o u s and i o n t o p h o r e t i c f l u o x e t i n e on the responses of nGC neurones t o noxious p e r i p h e r a l s t i m u l i and p e r i a q u e d u c t a l gray s t i m u l a t i o n were s t u d i e d . The s t u d i e s r e p o r t e d i n t h i s t h e s i s were d i r e c t e d a t e s t a b l i s h i n g evidence f o r a monosynaptic pathway from the PAG to the NGC and f o r the involvement of the nGC i n the mediation of the a n t i n o c i c e p t i v e e f f e c t s of p e r i a q u e d u c t a l gray s t i m u l a t i o n v i a i h i b i t i o n of n o c i c e p t i v e i n f o r m a t i o n i n e i t h e r the s p i n a l cord or the nucleus g i g a n t o c e l l u l a r i s . , 4 BACKGROUND A. N o c i c e p t o r s and t h e i r A f f e r e n t F i b r e s N o c i c e p t o r s are d e f i n e d as r e c e p t o r s which respond to noxious s t i m u l i , that i s , s t i m u l i which are p o t e n t i a l l y or a c t u a l l y damaging to the t i s s u e s t i m u l a t e d , N o c i c e p t o r s are c l a s s i f i e d by t h e i r responses to v a r i o u s s t i m u l i , the affe.re.nts s u p p l y i n g them, and t h e i r l o c a t i o n , Although the d i f f e r e n t c l a s s e s of n o c i c e p t o r s have d i s t i n c t i v e responses t o noxious s t i m u l i they a l l appear t o have f r e e endings and t o be s u p p l i e d by s m a l l - s i z e d a f f e r e n t f i b r e s . These f i b r e s i n c l u d e myelinated A - d e l t a and unmyelinated C f i b r e s ( P a i n t a l , 1960; Burgess and P e r l , 1S73), The major cutaneous n o c i c e p t o r s i n c l u d e the A-d e l t a mechanical, A - d e l t a heat, and C polymodal receptors.., A-d e l t a mechanical n o c i c e p t o r s are e x c i t e d best by damaging mechanical s t i m u l i and do not respond t o noxious heat or a l g e s i c chemicals {Burgess and P e r l , 1967; P e r l , 1968; Burgess e t a l . , 196 8). A - d e l t a heat n o c i c e p t o r s are e x c i t e d by noxious heat {> 15 "C) as w e l l as i n t e n s e mechanical s t i m u l i (Beck e t a l . , 1974; Georgopoulcs, 1976, 1977). The t h i r d major c l a s s of n o c i c e p t o r s are the C polymodal n o c i c e p t o r s . These are abundant and respond to noxious mechanical, thermal, and chemical s t i m u l i {Bessou and P e r l , 1969). Muscle n o c i c e p t o r s a r e a l s o a c t i v a t e d by noxious mechanical, t h e r m a l , or a l g e s i c c h e m i c a l s t i m u l i { P a i n t a l , 1960; Stacey, 1969)., 5 B,. . S p i n a l Cord S u b s t r a t e s o f N o c i c e p t i o n Laminae o f the S p i n a l Cord Gray_Matter The anatomy and to a l a r g e extent the e l e c t r o p h y s i o l o g y of the s p i n a l cord i s d e s c r i b e d i n the terms i n t r o d u c e d by C a j a l (1909) and fiexed (1952)., fiexed d i v i d e d the s p i n a l c o r d gray matter i n t o nine d i s c r e t e laminae. These are d e s c r i b e d i n terms of t h e i r c e l l t y p e s , i n p u t s , and outputs i n the f o l l o w i n g s e c t i o n s . Lamina I - the Marginal Layer The most prominent f e a t u r e of lamina I i s the 'marginal c e l l * of Haldeyer (1888),, These sparse l a r g e neurones have ex t e n s i v e o v e r l a p p i n g d e n d r i t i c t r e e s made up o f r e l a t i v e l y long unbranched axons. Most of the axons remain i n s i d e the marginal l a y e r but a few penetrate i n t o deeper laminae (Saldeyer,1888; C a j a l , 1909; S c h e i b e l and S c h e i b e l , 1968). In a d d i t i o n t o these c e l l s t h e r e are two other types of neurones i n t h i s lamina: very s m a l l and medium s i z e d c e l l s . The l a t t e r have l a r g e d e n d r i t i c t u f t s p r o j e c t i n g i n t o the s u b s t a n t i a g e l a t i n o s a and axons p r o j e c t i n g to the f a s c i c u l u s p r o p r i u s or L i s s a u e r ' s t r a c t ( C a j a l , 1 909) . The marginal l a y e r r e c e i v e s f i n e a f f e r e n t s from the t r a c t of L i s s a u e r , some of which may be primary a f f e r e n t s , but most are probably p r o p r i o s p i n a l (Kerr, 1975a). P e r i p h e r a l nerve s t i m u l a t i o n s t u d i e s i n c a t s and monkeys have i n d i c a t e d lamina I neurones r e c e i v e i n p u t from both A - d e l t a and C f i b r e s 6 {Christensen and P e r l , 1970; Kumazawa e t a l . , 1975).., Recent a u t o r a d i o g r a p h i c a l s t u d i e s i n the c a t and monkey ( R e t h e l y i e t a l . , 1979) and h o r s e r a d i s h peroxidase (HRP) s t u d i e s i n the cat {Light et a l . , 1979; L i g h t and P e r l , 1979a,b) a l s o i n d i c a t e lamina I r e c e i v e s s m a l l diameter myelinated n o c i c e p t i v e f i b r e s . Many neurones i n lamina I respond s p e c i f i c a l l y to noxious mechanical and/or noxious thermal s t i m u l i {Christensen and P e r l , 1970; Cervero e t a l . # 1976, 1979), but a few can be c l a s s i f i e d as wide-dynamic range neurones as they respond to non-noxious as w e l l as noxious s t i m u l i {Cervero et a l . . , 1976)., Marginal l a y e r neurones probably c o n t r i b u t e t o the sp i n o t h a l a m i c t r a c t . They have been a n t i d r c m i c a l l y a c t i v a t e d by s t i m u l a t i o n of the s p i n o t h a l a m i c t r a c t from the d i e n c e p h a l i c l e v e l i n the c a t { W i l l i s e t a l . , 1974) and c e r v i c a l l e v e l i n the cat and monkey (Kumazawa e t a l . , 1975). Ho r s e r a d i s h peroxidase s t u d i e s have a l s o l o c a t e d neurones i n t h i s lamina i n the c a t (Trevino and C a r s t e n s , 1975; Ca r s t e n s and T r e v i n o , 1978) and the monkey {Trevino and Ca r s t e n s , 1975; W i l l i s e t a l . , 1979).: In a d d i t i o n , marginal l a y e r neurones may p r o j e c t p r o p r i o s p i n a l l y to more r o s t r a l s i t e s v i a the t r a c t of L i s s a u e r or the l a t e r a l f a s c i c u l i p r o p r i u s ( S c h i e b e l and S c h i e b e l , 1968; Cervero et a l . , 1979) . Laminae I I and I I I -the S u b s t a n t i a S e l a t i n o s a The neurones i n lamina I I are s m a l l but have g o i t e e x t e n s i v e r a d i a l d e n d r i t i c t r e e s extending from t h e i r a p i c a l and b a s a l p o l e s ( C a j a l , 1909; S c h i e b e l and S c h i e b e l , 1968). The d e n d r i t e s reach f a i r l y e x t e n s i v e l y d o r s o m e d i a l l y but u s u a l l y do 7 not leave laminae I I or I I I , The neurones i n lamina I I I are s l i g h t l y l a r g e r and more d i s p e r s e d and have l a r g e r and more complex d e n d r i t i c a r b o r i z a t i o n s ( S c h i e b e l aad S c h i e b e l , 1968). Lamina I I and I I I appear t o be d i s t i n g u i s h a b l e by t h e i r a f f e r e n t i n p u t . Lamina I I neurones r e c e i v e f i n e f i b r e s , most of which are c o l l a t e r a l s of axons running i n the t r a c t of L i s s a u e r or the nearby white matter, although other areas might c o n t r i b u t e to them ( C a j a l , 1909; S z e n t a g o t h a i , 1964; S c h i e b e l and S c h i e b e l , 1968). These f i b r e s are probably n o c i c e p t i v e unmyelinated f i b r e s as lamina I I neurones are r e p o r t e d to r e c e i v e t h i s c l a s s of f i b r e s (Kurazawa and P e r l , 1976; Yaksh e t a l . , 1977; L i g h t e t a l . , 1979a,b),, Co n v e r s e l y , lamina I I I neurones are r e p o r t e d to r e c e i v e coarse c o l l a t e r a l s of primary a f f e r e n t o r i g i n from l a r g e f i b r e s which pass v e n t r a l l y along the medial edge of the d o r s a l horn before t u r n i n g and e n t e r i n g lamina I I I from below i n a flame-shaped a r b o r i z a t i o n ( C a j a l , 1909; S z e n t a g o t h a i , 1964). These were reported to be innocuous myelinated a f f e r e n t f i b r e s i n s t u d i e s i n v o l v i n g : e l e c t r i c a l s t i m u l a t i o n of nerves i n the monkey (Kumazasa and P e r l , 1976, 1978) and the c a t (Yaksh e t a l . , 1977; B a l l e t a l . , 1979); autoradiography i n the c a t and monkey ( B e t h e l y i e t a l . , 1979); and HBP i n the c a t (Light and P e r l , 1979a,b), The axonal p r o j e c t i o n s of the neurones i n these laminae e i t h e r never l e a v e the s u b s t a n t i a g e l a t i n o s a or t e m p o r a r i l y run i n the t r a c t of L i s s a u e r or f a s c i c u l i p r o p r i u s before t e r m i n a t i n g back i n the s u b s t a n t i a g e l a t i n o s a ( C a j a l , 1909; S z e n t a g o t h a i , 1964; S c h i e b e l and S c h i e b e l , 1968). T h i s suggests t h a t the s u b s t a n t i a g e l a t i n o s a i s a c l o s e d system. However, 8 outflow from the s u b s t a n t i a g e l a t i n o s a probably e x i s t s v i a lamina IV antenna neurones which have d e n d r i t e s p r o j e c t i n g i n t o the s u b s t a n t i a g e l a t i n o s a (Szentagothai, 1964; B e t h e l y i and Sze n t a g o t h a i , 1973) or by say of axons from lamina I I I neurones, some of which are r e p o r t e d t o d i p i n t o deeper laminae before r e t u r n i n g to the s u b s t a n t i a g e l a t i n o s a (Mannen and Sugiura, 1976) . Neurones i n the s u b s t a n t i a g e l a t i n o s a respond t o noxious as w e l l as innocuous cutaneous s t i m u l i . Many are a c t i v a t e d by polymodal or mechanical n o c i c e p t o r s (Kumazawa and P e r l , 1976, 1978), by innocuous s t i m u l i or by both {Yaksh e t a l . , 1977; H a l l e t a l . , 1979). A l s o , some neurones d i s p l a y extended d i s c h a r g e s a f t e r only b r i e f s t i m u l i {Yaksh e t a l . , 1977; Cervero et a l . , 1977; H a l l et a l . , 1979). Laminae IV, V, and VI - the Nucleus P r o p r i u s These laminae have neurones of v a r i e d s i z e i n t e r s p e r s e d between myelinated f i b r e s ( C a j a l , 1909). Lamina IV neuronal d e n d r i t e s are o r i e n t a t e d l o n g i t u d i n a l l y , whereas the d e n d r i t e s of neurones i n lamina V are o r i e n t a t e d i n the d o r s o v e n t r a l plane ( B e t h e l y i and Sz e n t a g o t h a i , 1973)., The d o r s a l d e n d r i t e s of many of these neurones, e s p e c i a l l y those i n lamina IV, penetrate i n t o the s u b s t a n t i a g e l a t i n o s a and t h e r e f o r e probably r e c e i v e a f f e r e n t i n p u t s i m i l a r t o t h a t of s u b s t a n t i a g e l a t i n o s a neurones (Szentagothai, 1964; B e t h e l y i and Sz e n t a g o t h a i , 1973). These neurones are termed antenna neurones and are suggested t o be the major neuronal outflow of the s u b s t a n t i a g e l a t i n o s a ( B e t h e l y i and S z e n t a g o t h a i , 1973). 9 Becent HBP s t u d i e s have r e p o r t e d n o c i c e p t i v e a f f e r e n t f i b r e s t e r m i n a t i n g i n these laminae. L i g h t and P e r l {1979a,b) r e p o r t that mechanical n o c i c e p t i v e s l o w l y conducting a f f e r e n t f i b r e s end i n lamina V, and B e t h e l y i e t a l . (1979) r e p o r t t h a t s m a l l to medium a f f e r e n t s responding to mechanical noxious s t i m u l i end i n the l a t e r a l p a r t s of lamina V. Many neurones i n these laminae are wide-dynamic range neurones but narrow range neurones responding t o j u s t innocuous or j u s t noxious s t i m u l i have a l s o been r e p o r t e d ( H a l l , 1960, 1967; Mendell, 1966; W i l l i s e t a l . , 1974; F i e l d s e t a l . , 1977a) . . The axonal p r o j e c t i o n s of these laminae have been suggested to c o n t r i b u t e to the s p i n o t h a l a m i c , s p i n o r e t i c u l a r , s p i n o c e r v i c a l , and p r o p r i o s p i n a l systems (Hexed, 1952; B e t h e l y i and S z e n t a g o t h a i , 1973). Indeed, a n t i d r o m i c a c t i v a t i o n from the brainstem ( W i l l i s et a l . , 1974; G i e s l e r et a l . , 1977; F i e l d s e t a l . , 1977b) and HBP s t u d i e s i n the r a t ( G i e s l e r e t a l . , 1977) and the monkey ( H i l l i s e t a l . , 1979) both i n d i c a t e t h a t these laminae are the o r i g i n of the s p i n o t h a l a m i c and s p i n o r e t i c u l a r t r a c t s . Ascending... N o c i c e p t i v e Pathways Many ascending pathways e x i s t i n the s p i n a l c o r d which convey n o c i c e p t i v e i n f o r m a t i o n . In man, the major pathway f o r pain i s the c r o s s e d s p i n o t h a l a m i c t r a c t (Kuru, 1949; White and Sweet, 1969), however, other pathways are undoubtedly i n v o l v e d because s u r g i c a l procedures which l e s i o n t h i s t r a c t produce only temporary c e s s a t i o n of p a i n s e n s a t i o n (White and Sweet, 1969). There i s a l s o a wide v a r i a t i o n i n the d i s t r i b u t i o n of 10 n o c i c e p t i v e pathways among s p e c i e s . For i n s t a n c e , the cat has a r e l a t i v e l y poorly developed s p i n o t h a l a m i c t r a c t . The f o l l o w i n g s e c t i o n s w i l l d i s c u s s the major n o c i c e p t i v e pathways i n g r e a t e r d e t a i l . The ..Spinothalamic T r a c t The s p i n o t h a l a m i c t r a c t i s the c l a s s i c a l pathway f o r c a r r y i n g pain i n man. Neurones of o r i g i n f o r t h i s t r a c t are l o c a t e d i n the gray matter of the s p i n a l c o r d . . T h e i r axons c r o s s the s p i n a l cord v i a the v e n t r a l white commissure and run r o s t r a l l y i n the v e n t r o l a t e r a l quadrants. although there appears t o be a d i f f e r e n c e i n the route taken by the l a t e r a l versus the v e n t r a l s p i n o t h a l a m i c t r a c t s , t h e i r f i n a l d e s t i n a t i o n s i n the thalamus appear t o be i d e n t i c a l i n the monkey (Kerr, 1975b). These two t r a c t s terminate i n the v e n t r o b a s a l , p o s t e r i o r , and i n t r a l a m i n a r n u c l e i of the thalamus i n the monkey (Jiehler et a l . , 1960; Kerr and Lippman, 1974; B o i v i e , 1979) and the c a t (Holloway e t a l . , 1978; Fox e t a l . , 1980). The i n t r a l a m i n a r t e r m i n a t i o n i s suggestive of a f u n c t i o n a l r o l e i n a r o u s a l mechanisms. The o r i g i n of the s p i n o t h a l a m i c t r a c t has been s t u d i e d e x t e n s i v e l y with r e t r o g r a d e and e l e c t r o p h y s i o l o g i c a l methods. The r e t r o g r a d e s t u d i e s i n d i c a t e the g r e a t e s t c o n c e n t r a t i o n of s p i n o t h a l a m i c t r a c t neurones i n monkeys and r a t s t o be i n the d o r s a l horn laminae I , IV, and V, with l e s s e r c o n c e n t r a t i o n s i n the more v e n t r a l p a r t s of the gray matter (Kerr, 1975b; T r e v i n o and C a r s t e n s , 1975; C a r s t e n s and T r e v i n o , 1978; G i e s l e r e t a l . , 1977). However, HHP s t u d i e s i n the c a t i n d i c a t e d most 11 s p i n o t h a l a m i c t r a c t neurones o f o r i g i n were i n the v e n t r a l horn (Trevino and C a r s t e n s , 1975). S i m i l a r l y , e l e c t r o p h y s i o l o g i c a l s t u d i e s i n v o l v i n g the a n t i d r o m i c a c t i v a t i o n of s p i n a l cord neurones by e l e c t r i c a l s t i m u l a t i o n of the s p i n o t h a l a m i c t r a c t at e i t h e r i t s terminus o r at more caudal s i t e s s u b s t a n t i a t e the retrogade f i n d i n g s i n the cat f D i l l y et a l . , 1968; T r e v i n o et a l . , 1972), r a t ( D i l l y e t a l . , 1968; G i e s l e r et a l . , 1977), and the monkey (Trevino e t a l . , 1973; Albe-Fessard e t a l . , 1974; W i l l i s e t a l . , 1974). Spinothalamic t r a c t neurones have been shown t o respond to non-noxious and/or noxious s t i m u l i . E f f e c t i v e noxious s t i m u l i i n c l u d e mechanical, thermal, and chemical a l g e s i c s , , N o c i c e p t i v e - s p e c i f i c s p i n o t h a l a m i c neurones are l o c a t e d mainly i n lamina I, but some occur i n the deeper laminae ( D i l l y e t a l . , 1968; T r e v i n o and C a r s t e n s , 1975; W i l l i s e t a l . , 1974; Kumazawa et a l . , 1975; Levante e t a l . , 1975). Foreman e t a l . (1979) r e p o r t t h a t mechanical and a l g e s i c c h e m i c a l (bradykinin,. s e r o t o n i n , and KC.1) s t i m u l a t i o n of f i n e muscle af f e r e n t s e x c i t e d many s p i n o t h a l a m i c t r a c t neurones but d i d not a f f e c t those i n lamina I. Most s p i n o t h a l a m i c neurones are r e p o r t e d t o be wide-dynamic range neurones, t h a t i s , they respond t o both innocuous and noxious s t i m u l i ( H i l l i s e t a l . , 1974; Albe-Fessard, 1974; McCreery and B l o e d e l , 1975)., 12 The S p i n o r e t i c u l a r T r a c t The s p i n o r e t i c u l a r t r a c t a r i s e s i n the s p i n a l cord gray matter and p r o j e c t s i p s i l a t e r a l l y t o the brainstem i n the l a t e r a l and v e n t r a l f u n i c u l i . I t i s d i v i d e d i n t o two p a r t s : the f i r s t t e r m i n a t e s i n the l a t e r a l r e t i c u l a r nucleus and i s p a r t of the s p i n o r e t i c u l a r c e r e b e l l a r pathway (Oscarrson, 197 3}; the second p a r t t e r m i n a t e s i n the medial r e t i c u l a r formation and w i l l be d e a l t with f u r t h e r below.. The t e r m i n a t i o n s i n the medial r e t i c u l a r f o r m a t i o n i n c l u d e the nucleus medullae oblongatae c e n t r a l i s , nucleus r e t i c u l a r i s g i g a n t o c e l l u l a r i s (nGC), n u c l e i r e t i c u l a r i s p o n t i s c a u d a l i s and o r a l i s , n u c l e i p a r a g i g a n t o c e l l u l a r i s d o r s a l i s and l a t e r a l i s , nucleus s u b c o e r u l e u s , and n u c l e i raphe p a l l i d a s and magnus (Healer .et a l . , 1960; Bowsher and Westman, 1970; K e r r , 1975b; Br o d a l et a l . , 1960). Although t h i s pathway i s r e p o r t e d t o be uncrossed by s t u d i e s i n v o l v i n g commissural myelotomy i n the monkey (Kerr and Lippman* 1974), i t appears to be b i l a t e r a l i n the c a t ( F i e l d s et a l . , 1975, 1977b). Antidromic a c t i v a t i o n of s p i n o r e t i c u l a r t r a c t neurones by s t i m u l a t i o n of the r e t i c u l a r formation i n d i c a t e s a c o n c e n t r a t i o n o f c e l l s of o r i g i n i n laminae VI to VIII of the s p i n a l cord (levante and A l b e - F e s s a r d , 1972; F i e l d s e t a l . , 1975, 1977b). F i e l d s et a l . (1975, 1977b) noted that s t i m u l a t i o n i n the nGC r e s u l t e d i n the a n t i d r o m i c a c t i v a t i o n of neurones i n the more s u p e r f i c i a l l a y e r s of the d o r s a l horn, p a r t i c u l a r l y lamina I . The neurones p r o j e c t i n g to the nGC o f t e n only responded to s t i m u l a t i o n of deep s t r u c t u r e s , although some responded to both innocuous and noxious or only noxious cutaneous s t i m u l i ( F i e l d s 13 e t a l . , 1975). These neurones d i s p l a y e d c o n t r a - , i p s i - , and b i l a t e r a l r e c e p t i v e f i e l d s . The neurones responding t o noxious s t i m u l i were o f t e n l o c a t e d i n the v e n t r a l horn i n laminae IV to IX ( F i e l d s et a l . , 197 5). The S p i n o c e r v i c o t h a l a m i c T r a c t The s p i n o c e r v i c a l t r a c t (SCT) ascends i p s i l a t e r a l l y i n the d o r s o l a t e r a l f a s c i c u l u s of the s p i n a l c o r d to t erminate i n the l a t e r a l c e r v i c a l nucleus ( C a j a l , 1909; Ha and L i u , 1966).. Axonal p r o j e c t i o n s from the l a t e r a l c e r v i c a l nucleus (LCN) decussate and ascend t o the v e n t r o p o s t e r o l a t e r a l , p o s t e r i o r , and medial g e n i c u l a t e n u c l e i of the thalamus ( B o i v i e , 1970). Hexed (1951) i d e n t i f i e d the l a t e r a l c e r v i c a l nucleus i n a wide range of s p e c i e s , i n c l u d i n g the c a t and monkey, but concluded t h a t i t does not e x i s t as such i n the r a t . Recent HBP s t u d i e s by G i e s l e r et a l . (1979) have demonstrated a d i f f u s e group of c e l l s i n the r a t which i s s h i f t e d somewhat d o r s a l l y and l a t e r a l l y from the p o s i t i o n of the w e l l - d e f i n e d LCN of the c a t . T h i s c e l l group i n the r a t may be e q u i v a l e n t t o t h e LCN as i t responds t o s i m i l a r s t i m u l i . . However, G i e s l e r e t a l . (1979) noted t h a t the conduction v e l o c i t y f o r the c e r v i c o - t h a l a m i c t r a c t i n the r a t was about a q u a r t e r of t h a t i n the c a t and they suggested t h a t t h i s pathway may serve a d i f f e r e n t f u n c t i o n from t h a t i n the c a t . The c e l l s of o r i g i n of the s p i n o c e r v i c a l t r a c t i n the c a t and monkey have been l o c a t e d both e l e c t r o p h y s i p l o g i c a l l y (Bryan et a l . , 1973; Brown e t a l . , 1976; Cervero et a l . , 1977) and by l a b e l l i n g s t u d i e s using p r o c i o n yellow or HEP ( C r a i g , 1976; Snow 14 e t a l . , 1976). Most c e l l s bodies were l o c a t e d i n lamina IV, with some a l s o i n laminae I and V t o V I I I . . Most SCT neurones responded to A f i b r e s t i m u l a t i o n with or without responding to C f i b r e s t i m u l a t i o n (Mendell, 1966; Brown e t a l . , 1973a, 1975). Some s p i n o c e r v i c a l t r a c t neurones a l s o respond to group I I I and IV muscle a f f e r e n t s t i m u l a t i o n { K n i f f i et a l . , 1977; Hamann et a l . , 1978). Most SCT neurones respond t o h a i r movement, but many a l s o respond t o noxious mechanical, thermal, or a l g e s i c c hemical s t i m u l i (Brown e t a l . , 1975; Cervero e t a l . , 1977; K n i f f i et a l . , 1977; G i e s l e r e t a l . , 1979), The S p i n o t e c t a l T r a c t The term s p i n o t e c t a l t r a c t i s a p p l i e d g e n e r a l l y t o s p i n a l cord p r o j e c t i o n s to the midbrain which ascend i n the v e n t r a l quadrants (Anderson and Berry, 1959; Mehler e t a l . , 1960),, T h i s t r a c t t e r m i n a t e s i n the s u p e r i o r c o l l i c u l u s , the e x t e r n a l nucleus of the i n f e r i o r c o l l i c u l u s , the p e r i a q u e d u c t a l gray matter, and the midbrain r e t i c u l a r f o r m a t i o n (Anderson and Berry, 19 59; Mehler e t a l . , 1960; Schroeder and Jane, 1971; Kerr, 1975b). T r e v i n o (1976) r e p o r t e d c e l l s o f o r i g i n i n laminae I and IV to VII i n the monkey. Other s p e c i e s have not been s t u d i e d but i t i s assumed t h a t the o r i g i n of t h i s t r a c t i s s i m i l a r t o t h a t o f the s p i n o t h a l a m i c and s p i n o r e t i c u l a r t r a c t s . No r e c o r d i n g s from s p i n o t e c t a l t r a c t neurones have been done but responses of midbrain r e t i c u l a r and PAG neurones suggest t h a t t h i s pathway i s a c t i v a t e d best by A - d e l t a and C f i b r e s , with a d d i t i o n a l i n p u t from A-alpha and A-beta f i b r e s and group I I and I I I muscle a f f e r e n t f i b r e s ( C o l i n s and Handt, 1960; Pompieano 15 and S s e t t , 1963) . C. Mesencephalic N u c l e i The P e r i a q u e d u c t a l Gray Matter The p e r i a q u e d u c t a l gray matter (PAG) c o n s i s t s of a volume of f a i r l y s m a l l neurones f r e e from myelinated f i b r e s t h a t surrounds the agueduct o f S y l v i u s . I t c o n t a i n s a high d e n s i t y o f o p i a t e r e c e p t o r s (Pert and Yaksh, 1974) and has been i m p l i c a t e d i n a n t i n o c i c e p t i o n . A n a l g e s i a has been induced both by e l e c t r i c a l s t i m u l a t i o n of and by m i c r o i n j e c t i o n of o p i a t e s and o p i o i d s i n t o the PAG {Reynolds, 1969; Mayer e t a l , 1971; Pert and Yaksh, 1974; Yaksh et a l . , 1976a-c). The PAG r e c e i v e s p e r i p h e r a l sensory input e i t h e r d i r e c t l y or v i a other brainstem n u c l e i . Observation of denervation o f the PAG a f t e r cordotomies suggests that the PAG r e c e i v e s ascending a f f e r e n t s d i r e c t l y v i a the s p i n o t e c t a l t r a c t {Mehler e t al», 1960; Kerr, 1975b). The r e c o r d i n g of evoked p o t e n t i a l s a l s o i m p l i e s a p e r i p h e r a l sensory i n p u t t o the PAG. L i e b e s k i n d and Mayer (1971) repo r t e d that s t i m u l a t i o n of the s k i n of any limb, the t a i l , or the f a c e caused s h o r t l a t e n c y evoked p o t e n t i a l s i n the PAG of r a t s under deep Nembutal a n a e s t h e s i a . S i m i l a r l y , r e c o r d i n g s made i n the PAG of c u r a r i z e d , a r t i f i c i a l l y r e s p i r a t e d r a t s showed evoked p o t e n t i a l s from e l e c t r i c a l s t i m u l a t i o n of the s c i a t i c nerve (Palmer and Klemm, 1976). The PAG a l s o r e c e i v e s a f f e r e n t s from other b r a i n a r e a s . B e i t z (1979) r e p o r t s a major a f f e r e n t i n p u t t o the PAG from the hypothalamus, with a d d i t i o n a l minor p r o j e c t i o n s from the c a u d a l 16 raphe. The p e r i a q u e d u c t a l gray matter has axonal p r o j e c t i o n s both r o s t a l l y to the diencephalon and c a u d a l l y to the brainstem...„ a u t o r a d i o g r a p h i c a l s t u d i e s i n d i c a t e p r o j e c t i o n s t o the r e t i c u l a r f ormation and the nucleus raphe magnus (Ruda, 1975). Hor s e r a d i s h peroxidase (HBP) s t u d i e s by G a l l a g e r and P e r t (197 8) r e p o r t p r o j e c t i o n s to the nucleus r e t i c u l a r i s g i g a n t o c e l l u l a r i s as well as to the c a u d a l raphe. E l e c t r o p h y s i o l o g i c a l s t u d i e s have a l s o been completed which i n d i c a t e input t o the nucleus raphe magnus from the PAG. Pomeroy and Behbehani (1979a,b) e l e c t r i c a l l y s t i m u l a t e d i n the PJG and recorded a c t i v a t i o n of s i n g l e u n i t s i n the nucleus raphe magnus. although t h i s study could not i n d i c a t e i f t h i s was a d i r e c t pathway, a n t i d r o m i c a c t i v a t i o n of PAG neurones by s t i m u l a t i o n of the nucleus raphe magnus i n d i c a t e there i s a monosynaptic conne c t i o n (Dostrovsky and Shah, 1979). Although e l e c t r i c a l s t i m u l a t i o n of the PAG has been repo r t e d t o i n h i b i t s p i n a l c o r d neurones, the PAG has not been r e p o r t e d t o have d i r e c t p r o j e c t i o n s to the cord by e i t h e r d e n e r v a t i o n s t u d i e s (Nyberg-Hansen, 1965), HRP s t u d i e s (Kuypers and Haisky, 1975; Basbaum and F i e l d s , 1977b), o r a u t o r a d i o g r a p h i c a l s t u d i e s (Buda, 1975)..,, There have been numerous r e p o r t s of e l e c t r i c a l s t i m u l a t i o n of the PAG causing a n a l g e s i a . The f i r s t r e p o r t of PAG s t i m u l a t i o n produced a n t i n o c i c e p t i o n (SPA) was p u b l i s h e d by D.. V. Reynolds i n 1969. He reported t h a t laparotomies c o u l d be performed on unanaesthetized r a t s without any o v e r t r e a c t i o n s t o the procedure while the PAG was being f o c a l l y s t i m u l a t e d . A n t i n o c i c e p t i o n induced by s t i m u l a t i o n i n the PAG has been found 17 by other workers i n the r a t (Mayer et a l . r 1971; A k i l and Mayer, 1972; Mayer and L i e b e s k i n d , 1974; Mayer and Hayes, 1975; A k i l et a-l..-,' 1976; Oleson e t a l . , 1978), i n the c a t {Liebeskind et a l . , 1973; O l i v e r a s e t a l . , 1974a,b), and i n other s p e c i e s i n c l u d i n g man (Richardson and A k i l , 1977a,b). P e r i a q u e d u c t a l gray SPA has been shown t o reduce both the r e f l e x and b e h a v i o u r a l responses to noxious s t i m u l i . S t i m u l a t i o n of the PAG i n c r e a s e s the l a t e n c y of the t a i l - f l i c k r e f l e x ( A k i l and Mayer, 1972; Mayer and L i e b e s k i n d , 1974; A k i l et a l . , 1976; faksh e t a l . , 1976c), the t h r e s h o l d f o r jump t o e l e c t r i c shock ( Mayer and L i e b e s k i n d , 1974), l e g f l e x i o n to footpad shock (Oleson e t a l . , 1978), and the jaw-opening r e f l e x t o tooth pulp s t i m u l a t i o n ( O l i v e r a s et a l . , 1974b)., The p e r i a q u e d u c t a l gray SPA i s r e p o r t e d to i n c l u d e l a r g e areas of the body, u s u a l l y at l e a s t one guadrant and o f t e n the whole body (Mayer e t a l . , 1971; L i e b e s k i n d e t a l . , 1973; Richardson and A k i l , 1977b). The a n a l g e s i a produced i s approximately e q u i v a l e n t to t h a t produced by 10 - 50 mg/kg of morphine admi n i s t e r e d i n t r a v e n o u s l y (Mayer and L i e b e s k i n d , 1974; Oleson et a l , 1978) . While t h i s amount of morphine i s s u f f i c i e n t t o produce obvious s i d e e f f e c t s , such as extensor r i g i d i t y , the a c t i o n of PAG s t i m u l a t i o n appears t o be s p e c i f i c f o r a n a l g e s i a . During s t i m u l a t i o n , animals respond normally t o f r i g h t evoking s t i m u l i or to noxious s t i m u l i a p p l i e d to non-a n a l g e s i c areas of the body (Mayer et a l , 1971; Mayer and L i e b e s k i n d , 1974). However, i n man s t i m u l a t i o n of the PAG produces s u f f i c i e n t s i d e e f f e c t s t o d i s a l l o w i t s c h r o n i c use. More r o s t r a l a r e a s , such as the p e r i v e n t r i c u l a r gray, are 18 u s u a l l y used (Bichardson and A k i l , 1977a). In r a t s and c a t s the best area of the p e r i a q u e d u c t a l gray f o r SPA i n terms of e f f i c a c y and l a c k of s i d e e f f e c t s i s r e p o r t e d t o be the v e n t r a l PAG, i n or around the d o r s a l raphe* nucleus. S t u d i e s have been done to examine the e f f e c t s of PAG s t i m u l a t i o n on s i n g l e and m u l t i p l e u n i t s i n the c e n t r a l nervous system, S t i m u l a t i o n of the PAG (excluding the d o r s a l raphe) has been repo r t e d to reduce noxious s t i m u l i evoked p o t e n t i a l s i n the d o r s a l and median raphe n u c l e i and the nucleus raphe magnus (Oleson e t a l , 1978). S i m i l a r l y , 55% of a l l medial r e t i c u l a r neurones (probably i n the nucleus r e t i c u l a r i s g i g a n t o c e l l u l a r i s ) which were d r i v e n by t a i l shock and 40X of neurones that responded to noxious s t i m u l i were r e p o r t e d to be i n h i b i t e d by PAG s t i m u l a t i o n (Morrow and Casey, 1576). Mohrland and Gebhart (1979) a l s o r e p o r t e d the i n h i b i t i o n of nGC neurones by PAG s t i m u l a t i o n , S i m i l a r l y , neurones i n the d o r s a l horn of the s p i n a l c o r d responding to noxious s t i m u l i have been reported t o be i n h i b i t e d by PAG s t i m u l a t i o n . Guilbaud e t a l . , (1973a) r e p o r t e d that PAG s t i m u l a t i o n i n h i b i t e d most lamina V neurones, but was without e f f e c t on the m a j o r i t y of lamina IV neurones. Carstens e t a l . (1979a,b) found t h a t neurones responding t o noxious r a d i a n t heat i n or near lamina V were i n h i b i t e d by s t i m u l a t i o n of the PAG. T h i s i n h i b i t i o n has been repo r t e d to be s e l e c t i v e on wide-dynamic range neurones over neurones responding o n l y t o non-noxious s t i m u l i (Bennett and Mayer, 1979). C o n v e r s e l y , Duggan and G r i e r s m i t h (1979) r e p o r t t h a t s t i m u l a t i o n near the d o r s a l raphe nucleus i n h i b i t s e x c i t a t i o n of lamina I , IV, and V d o r s a l 19 horn neurones by both noxious and non-noxious s t i m u l i . although PAG s t i m u l a t i o n c l e a r l y a f f e c t s s p i n a l cord neurones as noted above, the PAG has no r e p o r t e d d i r e c t c o n n e c t i o n s to the c o r d . T h e r e f o r e , these e f f e c t s are probably mediated by ether brainstem n u c l e i . , For t h i s r e a s o n , the e f f e c t of the PAG s t i m u l a t i o n on l i k e l y c a n d i d a t e n u c l e i has been s t u d i e d e x t e n s i v e l y . The most o f t e n c i t e d candidate f o r t h i s mediatory nucleus i s the nucleus raphe" magnus. Most s t u d i e s have found t h a t PAG s t i m u l a t i o n f a c i l i t a t e s spontaneous raphe magnus neuronal f i r i n g ( f i e l d s and Anderson, 1978; Lovick et a l . , 1978a; Oleson et a l . , 1978) but some s t u d i e s have found i n h i b i t i o n or mixed e f f e c t s upon the spontaneous f i r i n g r a t e (Mohrland and Gebhart, 1979; Pomeray and Behbehani, 1979a,b), S i m i l a r l y , another c a n d i d a t e which may mediate p e r i a q u e d u c t a l gray SPA i s the nucleus g i g a n t o c e l l u l a r i s . Neurones w i t h i n t h i s nucleus are r e p o r t e d to be e x c i t e d , i n h i b i t e d , or both by PAG s t i m u l a t i o n (Morrow and Casey, 1976; Mohrland and Gebhart, 1979) , P e r i a q u e d u c t a l qray SPA has been antagonized by both l e s i o n s and pharmacological m a n i p u l a t i o n s . / S t u d i e s have demonstrated t h a t a n a l g e s i a due to s t i m u l a t i o n of PAG s i t e s i n the r a t i s t o t a l l y a b o l i s h e d i n the i p s i l a t e r a l h i n d l i m b c a u d a l to a d o r s o l a t e r a l f u n i c u l u s l e s i o n (Basbaum et a l . , 1976b). In t r a v e n o u s l y administered naloxone has been r e p o r t e d both to i n h i b i t and to f a i l t o i n h i b i t PAG s t i m u l a t i o n produced a n t i n o c i c e p t i o n . A k i l e t a l . (1976) rep o r t e d p a r t i a l blockade by naloxone of the i n c r e a s e d t a i l - f l i c k l a t e n c y caused by PAG s t i m u l a t i o n . Another study found t h a t naloxone p a r t i a l l y 20 reversed the PAG s t i m u l a t i o n produced r e d u c t i o n of l e g f l e x i o n and electrcmyograph changes due t o f o o t shock or pinch (Oleson et a l . , 1978). These s t u d i e s suggest t h a t p e r i a q u e d u c t a l gray SPA i s mediated by an endogenous o p i o i d system. However, other s t u d i e s have found no e f f e c t o f naloxone on PAG s t i m u l a t i o n induced i n c r e a s e s i n t a i l - f l i c k l a t e n c i e s or reduced b e h a v i o u r a l responses t o f o o t pinch (Yaksh e t a l . , 1976c), nor PAG i n h i b i t i o n of d o r s a l horn neurones e x c i t a t e d by noxious r a d i a n t heat (Carstens et a l , 1979a). Manipulation of monoamine l e v e l s a f f e c t s the e f f i c a c y of p e r i a q u e d u c t a l gray SPA. A k i l and L i e b e s k i n d (1975) r e p o r t e d t h a t r a t t a i l - f l i c k l a t e n c y i s i n c r e a s e d by: e l e v a t i o n of s e r o t o n i n or dopamine l e v e l s ; d e p l e t i o n of n o r a d r e n a l i n e l e v e l s ; or a d m i n i s t r a t i o n o f a dopamine a g o n i s t . , Conversely, the SPA was reduced by d e p l e t i o n of s e r o t o n i n or blockade of dopamine with an a n t a g o n i s t . L y s e r g i c a c i d d i e t h y l a m i d e (LSD), which presumably a l t e r s s e r o t o n i n s y n a p t i c t r a n s m i s s i o n although the mechanism i s u n c l e a r , has been r e p o r t e d t o block the i n h i b i t i o n of s p i n a l cord n o c i c e p t o r - d r i v e n neurones produced by s t i m u l a t i o n near the d o r s a l raphe (Guilbaud e t a l . , 1973a). Increased t a i l - f l i c k l a t e n c i e s i n r a t s due to PAG s t i m u l a t i o n have a l s o been found to be antagonized by LSD suggesting t h a t p e r i a q u e d u c t a l gray SPA i s mediated by s e r o t o n i n and/or dopamine. As w e l l as p e r i a q u e d u c t a l gray SPA t h e r e have been many r e p o r t s t h a t m i c r o i n j e c t i o n of drugs i n t o the PAG produces a n a l g e s i a . The most commonly s t u d i e d drug i s morphine and m i c r o i n j e c t i o n s t u d i e s have been done i n the mouse ( C r i s w e l l , 21 1976; Yaksh e t a l , , 1976b), the r a t (Sharpe et a l , , 1974; Lewis and Gebhart, 1977; Yaksh and Tyce, 1979; Levy and P r o u d f i t , 1979) and primates (Pert and Yaksh, 1974),/ As with p e r i a q u e d u c t a l gray SPA, m i c r o i n j e c t i o n - p r o d u c e d a n t i n o c i c e p t i o n can cause whole body or p a r t i a l body a n a l g e s i a and i s r e p o r t e d not to have s i d e e f f e c t s such as h y p e r r e a c t i v i t y (Yaksh and Tyce, 1979), The f i n d i n g s that there are o p i a t e r e c e p t o r s i n the PAG and t h a t the a n a l g e s i a i s a s t e r e o s p e c i f i c e f f e c t i n d i c a t e t h a t the a n a l g e s i a i s due to a s p e c i f i c i n t e r a c t i o n with o p i a t e r e c e p t o r s . For example, dextrorphan i s i n e f f e c t i v e i n producing a n a l g e s i a when m i c r o i n j e c t e d i n t o the p e r i a q u e d u c t a l gray (Pert and Yaksh, 1974), Again, the e f f e c t appears t o be s p e c i f i c , l i k e SPA, to a n t i n o c i c e p t i o n , In monkeys t r a i n e d t o d i s c r i m i n a t e between two a v e r s i v e e l e c t r o c u t a n e o u s s t i m u l i , m i c r o i n j e c t e d morphine produced a r e d u c t i o n i n p e r c e i v e d pain without reducing the monkeys' d i s c r i m i n a t i v e a b i l i t i e s (Bennett e t a l . , 1976), L i k e PAG s t i m u l a t i o n s t u d i e s , the e f f e c t of morphine m i c r o i n j e c t i o n has been s t u d i e d on s i n g l e u n i t s . Nucleus g i g a n t o c e l l u l a r i s and nucleus raphe magnus neuronal responses to noxious s t i m u l i are reduced by morphine m i c r o i n j e c t i o n i n t o the PAG (Mohrland and Gebhart, 1979). Bennett and Mayer (1976, 1979) r e p o r t e d that 55% of wide-dynamic range s p i n a l cord neurones responding t o noxious s t i m u l i were a l s o i n h i b i t e d by morphine i n j e c t e d i n t o the PAG. M i c r o i n j e c t i o n of morphine i n t o the PAG a l s o i n c r e a s e s the spontaneous f i r i n g of neurones i n the nucleus raphe magnus (Behbehani and Pomeroy, 1978; Behbehani, 1979; Mohrland and Gebhart, 1979) and nucleus g i g a n t o c e l l u l a r i s 22 {Mohrland and Gebhart, 1979). As with PAG s t i m u l a t i o n produced a n t i n o c i c e p t i o n , morphine m i c r o i n j e c t i o n a n a l g e s i a can be antagonized by l e s i o n s or chemical manipulations, Murfin e t a l . (1976) r e p o r t e d d o r s o l a t e r a l f u n i c u l a r l e s i o n s a b o l i s h e d the len g t h e n i n g o f t a i l -f l i c k l a t e n c i e s by m i c r o i n j e c t e d morphine* The i n h i b i t i o n of s p i n a l c o r d n o c i c e p t o r - d r i v e n neurones by morphine m i c r o i n j e c t i o n i n t o the PAG was r e v e r s e d by i n t r a v e n o u s l y a d m i n i s t e r e d naloxone (Bennett and Mayer, 1979). As w e l l , c i n a n s e r i n and methysergide, two p u t a t i v e s e r o t o n i n a n t a g o n i s t s , are r e p o r t e d t o antagonize a n a l g e s i a due t o PAG m i c r o i n j e c t e d morphine (Ifaksh e t a l . , 1976a) . M i c r o i n j e c t i o n o f other drugs i n t o the PAG has been r e p o r t e d t o produce a n a l g e s i a . „• The n a r c o t i c e t o r p h i n e i s more potent than morphine and i t i n h i b i t e d 801 of s p i n a l c o r d neurones e x c i t e d by noxious s t i m u l i (Bennett and Mayer, 1979). B a c l o f e n a l l e g e d l y i n c r e a s e s t a i l - f l i c k l a t e n c i e s i n r a t s when m i c r o i n j e c t e d i n t o the PAG (Levy and P r o u d f i t , 1979) and glutamate i s re p o r t e d to i n c r e a s e the t h r e s h o l d f o r f o o t pad shock evoked l e g f l e x i o n (Behbehani and F i e l d s , 1979). T h i s e f f e c t of glutamate was rev e r s e d by i n t r a v e n o u s l y a d m i n i s t e r e d naloxone or by nucleus raphe magnus l e s i o n s . . M i c r o i n j e c t i o n o f glutamate i n t o the PAG a l s o f a c i l i t a t e d the spontaneous f i r i n g of the m a j o r i t y of nucleus raphe magnus neurones. M i c r o i o n t o p h o r e s i s of morphine onto PAG neurones was re p o r t e d t o i n h i b i t about one-half and t o e x c i t e about o n e - f i f t h of the neurones t e s t e d while e t o r p h i n e i n h i b i t e d f o u r - f i f t h s o f the neurones t e s t e d ( U o l s t e n c r o f t e t a l . , 1977). However, none 23 of these e f f e c t s were blocked by naloxone suggesting a non-s p e c i f i c mode of a c t i c n f o r these two drugs. In agreement with these r e s u l t s , H a i g l e r (1978) r e p o r t e d t h a t i o n t o p h o r e s i s of morphine onto d o r s a l and medial raphe neurones i n h i b i t e d 34$ o f the neurones. However, t h i s i n h i b i t i o n was a l s o not s t e r e o s p e c i f i c and was r a r e l y blocked by naloxone. L e s i o n s of the p e r i a q u e d u c t a l gray were found by Deakin and Dostrovsky (1978) to reduce the le n g t h e n i n g of t a i l - f l i c k and hot p l a t e t e s t l a t e n c i e s due t o i n t r a v e n o u s l y administered morphine.. S i m i l a r l y , l e s i o n s of the cau d a l PAG i n c r e a s e d t h r e s h o l d s f o r caudal thalamus SPA and decreased the b a s e l i n e l a t e n c i e s f o r the t a i l - f l i c k r e f l e x (Rhodes and L i e b e s k i n d , 1978). The D o r s a l and Medial Baphe The d o r s a l raphe* nucleus (DB) and the medial raphe nucleus (MR) are two m i d l i n e n u c l e i i n the mesencephalon. , The d o r s a l raphe i s c o n t a i n e d i n the v e n t r a l most part of the p e r i a q u e d u c t a l gray; the medial raphe i s l o c a t e d v e n t r a l l y t o the d o r s a l raphe and runs c a u d a l l y from about the l e v e l of the PAG. Both n u c l e i c o n t a i n many s e r o t o n e r g i c neurones (Dahlstrom and Fuxe, 1964; P o i t r a s and Parent, 1978) but they a l s o c o n t a i n c a t e c h o l a m i n e r g i c neurones. C e l l bodies and axonal t e r m i n a t i o n s c o n t a i n i n g met-enkephalin were observed w i t h i n these n u c l e i and d e s c r i b e d by H o k f e l t e t a l . (1S77). Do r s a l raphe neurones r e c e i v e a f f e r e n t i n p u t from other d o r s a l raphe neurones and medial raph£ neurones (Mosko e t a l . , 1977). T h i s n u c l e u s i s r e p o r t e d to a l s o r e c e i v e i n p u t from the 24 s p i n a l c o r d , although i t may not be d i r e c t . Aghajanian e t a l . (1978) r e p o r t t h a t low frequency p e r i p h e r a l nerve s t i m u l a t i o n r e s u l t s i n i n h i b i t i o n of neurones i d e n t i f i e d as s e r o t o n e r g i c but i n e x c i t a t i o n of those neurones which were non-se r o t o n e r g i c . S t i m u l a t i o n of t h e pontine r e t i c u l a r formation has been found to i n h i b i t d o r s a l raphe neurones (Wang e t a l . , 1976). B o b i l l i e r et a l . (1975, 1976) r e p o r t a low amount of l a b e l l i n g i n both n u c l e i when ** C - l e u c i n e was m i c r o i n j e c t e d i n t o the nucleus raphe c e n t r a l i s s u p e r i o r i n the r a t . These two n u c l e i predominantly send axonal f i b r e s , with l a r g e numbers of t e r m i n a l c o l l a t e r a l s , t o the d i e n c e p h a l i c and t e l e n c e p h a l i c f o r e b r a i n r e g i o n s v i a the medial f o r e b r a i n bundle. D e s t i n a t i o n s i n c l u d e the hypothalamus, s t r i a t u m , septum, and c o r t e x (Dahlstrom and Fuxe, 1964; Anden e t a l . , 1966; P i e r c e e t a l . , 1976).. Descending axonal p r o j e c t i o n s terminate i n the s u b s t a n t i a n i g r a (Dray e t a l . , 1976; P i e r c e e t a l . , 1976), the c a u d a l raphe, and the nucleus g i g a n t o c e l l u l a r i s (Conrad e t a l . , 1974; P i e r c e e t a l . , 1976; B o b i l l i e r e t a l . , 1976, 1979).; The two mesencephalic n u c l e i do not appear to have d i r e c t p r o j e c t i o n s t o the s p i n a l cord (Conrad, 1974). As t h e d o r s a l raphe i s so i n t i m a t e l y a s s o c i a t e d with the p e r i a q u e d u c t a l qray, and indeed i s p a r t of i t , most of the r e p o r t s d e a l i n g with the e f f e c t s of s t i m u l a t i o n of and m i c r o i n j e c t i o n i n t o the d o r s a l raphe were i n c l u d e d i n the s e c t i o n on the PAG. M i c r o i o n t o p h o r e t i c s e r o t o n i n was r e p o r t e d t o be i n h i b i t o r y on almost a l l raphe" neurones i n s t u d i e s by Bramwell and Gonye (1976). T h i s i s c o n t r a s t e d with the e x c i t a t o r y e f f e c t t h a t s e r o t o n i n had on the non-raphe neurones 25 i n these s t u d i e s , L e s i o n i n g i n the v i c i n i t y of the d o r s a l raphe was r e p o r t e d to reduce the e f f i c a c y of i n t r a v e n o u s l y administered morphine a n a l g e s i a (Samanin e t a l . , 1970)., A r e c e n t study by Chance et a l . (1978) found morphine a n a l g e s i a was a l s o antagonized by l e s i o n i n g of the medial raphe nu c l e u s . However, i n the same study, methadone-induced a n a l g e s i a was u n a f f e c t e d by s i m i l a r l e s i o n s , suggesting t h a t methadone has a d i f f e r e n t s i t e of a c t i o n . D. Medullary N u c l e i The Nucleus R e t i c u l a r i s G i g a n t o c e l l u l a r i s The nucleus r e t i c u l a r i s g i g a n t o c e l l u l a r i s (nGC) i s a l a r g e i l l - d e f i n e d nucleus i n the medullary r e t i c u l a r formation l a t e r a l to the caudal raphe". As i t s name i m p l i e s , the c e l l s w i t h i n the nGC are very l a r g e . i t has been suggested t h a t the nGC may be a r e l a y nucleus f o r ascending i n f o r m a t i o n d e s t i n e d f o r the c e n t r e median nucleus of the thalamus (Bowsher et a l . , 1968). As the nGC a l s o p r o j e c t s t o the s p i n a l c o r d and has been shown to i n h i b i t s p i n a l cord neurones responding t o noxious s t i m u l i (McCreery and B l o e d e l , 1975), i t may a l s o be i n v o l v e d with descending i n h i b i t o r y c o n t r o l of n o c i c e p t i v e pathways at the s p i n a l c o r d l e v e l . L i k e the p e r i a q u e d u c t a l gray, the the nGC has been i m p l i c a t e d i n the mechanism of a n t i n o c i c e p t i o n by i t s responses t o noxious s t i m u l i and the e f f e c t s t h a t s t i m u l a t i o n of and m i c r o i n j e c t i o n of drugs i n t o the nGC have on the b e h a v i o u r a l and r e f l e x responses to noxious s t i m u l i . 26 Anatomical s t u d i e s i n v o l v i n g e i t h e r s p i n a l c o r d l e s i o n s , r e t r o g r a d e t r a n s p o r t of h o r s e r a d i s h peroxidase, or e l e c t r o p h y s i o l o g i c a l methods have demonstrated t h a t the nGC r e c e i v e s a f f e r e n t i n p u t from ascending sensory pathways. Mehler e t a l . (1960) documented the de n e r v a t i o n of b r a i n areas f o l l o w i n g l e s i o n s of the v e n t r o l a t e r a l guadrants of the s p i n a l c o r d and found dense t e r m i n a l degeneration i n the nGC.•, G a l l a g e r and P e r t (1978) iontophoresed h o r s e r a d i s h peroxidase i n t o the nGC and found HRP c o n t a i n i n g c e l l bodies i n the d o r s a l and v e n t r a l gray o f the c e r v i c a l s p i n a l c o r d . Antidromic a c t i v a t i o n of s p i n a l cord neurones by s t i m u l a t i o n of the nGC a l s o i n d i c a t e s an i n p u t t o the nGC from the c o r d . F i e l d s et a l . , (1975) s t i m u l a t e d the nGC of c a t s with b i p o l a r e l e c t r o d e s and recorded from s i n g l e u n i t s deep t o lamina V which c o u l d be a n t i d r o m i c a l l y a c t i v a t e d . These neurones e i t h e r responded to noxious s t i m u l a t i o n of deep s t r u c t u r e s or had l a r g e cutaneous f i e l d s which were r e s p o n s i v e to noxious s t i m u l i . S t u d i e s r e c o r d i n g from s i n g l e u n i t s i n the nGC a l s o support the e x i s t e n c e of noxious a f f e r e n t i n p u t t o t h i s n ucleus. Casey (1969) found t h a t t w o - t h i r d s of nGC neurones responding t o n a t u r a l s t i m u l i r e q u i r e d f i r m pinch or heavy pressure t o maximally a c t i v a t e them. / S i m i l a r l y , i n subsequent experiments 61% of nGC neurones responded t o noxious pinch or f o o t pad shock while the remainder of the u n i t s e i t h e r were non^responsive t o any s t i m u l i or responded to l i g h t touch (Keene and Casey, 1970). LeBlane and Gatipon (1974) repor t e d t h a t noxious e l e c t r i c a l or mechanical s t i m u l a t i o n of footpads caused most nGC neurones to i n i t i a l l y i n c r e a s e or decrease t h e i r f i r i n g r a t e , followed by a 27 prolonged a f t e r - d i s c h a r g e or an extended s i l e n t p e r i o d . S i m i l a r types of responses were reported when the s c i a t i c , r a d i a l , or t r i g e m i n a l (tooth pulp) nerves were s t i m u l a t e d ( P e a r l and Anderson, 1978). Most neurones responded with a s h o r t -l a t e n c y and sh o r t d u r a t i o n e x c i t a t o r y phase which was f o l l o w e d by a long suppression of a c t i v i t y . A l s o , most nGC neurones have been found t o respond to A - d e l t a f i b r e s t i m u l a t i o n (Goldman et a l . , 1972). Casey (1969) r e p o r t e d t h a t 70% of the maximal response t o p e r i p h e r a l nerve s t i m u l a t i o n remained when myelinated A f i b r e s were blocked by anodal c u r r e n t . I n t r a - a r t e r i a l a d m i n i s t r a t i o n of the a l g e s i c agent b r a d y k i n i n has a l s o been r e p o r t e d t o a f f e c t nGC neuronal f i r i n g . Guilbaud e t a l , (1973b) r e p o r t e d most nGC neurones i n decerebrate c a t s were e x c i t e d , a few were i n h i b i t e d , and a few had mixed responses t o i n t r a - a r t e r i a l b r a d y k i n i n , ^ Besson e t a l , (1974) recorded s i m i l a r r e s u l t s . fhe nGC a l s o r e c e i v e s a f f e r e n t i n p u t from other b r a i n a r e a s . M i c r o i o n t o p h o r e t i c h o r s e r a d i s h peroxidase s t u d i e s found c e l l bodies with axonal p r o j e c t i o n s t o the nGC i n the s u p e r i o r c o l l i c u l u s , the d o r s a l and v e n t r a l tegmentum, the brainstem raphe and o c c a s i o n a l l y the PAG ( G a l l a g e r and P e r t , 1978). Denervation s t u d i e s i n d i c a t e an i n p u t from the c o n t r a l a t e r a l t r i g e m i n a l nucleus (Carpenter and Hanna, 1961; Stewart and King, 1963).. T r a c i n g s t u d i e s using »*C-leucine suggest some nGC a f f e r e n t f i b r e s o r i g i n a t e from the medial raphe nucleus ( B o b i l l i e r e t a l . , 1976)., S t i m u l a t i o n s t u d i e s i n d i c a t e t h a t the nGC a l s o r e c e i v e s a f f e r e n t s from the l a t e r a l hypothalamus, caudal raphe", and the PAG. S t i m u l a t i o n i n the l a t e r a l 2 8 hypothalamus e x c i t e d most nGC neurones responding to noxious s t i m u l i whereas only a few of those which responded only t o non-noxious s t i m u l i were e x c i t e d (Keene and Casey, 1970). S t i m u l a t i o n of the n u c l e i raphe magnus, obscurus, and p a l l i d u s a c t i v a t e d most nGC neurones and t h i s a c t i v a t i o n was reversed by LSD (Briggs, 1977). S t i m u l a t i o n of a n a l g e t i c s i t e s i n the PAG e x c i t e s , i n h i b i t s , or has mixed e f f e c t s on nGC neuronal spontaneous f i r i n g but i s r e p o r t e d to i n h i b i t most of the responses of nGC neurones to noxious s t i m u l i (Hohrland and Gebhart, 1979). , P e r i a q u e d u c t a l gray s t i m u l a t i o n a t a n a l g e t i c s i t e s were a l s o shown fay Borrow and Casey (1969) t o be i n h i b i t o r y on nGC neurones responding to noxious s t i m u l i . Neurones of the nucleus g i g a n t o c e l l u l a r i s p r o j e c t t o many areas of the b r a i n . Bowsher e t a l . (1968) suggested that the nGC i s a r e l a y nucleus as i t p r o j e c t s to the c e n t r e median nucleus o f the thalamus. There i s s u b s t a n t i a l evidence f o r the e x i s t e n c e of a descending p r o j e c t i o n from the nGC to the s p i n a l c o r d . Nyberg-Hansen (1965) l e s i o n e d the nGC and f o l l o w e d i t s degenerating axon courses by the s i l v e r impregnation technique. He noted t h a t the f i b r e s t r a v e l l e d c a u d a l l y i n the v e n t r a l white of the s p i n a l cord and terminated i n lamina V I I . A s i m i l a r study using t h i s technique together with h o r s e r a d i s h peroxidase methods recorded descending f i b r e s i n the l a t e r a l columns to a l l l e v e l s of the cord (Zemlan et a l . , 1979). H a r t i n (1979) repor t e d t h a t descending f i b r e s from the nGC terminate on the i n t e r m e d i a l c e l l column. Basbaum e t a l . (1976a,b, 1 9 7 8 ) , u s i n g t r i t i a t e d l e u c i n e t r a c i n g techniques, reported t h a t nGC r e t i c u l o s p i n a l f i b r e s descend i n the v e n t r a l and v e n t r o l a t e r a l 29 f u n i c u l i and terminate mainly i n the v e n t r a l horn i n the motor neuronal l a y e r s , They concluded t h a t any descending i n h i b i t o r y i n f l u e n c e s of the nGC are l i m i t e d t o the motor system. However, groups s t u d y i n g s p i n a l c o r d neurones responding t o noxious s t i m u l i , some of which are s p i n o t h a l a m i c t r a c t neurones, r e p o r t t h a t these neurones are i n h i b i t e d by s t i m u l a t i o n of the nGC (McCreery and B l o e d e l , 1975; Takagi e t a l . , 1975). The e f f e c t s of s t i m u l a t i o n of the nGC a l s o imply t h a t i t i s i n v o l v e d i n a n a l g e s i a . S t i m u l a t i o n of the nGC r e s u l t e d i n escape behavior i n c a t s and r a t s , suggesting t h a t the nGC r e c e i v e s and t r a n s m i t s p e r c e i v a b l e noxious i n f o r m a t i o n (Casey, 1971b, Keene and Casey, 1970; C a r r and Coons, 1979). These animals were t r a i n e d t o perform a s e t behavior to escape from a noxious s t i m u l u s . S t i m u l a t i o n of the nGC has a l s o been r e p o r t e d to i n h i b i t neurones r e s p o n s i v e to noxious s t i m u l i . McCreery and B l o e d e l (1975) recorded from s p i n o t h a l a m i c t r a c t neurones which responded to graded noxious mechanical s t i m u l i . They found that these responses were i n h i b i t e d by nGC s t i m u l a t i o n . Haber and Hagman (1974), on t h e other hand, r e p o r t e d f a c i l i t a t i o n of s p i n a l c o r d i n t e r n e u r o n e s by nGC s t i m u l a t i o n . However, t h i s was probably due to t h e i r use of much hi g h e r s t i m u l a t i o n f r e q u e n c i e s because McCreery et a l . (1979), i n another study, rep o r t e d t h a t s t i m u l a t i o n of the nGC r e s u l t e d i n a t r a n s i e n t e x c i t a t i o n f o l l o w e d by a subsequent i n h i b i t i o n of s p i n o t h a l a m i c t r a c t neurones. T h e r e f o r e , the high s t i m u l a t i o n f r e q u e n c i e s may have been causing summation of the e x c i t a t o r y responses. In a l a t e r r e p o r t , Haber et a l . (1978) a l s o found i n h i b i t i o n i n a d d i t i o n to f a c i l i t a t i o n . Nucleus g i g a n t o c e l l u l a r i s s t i m u l a t i o n i s a l s o 30 r e p o r t e d t o i n h i b i t b r a d y k i n i n evoked a c t i v a t i o n of lamina V neurones (Takagi et a l , , 19 75) and to produce both s y n a p t i c e x c i t a t i o n and marked i n h i b i t i o n of some lamina V s p i n o r e t i c u l a r neurones ( F i e l d s et a l . , 1975). S i m i l a r l y to the PAG, m i c r o i n j e c t i o n of, o p i a t e s i n t o the nGC r e s u l t s i n a n a l g e s i a . M i c r o i n j e c t i o n of morphine (Takagi e t a l , , 1976, 1977; K u r a i s h i e t a l . , 1979) and met- and l e u -enkepalin (Takagi e t a l . , 1978) i n t o the nGC reduced the b e h a v i o u r a l responses of r a t s t o t a i l pinch and t h i s i n h i b i t i o n was r e v e r s e d by i n t r a v e n o u s l y a d m i n i s t e r e d naloxone. A l s o , Chan (1979) r e p o r t s that i n j e c t i o n of morphine i n t o the nGC reduced to o t h pulp evoked responses i n the s p i n a l t r i g e m i n a l n u c l e u s . However, at l e a s t two groups have found l i t t l e a n a l g e t i c e f f e c t from m i c r o i n j e c t e d morphine (Dickenson et a l . , 1979; Levy and P r o u d f i t , 1979). Levy and P r o u d f i t (1979), however, d i d r e p o r t t h a t m i c r o i n j e c t i o n of b a c l o f e n i n t o the nGC d i d cause a n a l g e s i a because i t i n c r e a s e d r a t t a i l - f l i c k l a t e n c i e s . M i c r o i o n t o p h o r e s i s of morphine or met-enkephalin onto nGC neurones r e s u l t s i n an e x c i t a t i o n of about one-half of the neurones which i s r e v e r s i b l e by microiontophoresed naloxone (Mohrland and Gebhart, 1979).., These workers r a r e l y saw i n h i b i t i o n of nGC neuronal responses t o noxious s t i m u l i . In c o n t r a s t , other workers (Gent and B o l s t e n c r o f t , 1976; H o l s t e n c r o f t e t a l . , 1977) reported t h a t iontophoresed morphine, met- or l e u - e n k e p h a l i n , or endorphin i n the c a t r e s u l t e d i n i n h i b i t i o n of e x c i t a t i o n induced i n nGC neurones by noxious s t i m u l i . However, they r e p o r t e d t h a t these e f f e c t s were not antagonized by naloxone. Satoh e t a l , (1979) have a l s o 3 1 r e p o r t e d e x c i t a t i o n of nGC neurones by iontophoresed morphine and endorphin i n the r a t . L e s i o n s of the nGC are r e p o r t e d to a f f e c t the b e h a v i o u r a l responses to noxious s t i m u l i . Halpern and H a l v e r s t o n (1967, 1974) r e p o r t e d t h a t l e s i o n i n g of the nGC, nucleus p a r a g i g a n t o c e l l u l a r i s , and/or the magnocellular tegmental f i e l d of c a t s r e s u l t e d i n an i n c r e a s e i n l a t e n c y and t h r e s h o l d f o r escape from f o o t shock. S i m i l a r r e s u l t s were p u b l i s h e d by Anderson and P e a r l (1975); l e s i o n i n g of the nGC a b o l i s h e d escape responses evoked by noxious s t i m u l i . The Caudal Raphe The c a u d a l raphe i n c l u d e s the n u c l e i raph£ magnus, p a l l i d u s , and obscurus i n the r a t . , These n u c l e i are a c a u d a l extension of the mesencephalic raphe n u c l e i and s i m i l a r l y c o n t a i n a l a r g e p r o p o r t i o n of s e r o t o n e r g i c neurones (Taber e t a l . , 1960; Dahlstrom and Fuxe, 1964; P o i t r a s and Parent, 1978). However, as opposed to the mesencephalic raphe n u c l e i , these n u c l e i have q u i t e low o p i a t e r e c e p t o r b i n d i n g l e v e l s (Atweh and Kuhar, 19 77; P e r t e t a l . , 1976). Although the c a u d a l raphe n u c l e i only r e c e i v e a sparse d i r e c t a f f e r e n t i n p u t from ascending sensory pathways, as demonstrated by m i c r o i o n t o p h o r e t i c h o r s e r a d i s h peroxidase s t u d i e s ( G a l l a g e r and P e r t , 1978), there are many r e p o r t s of neurones i n these n u c l e i responding t o p e r i p h e r a l s t i m u l i . Approximately one-half of raphe magnus neurones, i n c l u d i n g many r a p h e s p i n a l neurones, are e x c i t e d by noxious p e r i p h e r a l s t i m u l i , and many are a c t i v a t e d by non-noxious forms of s t i m u l i (Lobatz 32 et a l , , 1976,1977; Moolenaar e t a l , , 1976;, Anderson e t a l . , 1977; F i e l d s and Anderson, 1978; Za r e t s k y et a l , , 1979). Z a r e t s k y et a l . , (1979) r e p o r t e d two types of neuronal responses to noxious r a d i a n t heat: the f i r s t was e x c i t a t i o n beginning j u s t before the occurrence of a t a i l - f l i c k i n response to heat which was not pr e s e n t i f a t a i l - f l i c k d i d not occur; the second response was i n h i b i t i o n of the neurone dur i n g the period of a p p l i c a t i o n of r a d i a n t heat. There have been numerous s t u d i e s of the PAG a f f e r e n t i n p u t t o the caudal raphe". Oleson and L i e b e s k i n d (1975, 1976) r e p o r t e d t h a t s t i m u l a t i o n of the PAG r e s u l t e d i n the r e d u c t i o n of e l e c t r i c a l p o t e n t i a l s and m u l t i u n i t f i r i n g i n the raphe magnus evoked by noxious s t i m u l i . Furthermore, p e r i a q u e d u c t a l gray s t i m u l a t i o n f a c i l i t a t e d spontaneous raphe" magnus neuronal a c t i v i t y , T h i s has been confirmed by other workers ( F i e l d s and Anderson, 1978; Hayes et a l . , 1977) , Although e x c i t a t i o n was the major response seen, p e r i a q u e d u c t a l gray s t i m u l a t i o n induced i n h i b i t i o n or mixed responses on raphe magnus neuronal f i r i n g were a l s o seen (Pcmeroy and Behbehbani, 1979a,b; Mohrland and Gebhart, 1979). Evidence to prove that these e f f e c t s are mediated by a d i r e c t monosynaptic pathway i s provided by h o r s e r a d i s h peroxidase and antidromic s t u d i e s . G a l l a g e r and Pe r t (1978) observed HEP c o n t a i n i n g c e l l bodies i n the p e r i a q u e d u c t a l gray a f t e r 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 HEP i n t o the c a u d a l raphe. They a l s o observed l a b e l l i n g of nGC neurones. In a d d i t i o n , Dostrovsky and Shah (1979) a n t i d r o m i c a l l y a c t i v a t e d neurones i n the PAG by e l e c t r i c a l s t i m u l a t i o n o f the raphe magnus. 3 3 Of t h e caudal raphe" n u c l e i , the raphe magnus has the l a r g e s t axonal p r o j e c t i o n t o the s p i n a l c o r d . Dahlstrom and Fuxe {1965) reported that l e s i o n s o f the caudal raph£ caused denervation of the d o r s a l , i n t e r m e d i a t e , and v e n t r a l horns of the s p i n a l c o r d . Antidromic a c t i v a t i o n of raphe" magnus neurones from the s p i n a l c o r d , together with d i s c r e t e l e s i o n s i n the r o s t r a l c o r d , v e r i f i e d the e x i s t e n c e of a r a p h ^ s p i n a l pathway descending v i a the d o r s o l a t e r a l f u n i c u l u s (DLF) and t e r m i n a t i n g i n laminae I , V, VI, and VIII ( A k i l and L i e b e s k i n d , 1975). C o n f i r m a t i o n of t h i s pathway was o b t a i n e d from a d d i t i o n a l s t u d i e s i n v o l v i n g : s p i n a l l e s i o n i n g (Basbaum and F i e l d s , 1977a,b; i i l l i s e t a l . , 1977), t r i t i a t e d l e u c i n e t r a c i n g s t u d i e s {Basbaum e t a l . , 1976a,b; F i e l d s and Basbaum, 1978), h o r s e r a d i s h peroxidase t r a c i n g s t u d i e s (Basbaum and F i e l d s , 1977a,b; L e i c h n e t z e t a l . , 1978; Batk i n s e t a l . , 1980), and a n t i d r o m i c a c t i v a t i o n s t u d i e s (West and W o l s t e n c r o f t , 1977). An e q u i v a l e n t p r o j e c t i o n to the s p i n a l t r i g e m i n a l nucleus has been r e p o r t e d by l o v i c k et a l . (1978a,b) . / They demonstrated t h a t the nucleus raphe magnus p r o j e c t s to both the lumbar cord and the t r i g e m i n a l nucleus v i a branched axons by c o l l i d i n g a n t i dromic a c t i o n p o t e n t i a l s o r i g i n a t i n g from e i t h e r s i t e . Other areas r e c e i v e axonal input from the caud a l raph€. Briggs (1977) found that raph£ magnus s t i m u l a t i o n caused the a c t i v a t i o n of nucleus g i g a n t o c e l l u l a r i s neurones which was c o r r e l a t e d with t h e i r responses to i o n t o p h o r e t i c a l l y a p p l i e d s e r o t o n i n , and which could be blocked by i o n t o p h o r e t i c LSD.< Mapping s t u d i e s with >*C-leucine i n d i c a t e a major p r o j e c t i o n from the raphe magnus to the i n t r a l a m i n a r n u c l e i of the 34 thalamus, and minor p r o j e c t i o n s to the nGC and the d o r s a l and medial raphe n u c l e i ( B o b i l l i e r et a l , 1976),. S t i m u l a t i o n of the nucleu s raphe magnus has been widely r e p o r t e d to produce a n a l g e s i a . I t has reduced b e h a v i o u r a l responses t o noxious s t i m u l i i n the c a t ( O l i v e r a s e t a l . , 1975, 1977), i n c r e a s e d t a i l - f l i c k l a t e n c i e s i n the r a t (Oleson e t a l . , 1975, 1S78), and reduced r e f l e x responses to noxious s t i m u l i i n the c a t (Engberg e t a l . , 1968; O l i v e r a s e t a l . , 1977) and the r a t (Oleson and L i e b e s k i n d , 1975). Naloxone was re p o r t e d t o antagonize the r e d u c t i o n of b e h a v i o u r a l responses t o pinch and the r e d u c t i o n of the jaw-opening r e f l e x produced by s t i m u l a t i o n of the raphe magnus ( O l i v e r a s e t a l . , 1977). An e q u i v a l e n t i n h i b i t i o n of the s p i n a l t r i g e m i n a l nucleus has been found i n decereb r a t e c a t s (Lovick and W o l s t e n c r o f t , 1979). Raph£ magnus s t i m u l a t i o n a b o l i s h e d the response of s p i n a l t r i g e m i n a l neurones t o noxious s t i m u l i while having a weak or i n s i g n i f i c a n t e f f e c t on responses t o non-?noxious i n p u t s . T h i s d e p r e s s i o n p a r a l l e l e d the i n c r e a s e i n t h r e s h o l d of the jaw-opening r e f l e x . Nucleus raphe magnus s t i m u l a t i o n has a l s o been r e p o r t e d to i n h i b i t the responses of s p i n o t h a l a m i c t r a c t neurones to mechanical and thermal noxious s t i m u l i ( B e a l l e t a l . , 1976; W i l l i s et a l . , 1976, 1977), and to d i f f e r e n t i a l l y i n h i b i t s p i n a l c o r d neuronal responses to noxious over non-noxious s t i m u l i (LeBars e t a l . , 1976; F i e l d s e t a l . , 1977a; Guilbaud e t a l . , 1977; Duggan and G r i e r s m i t h , 1979).. Duggan and G r i e r s m i t h (1979) found t h a t t h i s i n h i b i t i o n , i n the c a t , was not r e v e r s i b l e by i n t r a v e n o u s l y a d m i n i s t e r e d naloxone, suggesting t h a t the i n h i b i t i o n i s not mediated by endogenous o p i o i d s . 35 C o n v e r s e l y , Pearson e t a l . , (1979) re p o r t e d t h a t the i n h i b i t i o n was not p r e f e r e n t i a l f o r noxious s t i m u l i responses i n the r a t . They found, i n f a c t , t h a t the t h r e s h o l d f o r i n h i b i t i o n of the non^noxious responses was l e s s than that r e q u i r e d to i n h i b i t the responses to noxious s t i m u l i . , T h e i r data a l s o suggest t h a t the i n h i b i t i o n was p o s t - s y n a p t i c a l l y mediated as glutamate evoked u n i t f i r i n g c o u l d a l s o be i n h i b i t e d by s t i m u l a t i o n of the raphS magnus. Two groups have reported raphe magnus s t i m u l a t i o n induced d e p o l a r i z a t i o n of primary a f f e r e n t f i b r e s ( P r o u d f i t and Anderson, 1974a,b, 1975; Martin e t a l . , 1979)., The d e p o l a r i z a t i o n was blocked by i n t r a v e n o u s l y a dministered c i n a n s e r i n , methysergide, and LSD ( P r o u d f i t and Anderson, 1974a,b), sugge s t i n g t h a t i t i s s e r o t o n e r g i c a l l y mediated. The i n h i b i t o r y e f f e c t was not p r e f e r e n t i a l f o r noxious responses, and Martin e t a l . (1979) suggested t h a t the p r e f e r e n t i a l e f f e c t s r e p o r t e d above must occur a t s i t e s p o s t - s y n a p t i c t o the a f f e r e n t t e r m i n a l . I n t r a v e n o u s l y administered morphine has been shown to be e x c i t a t o r y on some nucleus raph£ magnus neurones, i n c l u d i n g r a p h e s p i n a l u n i t s ( F i e l d s and Anderson, 1978). T h i s e x c i t a t i o n was r e v e r s i b l e by naloxone (Anderson e t a l . , 1977; Deakin e t a l . , 1977; Oleson e t a l . , 1978). In f a c t , the naloxone i n h i b i t e d the spontaneous f i r i n g of some neurones suggesting t h a t there i s t o n i c a l l y a c t i v e o p i o i d i n p u t to the raphe magnus (Anderson e t a l . , 1977)., M i c r o i n j e c t i o n of morphine i n t o the nucleus raph£ magnus i s report e d t o produce a n a l g e s i a : i t i n c r e a s e s t a i l - f l i c k l a t e n c i e s 3 6 { P r o u d f i t , 1977; Levy and P r o u d f i t , 1979) and reduces v o c a l i z a t i o n upon t a i l shock (Dickenson e t al.., 1979). These e f f e c t s c o u l d be reversed by i n t r a v e n o u s naloxone ( P r o u d f i t , 1977; Dickenson e t a l . , 1979) and reduced by i n t r a v e n o u s c i n a n s e r i n (Dickenson et a l . , 1979). A l s o , m i c r o i n j e c t i o n of naloxone i n t o the raphe magnus rev e r s e d the a n a l g e s i a induced by i n t r a v e n o u s l y administered morphine ( P r o u d f i t , 1977; Dickenson et a l . , 1979). Levy and P r o u d f i t (1979) a l s o r e p o r t e d t h a t m i c r o i n j e c t i o n of b a c l o f e n i n t o the r a p h i magnus d i d not produce a n a l g e s i a as i t had done when i t was i n j e c t e d i n t o the nGC. Phentolamine, on the other hand, i n c r e a s e d t a i l - f l i c k and hot p l a t e l a t e n c i e s when m i c r o i n j e c t e d i n t o the raphe\ magnus, suggest i n g t o n i c a l l y a c t i v e a d r e n e r g i c i n p u t onto raphe magnus neurones (Hammond and Levy, 1979). There have been c o n f l i c t i n g r e p o r t s of the e f f e c t s of microiontophoresed morphine on the spontaneous a c t i v i t y of nucleus raphe magnus neurones. v I t was repor t e d t o e i t h e r not a f f e c t the a c t i v i t y o f most neurones ( H a i g l e r , 1977) or to i n h i b i t about one-half of them (Gent and H o l s t e n c r o f t , 1 9 7 6 ; Lobatz et a l . , 1976, 1S77; H o l s t e n c r o f t e t a l . , 1977). However, the i n h i b i t i o n of spontaneous a c t i v i t y was not r e v e r s e d by naloxone i n these s t u d i e s and t h e r e f o r e these e f f e c t s are u n l i k e l y to be mediated by o p i a t e r e c e p t o r s , Although approximately one-half o f a l l raphe magnus neurones were re p o r t e d t o be i n h i b i t e d by i o n t o p h o r e t i c morphine, the m a j o r i t y of neurones i d e n t i f i e d as r a p h e s p i n a l were u n a f f e c t e d by i t (Gent and W o l s t e n c r o f t , 1976; H o l s t e n c r o f t e t a l . , 1977). > In a d d i t i o n , although Lobatz e t a l . , (1976, 1977) found t h a t about 37 one-half of the neurones responding t o noxious s t i m u l i were i n h i b i t e d by i o n t o p h o r e t i c morphine, Mohland and Gebhart (1979) r a r e l y found i n h i b i t i o n of raphe magnus neurones which responded to noxious s t i m u l i . To t e s t the p o s s i b i l i t y t h a t morphine was working by m o d i f i c a t i o n of the a c t i o n of a c e t y l c h o l i n e , Lobatz e t a l . (1977) a l s o t e s t e d raphe* magnus neurones with i o n t o p h o r e t i c a c e t y l c h o l i n e . , They found t h a t 85% of the neurones were e x c i t e d by the a c e t y l c h o l i n e but t h a t t h i s e x c i t a t i o n was u n a f f e c t e d by i o n t o p h o r e t i c morphine. 38 MATERIALS AND METHODS A. S u r g i c a l P r e p a r a t i o n Male a l b i n o Wistar r a t s (150-300 g) were used i n these experiments. Each a n i s a l was a n a e s t h e t i z e d with an i n t r a p e r i t o n e a l i n j e c t i o n of urethane (1.4 g/kg, Sigma) admin i s t e r e d as a 20% s o l u t i o n by weight i n d i s t i l l e d water. The r e c t a l temperature was maintained w i t h i n p h y s i o l o g i c a l l i m i t s with an automatic animal h e a t e r - c o n t r o l l e r d r i v i n g a p l e x i g l a s s and wire h e a t i n g pad. T h i s temperature was a l s o monitored independently on a Yellow Springs Telethermometer. . To a l l o w subsequent i n t r a v e n o u s i n j e c t i o n s t o be made, the r i g h t f e m o r a l v e i n was cannulated with No. 10 p o l y e t h y l e n e t u b i n g (Clay Adams) p r e f i l l e d with normal s a l i n e . , I f the blood pressure was t o be recorded the r i g h t femoral a r t e r y was cannulated with No. 50 p o l y e t h y l e n e t u b i n g p r e f i l l e d with a d i l u t e s a l i n e - h e p a r i n (Sigma) s o l u t i o n and atta c h e d to a pressure transducer (Stratham Medical Instruments, Inc., model P23AC)• The r e s u l t i n g e l e c t r i c a l s i g n a l was recorded on one channel o f a f o u r channel polygraph (Grass Instruments, model 79D) . The s c i a t i c nerve was prepared f o r e l e c t r i c a l s t i m u l a t i o n by a t t a c h i n g a b i p o l a r e l e c t r o d e . T h i s e l e c t r o d e was c o n s t r u c t e d from two le n g t h s of 0,35 mm s i l v e r wire wrapped around a 2 cm long p i e c e of No. 90 p o l y e t h y l e n e t u b i n g and connected to two i n s u l a t e d wires which were l e d to the e x t e r i o r . The e l e c t r o d e was h e l d i n place and i s o l a t e d from the surrounding t i s s u e by a 10 mm pie c e of rubber t u b i n g placed 3 9 around the e l e c t r o d e and the nerve and secured with s u r g i c a l t h r e a d . , The p r o t r u d i n g wires were f u r t h e r secured by being t i e d to a s k i n f l a p with t h r e a d . The animal's head was then p o s i t i o n e d i n a s t e r e o t a x i c animal frame (Narashige S c i e n t i f i c Instrument L a b o r a t o r y ) . The toothfcar was p o s i t i o n e d such t h a t i t s top s u r f a c e was l e v e l with the c e n t r e of the e a r b a r s , a c c o r d i n g t o the method of &bad-A l e g r i a (1971) . The s k i n over the s k u l l was c u t and r e f l e c t e d , the s k u l l scraped c l e a r of t i s s u e , and a b u r r h o l e d r i l l e d with a H o t o t o o l (Dremel). The h o l e , about 5 mm i n diameter, was placed approximately 9 mm l e f t of the s a g i t t a l suture and l e v e l with the s k u l l landmark 'lambda' i n the a n t e r i o r - p o s t e r i o r plane. T h i s l o c a t i o n avoided damage t o the d u r a l venous s i n u s e s . a f u r t h e r s m a l l s e c t i o n of s k u l l over the cerebellum was removed with a p a i r of rongeurs t o allow an e l e c t r o d e to be placed i n t o the nucleus g i g a n t o c e l l u l a r i s . , B. . E l e c t r o d e P r e p a r a t i o n and P o s i t i o n i n g P r e p a r a t i o n S t i m u l a t i n g e l e c t r o d e s were c o n s t r u c t e d by i n s e r t i n g a l e n g t h of t h i n i n s u l a t e d copper wire i n t o a 1 . 5 mm g l a s s c a p i l l a r y tube (Kimble Products) and p u l l i n g the e l e c t r o d e with a m i c r o e l e c t r o d e p u l l e r (Narashige). The r a t e of p u l l was slowed by hand to achieve a l o n g well-formed e l e c t r o d e . The wire p r o t r u d i n g from the b l u n t end was glued i n p l a c e with a spot of epoxy, the t i p broken and the exposed wire s t r i p p e d of 40 i n s u l a t i o n and trimmed with a s c a l p e l blade l e a v i n g 0.1 ram of s t r i p p e d wire. I f a b i p o l a r e l e c t r o d e was being c o n t r u c t e d then a s h o r t e r p i e c e of c a p i l l a r y tube bent i n t o the shape of a hockey s t i c k was glued p a r a l l e l to the f i r s t and a wire i n s e r t e d i n t o each tube, a l s o , p r i o r to p u l l i n g , the e l e c t r o d e was heated and t w i s t e d 180° to bind the c a p i l l a r i e s together and ensure t h a t the two b a r r e l s would form one t i p when the e l e c t r o d e was p u l l e d . S i n g l e b a r r e l r e c o r d i n g e l e c t r o d e s were made from 1.5 mm diameter g l a s s m i c r o p i p p e t t e s c o n t a i n i n g a s i n g l e strand of f i b r e g l a s s t o ease the f i l l i n g , by c a p i l l a r y a c t i o n , of the r e s u l t i n g e l e c t r o d e s (Glass Company of America, Omega Dot). The e l e c t r o d e s were p u l l e d i n the c o n v e n t i o n a l manner on a commercial e l e c t r o d e p u l l e r (Narashige) and the t i p s broken a g a i n s t a g l a s s rod under a microscope t o about one um. They were then f i l l e d with 2% pontamine sky blue 6BX (ICN Pharmaceuticals, Inc.) i n 0.5 M sodium a c e t a t e , a l l o w i n g both r e c o r d i n g and the h i s t o l o g i c a l marking of r e c o r d i n g s i t e s by i o n t o p h o r e t i c e j e c t i o n o f the dye ( H e l l o n , 1971). E l e c t r o d e r e s i s t a n c e s were deemed a c c e p t a b l e i f they were between 10-30 megohms,. M u l t i b a r r e l e l e c t r o d e s were c o n s t r u c t e d by g l u e i n g a number of sho r t hockey s t i c k shaped c a p i l l a r y tubes (Omega Dot) around a f u l l l e ngth m i c r o p i p e t t e . The r e s u l t i n g m u l t i b a r r e l assemblage was placed i n the m i c r o e l e c t r o d e p u l l e r , heated, and tw i s t e d 180° t o bond the b a r r e l s t o g e t h e r , a f t e r a delay t o allow f o r c o o l i n g , the e l e c t r o d e was p u l l e d i n the c o n v e n t i o n a l manner. The t i p was broken back t o a diameter of about 10 um to prevent blockage of the drag b a r r e l s d u r i n g i o n t o p h o r e s i s . The ce n t r e b a r r e l was f i l l e d with dye s o l u t i o n , as was done above f o r the s i n g l e b a r r e l e l e c t r o d e s , and the surrounding b a r r e l s were f i l l e d with t e s t drugs (see below) . The v a r i o u s drugs were made up i n s o l u t i o n s a t optimum pH or with a p o l a r v e h i c l e i n order t o achieve s a t i s f a c t o r y r e l e a s e of the drug d u r i n g i o n t o p h o r e s i s . , The pH was s e l e c t e d such t h a t the drug was i n a h i g h l y p o l a r s t a t e . For drugs which are non-p o l a r the s o l u t i o n was made with a p o l a r s o l u t e , u s u a l l y sodium c h l o r i d e , as a v e h i c l e to enable the drug t o be r e l e a s e d by e l e c t r o - o s m o s i s . The drugs used i n these experiments were: s e r o t o n i n c r e a t i n i n e s u l f a t e (Sigma), 0.04 M; f l u o x e t i n e ( L i l l y ) , 0 .1 fi i n 200 mM sodium c h l o r i d e , pH 6.5; glutamate (Sigma), 0.2 M, pH 7.5; and sodium c h l o r i d e (BDH Ch e m i c a l s ) , 2.0 M. The s o l u t i o n s were f i l t e r e d with a 0.45 um M i l l i p o r e f i l t e r i n g system and s t o r e d f r o z e n i n 1.0 ml l o t s between experiments. P o s i t i o n i n g The e l e c t r o d e to be p o s i t i o n e d i n the p e r i a q u e d u c t a l gray matter, whether i t was a s t i m u l a t i n g or r e c o r d i n g e l e c t r o d e , was he l d by a standard e l e c t r o d e c a r r i e r (Narashige) with an a d d i t i o n a l s l i d i n g rack and p i n i o n a t t a c h e d . The added rack was i n c l i n e d 30° l e f t o f v e r t i c a l i n the l a t e r a l plane t o all o w the PAG to be reached without damaging the d u r a l venous s i n u s e s . The e l e c t r o d e t i p was centred on a s t e r e o t a x i c c a l i b r a t o r frame (Narashige) i n order t o record the a n t e r i o r - p o s t e r i o r and v e r t i c a l zero c o o r d i n a t e s . The l a t e r a l zero c o o r d i n a t e was 42 obtained by d i r e c t comparison of the e l e c t r o d e t i p to the s a g g i t a l s u t u r e of the r a t * s s k u l l . T h i s a l l o u e d f o r any assymetry i n the l a t e r a l placement of the head i n the s t e r e o t a x i c frame. The f i n a l c o o r d i n a t e s were c a l c u l a t e d u s i n g the c o o r d i n a t e s of the PAG s p e c i f i e d by a s t e r e o t a x i c a t l a s (Abad-Alegria, 1971): 0.0-0.5 mm p o s t e r i o r ; 0.6-0.9 mm l a t e r a l ; and 4.0-5.0 mm d o r s a l . The e l e c t r o d e p o s i t i o n e d i n the nucleus r e t i c u l a r i s g i g a n t o c e l l u l a r i s was held i n another standard c a r r i e r i n c l i n e d 25° p o s t e r i o r of v e r t i c a l i n the s a g g i t a l plane. I t was mounted on the arm of the s t e r e o t a x i c frame o p p o s i t e t h a t of the f i r s t c a r r i e r t o a v o i d c o l l i s i o n of t h e two and to allow f r e e access of a ste r e o s c o p e (Olympus). C a l i b r a t i o n and p o s i t i o n i n g o f t h i s e l e c t r o d e was s i m i l a r t o that f o r the PAG e l e c t r o d e , except t h a t the a t l a s c o o r d i n a t e s used were: 3.0-4.0 mm p o s t e r i o r ; 0,6-1.0 mm l a t e r a l ; and ±0.5 mm d o r s a l . , C, Equipment I n t e r c o n n e c t i o n The s c i a t i c nerve e l e c t r o d e and the b r a i n s t i m u l a t i n g e l e c t r o d e were connected v i a a switch box to a constant v o l t a g e s t i m u l a t o r (Ortec) a l l o w i n g s e l e c t i o n of e i t h e r s i t e f o r e l e c t r i c a l s t i m u l a t i o n . , A noxious r a d i a n t heat s t i m u l a t o r (see Appendix) was focused on a s m a l l area o f the t a i l (4x4 mm) . T h i s s t i m u l a t o r was under temperature feedback c o n t r o l v i a a thermocouple which r e g u l a t e d both the noxious and basal non-noxious temperatures of the s u r f a c e o f the t a i l . , The b a s a l temperature, which was kept c o n s t a n t between noxious t r i a l s , was s e t at 30-32 °C and the noxious a p p l i c a t i o n s were set at 48-43 54 °C. The noxious t r i a l s were a u t o m a t i c a l l y l i m i t e d to 15 s to prevent s k i n trauma. The temperature of the i r r a d i a t e d thermocouple was recorded on one channel of the polygraph. S i n g l e u n i t a c t i v i t y was recorded by i n s e r t i n g a s i l v e r wire i n t o the r e c o r d i n g e l e c t r o d e with the other end a t t a c h e d to the probe of a u n i t y gain ;voltage f o l l o w e r (8., P. Instruments, I n c . ) . The r e s u l t i n g low l e v e l s i g n a l was fed d i f f e r e n t i a l l y to one channel of a storage o s c i l l o s c o p e {Tektronix) which a m p l i f i e d and f i l t e r e d the s i g n a l i n a d d i t i o n t o d i s p l a y i n g i t on the s c r e e n . The d i f f e r e n t i a l c o n n e c t i o n and the f i l t e r s were used t o reduce unwanted n o i s e . The low-pass f i l t e r ' s 3 db c u t o f f was s e t at 1 kHz and the high-pass f i l t e r ' s 3 db c u t o f f was s e t a t 1 kHz., The output of the o s c i l l o s c o p e p r e a m p l i f i e r was fed t o an FM tape recorder (Hewlett-Packard) a l l o w i n g the r e c o r d i n g and subsequent playback o f u n i t a c t i v i t y . A t r i g g e r pulse from the s t i m u l a t o r was recorded on another channel of the tape r e c o r d e r f o r s y n c r o n i z a t i o n a l purposes. The p r e a m p l i f i e r output was a l s o f e d t o a window d i s c r i m i n a t o r (see Appendix) th a t allowed the s e l e c t i o n of s p i k e s of i n t e r e s t while e x c l u d i n g background n o i s e and unwanted s p i k e s . P e r i a q u e d u c t a l Gray Neurones - Antidromic A c t i v a t i o n In the experiments performed t o t e s t f o r a n t i d r o m i c a c t i v a t i o n of PAG neurones by s t i m u l a t i o n of the nucleus g i g a n t o c e l l u l a r i s the f o l l o w i n g i n t e r c o n n e c t i o n s were made i n a d d i t i o n to those l i s t e d above (see F i g . 1). The output from the window d i s c r i m i n a t o r was connected to the t r i g g e r i n p u t of a constant v o l t a g e s t i m u l a t o r ( O r t e c ) , which i t s e l f was connected 44 F i g u r e 1: Schematic diagram of equipment i n t e r c o n n e c t i o n used i n experiments st u d y i n g the e f f e c t of e l e c t r i c a l s t i m u l a t i o n of the nucleus g i g a n t o c e l l u l a r i s on p e r i a q u e d u c t a l gray neurones. The s t i m u l a t o r uas connected so that i t c o u l d be t r i g g e r e d by spontaneously or evoked a c t i o n p o t e n t i a l s . A f t e r a s u i t a b l e delay f o l l o w i n g t r i g g e r i n g , the s t i m u l a t o r would d i s c h a r g e . PREAMP VOLTAGE AND FOLLOWER * 4 FILTERS 1 X RECORDING ELECTRODE STIMULATING ELECTRODE OSCILLOSCOPE DISPLAY WINDOW DISCRIM-INATOR F.M. TAPE RECORDER CONSTANT VOLTAGE STIMULATOR 46 to the b i p o l a r s t i m u l a t i n g e l e c t r o d e p o s i t i o n e d i n the nGC. , The s t i m u l a t o r c o u l d be used i n two ways: i n a f r e e - r u n n i n g mode with a s t i m u l a t i o n every 2 s, or i n a t r i g g e r e d mode with the s t i m u l a t o r being t r i g g e r e d i r r e g u l a r l y by the d i s c r i m i n a t o r output. The t r i g g e r e d mode allowed the s t i m u l a t i o n to be p o s i t i o n e d a t a s e l e c t e d time i n t e r v a l a f t e r a spontaneous a c t i o n p o t e n t i a l , See the p r o t o c o l s e c t i o n f o r f u r t h e r d e t a i l s . The taped responses were processed with the f o l l o w i n g equipment setup i l l u s t r a t e d i n Figu r e 2, The output of the tape r e c o r d e r was f i l t e r e d and passed to the window d i s c r i m i n a t o r and a delay l i n e (see Appendix), The d i s c r i m i n a t o r allowed the s t i m u l a t o r to be t r i g g e r e d by e i t h e r the recorded t r i g g e r p u l s e s , the a r t i f a c t s , or the recorded a c t i o n p o t e n t i a l s . , The s t i m u l a t o r was used t o s e l e c t every nth pulse which then t r i g g e r e d the r a s t e r scan u n i t (see Appendix), The delay l i n e was used to allow the t r i g g e r i n g s i g n a l to be placed i n the ce n t r e of the o s c i l l o s c o p e screen* The delayed s i g n a l was a l s o ^ passed to a another d i s c r i m i n a t o r whose output f e d the s i g n a l i n p u t s of the r a s t e r scan u n i t . The r a s t e r scan u n i t c o n s t r u c t e d a d i s p l a y on the o s c i l l o s c o p e c o n s i s t i n g of n t r i a l s . Each t r i a l c o n s i s t e d of one o s c i l l o s c o p e t r a c e p o s i t i o n e d at a p r o g r e s s i v e l y lower p o s i t i o n on the o s c i l l o s c o p e s c r e e n . Each sweep c o u l d be e i t h e r the o r i g i n a l s i g n a l or t h a t s i g n a l processed i n t o a s e r i e s of dots where each dot represented an a c t i o n p o t e n t i a l or a st i m u l u s a r t i f a c t . 47 F i g u r e 2: Schematic diagram of the equipment setup used t o process the recorded neuronal u n i t a c t i v i t y . Mote the use of a delay l i n e t o allow the t r i g g e r i n g t o be from s i g n a l s i n the middle of a sweep. F.M. TAPE RECORDER PREAMP AND FILTERS WINDOW DISCRIM-INATOR STIMULATOR ( LOGIC ) DELAY LINE WINDOW DISCRIM-INATOR t l RASTER 0 SCAN to PREAMP AND FILTERS DISPLAY T r i g 49 Nucleus G i g a n t o c e l l u l a r i s Neurones - E f f e c t of PAG S t i m u l a t i o n The eguipment i n t e r c o n n e c t i o n f o r the experiments s t u d y i n g the e f f e c t of PAG s t i m u l a t i o n on nGC neurones i n a d d i t i o n to tha t above i s d i s c u s s e d i n t h i s s e c t i o n (see F i g . 3 ) . The output of the d i s c r i m i n a t o r was connected t o : a s p i k e i n t e g r a t o r (see Appendix) which allowed the c e l l f i r i n g r a t e during p r e s e t time epochs to be recorded on one channel of the polygraph; and to a time histogram a n a l y s i s module (Ortec) t h a t allowed the c o n s t r u c t i o n o f p e r i s t i m u l u s time histograms (PSTHs) and i n t e r s p i k e i n t e r v a l histograms ( I S I ) . The r e s u l t i n g histograms were viewed on a dual beam o s c i l l o s c o p e {Tektronix) and hard c o p i e s of them made on a X-Y p l o t t e r (Houston Instruments). The drug b a r r e l s of m u l t i b a r r e l e d e l e c t r o d e s were connected to a s i x channel programmable m i c r o i o n t o p h o r e t i c c u r r e n t c o n t r o l l e r (Dagan). The a p p l i c a t i o n s of drugs were monitored on one channel of the polygraph. Taped responses were processed by connecting the tape r e c o r d e r ' s output t o the f i r s t o s c i l l o s c o p e ' s p r e a m p l i f i e r i n p u t . D. P r o t o c o l P e r i a q u e d u c t a l Gray Neurones - Antidromic A c t i v a t i o n A search was made f o r s i n g l e u n i t s which responded t o s t i m u l a t i o n (1-5 V, 0.1 ms, 2.0 Hz) of the nucleus g i g a n t o c e l l u l a r i s . Once such a neurone was found i t was c l a s s i f i e d a c c o r d i n g to i t s response to noxious and non-noxious p e r i p h e r a l s t i m u l i and e l e c t r i c a l s t i m u l a t i o n of the nGC. The 50 F i g u r e 3: Schematic diagram showing the equipment i n t e r c o n n e c t i o n used i n experiments s t u d y i n g nucleus g i g a n t o c e l l u l a r i s neurones. SIX CHANNEL [PROGRAMMABLE] TWO BARREL GLASS COATED WIRE STIMULATING ELECTRODE SPIKE INTEGRATOR PERI-STIM-ULUS TIME HISTOGRAM UNIT SCIATIC NERVE BIPOLAR ELECTRODE PRESSURE TRANSDUCER X-Y PLOTTER FOCUSED LIGHT SOURCE RADIANT HEAT CONTROLLER POLYGRAPH 5 2 noxious p e r i p h e r a l s t i m u l i i n c l u d e d : noxious pinch and p r e s s u r e to the t a i l , hind paws, and e a r s ; e l e c t r i c a l s t i m u l a t i o n of the s c i a t i c nerve {1-25 V, 0 . 1 ras, 0.5-2.0 Hz); and r a d i a n t heat focused on the t a i l . These s t i m u l i were c l e a r l y noxious t o the experimenter. Non-noxious s t i m u l i i n c l u d e d a i r p u f f s t o or g e n t l e mechanical movement of the animal^s f u r and j o i n t movement. The response of a c e l l t o s t i m u l a t i o n of the nGC was c l a s s i f i e d as "suspected antidromic™ or "orthodromic". The response was c l a s s i f i e d as orthodromic i f i t f a i l e d to f u l f i l one or more of the f o l l o w i n g c r i t e r i a : (1) demonstration of constant l a t e n c y at t h r e s h o l d ; {2) demonstration t h a t the response was a b l e t o f o l l o w s t i m u l a t i o n f r e q u e n c i e s t o g r e a t e r than 200 Hz; and {3) demonstration of c o l l i s i o n between the evoked a c t i o n p o t e n t i a l and an orthodromic a c t i o n p o t e n t i a l w i t h i n the c r i t i c a l time p e r i o d : the l a t e n c y plus the r e f r a c t o r y p e r i o d , or i f the l a t e n c y o f the response was g r e a t e r than 7 ms. The value of 7 ms was chosen because the d i r e c t d i s t a n c e , s t e r e o t a x i c a l l y , between the two n u c l e i i s about 3.5 mm. T h e r e f o r e , a c c e p t i n g the slowest conduction v e l o c i t y of unmyelinated f i b r e s t o be 0.5 m/s, an a c t i o n p o t e n t i a l would t r a v e l between the n u c l e i i n l e s s than 7 ms. A p a i r e d pulse was used t o t e s t the f o l l o w i n g frequency i n most cases but longer t r a i n s were sometimes used. Responses were c l a s s i f i e d as 53 •suspected a n t i d r o m i c ' i f they demonstrated a, c o n s t a n t l a t e n c y of l e s s than 7 ms and i f they passed a l l of the c r i t e r i a f o r which they were t e s t e d . C o l l i s i o n was t e s t e d i n two ways. I f the neurone's spontaneous a c t i v i t y was f a i r l y high then the nGC was s t i m u l a t e d r e g u l a r l y at 0.5 Hz and the i n t e r a c t i o n of the evoked p o t e n t i a l s with the spontaneous p o t e n t i a l s was observed. I f the spontaneous a c t i v i t y was slow or n o n - e x i s t a n t then an attempt was made to i n c r e a s e i t , i f p o s s i b l e , with noxious s t i m u l i . The other method used was to t r i g g e r the s t i m u l a t o r from spontaneous s p i k e s . T h i s allowed the s t i m u l a t i o n t o be placed at v a r y i n g p e r i o d s a f t e r the spontaneous s p i k e , a l l o w i n g c o l l i s i o n to be s t u d i e d more c l o s e l y and having the added advantage of making each spontaneous a c t i o n p o t e n t i a l count. Nucleus G i g a n t o c e l l u l a r i s Neurones - E f f e c t of PAG S t i m u l a t i o n While s e a r c h i n g f o r c e l l s i n these experiments i o n t o p h o r e s i s of glutamate was f r e q u e n t l y used t o e x c i t e c e l l s not spontaneously a c t i v e . Neurones were then c h a r a c t e r i z e d by t h e i r responses to p e r i p h e r a l s t i m u l i , i o n t o p h o r e s i s of drugs, and to s t i m u l a t i o n of the PAG. The e f f e c t s of i o n t o p h o r e s i s were recorded as changes i n the spontaneous or glutamate evoked f i r i n g r a t e as w e l l as changes i n the PSTHs c o n s t r u c t e d from responses t o e l e c t r i c a l s t i m u l a t i o n of the PAG or the s c i a t i c nerve. To check t h a t the responses were not due to the i o n t o p h o r e t i c c u r r e n t , c o n t r o l a p p l i c a t i o n s o f NaCl, at c u r r e n t s g r e a t e r than those used i n the drug a p p l i c a t i o n s , were performed, I f the m u l t i b a r r e l e l e c t r o d e s sere u n s a t i s f a c t o r y , i . e . they became blocked or had poor r e c o r d i n g c h a r a c t e r i s t i c s , the experiment was continued with s i n g l e b a r r e l e l e c t r o d e s . Towards the end of most experiments a neurone was s e l e c t e d f o r t e s t i n g with an i n t r a v e n o u s l y administered drug. The e f f e c t s of the drug were noted on the spontaneous f i r i n g r a t e of the neurone and the response of the neurone to PAG s t i m u l a t i o n as recorded i n PSTHs. E. H i s t o l o g y Recording s i t e s of i n t e r e s t were marked by p a s s i n g up t o 15 uA of d i r e c t n e g a t i v e c u r r e n t through the r e c o r d i n g b a r r e l f o r up to 15 min. T h i s procedure l e f t a spot of the blue dye contained i n the r e c o r d i n g b a r r e l a t the r e c o r d i n g s i t e . These spots were subsequently l o c a t e d a f t e r the b r a i n had been s e c t i o n e d on a microtome. S t i m u l a t i o n s i t e s were marked by l e s i o n i n g the t i s s u e a t the e l e c t r o d e t i p with d i r e c t c u r r e n t (0.3-0.9 mA, 30 s) passed through the e l e c t r o d e . At the end o f the experiment the animal's c h e s t was opened and a needle, att a c h e d t o a 50 ml s y r i n g e f i l l e d with normal s a l i n e , was i n t r o d u c e d i n t o the l e f t v e n t r i c l e of the h e a r t . The r i g h t v e n t r i c l e was then s l i t and the animal perfused with the s a l i n e . The animal was d e c a p i t a t e d with a g u i l l o t i n e , the b r a i n removed and s t o r e d i n 10% formaldehyde s o l u t i o n f o r at l e a s t t h r e e days. The b r a i n was then s l i c e d i n t o 40 um s e c t i o n s with a f r e e z i n g microtome (Damon/IEC D i v i s i o n ) at -20 °C and mounted onto g l a s s s l i d e s using an a l c o h o l - g e l a t i n (2 g of g e l a t i n i n 2 1 of 40% a l c o h o l ) s o l u t i o n . The b r a i n s l i c e s were then used t o map the r e c o r d i n g and s t i m u l a t i o n s i t e s onto composite diagrams. Recording s i t e s t h a t were not marked were i n t e r p o l a t e d from th l o c a t i o n s of marked s i t e s i n the same e l e c t r o d e t r a c k . , Responses t o e l e c t r i c a l s t i m u l a t i o n s i n which the s t i m u l a t i o n s i t e was l o c a t e d o u t s i d e i t s t a r g e t area were d i s c a r d e d . , 5 6 RESULTS A. P e r i a q u e d u c t a l Gray Neurones A t o t a l of f o r t y - f i v e experiments were performed to study the responses c f neurones i n the v i c i n i t y of t h e p e r i a q u e d u c t a l gray to noxious p e r i p h e r a l s t i m u l i and t o e l e c t r i c a l s t i m u l a t i o n of the nucleus g i g a n t o c e l l u l a r i s . The r e s u l t s of these experiments are presented i n the f o l l o w i n g two s e c t i o n s . The recorded a c t i o n p o t e n t i a l s were probably from c e l l bodies r a t h e r than axons as they c o u l d be recorded f o r a d i s t a n c e o f 40-100 pm and the a c t i o n p o t e n t i a l s were b i p h a s i c . Host u n i t s had slow spontaneous f i r i n g r a t e s of l e s s than 15 Hz although r a t e s as high as 80 Hz were a l s o seen., Responses t o Noxious P e r i p h e r a l S t i m u l i The noxious p e r i p h e r a l s t i m u l i used to t e s t the P&G area u n i t s i n c l u d e d pinch and pressure a p p l i e d to the paws, e a r s , and t a i l , r a d i a n t heat a p p l i e d t o the t a i l , and e l e c t r i c a l s t i m u l a t i o n o f the l e f t s c i a t i c nerve, about o n e - t h i r d of the neurones were a f f e c t e d by the noxious s t i m u l i . ; Of the 61 u n i t s a f f e c t e d , 46 were e x c i t e d and 12 were i n h i b i t e d . In a d d i t i o n , 3 neurones were e x c i t e d by some s t i m u l i but i n h i b i t e d by other s t i m u l i . There d i d not appear t o be any c o r r e l a t i o n between the neuronal responses to the noxious s t i m u l i and t h e i r h i s t o l o g i c a l l y d e t e r l i n e d l o c a t i o n s . The neurones which were a f f e c t e d by noxious s t i m u l i were c l a s s i f i e d by t h e i r responses to noxious s t i m u l i ( F i g , 4). , The l a t e n c i e s of responses to e l e c t r i c a l s t i m u l a t i o n c f the s c i a t i c nerve were between 5 and 64 ms. The 57 Fi g u r e 4: Coronal s e c t i o n of r a t b r a i n showing l o c a t i o n of neurones i n the v i c i n i t y of the peragueductal gray c l a s s i f i e d by t h e i r responses t o : noxious pinch and pressure to the paws, e a r s and t a i l ; r a d i a n t heat a p p l i e d to the t a i l ; and e l e c t r i c a l s t i m u l a t i o n of the s c i a t i c nerve. The a c t u a l l o c a t i o n s were found at ±0.5 mm a n t e r i o r - p o s t e r i o r l y but are d i s p l a y e d on one s e c t i o n at s t e r e o t a x i c z e r c f o r convenience and c l a r i t y . Symbols: CI - i n f e r i o r c o l l i c u l u s ; nRP - n. r e t i c u l a r i s p a r v i -c e l l u l a r i s ; PAG - p e r i a q u e d u c t a l gray. The axes are numbered i n mm. Modified from Abad-Alegria (1971). 58 59 average l a t e n c y was 18±10 ms {mean t the standard e r r o r of the mean). The responses ranged from a s i n g l e s p i k e i n s i l e n t u n i t s to an i n c r e a s e of f i r i n g r a t e l a s t i n g up to 130 ms i n spontaneously a c t i v e u n i t s . Responses to S t i m u l a t i o n of the nGC N i n e t y - f i v e p e r i a q u e d u c t a l gray area neurones were a c t i v a t e d by e l e c t r i c a l s t i m u l a t i o n of the nucleus g i g a n t o c e l l u l a r i s . The h i s t o l o g i c a l l y determined s i t e s of the s t i m u l a t i o n e l e c t r o d e placed i n the nGC are i l l u s t r a t e d i n Figu r e 5. Host u n i t s {96%) a f f e c t e d by nGC s t i m u l a t i o n were a c t i v a t e d but 4 u n i t s were i n h i b i t e d . These i n h i b i t o r y responses were not i n c l u d e d with the o r t h o d r o m i c a l l y a c t i v a t e d responses. The a c t i v a t i o n responses were c l a s s i f i e d as •orthodromic' or 'suspected a n t i d r o m i c ' based on t h e i r f u l f i l m e n t of the c r i t e r i a f o r a n t i d r o m i c i t y and t h e i r l a t e n c i e s as o u t l i n e d i n the M a t e r i a l s and Methods. T o t a l numbers of u n i t s so c l a s s i f i e d are shown i n Table I . A t o t a l of 59 neuronal responses were c l a s s i f i e d as orthodromic because they f a i l e d one or more of the a n t i d r o m i c i t y c r i t e r i a or had l a t e n c i e s of g r e a t e r than 7 ms., The l o c a t i o n s of these u n i t s were d i v e r s e and i n c l u d e d the i n f e r i o r c o l l i c u l u s and midbrain r e t i c u l a r f o r m a t i o n i n a d d i t i o n t o the PAG (see F i g . , 6) ., Thi r t y - t w o neuronal responses were c l a s s i f i e d as 'suspected a n t i d r o m i c ' . They a l l demonstrated constant l a t e n c i e s at t h r e s h o l d of l e s s than 7 ms and passed a l l of the a n t i d r o m i c i t y c r i t e r i a f o r which they were t e s t e d . The numbers of neurones passing each t e s t i s given i n Table I and the u n i t s ' l o c a t i o n s 6 0 F i g u r e 5 : Coronal s e c t i o n showing the h i s t o l o g i c a l l y determined p o s i t i o n o f the s t i m u l a t i n g e l e c t r o d e s p l a c e d i n the nucleus g i g a n t o c e l l u l a r i s which were used i n experiments st u d y i n g the a c t i v a t i o n of PAG neurones by e l e c t r i c a l s t i m u l a t i o n o f the nGC. The a c t u a l s t i m u l a t i o n s i t e s were l o c a t e d at 3 . 0 ± 0 . 5 mm p o s t e r i o r but are d i s p l a y e d on one s e c t i o n a t 3 . 0 mm p o s t e r i o r f o r convenience and c l a r i t y . Symbols: V, V I I I , XII - n u c l e i of the corresponding c r a n i a l nerves; CR - c a u d a l raphe; nGC -nucleus g i g a n t o c e l l u l a r i s . The axes are numbered i n mm. M o d i f i e d from Abad-Alegria (1971). Table I: Numbers of neurones i n "".the v i c i n i t y of the periaqueductal gray f u l f i l l i n g or f a i l i n g the c r i t e r i a for antidromicity. Orthodromic Antidromic C r i t e r i a / i yes no yes no C o l l i s i o n 1 4 8 0 Following>200 Hz 0 23 11 0 Latency $ 7 ms 30 17 32 0 Constant Latency 35 14 32 0 T o t a l 59 32 F i g u r e 6: Coronal s e c t i o n showing the h i s t o l o g i c a l l y determined l o c a t i o n s of neurones i n the v i c i n i t y of the PAG c l a s s i f i e d as b e i n g o r t h o d r o m i c a l l y a c t i v a t e d by s t i m u l a t i o n of the nGC. For c l a s s i f i c a t i o n c r i t e r i a see t e x t and Table I . Symbols and comments as i n Figure 4. 64 65 are i l l u s t r a t e d i n Figure 7, E i g h t u n i t s with constant l a t e n c i e s of l e s s than 7 ms demonstrated c o l l i s i o n of evoked a c t i o n p o t e n t i a l s with spontaneously o c c u r r i n g orthodromic p o t e n t i a l s . Two examples of c o l l i s i o n are i l l u s t r a t e d i n F i g u r e s 8 and 9. One u n i t with a l a t e n c y o f 13 ms showed what appeared to be c o l l i s i o n between a s t i m u l a t i o n evoked a c t i o n p o t e n t i a l and a spontaneous p o t e n t i a l , However, as i t d i d not pass the s h o r t l a t e n c y c r i t e r i o n i t s response was c l a s s i f i e d as orthodromic, , B » Nucleus R e t i c u l a r i s G i g a n t o c e l l u l a r i s Neurones A t o t a l o f f o r t y experiments were performed t o study the e f f e c t of i c n t o p h o n e t i c s e r o t o n i n , i o n t o p h o r e t i c and i n t r a v e n o u s f l u o x e t i n e , which i s a s p e c i f i c s e r o t o n i n uptake b l o c k e r , noxious p e r i p h e r a l s t i m u l i , and PAG s t i m u l a t i o n on the a c t i v i t y of nGC neurones. As w e l l , the e f f e c t s of s e r o t o n i n and f l u o x e t i n e on the nGC neuronal responses to s t i m u l a t i o n of the s c i a t i c nerve and the PAG were s t u d i e d . „ The r e s u l t s of these experiments are d e s c r i b e d below. The r e c o r d i n g s made were almost c e r t a i n l y from c e l l bodies and not axonal f i b r e s because 97% of the neurones t e s t e d with iontophoreses glutamate were e x c i t e d . Glutamate i s re p o r t e d t o produce e x c i t a t o r y e f f e c t s s p e c i f i c a l l y on c e l l bodies ( C u r t i s e t a l . , 1960; K r n j e v i c and P h y l l i s , 1963).. A l s o , the recorded a c t i o n p o t e n t i a l s had constant s i z e and were b i p h a s i c . Most neurones could be recorded over a d i s t a n c e of 50-100 pm. . 66 F i g u r e 7: Coronal s e c t i o n showing the h i s t o l o g i c a l l y determined l o c a t i o n s of PAG area neurones which were suspected of being a n t i d r o r a i c a l l y a c t i v a t e d by nGC s t i m u l a t i o n . See Table I and t e x t f o r c l a s s i f i c a t i o n d e s c r i p t i o n s . Symbols: O _ d i s p l a y e d a contant l a t e n c y of l e s s than 7 ms; Q - f o l l o w e d s t i m u l a t i o n to a freguency g r e a t e r than 200 Hz and had constant l a t e n c y of l e s s than 7 ms; • - demonstrated c o l l i s i o n and had a constant l a t e n c y of l e s s than 7 ms. See F i g u r e 4 f o r the other symbols and f o r comments. 68 F i g u r e 8: O s c i l l o s c o p e t r a c i n g s of a p e r i a g u e d u c t a l gray neurone showing c o l l i s i o n o f spontaneous a c t i o n p o t e n t i a l s and a c t i o n p o t e n t i a l s evoked by s t i m u l a t i o n o f the n u c l e u s g i g a n t o c e l l u l a r i s . (a) R a s t e r scan o f c o n s e c u t i v e s t i m u l a t i o n s o f t h e nGC, at r e g u l a r i n t e r v a l s , which caused the a c t i v a t i o n o f t h e neurone t o the r i g h t o f t h e s t i m u l a t i o n a r t i f a c t (below t h e d o t ) . Note t h a t c o l l i s i o n a p p a r e n t l y o c c u r r e d when a spontaneous a c t i o n p o t e n t i a l o c c u r r e d w i t h i n a c e r t a i n d i s t a n c e of the a r t i f a c t , (b) T h i s t r a c e i s i d e n t i c a l t o t h e f i r s t t r a c e above ex c e p t i t was a t a h i g h e r a m p l i f i c a t i o n and had l e s s f i l t e r i n g . N o t i c e t h e prominent * i n i t i a l segment' o f t h e evoked p o t e n t i a l . 5 m s e c 70 F i g u r e 9: O s c i l l o s c o p e t r a c i n g s showing spontaneously o c c u r r i n g a c t i o n p o t e n t i a l s c o l l i d i n g with and c a n c e l l i n g a c t i o n p o t e n t i a l s a c t i v a t e d by t r i g g e r e d s t i m u l a t i o n i n the nGC. The s t i m u l a t o r was t r i g g e r e d by spontaneous p o t e n t i a l s , and, a f t e r a s e l e c t e d d e l a y , s t i m u l a t e d the nGC e l e c t r i c a l l y , (a) A t r a c e showing a spontaneous p o t e n t i a l to the l e f t of the s t i m u l a t i o n a r t i f a c t ( i n d i c a t e d by the dot) which t r i g g e r e d the s t i m u l a t o r a f t e r a delay of 6.6 ms. (b) S i m i l a r to above but with a delay of 6.8 ms, an a c t i v a t e d p o t e n t i a l i s seen at r i g h t as no c o l l i s i o n with the spontaneous p o t e n t i a l occurs as i n part (a). (c) A r a s t e r scan of 32 sweeps or t r i a l s . Each dot r e p r e s e n t s an a c t i o n p o t e n t i a l or a s t i m u l a t i o n a r t i f a c t (under the d o t ) . The s t i m u l a t o r was t r i g g e r e d by the spontaneously o c c u r r i n g p o t e n t i a l to the l e f t , and the delay u n t i l s t i m u l a t i o n s e t s u c c e s s i v e l y t o 6.6, 6,7, and 6.8 ms, but f a i l e d at the 6.6 ms s e t t i n g because of c o l l i s i o n . 2 m s e c 72 E f f e c t of Drugs on Unit A c t i v i t y The e f f e c t o f intravenous f l u o x e t i n e {3-10 mg/kg) was t e s t e d on 16 nGC neurones. Nine of these were u n a f f e c t e d by the drug, Of the t h r e e neurones i n h i b i t e d by the f l u o x e t i n e , the i n h i b i t i o n was minimal i n one ca s e , Two of the 4 e x c i t a t o r y responses were a l s o s l i g h t and another was t r a n s i t o r y . H i c r o i c n t o p h o r e t i c a p p l i c a t i o n of glutamate e x c i t e d a l l but two, which were not a f f e c t e d , of the 59 neurones on which i t was t e s t e d . Forty-two neurones were t e s t e d with iontophoresed s e r o t o n i n . Approximately t w o - t h i r d s of these responded to t h i s treatment: 45% were i n h i b i t e d and 21% e x c i t e d , , S i m i l a r l y , 50% of the 28 neurones t e s t e d with iontophoresed f l u o x e t i n e were i n h i b i t e d while 14% sere e x c i t e d and the remaining 36% were u n a f f e c t e d . These responses a r e l i s t e d i n Table I I and an example of a neurone's response t o i o n t o p h o r e t i c s e r o t o n i n , f l u o x e t i n e , and glutamate i s i n c l u d e d i n F i g u r e 10. P e r i p h e r a l S t i m u l i Twenty-nine u n i t s were t e s t e d with non-noxious forms of p e r i p h e r a l s t i m u l i . These i n c l u d e d l i g h t touch and a i r - p u f f s a p p l i e d t o the f u r of the animal or g e n t l e j o i n t movement. Only 18% of the neurones t e s t e d were a f f e c t e d and these were e x c i t e d {see Table II) . In c o n t r a s t , 75% of the neurones t e s t e d with noxious s t i m u l i were a f f e c t e d . Over o n e - h a l f of the 224 neurones were e x c i t e d (53%) by noxious s t i m u l i which i n c l u d e d p i n c h and pressure a p p l i e d to the paws, ears and t a i l , r a d i a n t h e a t i n g of Table I I : Responses of neurones i n the nucleus g i g a n t o c e l l u l a r i s to various t e s t s . Resp onse Test E x c i t a t i o n Mixed I n h i b i t i o n None Total s c i a t i c nerve^ 43 7 9 46 105 2 noxious 118 15 35 56 224 3 non-noxious 7 0 0 32 39 4 i- • • u serotonin 9 - 19 14 42 u fluoxetine 4 - 14 10 28 u glutamate 57 - 0 2 59 PAG 5 17 12 58 41 128 1 - e l e c t r i c a l stimulation of the s c i a t i c nerve 2 - included pinch and pressure to the paws, ears, and t a i l , radiant heating of t a i l , and e l e c t r i c a l stimulation of the s c i a t i c nerve 3 - included a i r - p u f f and stroking applied to the animal's h a i r and gentle j o i n t movement 4 - microiontophoretic a p p l i c a t i o n of ... 5 - e l e c t r i c a l stimulation of the periaqueductal gray matter F i g u r e 10: Examples of nucleus g i g a n t o c e l l u l a r i s neuronal responses t o i o n t o p h o r e s i s and p e r i p h e r a l s t i m u l i , (a) Batemeter output d i s p l a y i n g a nGC neurone*s f i r i n g r a t e changes i n response to i o n t o p h o r e t i c s e r o t o n i n (S), F l u o x e t i n e ( F ) , glutamate (G), and NaCl, used as a c u r r e n t c o n t r o l , (C) . The a b s c i s s a r e p r e s e n t s time and the bars and numbers below i t r e p r e s e n t the p e r i o d s of and the c u r r e n t s (nA) used f o r the drug a p p l i c a t i o n s , r e s p e c t i v e l y . The o r d i n a t e r e p r e s e n t s u n i t a c t i v i t y i n s p i k e s per epoch, where each epoch i s 1.0 s i n d u r a t i o n . Notice the c e l l was e x c i t e d by the glutamate and was i n h i b i t e d by the s e r o t o n i n and f l u o x e t i n e but was not a f f e c t e d by the c u r r e n t c o n t r o l . Also note t h a t the i n h i b i t i o n by s e r o t o n i n was enhanced by the simultaneous e j e c t i o n of f l u o x e t i n e , (b) A c t i v i t y of another nGC neurone i n response to p e r i p h e r a l s t i m u l i . I t was e x c i t e d by noxious pinch a p p l i e d to the r i g h t paw (LP) and both ears (LE, HE) but not to the l e f t paw (LP) or t a i l (T) nor by non-noxious s t r o k i n g of the f u r (S). (c) The top t r a c e shows the temperature (ordinate) of a thermocouple mounted on the s u r f a c e of the t a i l i n the focus of the r a d i a n t heat source, The bottom t r a c e shows another nGC neurone which was a c t i v a t e d by noxious r a d i a n t heat pulses (47 oc, 15 s ) , 75 80-N 401 N G S F C F S F S S+F -10 30 15 5 0 20 25 20 23 23 20 T L P ' R P " LE RE RE S 1min 50i O o 30 80 N L_ l _ 76 the t a i l , and e l e c t r i c a l s t i m u l a t i o n of the l e f t s c i a t i c n e rve. In a d d i t i o n , some u n i t s (16%) were i n h i b i t e d and others responded with mixed e x c i t a t i o n - i n h i b i t i o n ( 7 % ) , Examples of two neurones* response t o p e r i p h e r a l s t i m u l i a r e i l l u s t r a t e d i n F i g u r e 10., The h i s t o l o g i c a l l y determined l o c a t i o n s of the neurones d i d not appear t o be c o r r e l a t e d t o t h e i r responses t o noxious p e r i p h e r a l s t i m u l i as can be seen i n F i g u r e 11. The responses mentioned above i n c l u d e d s t i m u l a t i o n of the s c i a t i c nerve, however, s t i m u l a t i o n of the s c i a t i c nerve alone produced s i m i l a r r e s u l t s . Of the 105 u n i t s t e s t e d with s c i a t i c nerve s t i m u l a t i o n , 41? were e x c i t e d while approximately the same p r o p o r t i o n were u n a f f e c t e d . A few neurones were i n h i b i t e d (9%) or had mixed responses ( 7 % ) . The responses are enumerated i n Table I I and t h r e e examples of t y p i c a l responses are i l l u s t r a t e d i n F i g u r e 12(d-f) . The e x c i t a t o r y responses had l a t e n c i e s r anging from 5 to more than 200 ms and d u r a t i o n s ranging over 4-52 ms. The e x c i t a t o r y responses with l a t e n c i e s o f l e s s than 200 ms c o u l d be d i v i d e d i n t o two groups: s h o r t - l a t e n c y responses having a mean l a t e n c y o f 10.2±1.3 ms and l o n g - l a t e n c y responses having a mean l a t e n c y of 70±11 ms. The i n h i b i t o r y responses* l a t e n c i e s and d u r a t i o n s ranged over 5-35 ms and 4-85 ms, r e s p e c t i v e l y . The mean i n h i b i t o r y response l a t e n c y was 13.8±4.7 ms, while the mean d u r a t i o n o f the i n h i b i t o r y responses was 30±11 ms. In those u n i t s t e s t e d with both noxious p e r i p h e r a l s t i m u l i and i o n t o p h o r e t i c s e r o t o n i n and/or f l u o x e t i n e t h e r e d i d not appear to be a c o r r e l a t i o n between the responses t o the two t e s t s . For example, of the 14 neurones which were e x c i t e d by 77 Figu r e 1 1 : Coronal s e c t i o n s e c t i o n showing h i s t o l o g i c a l l y determined l o c a t i o n s cf nGC area u n i t s which responded to noxious p e r i p h e r a l s t i m u l i . S t i m u l i i n c l u d e d pinch and pressure to the paws, e a r s , and t a i l , r a d i a n t h e a t i n g of the t a i l , and e l e c t r i c a l s t i m u l a t i o n o f the l e f t s c i a t i c nerve. Symbols and comments as i n F i g u r e 5 . 78 7 9 F i g u r e 12: Examples of nucleus g i g a n t o c e l l u l a r i s neuronal responses t o e l e c t r i c a l s t i m u l a t i o n of the p e r i a q u e d u c t a l gray (a-c) and the s c i a t i c nerve ( d - f ) , The responses are i n the form of p e r i s t i m u l u s time histograms (PSTH) and demonstrate neurones which responded with e x c i t a t i o n only (a,d), mixed e x c i t a t i o n -i n h i b i t i o n (b,e), and i n h i b i t i o n only ( c , f ) . The a b s c i s s a e r e p r e s e n t time and the o r d i n a t e s the number of s p i k e s per b i n (or epoch), The bars above the responses i n d i c a t e the p e r i o d s o f s t i m u l a t i o n and the l a r g e peaks below them are t r u n c a t e d s t i m u l a t i o n a r t i f a c t s . S t i m u l a t i o n of e i t h e r a s i n g l e pulse (a,e,f) (0.1 ms, 2-5 V) or a t r a i n of p u l s e s (b-d) (0.05 ms, 2-5 V) and. Each .PSTH i s c o n s t r u c t e d from 32 or 64 sweeps. Bin width = 1.0 ms (a-e) or 2.0 ms ( f ) . . 80 sa>)!ds |0 O N 81 noxious s t i m u l i only 4 were e x c i t e d by s e r o t o n i n (Table I I I ) . Six u n i t s were t e s t e d with both i n t r a v e n o u s l y a d m i n i s t e r e d f l u o x e t i n e and s c i a t i c nerve s t i m u l a t i o n . I n 4 of these, drug a d m i n i s t r a t i o n s d i d not a f f e c t the response of the neurone to nerve s t i m u l a t i o n . I n the other two t r i a l s the e x c i t a t o r y response of the u n i t s was i n c r e a s e d by the f l u o x e t i n e , as can be seen i n Tab l e IV. S t i m u l a t i o n of the P e r i a q u e d u c t a l Gray The predominant response of the 128 nucleus g i g a n t o c e l l u l a r i s neurones t e s t e d with s t i m u l a t i o n of the pe r i a q u e d u c t a l gray was i n h i b i t i o n (45%) . Some e x c i t a t o r y (13%) and mixed e x c i t a t o r y - i n h i b i t o r y (9%) e f f e c t s were a l s o seen while approximately a t h i r d of the neurones were non-responsive (see Table I I ) . An example of each type of response i s i n c l u d e d i n F i g u r e 12(d-f) and the h i s t o l o g i c a l l y determined l o c a t i o n s of the s t i m u l a t i o n s i t e s i n the p e r i a q u e d u c t a l gray are mapped i n Fi g u r e 13. The e x c i t a t o r y responses of nGC neurones t o PAG s t i m u l a t i o n had l a t e n c i e s of 0-75 ms and d u r a t i o n s of 0-110 ms. T h e i r mean l a t e n c y and d u r a t i o n were 23±6 ms and 33±8 ms, r e s p e c t i v e l y , , . The i n h i b i t o r y responses* l a t e n c i e s and d u r a t i o n s ranged over 0-45 ms and 15-200 ms, r e s p e c t i v e l y . The mean i n h i b i t o r y response was 11.6±1.3 ms, while the mean d u r a t i o n of the i n h i b i t o r y responses was 51±8 ms. To see i f t h e r e was a c o r r e l a t i o n between a neurone's response t o noxious p e r i p h e r a l s t i m u l i and i t s response t o PAG s t i m u l a t i o n , the numbers of neurones responding i n each way to 82 Table I I I : Comparison of the responses of nucleus g i g a n t o c e l l u l a r i s neurones to noxious peripheral s t i m u l i ;and iontophoretic a p p l i c a t i o n of serotonin or fluoxetine. Responses to iontophoretic a p p l i c a t i o n of: Serotonin Fluoxetine s t i m u l i Excited Inhibited None Excited Inhibited None Ex c i t a t i o n 4 9 1 1 .7 1 Mixed 0 1 0 1 Q 0 I n h i b i t i o n 0 1 1 0 2 0 None 0 1 0 0 1 0 Column t o t a l 4 12 2 2 10 1 1— included pinch and pressure to paws, ears, and t a i l ; radiant heating of t a i l ; and e l e c t r i c a l stimulation of the s c i a t i c nerve. Response to noxious peripheral 83 Table IV: The effect of intravenously applied fluoxetine on the responses of nucleus gigantocellularis neurones to electrical stimulation of the periaqueductal gray of the sciatic nerve. Response to stimulation was:  Enhanced Reduced None Total Response to stimulation of the periaqueductal gray: Excitation 0 4 0 4 Inhibition 3 0 4 7 Response to stimulation of the sciatic nerve: Excitation 2 0 4 6 Inhibition 0 0 0 0 84 F i g u r e 13: Coronal s e c t i o n showing the h i s t o l o g i c a l l y determined l o c a t i o n of the s t i m u l a t i n g e l e c t r o d e s used i n experiments which s t u d i e d the e f f e c t of e l e c t r i c a l s t i m u l a t i o n of the p e r i a q u e d u c t a l gray on the a c t i v i t y of nucleus g i g a n t o c e l l u l a r i s neurones. Symbols and comments as i n F i g u r e 4, 86 the two t e s t s were enumerated. As can be seen from Table V the l a r g e s t group of neurones {23) were e x c i t e d by the noxious s t i m u l i and i n h i b i t e d by the PAG s t i m u l a t i o n . T h i s group was equal t o 37% of those neurones a f f e c t e d by both treatments or 18% of the t o t a l neurones t e s t e d (128). Although few neurones were t e s t e d with both i o n t o p h o r e r e t i c s e r o t o n i n o r f l u o x e t i n e and PAG s t i m u l a t i o n , a comparison of the responses t o these two t e s t s r e v e a l s a modest c o r r e l a t i o n (see Table V I ) . Both of the u n i t s e x c i t e d by PAG s t i m u l a t i o n were a l s o e x c i t e d by i o n t o p h o r e t i c s e r o t o n i n , , S i m i l a r l y , 5 of the 7 neurones i n h i b i t e d by PAG s t i m u l a t i o n were a l s o i n h i b i t e d by s e r o t o n i n * The e f f e c t of intravenous and i o n t o p h o r e t i c f l u o x e t i n e on the responses of nGC neurones t o PAG s t i m u l a t i o n were a l s o t e s t e d . The r e s u l t s of i n t r a v e n o u s f l u o x e t i n e are shown i n Table IV, The i n h i b i t o r y response produced by PAG s t i m u l a t i o n i n 4 u n i t s was enhanced by the f l u o x e t i n e . Another seven u n i t s were e x c i t e d by PAG s t i m u l a t i o n . In a l l 3 of these e x c i t a t o r y responses which were modified by the f l u o x e t i n e , the responses were reduced. Three examples of the e f f e c t o f f l u o x e t i n e , both i o n t o p h o r e t i c and i n t r a v e n o u s l y a d m i n i s t e r e d , on the response of nGC neurones t o PAG s t i m u l a t i o n are i l l u s t r a t e d i n F i g u r e s 14 and 15. , Table.Vr. Comparison of nucleus g i g a n t o c e l l u l a r i s neuronal responses to noxious peripheral s t i m u l i ^ and stimulation of the periaqueductal gray. Response to noxious peripheral Response to e l e c t r i c a l stimulation of the periaqueductal gray matter Row s t i m u l i E x c i t a t i o n Mixed I n h i b i t i o n None Tota l E x c i t a t i o n 14 8 23 17 62 Mixed 0 1 3 1 5 I n h i b i t i o n 1 2 11 3 17 None 2 1 21 20 44 Column Total 177 12 58 41 128 1 - included pinch and pressure to the paws, ears, and t a i l , radiant heating of the t a i l , and e l e c t r i c a l stimulation of the s c i a t i c nerve. 8 8 Table VI: Comparison of the responses of nucleus g i g a n t o c e l l u l a r i s neurones to periaqueductal gray stimulation and iontophoretic a p p l i c a t i o n of serotonin or fluoxetine. Response to periaqueductal Responses to iontophoretic a p p l i c a t i o n of: gray Serotonin Fluoxetine  stimulation Excited Inhibited None Excited Inhibited None E x c i t a t i o n 2 0 0 1 2 0 Mixed 0 3 0 0 1 0 I n h i b i t i o n 0 5 2 0 3 2 None 1 2 0 . 0 1 0 Column t o t a l 3 10 2 1 7 2 89 F i g u r e 14: Examples of two neurones (a-b, c-f) i n h i b i t e d by pe r i a q u e d u c t a l gray s t i m u l a t i o n and the enhancement of t h i s i n h i b i t i o n by f l u o x e t i n e , a s p e c i f i c s e r o t o n i n uptake b l o c k e r . , Sesponses are shown i n the form of p e r i s t i m u l u s time histograms with time on the a b s c i s s a e and the number of s p i k e s per b i n (or epoch) on the o r d i n a t e s . The bars above the responses represent the p e r i o d s of s t i m u l a t i o n and the l a r g e responses below them are t r u n c a t e d s t i m u l a t i o n a r t i f a c t s , (a) C o n t r o l response of f i r s t u n i t to PAG s t i m u l a t i o n (10 p u l s e s , 0.05 ms, 4 V) . (b) Enhancement of the i n h i b i t o r y p e r i o d 15 min a f t e r i n t r a v e n o u s f l u o x e t i n e (3 mg/kg). (c) C o n t r o l response of the second neurone to PAG s t i m u l a t i o n (10 p u l s e s , 0,05 ms, 5 V) . (d) Enhancement of response dur i n g i o n t o p h o r e s i s of f l u o x e t i n e (40 nA, 1 min) . (e) E i g h t min a f t e r iontophoresed f l u o x e t i n e , the response was comparable t c the c o n t r o l i n (c) . (f) S l i g h t enhancement of response 6 min a f t e r i n t r a v e n o u s f l u o x e t i n e (5 mg/kg).,Bin width = 2 ms, 64 sweeps per response. 90 91 F i g u r e 15: An example of a nucleus g i g a n t o c e l l u l a r i s neurone which was e x c i t e d by p e r i a q u e d u c t a l gray s t i m u l a t i o n and the r e d u c t i o n cf t h i s response by f l u o x e t i n e , a s p e c i f i c s e r o t o n i n uptake b l o c k e r . See F i g u r e 14 f o r a d e s c r i p t i o n of the p e r i s t i m u l u s time histograms, (a) C o n t r o l response, (b-d) 9.5, 23, and 25 rain a f t e r i o n t o p h o r e s i s of f l u o x e t i n e (60 nA, 1 min) , r e s p e c t i v e l y . (e,f) 30 and 40 min a f t e r i n t r a v e n o u s f l u o x e t i n e (two 5 mg/kg d o s e s ) . Bin width = 1.0 ms, 64 sweeps per response. 92 93 DISCUSSION A • - • P e r i a q u e d u c t a l Gray Neurones Noxious P e r i p h e r a l S t i m u l i Se found many neurones i n the v i c i n i t y of the p e r i a q u e d u c t a l gray which responded t o noxious p e r i p h e r a l s t i m u l i . T h i s was expected as the s p i n o t e c t a l t r a c t i s known to terminate i n t h i s area (Mehler, 1960; Kerr, 1975)., a l s o , evoked p o t e n t i a l s have been recorded from t h i s area i n r a t s i n response to e l e c t r i c a l s t i m u l a t i o n of the s k i n (Mayer and L i e b e s k i n d , 1971) and the s c i a t i c nerve (Palmer and Klemm, 1976), Recent s t u d i e s , which recorded u n i t a c t i v i t y from neurones i n the same area, a l s o found e x c i t a t o r y and i n h i b i t o r y responses to noxious s t i m u l i which i n c l u d e d p i n c h , s t i m u l a t i o n of p e r i p h e r a l n e r v e s , and r a d i a n t heat (Aghajanian e t a l . , 1978; H a i g l e r , 1978; Sanders e t a l . , 1980.). Aghajanian e t a l . (1978) r e p o r t e d t h a t s e r o t o n e r g i c neurones were i n h i b i t e d and n o n - s e r o t o n e r g i c neurones were e x c i t e d by noxious p e r i p h e r a l s t i m u l i . Sanders e t a l . , (198 0) found t h a t most neurones with e x c i t a t o r y responses were l o c a t e d i n the p e r i a q u e d u c t a l gray, while those i n h i b i t e d were l o c a t e d i n the d o r s a l raphe. T h i s agrees with the p r e v i o u s study as the d o r s a l raphe i s a major s e r o t o n e r g i c nucleus. , we d i d not f i n d any correspondence between c e l l l o c a t i o n s and responses to noxious s t i m u l i . However, d o r s a l raphe neurones are r e p o r t e d to have slow spontaneous f i r i n g r a t e s ( l e s s than 2 Hz) which makes i n h i b i t o r y responses very hard t o r e c o g n i z e . T h e r e f o r e , our r e s u l t s are very probably b i a s e d towards neurones with r e l a t i v e l y f a s t e r spontaneous f i r i n g r a t e s and those responding with e x c i t a t i o n . T h i s a l s o probably r e s u l t e d i n many of the i n h i b i t o r y responses c f d o r s a l raphe neurones being c l a s s i f i e d as non-responsive. S t i m u l a t i o n o f the Nucleus G i g a n t o c e l l u l a r i s The orthodromic responses of neurones i n the v i c i n i t y of the PAG e l i c i t e d by s t i m u l a t i o n i n the nGC were u s u a l l y e a s i l y i d e n t i f i e d as most showed m u l t i p l e f i r i n g a t i n c r e a s e d s t i m u l u s i n t e n s i t i e s , very long l a t e n c i e s , or very i n c o n s i s t e n t l a t e n c i e s . However, d i s t i n g u i s h i n g between orthodromic and a n t i dromic responses with c o n s t a n t l a t e n c i e s of l e s s than 7 ms was more d i f f i c u l t . Constant l a t e n c y i s a f a i r l y poor c r i t e r i o n f o r a n t i d r c m i c i t y except at t h r e s h o l d . , However, even at t h r e s h o l d o r t h o d r o m i c a l l y a c t i v a t e d neurones might have shown constant l a t e n c i e s i f the s y n a p t i c i n p u t on to them was i t s e l f c o n s t a n t . In such a case the neurone would have been e x c i t a b l e t o the same degree f o r each s y n a p t i c a l l y evoked response and t h e r e f o r e would have d i s p l a y e d a constant l a t e n c y . C onversely, a n t i d r o m i c a l l y a c t i v a t e d neurones can have l a t e n c i e s v a r y i n g up t o 0.5 ms due to delays of the a c t i o n p o t e n t i a l c r o s s i n g the axonal h i l l o c k and i n v a d i n g the c e l l body ( E c c l e s , 1955). T h i s a g a i n depends upon the e x c i t a b i l i t y of the c e l l body membrane and a l s o upon the r a t e of i n c r e a s e i n membrane area t h a t the p o t e n t i a l encounters as i t t r a v e l s from the axon to the c e l l body. In t h i s study only 29% of the H9 neurones which were c l a s s i f i e d as 95 orthodromic had i n c o n s i s t e n t l a t e n c i e s . T h e r e f o r e , i n c o n s t a n t l a t e n c y appears t o be a good i n d i c a t i o n of orthodromic a c t i v a t i o n but constant l a t e n c y not t o be a good c r i t e r i o n f o r an t i d r o m i c a c t i v a t i o n . , The second c r i t e r i o n , the maximum frequency a t which the response can f o l l o w the s t i m u l u s , i s o f t e n c i t e d as good evidence f o r a n t i d r o m i c i t y . However, o r t h o d r o m i c a l l y a c t i v a t e d neurones can sometimes f o l l o w a t high f r e q u e n c i e s (Darian-Smith e t a l . , 1963) and, c o n v e r s e l y , a n t i d r o m i c a l l y a c t i v a t e d neurones can sometimes f a i l to f e l l o w h i g h s t i m u l a t i o n f r e q u e n c i e s ( E c c l e s , 1S55). Again, t h i s f a i l u r e i s due t o the blockade of the a c t i o n p o t e n t i a l at the axonal h i l l o c k . In the present study, because the d i s t a n c e between the s t i m u l a t i o n s i t e and the r e c o r d i n g s i t e was so s m a l l , i t was o f t e n d i f f i c u l t t o t e s t f o l l o w i n g frequency because of the l a r g e s t i m u l a t i o n a r t i f a c t . In a d d i t i o n , i f long t r a i n s of s t i m u l i were attempted, an i n c r e a s i n g l y l a r g e a r t i f a c t o c c u r r e d due t o both summation of m u l t i - u n i t evoked p o t e n t i a l s and muscle twi t c h . T h e r e f o r e , because o f these d i f f i c u l t i e s i n t e s t i n g f o r f o l l o w i n g , some neurones which were a c t u a l l y a n t i d r o m i c a l l y a c t i v a t e d may have been e r r o n e o u s l y c l a s s i f i e d as orthodromic i n t h i s study. Even so, high frequency f o l l o w i n g would appear, a t l e a s t i n t h i s study, to be a good i n d i c a t i o n of a n t i d r o m i c i t y because none of the responses c l a s s i f i e d as orthodromic f o l l o w e d the s t i m u l a t i o n at high f r e q u e n c i e s . , The f i n a l c r i t e r i o n of a n t i d r o m i c i t y , c o l l i s i o n of orthodromic a c t i o n p o t e n t i a l s with a n t i d r o m i c ones, i s g e n e r a l l y c o n s i d e r e d t o be the most robust of the c r i t e r i a . However, some 9 6 problems do occur i n a c t u a l p r a c t i c e . F i r s t l y , t o t e s t f o r c o l l i s i o n , the c e l l under study must be spontaneously a c t i v e or capable o f being a c t i v a t e d . , A c t i v a t i o n can occur by the 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 an e x c i t a t o r y drug, such as glutamate, onto the neurone or by i n c r e a s i n g the e x c i t a t o r y s y n a p t i c i n p u t to the c e l l . In t h i s study some neurones were a c t i v a t e d by a p p l y i n g noxious pressure t o the t a i l . Secondly, the recorded a c t i o n p o t e n t i a l had t o be l a r g e enough to g i v e a s i g n a l - t o - n o i s e r a t i o which was s u f f i c i e n t t o allow the c o n s i s t e n t t r i g g e r i n g of the s t i m u l a t o r . T h i r d l y , the c r i t i c a l p e r i o d i s t h e o r e t i c a l l y the sum of the l a t e n c y and the r e f r a c t o r y p e r i o d . However, the measurement of these two v a l u e s i s s u b j e c t to e r r o r . The measurement of a r e f r a c t o r y p e r i o d i n v o l v e s observing the maximum frequency at which the response c o n s i s t e n t l y f o l l o w s the s t i m u l a t i o n . U n f o r t u n a t e l y t h i s a c t u a l l y measures the ' l e a s t i n t e r v a l f o r two propragated s p i k e s ' ( c f . F u l l e r and S c hlag, 1975) t r a v e l l i n g the same d i r e c t i o n along the axon, the second s p i k e f o l l o w i n g immediately a f t e r the f i r s t ' s r e f r a c t o r y p e r i o d . For a n t i d r o m i c c o l l i s i o n the two s p i k e s are t r a v e l l i n g i n o p p o s i t e d i r e c t i o n s and t h e r e f o r e the value r e q u i r e d t o c a l c u l a t e the c r i t i c a l p e r i o d w i l l be l e s s than t h a t measured., The measurement of the l a t e n c y o f response i s s u b j e c t to two e r r o r s . There i s a delay between the onset of the s t i m u l a t i o n pulse and the a c t i v a t i o n of the f i b r e . T h i s i s termed u t i l i z a t i o n time and can range over 0.2-0.5 ms ( B l a i r and E r l a n g e r , 1936; c f . F u l l e r and S c h l a g , 1976).. There i s a l s o a delay between the a r r i v a l of the s p i k e at the c e l l body and the i n v a s i o n of the soma, which i s what i s 97 u s u a l l y r e c o r d e d e x t r a c e l l u l a r l y . T h i s delay can t y p i c a l l y be 0.2 ms longer than the e q u i v a l e n t delay i n orthodromic a c t i v a t i o n s {cf. F u l l e r and S c h l a g , 1976). The f i r s t two e r r o r s can be le s s e n e d by i n c r e a s i n g the s t i m u l u s i n t e n s i t y . U n f o r t u n a t e l y , the maximum st i m u l u s l e v e l s which c o u l d be used i n t h i s study were d i c t a t e d by the s i z e of the s t i m u l a t i o n a r t i f a c t , the presence of an evoked muscle movement, and s a t u r a t i o n o f the u n i t y g a i n p r e a m p l i f i e r * The e r r o r s d i s c u s s e d i n the p r e v i o u s paragraph are f a i r l y c onstant and do not vary with l a t e n c y or r e f r a c t o r y p e r i o d . . T h e r e f o r e , the e r r o r i n the c a l c u l a t i o n of the c r i t i c a l p e r i o d f o r c o l l i s i o n becomes r e l a t i v e l y l a r g e a t short l a t e n c i e s and i s t y p i c a l l y 0.5-2 ms. The neurones i n t h i s study t y p i c a l l y showed c o l l i s i o n i n a p e r i o d approximately e q u a l t o t h e l a t e n c y . I f the e r r o r i n the measurement of l a t e n c y i s not taken i n t o c o n s i d e r a t i o n then the r e f r a c t o r y p e r i o d would appear t o be very s h o r t or n o n - e x i s t e n t and the e r r o r t o be about as l a r g e as the r e f r a c t o r y p e r i o d . I f the l a t e n c y of a response i s very s h o r t (<2 ms) , i t would be p o s s i b l e to see what appears to be c o l l i s i o n but what i s a c t u a l l y due to the a r r i v a l of an evoked orthodromic p o t e n t i a l during t h e r e f r a c t o r y p e r i o d of a preceding spontaneous p o t e n t i a l . However, f o r t h i s t o happen would r e q u i r e the r e f r a c t o r y p e r i o d to be grea t e r than two times the l a t e n c y . Some anomalies were observed i n the responses of some neurones. One orthodromic neurone, c l a s s i f i e d as such because i t had a l a t e n c y of 13 ms, appeared to demonstrate c o l l i s i o n . I f t h i s neurone was a c t u a l l y a c t i v a t e d a n t i d r o m i c a l l y t h i s would 98 imply t h a t the conduction v e l o c i t y of t h i s neurone was about 0,3 ms. I f i t was i n f a c t o r t h o d r o m i c a l l y a c t i v a t e d then the apparent c o l l i s i o n might have been due t o an extremely long r e f r a c t o r y p e r i o d (in the order of 20 ms) , r e c u r r e n t i n h i b i t i o n , or i n h i b i t i o n v i a a slower pathway, Some neurones demonstrated c o l l i s i o n where the c o l l i s i o n i n t e r v a l was much g r e a t e r than a reasonable c r i t i c a l p e r i o d . Again, t h i s may be due to an abnormally long r e f r a c t o r y p e r i o d or r e c u r r e n t i n h i b i t i o n . , . As w e l l as the above c r i t i c i s m s and d i f f i c u l t i e s with the i n d i v i d u a l c r i t e r i a , other g e n e r a l d i f f i c u l t i e s must be d i s c u s s e d . . Because o f the f i l t e r i n g which was necessary i n these experiments t o attenuate unwanted n o i s e , the shape of the a c t i o n p o t e n t i a l s was d i s t o r t e d . T h i s made o b s e r v a t i o n of the i n i t i a l segment of the a c t i o n p o t e n t i a l s d i f f i c u l t except f o r e x c e p t i o n a l l y l a r g e s p i k e s . The f i l t e r i n g a l s o lengthened the time c o n s t a n t of the stim u l u s a r t i f a c t s , i n c r e a s i n g t h e i r d u r a t i o n to 1-2 ms., T h i s meant t h a t a c t i o n p o t e n t i a l s with l a t e n c i e s of l e s s than 1 ms, or c o n v e r s e l y , with c o n d u c t i o n v e l o c i t i e s g r e a t e r than 3.5 m/s, were very d i f f i c u l t or im p o s s i b l e to d i s t i n g u i s h from the a r t i f a c t . For t h i s reason, the r e s u l t s of these experiments a r e bia s e d toward long l a t e n c y responses and many a n t i d r o m i c a l l y a c t i v a t e d neurones may have been missed. However, there are a few a d d i t i o n a l f e a t u r e s of a n t i d r o m i c a c t i v a t i o n s which are u s e f u l i n t h e i r i d e n t i f i c a t i o n . F r a c t i o n a t i o n of an antidromic a c t i o n p o t e n t i a l may occur due t o blockade of the p o t e n t i a l at t h e axonal h i l l o c k . The blockade may occur at any s t i m u l u s i n t e n s i t y as the a c t i v a t e d f i b r e 99 p o t e n t i a l has an a l l - o r - n o t h i n g nature {Bishop e t a l . , 1962). I f the p o t e n t i a l f a i l s to invade the c e l l body then the p o t e n t i a l observed i s r e m i n i s c e n t o f the i n i t i a l segment of a s y n a p t i c a l l y a c t i v a t e d p o t e n t i a l , but i s much l a r g e r and can be d i s t i n g u i s h e d e a s i l y . A l s o as opposed t o orthodromic s t i m u l i which can cause m u l t i p l e p o s t - s y n a p t i c p o t e n t i a l s from one sti m u l u s , a n t i d r o m i c s t i m u l i cause only one a c t i v a t i o n per stimulus. , A f i n a l d i f f i c u l t y i n the i n t e r p r e t a t i o n o f these r e s u l t s i s the p o s s i b i l i t y t h a t the a n t i d r o m i c a c t i o n p o t e n t i a l s are due to the a c t i v a t i o n o f f i b r e s which do not terminate i n the nGC. While no assurance can be given t h a t t h i s i s not i n f a c t the case, anatomical s t u d i e s using a u t o r a d i o g r a p h i c a l { B o b i l l i e r e t a l . , 1976) and HBP (G a l l a g e r and P e r t , 1978) techniques a l s o i n d i c a t e that the nGC r e c e i v e s d i r e c t axonal i n p u t from t h i s r e g i o n . , While we found i t d i f f i c u l t t o t e s t every c r i t e r i o n on every neurone i t i s very l i k e l y t h a t p e r i a q u e d u c t a l gray neurones were a n t i d r o m i c a l l y a c t i v a t e d from the nGC. In a d d i t i o n t o neurones which demonstrated c o l l i s i o n , neurones which met the high frequency c r i t e r i o n were a l s o probably a n t i d r o m i c a l l y a c t i v a t e d . As w e l l , some neurones c l a s s i f i e d as orthodromic may have a c t u a l l y been a n t i d r o m i c a l l y a c t i v a t e d as suggested above. Some neurones may have f a i l e d the c o l l i s i o n c r i t e r i o n because of lack of spontaneous a c t i v i t y or a c t i o n p o t e n t i a l s which were too s m a l l t o t r i g g e r the s t i m u l a t o r . , Some neurones may have f a i l e d the f o l l o w i n g c r i t e r i o n due to the t e c h n i c a l d i f f i c u l t i e s . . In a d d i t i o n , some long l a t e n c y 100 responses may i n f a c t be a ntidromic i f the conduction v e l o c i t i e s of the f i b r e s i n v o l v e d were l e s s than 0.5 ms or i f the f i b r e s took c i r c u i t o u s r o u t e s . For example, the neurone which app a r e n t l y showed c o l l i s i o n with a l a t e n c y of 13 ms d i d have a c o n s t a n t l a t e n c y and d i s p l a y e d f r a c t i o n a t i o n . . F i n a l l y , a n t i d r c m i c a l l y a c t i v a t e d neurones may have been missed because they had very s h o r t l a t e n c i e s <<1 ms) or because they p r o j e c t e d v i a t h i n non-invadable axons (Sanders e t a l . , 1980). The neuronal p o p u l a t i o n s t u d i e d i s very probably b i a s e d towards l a r g e r neurones and t h i s may a l s o have i n f l u e n c e d the numbers of orthodromic versus antidromic responses. T h e r e f o r e , i t i s very l i k e l y t h a t there i s a monosynaptic pathway from the PAG to the nGC. B» Nucleus G i g a n t o c e l l u l a r i s Neurones E f f e c t s of Drugs Intravenous f l u o x e t i n e i n c r e a s e d (4), decreased (3), or d i d not e f f e c t (9) t h e spontaneous a c t i v i t y of the 16 nGC neurones on which i t was t e s t e d . The e f f e c t s of i n t r a v e n o u s l y a d m i n i s t e r e d f l u o x e t i n e on the spontaneous f i r i n g of nGC neurones i s d i f f i c u l t to i n t e r p r e t f o r a number of reasons., F i r s t l y , most of the responses were s l i g h t or t r a n s i t o r y i n nature. Secondly, PSTHs of the responses of nGC neurones to PAG and s c i a t i c nerve s t i m u l a t i o n were being c o n s t r u c t e d c o n c u r r e n t l y with the drug a d m i n i s t r a t i o n making i t d i f f i c u l t to separate the e f f e c t s of the s t i m u l a t i o n on the neuronal a c t i v i t y from those of the drug. T h i r d l y , the nGC r e c e i v e s d i f f u s e i n p u t 101 from many areas making i t i m p o s s i b l e to know i f the responses of the nGC neurones were due to d i r e c t or i n d i r e c t e f f e c t s . Many other workers have r e p o r t e d e x c i t a t o r y and i n h i b i t o r y responses of neurones i n the v i c i n i t y of the nucleus g i g a n t o c e l l u l a r i s t o i o n t o p h o r e t i c a l l y a p p l i e d s e r o t o n i n i n the c a t {Bradley and Wo l s t e n c r o f t , 1965; Boakes et a l . , 1970) and the r a t (Bradley and Dray, 1973; Boakes e t a l . , 1974; H a i g l e r and Aghajanian, 1974; Br i g g s , 1977)., In c o n t r a s t to t h i s study these workers g e n e r a l l y found a high e r percentage of e x c i t a t o r y than i n h i b i t o r y responses. T h i s may have been due to the use of urethane as an a n a e s t h e t i c agent i n t h i s study as Bradley and Dray (1973) reported t h a t urethane i n c r e a s e d t h e p r o p o r t i o n of i n h i b i t o r y responses t o iontophoresed s e r o t o n i n . , A l s o , as c r e a t i n i n e i s i t s e l f reported to be e x c i t a t o r y on some neurones, some of the e x c i t a t o r y responses t h a t these workers saw may have been due t o the s e r o t o n i n - c r e a t i n i n e complex used (Bradley and B o l s t e n c r o f t , 1965). Some neurones responding t o s e r o t o n i n with e x c i t a t i o n were excluded from t h i s study because they were due t o the d i r e c t e x c i t a t o r y e f f e c t of the e j e c t i o n c u r r e n t . Not a l l of the p r e v i o u s s t u d i e s were c o n t r o l l e d f o r c u r r e n t e f f e c t s . L a s t l y , t h e p o p u l a t i o n of neurones t e s t e d i n t h i s study i s q u i t e s m a l l and t h e r e f o r e may not be r e p r e s e n t a t i v e of the e n t i r e neuronal p o p u l a t i o n o f the nGC. A l s o , the group of neurones s t u d i e d w i l l be bia s e d towards l a r g e neurones with s t a b l e background f i r i n g r a t e s as these are the e a s i e s t t o r e c o r d and t e s t . 102 Besponses to P e r i p h e r a l S t i m u l i Most neurones i n the v i c i n i t y of the nGC responding t o p e r i p h e r a l noxious s t i m u l i were e x c i t e d although some i n h i b i t o r y or mixed e x c i t a t o r y - i n h i b i t o r y responses were a l s o seen., These r e s u l t s a r e i n g e n e r a l agreement with the f i n d i n g s of other workers. The m a j o r i t y of nGC neurones i n the c a t are r e s p o n s i v e to noxious p e r i p h e r a l s t i m u l i . These i n c l u d e noxious pinch and pressure ( W o l s t e n c r o f t , 1964; Casey, 1969, 1971c), i n t r a -a r t e r i a l l y a d ministered b r a d y k i n i n ( G u i l l b a u d e t a l . , 1973b; Besson e t a l . , 1974), p e r i p h e r a l nerve s t i m u l a t i o n (Goldman e t a l . , 1972; Casey, 1969, 1971c; E c c l e s , 1975; Fox and B o l s t e n c r o f t , 1976; P e a r l and Anderson, 1978), and cutaneous s t i m u l a t i o n (Peterson e t a l . , 1974; LeBlanc and Gatipon, 1974). S t i m u l a t i o n of the s c i a t i c nerve was c l a s s i f i e d as a noxious s t i m u l u s i n t h i s study. T h i s was probably j u s t i f i a b l e because Goldman e t a l . (1972) r e p o r t t h a t the t h r e s h o l d o f A-d e l t a f i b r e a c t i v a t i o n i n the f e l i n e s c i a t i c nerve was 0.14-2.5 V. The s t i m u l a t i o n i n t e n s i t i e s used i n t h i s study ranged between 2 and 25 V with the m a j o r i t y l y i n g between 5 and 10 V. In a d d i t i o n , Goldman et a l . (1972) s t a t e d t h a t few neurones i n the nGC were a f f e c t e d by j u s t A-beta s t i m u l a t i o n , most a l s o r e q u i r e d A - d e l t a f i b r e a c t i v a t i o n . .... S i m i l a r l y , Casey (1969) found t h a t 70% of the neuronal responses were due t o A - d e l t a a c t i v a t i o n . The p r o p o r t i o n o f e x c i t a t o r y , i n h i b i t o r y , and mixed responses to noxious s t i m u l i vary among the r e p o r t s , depending upon the techniques and s t i m u l i used, but are g e n e r a l l y comparable to those r e p o r t e d here. M u l t i p l e e x c i t a t i o n s and 103 i n h i b i t i o n s have been reported and were a l s o observed i n t h i s study. They made the measurement and i n t e r p r e t a t i o n of the responses d i f f i c u l t as i t was hard to decide where one response ended and another began* E x c i t a t o r y responses are r e p o r t e d t o have l a t e n c i e s of 5-20 ms and d u r a t i o n s o f 8-60 ms, which are i n agreement with our f i n d i n g s . In a d d i t i o n we found some responses with d u r a t i o n s of g r e a t e r than 200 ms. I n t e r e s t i n g l y , Peterson e t a l . (197 4) r e p o r t e d t h a t d e p o l a r i z a t i o n of some u n i t s , which were recorded i n t r a c e l l u l a r l y , l a s t e d f o r over 200 ms. I n h i b i t o r y responses were r e p o r t e d t o have l a t e n c i e s of 5-70 ms and d u r a t i o n s comparable t o those reported here. Again, i n t r a c e l l u l a r h y p e r p o l a r i z a t i o n s l a s t g r e a t e r than 200 ms a f t e r paw pad cutaneous shocks i n the c a t (Peterson e t a l , 1974)., Summation of e x c i t a t o r y responses over m u l t i p l e s t i m u l i has been observed (Goldman e t a l . , 1972; LeBlanc and Gatipon, 1974) and may e x p l a i n the d i f f e r e n c e s i n response d u r a t i o n s between the responses due to n a t u r a l s t i m u l i versus e l e c t r i c a l s t i m u l a t i o n * F i n a l l y i t must be noted t h a t a l l of the above s t u d i e s were done i n the c a t and t h e r e f o r e may not be d i r e c t l y comparable to the r a t . In any event, i t i s reasonable t o conclude that nGC neurones i n the r a t r e c e i v e noxious p e r i p h e r a l i n p u t and t h e r e f o r e are probably i n v o l v e d i n n o c i c e p t i v e p r o c e s s i n g . The apparent enhancement of the e x c i t a t o r y response of the nGC neurones to s t i m u l a t i o n of the s c i a t i c nerve by i n t r a v e n o u s l y a dministered f l u o x e t i n e i s d i f f i c u l t t o i n t e r p r e t . Because the nGC has d i f f u s e i n p u t s the f l u o x e t i n e may not be a c t i n g d i r e c t l y on the nGC neurones. To support t h i s argument 104 there i s no c l e a r c o r r e l a t i o n between the response of nGC neurones t o p e r i p h e r a l noxious s t i m u l i and t h e i r response to i o n t o p h o r e t i c s e r o t o n i n or f l u o x e t i n e i n those neurones t e s t e d with both the noxious s t i m u l i and the i o n t o p h o r e t i c drugs. C l e a r l y , a g r e a t e r number of neurones need to be looked a t to d i s t i n g u i s h any r e l a t i o n s h i p between these two treatments. Responses to PAG S t i m u l a t i o n Most nGC neurones were i n h i b i t e d or u n a f f e c t e d by p e r i a q u e d u c t a l gray s t i m u l a t i o n . , Some e x c i t a t o r y and mixed e x c i t a t o r y - i n h i b i t o r y responses were a l s o seen.. Other workers have r e p o r t e d s i m i l a r responses of nGC neurones t o e l e c t r i c a l s t i m u l a t i o n of the PAG. In p r e l i m i n a r y experiments Casey and Morrow (1976) found t h a t 55% of r a t nGC neurones d r i v e n by t a i l shock at 10-15 ms l a t e n c i e s and 40% of the u n i t s responding t o noxious mechanical or thermal s t i m u l i were i n h i b i t e d by PAG s t i m u l a t i o n . T h i s compares f a v o u r a b l y with the 40% of neurones i n t h i s study that responded to noxious p e r i p h e r a l s t i m u l i and which were a l s o i n h i b i t e d by PAG s t i m u l a t i o n . Morrow and Casey (1976) c a r r i e d out f u r t h e r experiments i n awake r a t s . They found t h a t 65% of neurones t e s t e d with PAG s t i m u l a t i o n were i n h i b i t e d as compared to the 45% of the neurones t e s t e d i n t h i s study. In a d d i t i o n , they reported t h a t s t i m u l a t i o n of a n a l g e t i c s i t e s i n the PAG, t h a t i s those s i t e s t h a t supported SPA as measured by t a i l - f l i c k l a t e n c i e s , i n h i b i t e d approximately 90% of nGC neurones which were e x c i t e d by noxious p e r i p h e r a l s t i m u l i but only 48% of the neurones e x c i t e d s o l e l y by non-noxious s t i m u l i . These r e s u l t s suggest a 105 s e l e c t i v e i n h i b i t i o n of nGC n o c i c e p t i v e neurones over non-n o c i c e p t i v e neurones. In agreement with t h i s study Mohrland and Gebhart (1979) found t h a t PAG s t i m u l a t i o n produced e x c i t a t o r y , i n h i b i t o r y , and mixed e f f e c t s on the spontaneous f i r i n g r a t e s of nGC neurones. They a l s o found t h a t 80% of nGC neuronal responses t o noxious s t i m u l i were i n h i b i t e d by s t i m u l a t i o n of a n a l g e t i c PAG s i t e s , but t h a t n o n - a n a l g e t i c s i t e s were i n e f f e c t i v e i n t h i s regard. He found that s i t e s i n the PAG which a f f e c t e d nGC neuronal spontaneous f i r i n g i n h i b i t e d 58% of neurones responding t o noxious p e r i p h e r a l s t i m u l i were i n h i b i t e d . Probably s e l e c t i o n of PAG s t i m u l a t i o n s i t e s , i . e . SPA s i t e s , i n t h i s study would have i n c r e a s e d the percentage of neurones i n h i b i t e d by PAG s t i m u l a t i o n . The a n t i d r o m i c experiments i n d i c a t e t h a t t h e r e i s very l i k e l y a monosynaptic pathway from the PAG to the nGC. However, the l a t e n c i e s c f the responses of some nGC neurones to PAG s t i m u l a t i o n suggest t h a t they were probably mediated by p o l y s y n a p t i c mechanisms. P o s s i b l y these e f f e c t s of PAG s t i m u l a t i o n are mediated by the medial raphe n u c l e u s , the c a u d a l raphe nucleus, or the d o r s a l tegmentum. A l l of these areas have been r e p o r t e d t o send i n p u t s t o the nGC ( B o b i l l i e r e t a l . , 1976; B r i g g s , 1976; G a l l a g e r and P e r t , 1978). A l s o , as the raphe n u c l e i are s e r o t o n e r g i c , t h e i r involvement would e x p l a i n the c o r r e l a t i o n observed i n t h i s study between the responses of nGC neurones to PAG s t i m u l a t i o n and t h e i r responses t 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 cf s e r o t o n i n or f l u o x e t i n e . I n t r a v e n o u s l y administered f l u o x e t i n e reduced a l l f o u r PAG 106 s t i m u l a t i o n induced e x c i t a t i o n s , and enhanced t h r e e of seven i n h i b i t o r y responses of nGC spontaneously f i r i n g neurones. A l s o , t h e r e was a moderate c o r r e l a t i o n between the e f f e c t s of i o n t o p h o r e t i c s e r o t o n i n and f l u o x e t i n e and the e f f e c t s of PAG s t i m u l a t i o n cn nGC neuronal f i r i n g . , These two r e s u l t s t o g e t h e r suggest t h a t the i n h i b i t o r y a f f e c t s o f PAG s t i m u l a t i o n on the nGC may be mediated by s e r o t o n i n . C. C o n c l u s i o n In the i n t r o d u c t i o n we d e s c r i b e d two p o s s i b l e schemes by which the nGC may mediate p e r i a q u e d u c t a l SPA. , The r e s u l t s of t h i s study w i l l be d i s c u s s e d i n terms of these schemes i n the f o l l o w i n g paragraphs., P e r i a q u e d u c t a l gray SPA may be mediated by the i n h i b i t i o n of nGC neurones i n v o l v e d i n an ascending n o c i c e p t i v e pathway {see F i g . 16c). These nGC neurones would be p r e d i c t e d to be e x c i t e d by noxious p e r i p h e r a l s t i m u l i and to be i n h i b i t e d by PAG s t i m u l a t i o n . Indeed, i n t h i s study the m a j o r i t y of neurones i n the nGC were e x c i t e d by noxious p e r i p h e r a l s t i m u l i {70% of the 168 neurones a f f e c t e d ) and many were i n h i b i t e d by s t i m u l a t i o n of the PAG (67% of the 87 neurones a f f e c t e d ) . Of those neurones t e s t e d with both PAG s t i m u l a t i o n and noxious p e r i p h e r a l s t i m u l i , 37% were i n h i b i t e d by the PAG s t i m u l a t i o n and e x c i t e d by the noxious s t i m u l i . These data suggest t h a t many of the nGC neurones are l i k e l y t o be i n v o l v e d i n an ascending pathway and by under the i n h i b i t o r y c o n t r o l of the PAG. Co n v e r s e l y , neurones w i t h i n the nGC may be mediating p e r i a q u e d u c t a l SPA by itodulating s p i n a l cord n o c i c e p t o r - d r i v e n 1 0 7 F i g u r e 16; Very s i m p l i f i e d diagrams of the nervous system showing the proposed schemes by which the nucleus g i g a n t o c e l l u l a r i s may mediate p e r i a q u e d u c t a l gray s t i m u l a t i o n produced a n t i n o c i c e p t i o n . Mediation of the SPA by: (a) nGC i n h i b i t i o n of s p i n a l cord n o c i c e p t o r - d r i v e n neurones; (b) t o n i c nGC e x c i t a t i o n of s p i n a l cord neurones; or {c) nGC neurones a c t i n g as r e l a y s i n an ascending n o c i c e p t i v e pathway. Symbols: nGC - nucleus g i g a n t o c e l l u l a r i s ; PAG - p e r i a q u e d u c t a l gray; SC -s p i n a l cord; T - thalamus. 108 109 neurones. T h i s might occur by PAG s t i m u l a t i o n e x c i t i n g nGC neurones which are i n h i b i t o r y to the s p i n a l c o r d neurones (see F i g . 16a)., However, only a m i n o r i t y of nGC neurones i n t h i s study were found to be e x c i t e d by PAG s t i m u l a t i o n (19% o f the 87 neurones a f f e c t e d ) . On the other hand, t h i s modulation may occur by the PAG s t i m u l a t i o n i n h i b i t i n g t o n i c a l l y a c t i v e nGC neurones which are e x c i t a t o r y t o the s p i n a l c o r d neurones (see F i g . 16b). , However, most nGC neurones i n the nGC d i d not have very f a s t spontaneous f i r i n g r a t e s and t h e r e f o r e , although i t i s a p o s s i b i l i t y , they probably do not mediate the p e r i a q u e d u c t a l gray SPA i n t h i s way. I t has been proposed t h a t brainstem l e v e l neurones might mediate the o b s e r v a t i o n t h a t noxious s t i m u l i a p p l i e d to one area of the body can i n h i b i t the responses of s p i n a l cord neurones to noxious s t i m u l i a p p l i e d to other body areas (LeBars et a l . , 1979)., Nucleus g i g a n t o c e l l u l a r i s neurones i n v o l v e d i n t h i s e f f e c t may mediate p e r i a q u e d u c t a l gray SPA at the s p i n a l l e v e l as w e l l . I f t h i s was the case then one would p r e d i c t that some nGC neurones should respond i n s i m i l a r ways to both PAG s t i m u l a t i o n and to noxious p e r i p h e r a l s t i m u l i . In f a c t some neurones i n t h i s study d i d respond t h i s way (22% of the 63 neurones a f f e c t e d by both t e s t s were e x c i t e d and 17% were i n h i b i t e d ) and t h e r e f o r e there i s a p o s s i b i l i t y t h a t some nGC neurones may be i n v o l v e d i n these e f f e c t s . C l e a r l y , experiments on neurones which are i d e n t i f i e d as to t h e i r axonal d e s t i n a t i o n s w i l l be r e q u i r e d to f u r t h e r c l a r i f y the involvement of nGC neurones i n these e f f e c t s . , 110 SUMMARY (1) Antidromic a c t i v a t i o n of PAG neurones by e l e c t r i c a l s t i m u l a t i o n of the nGC i n d i c a t e d there i s a monosynaptic pathway from the PAG t o the nGC. (2) Although the ma j o r i t y of nGC neurones responded with e x c i t a t i o n to noxious p e r i p h e r a l s t i m u l i , mixed and i n h i b i t o r y responses were a l s o seen. These r e s u l t s suggest the nGC p l a y s some r o l e i n n o c i c e p t i v e p r o c e s s i n g . (3) The major e f f e c t of PAG s t i m u l a t i o n on nGC neuronal a c t i v i t y was i n h i b i t i o n , although mixed and e x c i t a t o r y responses were a l s o seen. {4) The responses of nGC neurones t o s e r o t o n i n and f l u o x e t i n e , a s p e c i f i c uptake b l o c k e r , and the e f f e c t of f l u o x e t i n e on the responses of nGC neurones to PAG s t i m u l a t i o n suggest that the i n h i b i t o r y e f f e c t s of PAG s t i m u l a t i o n may be mediated by s e r o t o n i n . (5) The l a r g e s t group of nGC neurones responding t o noxious p e r i p h e r a l s t i m u l i and/or PAG s t i m u l a t i o n was e x c i t e d by the former and i n h i b i t e d by the l a t t e r , suggesting t h a t many nGC neurones are i n v o l v e d i n an ascending n o c i c e p t i v e pathway and under i n h i b i t o r y c o n t r o l of the PAG. ( 6 ) Other neurones i n the nGC had responses t o noxious p e r i p h e r a l s t i m u l i and PAG s t i m u l a t i o n which suggest t h a t they may mediate PAG s t i m u l a t i o n produced i n h i b i t i o n by descending c o n t r o l of s p i n a l c o r d neurones. <7) The r e s u l t s of the study suggest t h a t nGC neurones mediate at l e a s t part of the a n t i n o c i c e p t i v e a f f e c t s of PAG s t i m u l a t i o n . 111 APPENDIX In order to do these experiments some pieces of e l e c t r o n i c equipment were designed by the author and c o n s t r u c t e d by members of the l a b o r a t o r y . T h i s procedure allowed the f i n a l d e s i gns t o be f i t t e d to the requirements' of the l a b o r a t o r y i n a f a i r l y easy and i n e x p e n s i v e manner. Recent developments i n i n t e g r a t e d c i r c u i t technology allows r e l a t i v e l y u n s o p h i s t i c a t e d designs and techniques to produce p r o f e s s i o n a l products. , The d i f f e r e n t p i e c e s of equipment were desiqned i n a modular format which allowed the u n i t s to be designed and b u i l t as they were r e q u i r e d . The f o l l o w i n g pages c o n t a i n the schematic diagrams of the v a r i o u s modules. 112 F i g u r e 17: Schematic diagram o f the power s u p p l y module,.This module s u p p l i e d *5 and ±15 V r e g u l a t e d D.C. t o t h e o t h e r modules i n the s e r i e s . 113 114 Fi g u r e 18: Schematic diagram of the window d i s c r i m i n a t o r module. T h i s module allowed the d i f f e r e n t i a t i o n of a c t i o n p o t e n t i a l s from the surrounding noise and from unwanted s p i k e s . Each recog n i z e d s p i k e was output from the module as a standard TTL l e v e l p u l s e . The window was v a r i a b l e i n both width and amplitude a l l o w i n g the s e l e c t i o n of s p i k e s on the b a s i s of t h e i r minimum and maximum height as we l l as t h e i r minimum and maximum d u r a t i o n . The p o r t i o n of the s p i k e w i t h i n the window c o u l d be i n t e n s i f i e d . The window was d i s p l a y e d on the o s c i l l o s c o p e by m u l t i p l e x i n g the window s i g n a l s with the in p u t s i g n a l . . 1 1 5 1 16 F i g u r e 19: Schematic diagram of the s p i k e i n t e g r a t o r module. T h i s module counted s p i k e s during s u c c e s s i v e s e l e c t a b l e epochs (0.5-20 s ) . This count was then output as an analogue s i g n a l p r o p o r t i o n a l t o the number of counts while the s p i k e s were counted i n the next epoch. The count of the previous epoch was a l s o d i s p l a y e d d i g i t a l l y . 117 118 F i g u r e 20: The s c h e m a t i c diagram o f the d e l a y l i n e module. T h i s module a l l o w e d an analogue s i g n a l t o be d e l a y e d i n each of two c h a n n e l s . I f t h e d e l a y i s net r e q u i r e d i t c o u l d be s w i t c h e d out of t h e c i r c u i t , The c i r c u i t uses a c o m m e r c i a l l y a v a i l a b l e b u c k e t - b r i g a d e type i n t e g r a t e d c i r c u i t and i t s a s s o c i a t e d development board. 120 F i g u r e 21: The schematic diagram of the r a s t e r scan module. T h i s module allows a r e p e t i t i v e s i g n a l t o be d i p l a y e d on an o s c i l l o s c o p e with each succeeding sweep d i s p l a c e d i n the v e r t i c a l d i r e c t i o n . The module a l s o i n c l u d e d c i r c u i t r y t o allow the s p i k e s t o be d i s p l a y e d as dots. 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