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The role of the brain stem in the development of inhibition of spinal interneuronal activity Smith, Wayne Michael 1978

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THE ROLE OF THE BRAIN STEM IN THE DEVELOPMENT OF INHIBITION OF SPINAL INTEBN EURONAL ACTIVITY by WAYNE MICHAEL SMITH B . S c , U n i v e r s i t y o f B r i t i s h Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f P h y s i o l o q y ) We a c c e p t t h i s t h e s i s as co n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1978 Wayne M i c h a e l Smith, 1978 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Depa rtment The University of British Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1WS ABSTRACT Repeated, i n t e n s e , cutaneous s t i m u l a t i o n r e s u l t s i n the gradual development of i n h i b i t i o n of s p i n a l i n t e r n e u r o n e s . T h i s change i n neuronal a c t i v i t y c o u l d not be demonstrated i n r a t s whose s p i n a l cords had been t r a n s e c t e d , and was con s i d e r e d to be the consequence of s u p r a s p i n a l mechanisms. . Experiments sere c a r r i e d out to determine which areas of the b r a i n were i n v o l v e d . U n i t a r y r e c o r d i n g s from neurones s i t u a t e d i n nucleus r e t i c u l a r i s p o n t i s - c a u d a l i s , 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 , n u c l e u s r e t i c u l a r i s p a r v o c e l l u l a r i s and nucleus medulla oblongata pars v e n t r a l i s demonstrated a p r o g r e s s i v e l y i n c r e a s i n g e x c i t a t o r y response t o repeated i n t e n s e cutaneous s t i m u l a t i o n . These areas were shown 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 c o r d , by re t r o g r a d e t r a n s p o r t o f h o r s e r a d i s h peroxidase. C e l l s i n 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 demonstrated a p r o g r e s s i v e l y i n c r e a s i n g e x c i t a t o r y response, could a l s o be 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 s p i n a l c o r d . Repeated s t i m u l a t i o n o f some of these areas produced a p r o g r e s s i v e i n h i b i t i o n of s p i n a l i n t e r n e u r o n e s which was s i m i l a r to t h a t r e s u l t i n g from cutaneous s t i m u l a t i o n . I t would appear t h a t 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 and nucleus r e t i c u l a r i s p o n t i s - c a u d a l i s are i n v o l v e d i n the development of a p r o g r e s s i v e i n h i b i t i o n of s p i n a l i n t e r n e u r o n e s . A s i m i l a r r o l e f o r other r e t i c u l a r and raphe n u c l e i can not be excluded on the b a s i s of evidence p r e s e n t l y a v a i l a b l e . J . a , Pearson TABLE Cf CONTENTS Abs t r ac t «» .;«««.-..« •• * • •. • • • . « .<. • • * •<••'•-. . • * ••• .. * 2 L i s t Of Figures ........................................... 5 Ac know led qe men t ................. . . •.. • •. . • • ... • •.•• *. •••. • • • 6 Introduction ........... 1 D e f i n i t i o n Of Habituation .............................. 1 Habituation As A Form Of Learning ...................... <4 Proposed Mechanisms Of Habituation ..................... 5 Neurophysiological Studies ............................ .6 Met hods ......................... .......... ..... . •.... . .... 10 AEimal Subjects .............. ........ .. ••# .............. 10 Animal S t a b i l i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • • * 10 Anaesthetics ........................... .. .... .......... 11 Surgical Procedure .....................................11 Stimulation ............................................ 12 Ext r a c e l l u l a r fiecording . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Recording Sites ........................................ 14 fiicroelectrodes ........................................ 15 Preparation Of Pontamine Sky Blue Solution ............. 15 Cleaning Of Glass Tubing .16 Fixation Of The Brain And Spinal Cord .................. 16 Betrograde Axonal Transport Of HBP From Spinal Cord To NGc ...................... ........... ..... ........... 17 Injection . . . . . . . . . . . . . . . . . . . . . . . . .« . . . • . . . . . . . . . . . . . . . . 17 S taining ............................•.................. 18 Results *. •'»••'•..•..). •..-. . . »•• ...•....'.»'... . . »•••••> .'. '«•.'•» 19 Experiment I ........................................... 19 A/ Intact Spinal Cords 19 B/ Spinal Animals ...................................... 29 Experiment II ...... .... ........................ ... ... .....33 A/ Betrograde Transport Of Horseradish Peroxidase . . . . . . 33 B/ Besponses Of C e l l s In The Brain Steia To Repeated Noxious Periperal Stimulation. . . . . . . . . . . . . . . . . . . . . . 3 5 C/ Antidromic Activation From The Spinal Cord Of Wind Up Cel l s In The Brain Stem ............................. 39 Experiment III ............................................43 Discussion ....... •.....................................•••'4 9 Reticular Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51 Raphe Nuclei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Beferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 V L I S T O F F I G O B E S F I G O R E P A G E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 20 23 25 27 28 29 30 32 34 36 38 39 40 42 44 46 47 48 v i ACKNOWLEDGEMENT I wish t o thank Dr. J . A. Pearson f o r h i s s u p e r v i s i o n and support which made t h i s t h e s i s p o s s i b l e . , I a l s o wish to thank Mrs. H. Brandejs f o r her t e c h n i c a l a s s i s t a n c e with the h o r s e r a d i s h peroxidase experiments, and Mr. K. Henze f o r p r e p a r i n g the f i g u r e s . Mr. T. L. fiichardson*s kind and unsparing h e l p with a l l forms of computing i n v o l v e d was most a p p r e c i a t e d . I d e d i c a t e t h i s t h e s i s t o my parents, and my wife Frances. 1 IIJTROD OCTION DEFINITION OF HABITUATION S u r v i v a l o f an o r g a n i s m d epends p r i m a r i l y upon i t s a b i l i t y t o a d a p t t o t h e e n v i r o n m e n t . Many f o r m s o f a d a p t a t i o n h a v e b e e n s t u d i e d s i n c e P a v l o v (1927) n o t e d , i n t h e f i r s t w e l l c o n t r o l l e d s t u d y on b e h a v i o u r , t h a t t h e r e l a t i o n s h i p b e t w e e n s t i m u l u s a n d r e s p o n s e i s n o t n e c e s s a r i l y a r i g i d o n e , b u t c a n be m o d i f i e d a s a c o n s e q u e n c e o f t h e i n t r o d u c t i o n o f o t h e r f a c t o r s . S h e r r i n g t o n ( 1 9 0 6 ) , i n h i s s t u d y o f t h e c h a r a c t e r i s t i c s o f t h e m e c h a n i s m s u n d e r l y i n g r e f l e x a c t i o n , a l s o r e c o g n i z e d t h e f a c t t h a t a r e f l e x i s c a p a b l e o f u n d e r g o i n g c h a n g e . B a s e d upon t h e f i n d i n g s o f h i s e x p e r i m e n t s on t h e f l e x o r r e f l e x o f t h e s p i n a l d o g he s t a t e d "A s p i n a l r e f l e x u n d e r c o n t i n u o u s e x c i t a t i o n o r f r e q u e n t r e p e t i t i o n becomes v e a k e r , and may c e a s e a l t o g e t h e r ; t h i s d e c l i n e i s p r o g r e s s i v e , and t a k e s p l a c e e a r l i e r i n some k i n d s o f r e f l e x e s t h a n i t d o e s i n o t h e r s " . S h e r r i n g t o n r e f e r r e d t o t h i s r e s p o n s e d e c r e m e n t a s f a t i g u e b u t was c a r e f u l t o p o i n t o u t t h a t " i t i s n o t t h e f l e x o r m u s c l e s t h e m s e l v e s w h i c h t i r e o u t , f o r t h e s e , when t h e y c o n t r a c t no l o n g e r d u r i n g t h e f l e x i o n r e f l e x , s t i l l c o n t r a c t i n r e s p o n s e t o t h e s c r a t c h r e f l e x w h i c h a l s o e m p l o y s them." The f i r s t p u b l i s h e d o b s e r v a t i o n o f a phenomenon d e m o n s t r a t i n g a p r o g r e s s i v e d e c l i n e was by Peckham and Peckham 2 (1887), who noted t h a t a s p i d e r , s t i m u l a t e d to drop from i t s web by the sound of a tuning f o r k ceased t o respond i f the s t i m u l a t i o n was repeated. Many phenomena have been i n d i s c r i m i n a t e l y c l a s s i f i e d as ' a d a p t a t i o n 1 t h e r e f o r e a more s p e c i f i c term, h a b i t u a t i o n , was i n t r o d u c e d by Dodge (1923). T h i s was o p e r a t i o n a l l y d e f i n e d as "a response decrement as a r e s u l t of repeated s t i m u l a t i o n * ' by H a r r i s (1943) and has become widely accepted (Thompson and Spencer, 1966). More r e c e n t l y , experiments have been undertaken t o i n v e s t i g a t e the g e n e r a l c h a r a c t e r i s t i c s of h a b i t u a t i o n o f the f l e x o r withdrawal r e f l e x . Spencer, Thompson and N e i l s o n (1966a, 1966b, 1966c) s p e c i f i e d i n g r e a t d e t a i l the parameters of the o v e r a l l i nput-output f u n c t i o n of h a b i t u a t i o n o f t h i s r e f l e x i n the acute s p i n a l c a t . Based on t h i s work they (Thompson and Spencer, 1966) suggested an o p e r a t i o n a l d e f i n i t i o n of h a b i t u a t i o n which c o n s i s t s of nine general c h a r a c t e r i s t i c s ; "1) Given t h a t a p a r t i c u l a r s t i m u l u s e l i c i t s a response, repeated a p p l i c a t i o n o f the s t i m u l u s r e s u l t s i n decreased response ( h a b i t u a t i o n ) . The decrease i s u s u a l l y a negative e x p o n e n t i a l f u n c t i o n of the number of s t i m u l u s p r e s e n t a t i o n s . 2) I f the s t i m u l u s i s withheld, the response tends to r e t u r n g r a d u a l l y to c o n t r o l l e v e l s (spontaneous r e c o v e r y ) . 3) I f repeated p e r i o d s of h a b i t u a t i o n and spontaneous recovery are permitted, h a b i t u a t i o n becomes p r o g r e s s i v e l y more r a p i d ( t h i s might be c a l l e d p o t e n t i a t i o n of 3 h a b i t u a t i o n ) . 4) Other parameters being equal, the higher the frequency of s t i m u l a t i o n , the more r a p i d and/or more pronounced i s ha b i t u a t i o n . 5) The weaker the s t i m u l u s , the more r a p i d and/or more pronounced i s h a b i t u a t i o n . Strong s t i m u l i may y e i l d no s i g n i f i c a n t h a b i t u a t i o n . 6) The e f f e c t s of h a b i t u a t i o n t r a i n i n g may proceed beyond the zero asymptotic response l e v e l . 7) Habituation of a response t o a given stimulus e x h i b i t s stimulus g e n e r a l i z a t i o n to other s t i m u l i . 8) Pre s e n t a t i o n of another (usually strong) stimulus r e s u l t s i n recovery of the habituated response ( d i s h a b i t u a t i o n ) . 9) Upon repeated a p p l i c a t i o n of the d i s h a b i t u a t o r y s t i m u l u s , the amount of d i s h a b i t u a t i o n produced i t s e l f habituates (This might be c a l l e d h a b i t u a t i o n of dishabituation.)** This d e f i n i t i o n has become widely accepted. , I t has been noted that three of the nine p r o p e r t i e s are common to almost a l l preparations thought t o habituate: v i z response decrement, spontaneous recovery, and d i s h a b i t u a t i o n ( E i s e n s t e i n and P e r e t z , 1973). I t was a l s o suggested t h a t under most circumstances these three c h a r a c t e r i s t i c s are s u f f i c i e n t to i d e n t i f y a decrement i n response as being h a b i t u a t i o n , and not some ether phenomenon. 4 HABITUATION AS A FORM OF LEARNING T h i s o p e r a t i o n a l d e f i n i t i o n has c o n t r i b u t e d s i g n i f i c a n t l y towards s t a n d a r d i z i n g r e s e a r c h on h a b i t u a t i o n . Study o f h a b i t u a t i o n may y i e l d i n f o r m a t i o n which c o u l d be u s e f u l i n the e l u c i d a t i o n of the physi o l o g y of other types of b e h a v i o u r a l phenomena. Thorpe (1963) expressed the o p i n i o n t h a t h a b i t u a t i o n i s a form of l e a r n i n g . The authors of s e v e r a l reviews have s i n c e s p e c u l a t e d e x t e n s i v e l y as to the us e f u l n e s s o f h a b i t u a t i o n as a paradigm o f l e a r n i n g (Galambos, 1967; Kandel, 1967). The major o b j e c t i o n a g a i n s t t h i s , i n v o l v e s c r i t e r e a s e t by M i l l e r (1967) f o r the acceptance of any b e h a v i o u r a l process as an a u t h e n t i c l e a r n i n g event. He s t a t e s t h a t " l e a r n i n g i s a r e l a t i v e l y , permanent i n c r e a s e i n the response s t r e n g t h t h a t i s based upon pre v i o u s r e i n f o r c e m e n t . . . " . R e l a t i v e permanence i s def i n e d by M i l l e r as a matter of days or months as oppposed t o minutes or hours., There are r e p o r t s that h a b i t u a t i o n may l a s t f o r p e r i o d s o f days or months (Nesmeianova , 1957; Kozak, Macfarlane and Besterman, 1962). U n f o r t u n a t e l y the time s c a l e , upon which measurements of a l t e r e d a c t i v i t y i n the nervous system i s made, i s i n m i l l i s e c o n d s . T h i s i s orders of magnitude below the time s c a l e of days and months which M i l l e r used t o d e f i n e permanence, Perhaps h a b i t u a t i o n can be termed a type o f p r i m i t i v e l e a r n i n g . T h i s would ifflply t h a t any mechanisms shown to operate i n h a b i t u a t i o n must a l s o be shown t o operate i n other l e a r n i n g 5 s i t u a t i o n s before a u n i v e r s a l r o l e can be accepted. PROPOSED MECHANISMS OF HABITUATION Of the many neuronal mechanisms postulated to e x p l a i n the phenomenon of h a b i t u a t i o n , two have become prominent., The f i r s t , f o l l o w i n g the d o c t r i n e of parsimony, u t i l i z e s the fewest p o s s i b l e .number of neurons. I t proposes that w i t h i n a r e f l e x pathway, s y n a p t i c e f f i c a c y i s depressed as a consequence of continued a c t i v a t i o n (homosynaptic depression). This i m p l i e s that h a b i t u a t i o n can be demonstrated i n a monosynaptic r e f l e x with the r e f l e x arc i s o l a t e d from a l l other n e u r a l p r o j e c t i o n s . The second, more complex, theory suggests the involvement of a greater number of neurons., I t proposes t h a t i n h i b i t o r y a c t i v i t y i s impinging on the pathway, and i s increased with continued a c t i v a t i o n . The interneurons r e s p o n s i b l e f o r t h i s i n h i b i t i o n are e x t r i n s i c to the d i r e c t r e f l e x pathway and are a c t i v a t e d by the same s t i m u l i . 6 NEUBOPHYSIQLCjGIC&L STUDIES I n v e s t i g a t i o n s i n t o the neuronal c o r r e l a t e s of h a b i t u a t i o n have i n v o l v e d a v a r i e t y of simple r e f l e x e s i n v a r i o u s s p e c i e s . One of the l e a s t complex of these s p e c i e s i s A p l y s i a , a marine gastropod. Kandel and co-workers, using a s i m p l i f i e d p r e p a r a t i o n , showed t h a t h a b i t u a t i o n of the g i l l withdrawal r e f l e x can be a t t r i b u t e d , i n part and perhaps e n t i r e l y , to a decrement i n the complex e x c i t a t o r y p o s t - s y n a p t i c p o t e n t i a l (EPSP) recorded i n one of the main i d e n t i f i e d motor neurons (L7) (Pinsker, Kupfermann, C a s t e l l u c c i and Kandel, 1970; Kupfermann, C a s t e l l u c c i , P i nsker and Kandel, 1970). They went on t o show i n an elegant s e r i e s of experiments t h a t t h i s decrement of the EPSP occurred i n a monosynaptic r e f l e x a r c and t h a t i t i s the r e s u l t of a decrease i n the number of quanta of t r a n s m i t t e r r e l e a s e d from the p r e s y n a p t i c t e r m i n a l s i n response t o a s i n g l e s p i k e ( C a s t e l l u c c i and Kandel, 1974)., The r e p o r t s based on a p l y s i a s t u d i e s prompted a quest f o r s i m i l a r mechanisms i n mammals. The major t e c h n i c a l d i f f e r e n c e between i n v e r t e b r a t e and v e r t e b r a t e p r e p a r a t i o n s i s the r e l a t i v e d i f f i c u l t y of i d e n t i f y i n g the a f f e r e n t and e f f e r e n t neurons of a s i n g l e synapse i n a v e r t e b r a t e p r e p a r a t i o n . The approach to understanding n e u r a l mechanisms i n v e r t e b r a t e p r e p a r a t i o n s i s understandably q u i t e d i f f e r e n t . The chanqes r e s p o n s i b l e f o r h a b i t u a t i o n c o u l d occur i n three p o s s i b l e f u n c t i o n a l s i t e s : 1) the sensory nerve f i b r e s , 2) 7 the motor neurons, or 3) interneacons of the ref l e x arc. Dsing an unanesthetized, acute, spi n a l cat preparation, Thompson and co-workers have p o s i t i v e l y ruled out the f i r s t two p o s s i b i l i t e s (Groves and Thompson, 1973; Groves, Glanzman, Patterson, and Thompson. 1970). To study the t h i r d p o s s i b i l i t y , namely the interneuronal correlates of habituation, they (Groves and Thompson, 1970) recorded from a large number of s p i n a l interneurons, the functional c h a r a c t e r i s t i c s of which suggest they could be substrates of habituation. They described two groups of interneurons which are: 1) type H interneurons, which show only a progressive decrease in the number of action potentials evoked by repeated cutaneous stimulation and 2) type S interneurons, which show an increase i n a c t i v i t y which i s either maintained or decreased with continued stimulation. No evidence demonstating a build-up of i n h i b i t i o n was reported i n t h i s study. Groves and Thompson have therefore suggested that the changing responses of these H and S type neurons are due to the properties of certain excitatory synapses which are specialized to y i e l d either increased or decreased responses to repeated ac t i v a t i o n . These authors are hence supporting the homosynaptic depression theory as a mechanism responsible f o r habituation. More direct evidence that i n h i b i t i o n does not play a role i n habituation i n the spi n a l cat was provided by Spencer, Thompson and Neilson (1966c). They showed that intravenous administration of strychnine or picrotoxin, i n doses adequate to 8 reduce p o s t s y n a p t i c or p r e s y n a p t i c i n h i b i t i o n r e s p e c t i v e l y , does not prevent h a b i t u a t i o n . From t h i s evidence, some authors conclude t h a t homosynaptic depression of s p i n a l i n t e r n e u r o n s i s a t l e a s t p a r t i a l l y r e s p o n s i b l e f o r the development of h a b i t u a t i o n i n the acute s p i n a l c a t . T h i s however s t a t e s nothing about the mechanism of h a b i t u a t i o n i n the i n t a c t animal. In f a c t , repeated s t i m u l a t i o n d e l i v e r e d t o an i n t a c t animal produces neuronal and b e h a v i o u r a l responses which are q u i t e d i f f e r e n t t o those observed i n s p i n a l animals. Behavioural s t u d i e s (Pearson and Wenkstern, 1972) have shown t h a t h a b i t u a t i o n can be more e a s i l y demonstrated i n the i n t a c t r a t than i n the s p i n a l r a t . Since then, HacDonald (PhD t h e s i s - 1975) has shown t h a t s t r y c h n i n e does im p a i r h a b i t u a t i o n i n the i n t a c t r a t but not i n the s p i n a l r a t . These r e s u l t s suggest t h a t q u i t e d i f f e r e n t mechanisms c o u l d be i n v o l v e d i n h a b i t u a t i o n i n i n t a c t animals and that these might i n v o l v e i n h i b i t o r y i n f l u e n c e s which are dependent upon s u p r a - s p i n a l s t r u c t u r e s . T h i s might account f o r the f i n d i n g s of Spencer, Thompson and N e i l s o n (1966c) t h a t s t r y c h n i n e has no e f f e c t on h a b i t u a t i o n i n s p i n a l cats and s i m i l a r l y account f o r the f i n d i n g s of Groves and Thompson (1970) and o t h e r s (Buchwald, Hales and Schramm, 1975) t h a t no evidence o f i n h i b i t o r y b u i l d - u p could be obtained from experiments on s p i n a l animals. Neuronal r e c o r d i n g s i n i n t a c t animals have demonstrated d r a m a t i c a l l y d i f f e r e n t r e s u l t s from those seen i n s p i n a l 9 animals. MacDonald (PhD t h e s i s - 1975) and MacDonald and Pearson (1977) have recorded from interneurones which demonstrate i n h i b i t i o n to noxious p e r i p h e r a l s t i m u l a t i o n and have shown that t h i s i n h i b i t i o n increases with repeated s t i m u l a t i o n . T h i s type of a c t i v i t y occured only i n the i n t a c t r a t , not i n the s p i n a l r a t . The i n h i b i t i o n i s almost e n t i r e l y abolished by s t r y c h n i n e and i t s duration does not change with repeated s t i m u l a t i o n . , The f i r s t o b j e c t i v e of t h i s p r o j e c t was to f u r t h e r e l u c i d a t e the c h a r a c t e r i s t i c s of these interneurons i n the i n t a c t and s p i n a l animal. The second o b j e c t i v e was to determine which s u p r a s p i n a l areas are reguired f o r the development of i n h i b i t i o n demonstrated of these interneurons. Haber and wagman (1974) reported t h a t s t i m u l a t i o n of the medial pontomedullary r e t i c u l a r formation r e s u l t e d i n i n h i b i t i o n of s p i n a l neuronal a c t i v i t y t hat p r o g r e s s i v e l y increased with repeated s t i m u l a t i o n . This resembles the " i n h i b i t o r y b u i l d - u p " described by MacDonald (PhD t h e s i s - 1975; MacDonald and Pearson, 1977). I t i s p o s s i b l e that responses of c e l l s i n t h i s area of the b r a i n stem, to repeated p e r i p h e r a l s t i m u l a t i o n , could r e s u l t i n the development of progressive i n h i b i t i o n of s p i n a l interneurones. The t h i r d o b j e c t i v e was to i n v e s t i g a t e the e f f e c t s of repeated s t i m u l a t i o n of the r e t i c u l a r formation on s p i n a l interneurons that demonstrate a progressive i n c r e a s e i n i n h i b i t i o n to repeated p e r i p h e r a l s t i m u l a t i o n . 10 METHODS MIML S U B J E C T S Male albino rats of the Wistar s t r a i n , weight range 200 to 700 grams, were used as experimental subjects. ANIMAL STABILITY The use of micrcelectrodes to record e x t r a c e l l u l a r unitary a c t i v i t y reguires that the preparation be immobilized. The spinal column was held by two clamps which were constructed from Hagenbarth suture c l i p forceps. The clamps were attached to a heavy r i g i d frame such that t h e i r positions could be changed to accommodate rats of d i f f e r e n t sizes. The head was fixed i n a conventional head holder i n such a way that the l i n e between the ear bars and the i n c i s o r bar was maintained at a 5 degree angle below the horizontal, regardless of the size of the rat. The head holder was an i n t e g r a l part of the frame that supported the spinal cord. Two Narishige micromanipulators were also attached to t h i s frame i n such a way that electrodes could be inserted into the brain or s p i n a l cord. The body of the rat was suspended from a plexiglass ring sutured to the skin of the back of the rat. This ring also served to create an o i l pool over the exposed spinal cord. 11 ANAESTHETICS Eats were anaesthetized with urethane ( e t h y l carbamate) given i n t r a p e r i t o n e a l l y at a dose of 1.5 gm./kq. i n a 201 s o l u t i o n . Jugular and t r a c h e a l cannulae were then i n s e r t e d . When muscular movement presented a problem the animals were a l s o paralysed with gallamine t r i e t h i o d i d e ( F l a x e d i l , 10 mg/ml) to a t o t a l dosage of 60 mg/kg. No r e f l e x responses c o u l d be e l i c i t e d from these animals even with very intense s t i m u l i , f o l l o w i n g p a r a l y s i s with f l a x e d i l , a r t i f i c i a l r e s p i r a t i o n was d e l i v e r e d by a mechanical r e s p i r a t o r (Harvard Rodent R e s p i r a t o r ) . SORGICM. PROCEDURE M I j i n a l Cord A laminectomy was performed t o expose the lumbar enlargement. The vertebrae clamps were placed, one immediately r o s t r a l and the other caudal t o the laminectomy. The d u r a l sheath was then removed, and the exposed cord was immediately covered with o i l . The temperature of t h i s o i l bath was monitored and maintained at 37 degrees C. JZ Brainstem A s t r i p of o c c i p i t a l bone was removed, a f t e r the head was placed i n the head holder, t o expose the medial d o r s a l (0.5 cm on e i t h e r s i d e of the midline) surface of the cerebellum. The dura was them removed and the exposed b r a i n was kept moist with 12 s a l i n e , CZ S p i n a l T r a n s e c t i o n A laminectomy was performed of the e i g h t h t h o r a c i c vertebrae and. t r a n s e c t i o n was achieved by l i g a t i o n . T h i s procedure was c a r r i e d out l e s s than one hour p r i o r t o commencing re c o r d i n g s o f s p i n a l neurones, STIMULATION A/ Cutaneous S t i m u l a t i n g e l e c t r o d e s (21 gauge, hypodermic needles) were i n s e r t e d i n t o the s k i n of a hind-paw. §/ C e n t r a l B i p o l a r c o n c e n t r i c s t e e l e l e c t r o d e s (Rhodes Medical Instruments, model NE100) were i n s e r t e d i n t o the b r a i n c r s p i n a l cord using a micromanipulator. These e l e c t r o d e s were connected t o a Devices I s o l a t e d S t i m u l a t o r {Type 2533) which was t r i g g e r e d by a Devices D i g i t i m e r . 13 MM ACELL UL A R BE COB DING A microelectrode was placed into one of the micromanipulators and connected to an impedance converter by means of a s i l v e r wire. The impedance converter was grounded to the rat. The preparation was located inside a double-walled copper wire cage which was open at the front to allow access. The s i g n a l from the impedance converter was fed into a Tektronix 3A9 d i f f e r e n t i a l amplifier of a Tektronix 565A oscilloscope. The oscilloscope and stimulators were triggered by a Devices Digitimer. The output from the d i f f e r e n t i a l amplifier was connected to an audio amplifier and a spike height discriminator/step generator (both designed in t h i s department by H. Kohne)• The audio amplifier was connected to a loudspeaker to allow monitoring of the s i g n a l . The spike height discriminator/step generator was connected to a Tektronix, type RM 502 A, dual-beam oscilloscope to produce a raster-dot display i n which each dot represented the occurrence of an action p o t e n t i a l . The output of the spike height discriminator was connected to the cathode of the oscilloscope via a Grass Stimulator (type S6) to produce a bright spot each time a spike of a height greater than the preset threshold was detected. The step generator and sweep of the BM 502 A oscilloscope were simultaneously triggered by the Digitimer., The step generator was connected to the v e r t i c a l input of the oscilloscope and caused a step increase in the 14 v e r t i c a l position of the trace just before each sweep was i n i t i a t e d . This method of display allowed a precise acnitorinq of the chanqing responses of a c e l l to repeated stimulation. The signals from the spike heiqht discriminator and diqitimer were also connected to separate channels of a stereo tape recorder (Toshiba Cassette type KT-403D). This allowed further analysis of the data by use of a d i q i t a l computer (type PDP 11). RECORDING SITES The depth of penetration of the microelectrode into the the spinal cord was noted whenever a unit was recorded. S i m i l a r l y the depth of penetration, distance from midline and from lambda were noted i n the case of unitary recordings from the the brain stem. The locations of some s i t e s were marked by e l e c t i o n of the dye pontamine sky blue (10 uA of neqative current for 10 min.) 15 MICROELECTROPES Hard g l a s s t u b i n g (Pyrex Lab. Glassware:tubing l a b . Std. Wall-2mm.), was p u l l e d and broken back to t i p diameters ranging frcm 1 to 2 u. These e l e c t r o d e s were then f i l l e d with NaCl <4M) or a 4% s o l u t i o n o f pontamine sky blue i n NaCl (1M). F i l l i n g was accomplished by breaking the t i p t o the r e q u i r e d t i p diameter and then i n j e c t i n q the s o l u t i o n s i n t o the b a r r e l . C a p i l l a r y a c t i o n c a r r i e d the f l u i d to the t i p . Small bubbles could o f t e n by teased out of the e l e c t r o d e with f i n e q l a s s f i b r e s . E l e c t r o d e s f i l l e d with NaCl, tended to have lower t i p r e s i s t a n c e s (2-5 megaohms) than e l e c t r o d e s f i l l e d with the dye s o l u t i o n (4-7 meqaohms). PREPARATION OF PONTAMINE SKI BLUE jjOLJ2TI0JI (from Godfraind, 1969) To a 1 M s o l u t i o n of sodium c h l o r i d e a q u a n t i t y o f pontamine sky blue (ESBE L a b o r a t o r i e s ) s u f f i c i e n t to make a -i% s o l u t i o n , was added. The dye i s r e a d i l y s o l u b l e . T h i s s o l u t i o n was then f i l t e r e d (0.8 u M i l l i p o r e f i l t e r ) at l e a s t 3 times. 16 CLEANING OF GLASS TUBING The g l a s s was immersed i n a s o l u t i o n of 10$ NaOH f o r a period of 24 hours. The tubing was then r i n s e d at l e a s t 3 times with a s o l u t i o n of 0.IM a c e t i c a c i d f o l l o w e d by 10 r i n s e s with d i s t i l l e d water and f i n a l l y oven d r i e d at 100 degrees C, The tubing was s t o r e d i n a s e a l e d c o n t a i n e r u n t i l r e q u i r e d . Of THE BRAIN AND SPINAL CORD A thoracotomy was performed and the h e a r t f r e e d from surrounding t i s s u e . A hypodermic needle (18 gauge), connected to a pressure b o t t l e c o n t a i n i n g s a l i n e , was i n s e r t e d i n t o the l e f t v e n t r i c l e . The i n f e r i o r vena cava was severed to allow f l u i d t o escape from the v a s c u l a r system, A pressure of 200 t o 300 mm. Hg. was a p p l i e d to the presure b o t t l e u n t i l 300 ml. of s a l i n e was f l u s h e d through the v a s c u l a r system. Hithout removing the needle from the v e n t r i c l e i t was reconnected to a second pressure b o t t l e c o n t a i n i n g a s o l u t i o n of M)% f o r m o l -s a l i n e . Pressure was a p p l i e d t o the second b o t t l e and the r a t was perfused with 300 ml. f i x a t i v e . The r e q u i r e d p i e c e of the br a i n or cord was removed and pl a c e d i n 10% f o r m o l - s a l i n e f o r 24 hours. S e c t i o n s (50u) were then cut on a f r e e z i n g microtome and s t a i n e d with s a f r a n i n - 0 . The s i t e o f the s t i m u l a t i n g e l e c t r o d e was i d e n t i f i e d by use of the P r u s s i a n blue r e a c t i o n . A 10 uA p o s i t i v e c u r r e n t *as passed through the e l e c t r o d e f o r 1 minute \ 17 to deposit a sm a l l amount of i r o n . A f t e r p e r f u s i o n with NaCI and f o r m a l i n as described above, 50 ml. of 18 potassium ferrocyanide i n f o r m a l i n was i n j e c t e d . The appropriate parts of the b r a i n or cord were removed and immersed i n potassium f e r r o c y a n i d e , and stored a t 5 degrees C f o r 24 hours. 50u se c t i o n s were cut and stored i n 10% f o r m o l - s a l i n e f o r an a d d i t i o n a l 24 hours. They were then s t a i n e d with s a f r a n i n - 0 and mounted. BETfiCGBADE AXONAL TRANSPORT OF J3RP FSOH SPIRAL CORD TO NGC INJECTION Rats were anaesthetized with sodium p e n t o b a r b i t a l {50 mg./ kg. i . p . ) . The lower t h o r a c i c and lumbar vertebrae were exposed and r i g i d l y clamped. A burr hole was made on the r i g h t s i d e i n each of three adjacent vertebrae. The dura was then punctured with a 21 gauge needle. Horseradish peroxidase (Sigma type VI) was i n j e c t e d i n t o the cord using a 10 u l Hamilton syringe mounted i n a micromanipulator. Three mg. of HBP was d i s o l v e d i n 10 u l of 0.9% s t e r i l e s a l i n e t o give a 30% s o l u t i o n . An i n j e c t i o n was made i n t o each of the exposed s i t e s at a depth of approximately 1.5 mm. At each i n j e c t i o n s i t e 1.0 u l of HHP s o u l t i o n was d e l i v e r e d over 1 minute and the syr i n g e was l e f t i n 18 p l a c e f o r an a d d i t i o n a l 3 minutes f o l l o w i n g e a c h . i n j e c t i o n . The wound was c l o s e d and the r a t s allowed t o to s u r v i v e f o r 48 to 72 hours. STJINING Three days p o s t o p e r a t i v e l y the r a t s were r e a n a e s t h e t i z e d and perfused t r a n s c a r d i a l l y f i r s t with 0.05 M phosphate b u f f e r and then with a s o l u t i o n of 3% paraformaldehyde and 1$ g l u t a r a l d e h y d e i n 0.05 M phosphate b u f f e r (ph 7.4, 24 degrees C). The b r a i n and s p i n a l cords were removed, placed i n f r e s h f i x a t i v e c o n t a i n i n g 5% sucrose and l e f t o v e r n i g h t a t 5 degrees C. The f o l l o w i n g morning the b r a i n s were s e c t i o n e d on a f r e e z i n g microtome. The s e c t i o n s , 40 u t h i c k , were placed i n a s o l u t i o n of 5% sucrose and 0.05 M t r i s b u f f e r (ph 7.6, 4 degree C) f o r h a l f and hour. They were then t r a n s f e r r e d t o a f r e s h l y prepared, f i l t e r e d , 0.03U s o l u t i o n o f 3 , 3-diaminobenzidine f r e e base (Sigma) i n t r i s b u f f e r (ph 7.6) with 5% s u c r o s e a t room temperature f o r h a l f an hour. The s e c t i o n s were incubated f o r a f i n a l h a l f hour i n 0.03 % 3,3-diaminobenzadine i n t r i s b u f f e r with 1% hydrogen peroxide. The s e c t i o n s were r i n s e d thouroughly with d i s t i l l e d water and then mounted, dehydrated, c l e a r e d with xylene, covered and i n s p e c t e d using l i g h t f i e l d and dark f i e l d microscopy. 1 9 RESULTS EXPERIMENT I INHIBITION OF SPONTANEOUS ACTIVITI OF SPINAL I1IERNEURONES BY REPEATED CUT.ANJOOS ST1BULAT1QB IN RATS 1ITH 1/ INTACT AND M TRANSECTED SPINAL CORDS,. i Z INTACT SPINAL CORDS The a c t i v i t y of 22 c e l l s , i n 11 r a t s was s t u d i e d using e x t r a c e l l u l a r r e c o r d i n g s . These c e l l s were spontaneously a c t i v e and i n h i b i t e d by i n t e n s e cutaneous s t i m u l a t i o n . I n 18 of these c e l l s the period of i n h i b i t i o n i n c r e a s e d with r e p e t i t i o n of the st i m u l u s . In the f o u r other c e l l s no obvious or c o n s i s t e n t change was seen. The response of a c e l l demonstrating i n c r e a s i n g i n h i b i t i o n t o repeated s t i m u l a t i o n i s i l l u s t r a t e d i n f i g u r e 1 i n which the data are presented i n two d i f f e r e n t ways. In 1A (above) a r a s t e r dot d i s p l a y i s shown. Each h o r i z o n t a l row of dots r e p r e s e n t s a c t i o n p o t e n t i a l s o c c u r r i n g i n 1 second of a 1.3 second epoch. A t o t a l of 110 epochs i s shown. The f i r s t s t i m u l u s r e s u l t e d i n an i n h i b i t i o n l a s t i n g 110 msecs. and t h i s i n c r e a s e d t o 250 msecs. over 40 s t i m u l i . The sti m u l u s FACING PAGE 20 FIGURE 1 A c t i v i t y o f a s p i n a l i n t e r n e u r o n e of an i n t a c t r a t i n c l u d i n g a c t i v i t y p r i o r t o , during and a f t e r a p p l i c a t i o n o f repeated i n t e n s e cutaneous s t i m u l a t i o n <10 ma. I n t e n s i t y , 0.2 msec. d u r a t i o n given every 1.3 seconds). A/ A r a s t e r - d o t d i s p l a y showing an i n h i b i t i o n which l a s t e d approximately 100 msec, i n i t i a l l y and i n c r e a s e d t o 250 msec, by the 40th s t i m u l u s . The s t i m u l u s a r t e f a c t s are r e p r e s e n t e d by a v e r t i c a l l i n e of dots on the x a x i s p o s i t i o n of 100 msec. B/ A histogram of the same data as above, showing the number of a c t i o n p o t e n t i a l s i n 900 msec, of each epoch. The average f i r i n g r a t e p r i o r to s t i m u l a t i o n was 12 a c t i o n p o t e n t i a l s /900 msec. , and durin g s t i m u l a t i o n g r a d u a l l y decreased t o 5/900 msec. The a c t i v i t y a f t e r s t i m u l a t i o n was terminated returned t o 14/900 msec, w i t h i n 20 epochs. 20 FIGOfiE 1 A. Epochs no -.• • off Stim. on 0 J • . < . * | i | 1 1 1 | 1 1 1 | ( 1 1 | 1 | l 1 1 1 B. 20 -, Time in msec. on off \tStim. ^ 1000 Number of Action Potentials o -J T-0 Epochs no 21 artefacts can be recognized as a v e r t i c a l l i n e of dots in the same position on the x axis. This method of presenting the data has the advantage that i t displays the precise point i n time that each action p o t e n t i a l occurs, and th i s allows one to deternine whether an i n h i b i t i o n consists of merely a decrease i n f i r i n g rate or of a completely s i l e n t period. I t has the disadvantage that i t only gives a g u a l i t a t i v e impression of neural a c t i v i t y as indicated by the density of dots. The exact quantitative rate i s not given and thus the prestimulus rate cannot be accurately compared with the rate durinq or afte r stimulation. Such information i s available i n the histoqram i n fiqure 1B. This shows the number of spikes that occurred within each consecutive epoch*. The spikes were counted only durinq 900 msec, after every stimulus or durinq the correspondinq period of spontaneous a c t i v i t y . It can be seen that the c e l l f i r e d at an average rate of approximately 12 per second with a high of 20 and a low of 5 but usually within a ranqe of 10 to 18. During stimulation the average f i r i n q rate qradually declined to 3 per second. After the stimulation was terminated the rate returned abruptly to i t s o r i q i n a l value. I t could be *Histcqrams of this type may also show the t o t a l number of spikes that occurred in the f i r s t second of consecutive qroups of epochs. The number of epochs in each qroup may vary from qraph to qraph. (See fiqure 9B) 22 argued t h a t t h i s development of i n h i b i t i o n i s merely a r e s u l t of presentation of the s t i m u l i during a spontaneously occuring d e c l i n e i n bach ground a c t i v i t y . To insure that t h i s was no the case, spontaneous a c t i v i t y was recorded f o r a t l e a s t 5 minutes i n every c e l l demonstrating such a response. The progressive increase i n i n h i b i t i o n was shewn t o be not r e l a t e d to f l u c t a t i e n s i n background f i r i n g r a t e . The responses of the same c e l l t o a more intense stimulus are i l l u s t r a t e d i n f i g u r e 2A and 2B. I t can be seen (2ft) that the i n i t i a l i n h i b i t i o n l a s t e d about 150 msec,, i e . 50 msec, longer than the i n i t i a l i n h i b i t i o n caused by the l e s s intense stimulus. A f t e r 45 s t i m u l i the i n h i b i t o r y period reached 400 msec. (150 msec, longer than that to the 45th stimulus of the f i r s t s e r i e s ) . . A f t e r a t o t a l of 60 s t i m u l i the i n h i b i t o r y period had a duration of 750 msec. A l s o , during the l a s t few s t i m u l i the spontaneous ra t e of a c t i v i t y , as shown by the prestimulus a c t i v i t y was n o t i c e a b l y reduced. The histogram i n f i g u r e 2B shows that the spontaneous a c t i v i t y , before and a f t e r the second s e r i e s of s t i m u l i , i s comparable t o that of the f i r s t s e r i e s (compare with f i g u r e 1). Thus the o v e r a l l decrease i n f i r i n g r a t e which occurred during the second s e r i e s o f s t i m u l a t i o n was a d i r e c t r e s u l t of the s t i m u l a t i o n and was not r e l a t e d to the spontaneous r a t e . The q u a n t i t a t i v e information from epoch to epoch, shown i n the histograms, and w i t h i n the epochs, i l l u s t r a t e d i n the r a s t e r d i s p l a y s , has been combined i n a t h i r d method of presentinq the FACING PAGE 23 FIGUBE 2 Besponses of the same s p i n a l interneurone as shown i n f i g u r e 1, to more intense s t i m u l a t i o n (20 ma. i n t e n s i t y , 0.2 msec, d u r a t i o n , given every 1.3 seconds). A/ A r a s t e r - d o t d i s p l a y i l l u s t r a t i n g the increase i n i n h i b i t o r y p e r i o d from 150 msec, i n i t i a l l y to 750 msec, a f t e r 60 s t i m u l i have been given. B/ A histogram of the same data as above, showing the number of a c t i o n p o t e n t i a l s i n 900 msec.,of each epoch. The average f i r i n g r a t e r a t e p r i o r to s t i m u l a t i o n was 15 a c t i o n p o t e n t i a l s /900 msec, and during s t i m u l a t i o n g r a d u a l l y decreased to 0/900 msec. The a c t i v i t y a f t e r s t i m u l a t i o n was terminated returned t o 14/900 msec, w i t h i n 15 epochs. 23 FIGURE 2 24 same data ( f i g u r e 3). This i s a two dimensional graph which i s colour coded. The various c o l o u r s and shades represent the number of spikes o c c u r r i n g w i t h i n a p a r t i c u l a r time b i n . Each row of blocks along the abcissa represents the f i r s t 990 m i l l i s e c o n d s of 6 consecutive epochs. . Each separate block represents the sum of a l l the spikes w i t h i n a p a r t i c u l a r 45 msec, period i n each of the s i x epochs. The number of s p i k e s w i t h i n each time b i n i s represented by the col o u r and shade ranging from white as 0 spikes to black as 10 sp i k e s . The lowest s i x rows represent spontaneous a c t i v i t y . This has an average value of 6, represented by a medium blue shade. The next eleven rows show the e f f e c t of s t i m u l a t i o n on the f i r i n g rate of the c e l l . The v e r t i c a l black s t r i p represents the stimulus a r t e f a c t . The top 6 rows show the recovery of spontaneous a c t i v i t y . I t can be seen that not only does the period of t o t a l i n h i b i t i o n increase with repeated s t i m u l a t i o n but that the o v e r a l l r a t e of discharge decreases. A f t e r the s t i m u l a t i o n i s terminated the spontaneous a c t i v i t y returns to the rate that occurred p r i o r to s t i m u l a t i o n . This c o l o u r coded graph combines the advantages of both the r a s t e r - d o t d i s p l a y and histogram, r e s u l t i n g i n a much more informative p r e s e n t a t i o n . The a c t i v i t y of the c e l l represented i n f i g u r e s 1 to 3 exe m p l i f i e s the response demonstrated i n 18 out of 22 c e l l s studied i n i n t a c t animals. I t was observed t h a t the magnitude of i n h i b i t i o n and r a t e of increase of i n h i b i t i o n was d i r e c t l y r e l a t e d to the i n t e n s i t y and or du r a t i o n of the stimulus as seen FACING PAGE 25 FIGUEE 3 A two dimensional qraph of the same data displayed i n f i g u r e 2. The c o l o u r coding i n d i c a t e s the number of s p i k e s o c c u r r i n g i n each time b i n . The v e r t i c a l black s t r i p i n d i c a t e s the stimulus a r t e f a c t s . This method of d i s p l a y i n g the data shows th a t the spontaneous f i r i n g r a t e s , before and a f t e r s t i m u l a t i o n , are comparable. I t a l s o shows th a t the i n h i b i t i o n c o n s i s t s of a period of t o t a l s i l e n c e and a l s o a decrease i n the background f i r i n g rate over the remainder of the epoch. 25 26 i n 5 c e l l s . Further evidence f o r t h i s i s presented i n f i g u r e 4 where the responses of a s i n g l e c e l l to 2 d i f f e r e n t i n t e n s i t i e s (10 and 20 mA) are shown. These are the same data as i l l u s t r a t e d i n f i g u r e s one and two hut p l o t t e d i n a d i f f e r e n t manner. In 4 of the 22 c e l l s which were i n h i b i t e d by p e r i p h e r a l s t i m u l a t i o n the period of i n h i b i t i o n d i d not change with repeated s t i m u l a t i o n . An example ot t h i s "constant" i n h i b i t i o n i s i l l u s t r a t e d i n f i g u r e 5. This r a s t e r d i s p l a y shows the i n h i b i t o r y period to be approximately 300 msec. I t i s apparent that t h i s response d i d not change i n a c o n s i s t e n t manner with repeated s t i m u l a t i o n . The d i s t r i b u t i o n w i t h i n the s p i n a l cord, of the c e l l s demonstrating i n c r e a s i n g i n h i b i t i o n and constant i n h i b i t i o n i s i l l u s t r a t e d i n f i g u r e 6. 12 JFINAL ANIMALS Experiments were c a r r i e d out on eleven animals whose s p i n a l cords were transected at the l e v e l of the eighth t h o r a c i c vertebra. The responses of 26 c e i l s whose spontaneous a c t i v i t y was i n h i b i t e d by p e r i p h e r a l s t i m u l a t i o n (10 or 20 mA; 0.2 msec; i n t e r s t i m u l u s i n t e r v a l 1.3 s e c ) were s t u d i e d . Three d i s t i n c t patterns of response to repeated s t i m u l a t i o n were seen. In 18 cases the i n h i b i t i o n decreased (see f i g u r e 7)-. .. In 3 cases i t / FACING PAGE 27 F I G U R E 4 The i n f l u e n c e of stimulus i n t e n s i t y upon the r a t e of development of i n h i b i t i o n of a s p i n a l interneurone. This qraph i s constructed from the data that were shown i n f i g u r e s 1 and 2. I t can be seen that the qreater i n t e n s i t y s t i m u l a t i o n (20 ma. i n t e n s i t y , 0.2 msec, d u r a t i o n , given every 1.3 seconds) produced a l a r q e r period of i n h i b i t i o n i n i t i a l l y and t h a t t h i s period increased at a greater r a t e with repeated s t i m u l a t i o n than with the lower i n t e n s i t y stimulus (10 ma. i n t e n s i t y , 0.2 msec, d u r a t i o n , given every 1.3 seconds). FIGURE Ii Stimulus No. FACING PAGE 28 F I G U B I 5 A r a s t e r - d o t d i s p l a y of the a c t i v i t y of a s p i n a l i n t e r n e u r c n e of an i n t a c t r a t , i n c l u d i n g r e c o r d i n g s before, d u r i n g , and a f t e r repeated noxious p e r i p h e r a l s t i m u l a t i o n . The stimulus a r t e f a c t s are a s e r i e s of dots i n the 100 msec, p o s i t i o n along the X a x i s . Each stimulus r e s u l t e d i n an i n h i b i t i o n which l a s t e d approximately 300 msec. The d u r a t i o n of t h i s i n h i b i t o r y p e r i o d remained r e l a t i v e l y constant with repeated s t i m u l a t i o n . 28 Epochs 100 - i FIGURE 5 Off Stim. on 0 -J 0 -i 1 1 1 1 1 r 1 1 1 Time in msec. 1000 FACING PAGE 29 FIGUBE 6 The d i s t r i b u t i o n of s p i n a l i n t e r n e u r o n e s i n h i b i t e d by i n t e n s e , cutaneous s t i m u l a t i o n ; t h i s i s based upon the f i g u r e o f lumbar 4, S t e i n e r and Turner, 1972. The neurones are s i t u a t e d i n laminae I I , IV, V, VII, and V I I I . FACING PAGE 30 FIGUBE 7 Responses of a s p i n a l interneurone from a s p i n a l r a t . A/ A r a s t e r - d o t d i s p l a y showing a c t i v i t y before, during and a f t e r repeated intense cutaneous s t i m u l a t i o n . This i n h i b i t i o n decreased gradually u n t i l the 170th stimulus produced an i n h i b i t i o n of a duration of 400 msec, i . e . l e s s than h a l f the i n i t i a l i n h i b i t o r y period. B/ A histogram of the same data as displayed above. The number of a c t i o n p o t e n t i a l s i n the f i r s t 800 msec, f o l l o w i n g s t i m u l a t i o n increased from an average of 2.5 to an average of 6 a f t e r 170 s t i m u l i . F I G O H E 7 31 remained constant, and i n 5 the i n h i b i t i o n increased., In three of the l a s t 5 cases the build-up of i n h i b i t o n was followed by a subsequent decrease i n the i n h i b i t o r y period (see f i g u r e 8). These f i q u r e s c o n s i s t of r a s t e r - d o t d i s p l a y s and histoqrams as described p r e v i o u s l y . The d i s t r i b u t i o n of some of these c e l l s i s i l l u s t r a t e d i n f i g u r e 6. FACING PAGE 32 FIGUSE 8 A c t i v i t y of a s p i n a l i n t e r n e u r o n e from a s p i n a l i z e d r a t . , A/ A r a s t e r - d o t d i s p l a y showing an i n i t i a l i n h i b i t o r y response l a s t i n g 350 msec. The i n h i b i t i o n i n c r e a s e d over the next 12 s t i m u l i u n t i l i t reached a d u r a t i o n of 500 msec. At t h i s p o i n t the p e r i o d of i n h i b i t i o n decreased g r a d u a l l y t o a d u r a t i o n of 150 msec, a f t e r 35 s t i m u l i . B/ A histogram of the above data showing spontaneous a c t i v i t y b e f o r e s t i m u l a t i o n had a value of 5.5/400 msec, i n 3 c o n s e c u t i v e epochs. In response to repeated s t i m u l u a t i o n t h i s a c t i v i t y a t f i r s t decreased t o a value of 0 and then i n c r e a s e d t o 3. A f t e r s t i m u l a t i o n was terminated the a c t i v i t y i n c r e a s e d to a value of 6. 32 FIGO.BE 8 A . Epochs 100 -i off Stim. on 0 - i -B. i— 1— i—"— i—i— i— i— i i i •—r~>—i—•—r Time in msec. on off vy Stim. \/ i • i 1000 10 -. Number of Action Potentials 0-1 ruin rui Lr 0 Epochs i 100 33 EXPERIMENT I I RETICULO-SPINAL NEUBCNES ANJ) TflJIR JJSPONSES TO REPEATED NOXICOS PERIPBERAL STIMULATION ' A p r o g r e s s i v e i n c r e a s e i n i n h i b i t i o n t o repeated cutaneous s t i m u l a t i o n has been shown to be more r e a d i l y demonstrable i n i n t a c t r a t s than i n s p i n a l animals. I t i s p o s s i b l e t h e r e f o r e t h a t the phenomenon i s dependent upon s u p r a s p i n a l mechanisms. The o b j e c t i v e of t h i s experiment was to determine a) which areas of t h e b r a i n stem have a d i r e c t p r o j e c t i o n to the s p i n a l cord and b) i f the c e l l s i n these areas respond to p e r i p h e r a l s t i m u l a t i o n i n a manner l i k e l y to be r e s p o n s i b l e f o r a b u i l d up of i n h i b i t i o n o f s p i n a l i n t e r n e u r o n a l a c t i v i t y . kL Ml£££JADE TRANSPORT OF HORSERADISH PEROXIDASE Using r e t r o g r a d e t r a n s p o r t o f h o r s e r a d i s h peroxidase i t was found t h a t a number of areas i n the b r a i n stem p r o j e c t d i r e c t l y to t h e lumber enlargement of the s p i n a l cord. The areas i n which c e l l s were s t a i n e d by t h i s method are i l l u s t r a t e d i n f i g u r e 9. The r e s u l t s from only one r a t are shown, although f i n d i n g s from 5 other animals were i n a l l r e s p e c t s s i m i l a r . These areas, named a c c o r d i n g t o P a l k o v i t s and Jocobowitz (1974), FACING PAGE 34 FIGUBE 9 The d i s t r i b u t i o n of H.B.P.-labelled c e l l s , i n d i c a t e d by dots on diagrams of transverse s e c t i o n s of the b r a i n stem at A. 2.8 mm.; B, : 4.5 mm.; C, 6.0 mm.; and D, 7.0 mm. p o s t e r i o r t o the i n t e r a u r a l l i n e (modified from Palkowitz and Jocobowitz, 1974). A b b r e v i a t i o n s : l c , locus coeruleus; rpoc, nucleus r e t i c u l a r i s p o n t i s - c a u d a l i s ; p, pyramids; r p c , nucleus r e t i c u l a r i s p a r v o c e l l u l a r i s ; r g i , nucleus r e t i c u l a r i s g i g a n t c c e l l u l a r i s ; rm, nucleus raphe magnus; nrv, nucleus r e t i c u l a r i s pars v e n t r a l i s ; r o , nucleus raphe obscurus; i o , nucleus o l i v e r a s i n f e r i o r . 34 . . PIGOBE 9 i . i 35 i n c l u d e l o c u s c o e r u l e u s , nucleus raphe pont i s c a u d a l i s , nucleus r e t i c u l a r i s p a r v o c e l l u l a r i s , nucleus 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 , nucleus raphe magnus, nucleus r e t i c u l a r i s medulla oblongata pars v e n t r a l i s , nucleus r e t i c u l a r i s paramedianus, and nucleus raphe obscurus., MZ EJSPONSJS OF CELLS IN THE BRAIN STEM TO REPEATED NOXIOUS PEJIPERAL STIMULATIQ1x I t i s p o s s i b l e t h a t neurons s i t u a t e d i n the b r a i n stem a c t to e x c i t e i n h i b i t o r y i n t e r n e u r o n s i n the s p i n a l cord. I f t h i s were t r u e then p r o g r e s s i v e l y i n c r e a s i n g e x c i t a t o r y responses of c e l l s i n the b r a i n stem might be expected t o r e s u l t i n a b u i l d up o f i n h i b i t i o n i n the s p i n a l c o r d . In 12 r a t s , 28 c e l l s i n the c a u d a l b r a i n stem were s t u d i e d and these c e l l s responded to i n t e n s e p e r i p h e r a l s t i m u l a t i o n . Response l e v e l s of 14 c e l l s remained constant d u r i n g repeated s t i m u l a t i o n . The other f o u r t e e n of these c e l l s demonstrated a g r a d u a l l y i n c r e a s i n g e x c i t a t o r y response t o repeated s t i m u l a t i o n . Ten of these f o u r t e e n c e l l s responded only to s t i m u l a t i o n o f 1 paw, 1 responded t o s t i m u l a t i o n of 2 paws, 1 to s t i m u l a t i o n of 3 paws and 2 to s t i m u l a t i o n of a l l 4 paws. A t y p i c a l response of such a c e l l i s i l l u s t r a t e d i n f i g u r e 10. I t can be seen t h a t t h i s c e l l had no spontaneous a c t i v i t y and that i t was not u n t i l 25 s t i m u l i had been given t h a t the FACING PAGE 36 FIGOBE 10 Besponses of a neurone i n the brainstem p r i o r t o , during and a f t e r repeated i n t e n s e , cutaneous s t i m u l a t i o n . A/ Baster-dot d i s p l a y showing no prestimulus a c t i v i t y and an i n c r e a s i n g e x c i t a t o r y response which extended over the e n t i r e epoch a f t e r 80 s t i m u l u i . Neuronal a c t i v i t y ceased immediately upon termination of the s t i m u l u s . ; £•/ A histogram of the same data as above showing the response increased from nothing i n the f i r s t 5 s t i m u l i to 9/900 msec, f o r the l a s t 5 s t i m u l i . FIGUHE 10 A. Epochs I 7 0 T off Stim. on 0 J | i | i | i | i | i | i | i I i I ' I 1 1 IOOO Time in msec. on B. Stim. 15 i Number of Action Potentials 0 i i nl off 1/ r -0 Epochs 1 7 0 37 c e l l responded. The responses to subsequent repeated s t i m u l i gradually increased. The a c t i v i t y ceased abruptly after the stimulation was terminated, Figure 11 represents the response of t h i s c e l l to various strengths of stimulation and i t i s shown that the rate of build up varies d i r e c t l y with the i n t e n s i t y of stimulation. Attempts were made to mark the position of a l l 14 of the c e l l s but only 9 of these were successful. The d i s t r i b u t i o n of these c e l l s i s i l l u s t r a t e d i n figure 12. £Z iJlIDRCMIC ACTIVATION FROM THE SPINAL COlfi fiFHHIiJD UP CELLS IB U J BRAIN STEM It has been shown i n the previous experiments that areas within the brain stem have projections to the sp i n a l cord and respond in a manner which could r e s u l t i n a build up of i n h i b i t i o n i n the spin a l cord. The objective of t h i s experiment was to determine i f the c e l l s demonstrating a build-up of a c t i v i t y in response to repeated peripheral s t i m u l i have projections to the spinal cord. A t o t a l of 8 c e l l s which demonstrated increasing excitation to repeated peripheral stimuli were antidromically activated from the sp i n a l cord. The responses of one of these c e l l s are i l l u s t r a t e d i n figure 13. Antidromic activation was i d e n t i f i e d on the basis of at l e a s t three out of 4 c r i t e r i a l i s t e d below: FACING PAGE 38 FIG0BE 11 The influence of stimulus i n t e n s i t y upon the rate of development of excitatory response. F I G U R E 11 FACING PAGE 39 FIGOfiE 1 2 She d i s t r i b u t i o n of neurones which responded with a p r o g r e s i v e l y i n c r e a s i n g e x c i t a t i o n to repeated i n t e n s e , cutaneous s t i m u l a t i o n . These s i t e s i n c l u d e nucleus r e t i c u l a r i s p o n t i s - c a u d a l i s , rpoc; 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 , r q i ; nucleus r e t i c u l a r i s p a r v o c e l l u l a r i s , r p c ; and nucleus r e t i c u l a r i s medulla oblongata pars v e n t r a i l s , nrv; nucleus raphe magnus, rm. L e v e l VB1 shows the l o c a t i o n of two c e l l s , i n d i c a t e d by open c i r c l e s , which demonstrate a gradual development of e x c i t a t i o n to repeated p e r i p h e r a l s t i m u l a t i o n and which 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 s p i n a l cord. Transverse s e c t i o n s are redrawn from P a l k v i t s and Jocobowitz, 1971. A, 3.4 mm; B, 3.9 mm; C, 4.5 mm; and D, 5.5 mm. p o s t e r i o r to the i n t e r a u r a l l i n e , FIGURE 12 FACING PAGE .40 F I G U R E 1 3 Responses of an i d e n t i f i e d b u l b o - s p i n a l neurone to repeated cutaneous s t i m u l a t i o n . A/ A r a s t e r - d o t d i s p l a y showing a gradual development of e x c i t a t o r y response to repeated s t i m u l a t i o n . B/ A histogram showing e x c i t a t o r y response s t a r t i n g at the 25th stimulus which increased from 1 a c t i o n potential/950 msec, to 5/95 0 msec, over 50 s t i m u l i . 40 FIGURE 13 FACING PAGE 4 2 FIGUBE 14 Evoked responses from a bulbar neurone 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 a l cord. A/ Five superimposed traces showing an antidromic spike (A) occuring 2 msec, a f t e r the stimulus a r t e f a c t (SA). A spontaneous spike i l l u s t r a t e d by S. B/ fl s i n g l e t r a c e showing spontaneous spiJces (S) occuring 2 msec, before and 45 msec, a f t e r a stimulus a r t e f a c t (SA). C a n c e l l a t i o n of the antidromic spike (A) i s i l l u s t r a t e d . C,D/ Pulses generated by the r i s i n g phase of spontaneous a c t i o n p o t e n t i a l s (ST) were used to i n i t i a t e the osc i l o s c o p e sweep and allow s t i m u l i to be a p p l i e d at predetermined i n t e r v a l s f o l l o w i n g the spontaneous p o t e n t i a l . In f i g u r e C a stimulus given a f t e r 3 msec, evoked an a n t i d r o m i c a l l y a c t i v a t e d p o t e n t i a l . , I n f i g u r e D when a stimulus was given a f t e r 2 msec, the antidromic p o t e n t i a l di d not occur., 41 (1) constant latency of evoked action potential to threshold stimulation; (2) a b i l i t y to f o l i o s high frequency stimulation (above 100/sec); (3) c o l l i s i o n test (see Fiqure 14) i n which cancellation of the antidromic action potential can be demonstrated following c o l l i s i o n with a spontaneous action potential occurring during a c r i t i c a l period before the antidromically evoked event; (4) an i n f l e c t i o n on the p o s i t i v e phase of an action p o t e n t i a l , i n d i c a t i n g the separation between i n i t i a l segment spikes and the somatodendritic spike. , Two of these c e l l s were shown to be located 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 , (see figure 12). 42 43 EXPERIMENT I I I EFFECTS OF BULBAR STIMULATION CJ SJ9INAL NEURONES D£MQNSTRATING PROGRESSIVE INHIBITION TO REPEATED NCXIOUS STIMULAJICN I t i s suqqested from the r e s u l t s of experiment I of t h i s p r o j e c t t h a t i n h i b i t o r y b u i l d up of s p i n a l i n t e r n e u r o n a l a c t i v i t y i n response to repeated cutaneous s t i m u l a t i o n i s dependent upon s u p r a s p i n a l i n f l u e n c e s , Haber and Waqman (1S74) have shown p r o g r e s s i v e i n h i b i t i o n of s p i n a l a c t i v i t y i n response to s t i m u l a t i o n of the nucleus r e t i c u l a r i s g i q a n t o c e l l u l a r i s . The r e s u l t s of experiment I I show t h a t neurones i n t h i s area have p r o j e c t i o n s to the s p i n a l cord and t h a t they respond i n a manner which c o u l d be r e s p o n s i b l e f o r the i n c r e a s i n q i n h i b i t i o n of s p i n a l a c t i v i t y . I t i s the o b j e c t of t h i s experiment to determine the e f f e c t of repeated s t i m u l a t i o n of t h i s , and adjacent areas, on the a c t i v i t y of c e l l s which demonstrate p r o q r e s s i v e i n h i b i t i o n i n response to repeated cutaneous s t i m u l a t i o n . In 5 r a t s , 5 c e l l s were encountered whose a c t i v i t y was a f f e c t e d by both p e r i p h e r a l and bulbar s t i m u l a t i o n . Of these f i v e c e l l s , two responded to s t i m u l i a p p l i e d to both s i t e s with a p r o q r e s s i v e i n c r e a s e i n i n h i b i t i o n (see F i q u r e 15). Two c e l l s FACING PAGE 44 FIGURE 15 Responses of a s p i n a l interneurone responding to s t i m u l a t i o n of both c e n t r a l and p e r i p h e r a l s i t e s . . A 6 B/ A r a s t e r - d o t d i s p l a y and histogram showing a g r a d u a l l y i n c r e a s i n g i n h i b i t o r y response t o repeated s t i m u l a t i o n of nucleus p o n t i s - c a u d a l i s . C S D/ A r a s t e r - d o t d i s p l a y and histogram showing a g r a d u a l l y i n c r e a s i n g i n h i b i t o r y response to repeated s t i m u l a t i o n of the periphery. ,. 44 FIGUEE 15 45 demonstrated a proqressive increase in excitation to repeated central stimulation and a proqressive increase in inhibition to cutaneous stimulation (see Fiqure 16). One c e l l responded with a proqressive increase in inhibition to repeated medullary stimulation but was excited by peripheral input. The maqnitude of this latter response increased with repetition (see fiqure 17). The distribution of these cells and of the appropriate stimulation sites are illustrated in fiqure 18. FACING PACE 46 F i g u r e 16 Besponses of a s p i n a l i n t e r n e u r o n e demonstrating responses to both p e r i p h e r a l and c e n t r a l s t i m u l a t i o n . A 6 E/ A r a s t e r - d o t d i s p l a y and histogram shosning a g r a d u a l l y i n c r e a s i n g e x c i t a t o r y response t o repeated s t i m u l a t i o n o f nucleus r e t i c u l a r i s p a r v o c e l l u l a r i s . C 8 D/ A r a s t e r - d o t d i s p l a y and histogram showing a g r a d u a l l y i n c r e a s i n g i n h i b i t o r y response t o repeated p e r i p h e r a l s t i m u l a t i o n . , FIGUEE 16 FACING PAGE 47 FIGURE 17 Responses of a s p i n a l i n t e r n e r o n e responding t o s t i m u l a t i o n of both p e r i p h e r a l and c e n t r a l s i t e s , A & B/ A r a s t e r - d o t d i s p l a y and histogram showing a g r a d u a l l y i n c r e a s i n g i n h i b i t o r y response to repeated s t i m u l a t i o n of the i n f e r i o r o l i v e nucleus. C 6 D/ A r a s t e r - d o t d i s p l a y and histogram showing a g r a d u a l l y i n c r e a s i n g e x c i t a t o r y response to repeated p e r i p h e r a l s t i m u l a t i o n . 47 FIGURE 17 A. Epochs 8O-1 Stim. off i • i • i • i. ' i i 1 i 1 i Time in msec. 1000 B. on off ^Stim. | 15 Number of Action Potentials i i " V Epochs 80 C. off Epochs 70-on 1 — • i ' i * i * i * i * i * i 1 ^ Time in msec. D. on off ^ Stim. ^ 25 n 0-1 i ' i 1 i 1000 Epoch* 70 FACING PAGE 48 FIGUBE 18 An i d e a l i z e d schematic of a cross-section of s p i n a l cord at the l e v e l of the fourth lumbar vertebrae (based on Steiner and Turner, 1972) showning the d i s t r i b u t i o n of c e l l s demonstrating responses to stimulation of both central and peripheral s i t e s . Indication of the responses and of the central stimulation s i t e s are given. 48 PIG08B 18 Peripheral Stimulation • Inhibition A Inhibition • Excitation Central Stimulation Excitation Inhibition Inhibition 49 DISCUSSION The r e s u l t s of Experiment I support MacDonald*s demonstration (PhD t h e s i s - 1975, MacDonald and Pearson, 1977) of a build-up of i n h i b i t i o n i n s p i n a l interneurons of r a t s with i n t a c t s p i n a l cords. This response p a t t e r n , on the other hand, did not occur i n s p i n a l r a t s . In f a c t , i n s p i n a l r a t s , i n h i b i t o r y responses to repeated p e r i p h e r a l s t i m u l a t i o n p r o g r e s s i v e l y diminished. Neither these r e s u l t s nor those of MacDonald and Pearson (1977) c o n t r a d i c t Groves and Thompson*s (197C) c l a i m that there i s no evidence for i n h i b i t o r y b u i l d up i n s p i n a l c a t s . Nor do they c o n t r a d i c t the c o n c l u s i o n s of Spencer, Thompson and Neilson's work (1966c) who showed that intravenous a d m i n i s t r a t i o n of strychnine did not impair the development of h a b i t u a t i o n of the f l e x o r withdrawal r e f l e x i n s p i n a l animals. The present r e s u l t s , taken tcqether with the f i n d i n q of MacDonald and Pearson (1973), t h a t s t r y c h n i n e impairs the development of h a b i t u a t i o n of the f l e x o r withdrawal r e r l e x i n i n t a c t animals, suggest that h a b i t u a t i o n i s a consequence of d i f f e r e n t mechanisms i n i n t a c t animals than i s the case i n s p i n a l animals. Thus, h a b i t u a t i o n i n the i n t a c t animal may be dependent upon i n h i b i t o r y interneurons which are i n f l u e n c e d by s u p r a s p i n a l mechanisms. The source of t h i s s u p r a s p i n a l a c t i v i t y was i n v e s t i g a t e d i n the second s e r i e s of experiments. A qradual increase i n i n h i b i t i o n miqht conceivably be produced by neurons, the 50 a c t i v i t y of which progressively increase and exert (either d i r e c t l y or i n d i r e c t l y ) an in h i b i t o r y influence on the c e l l i n question. The r e t i c u l a r formation and raphe nuclei have been shown to contain neurones whose a c t i v i t y increase with repeated cutaneous stimulation. Moreover, the axons of c e l l s in these areas project to the spinal cord, as shown by retroqrade transport of horseradish peroxidase. Some neurones which showed both a build-up of a c t i v i t y and could be antidromically activated from the spinal cord were found to be situated 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 ( r q i ) . This nucleus therefore might well be involved in the qradual development of i n h i b i t i o n at the s p i n a l l e v e l . Such a p o s s i b i l i t y i s supported by the finding of Haber and Haqman (1974) that stimulation of r q i results i n a build-up of i n h i b i t i o n of spinal interneurones. However, i n the present study, i n h i b i t o r y build-up was e l i c i t e d by stimulation of the nucleus r e t i c u l a r i s pontis caudalis. I t must be stressed thouqh, that the i n t e n s i t y of stimulation was such that structures, adjacent to those i n which positive Prussian blue reaction was found, (eq. rqi) could well have been activated (Hanck, 1975). I t i s relevant therefore to consider several structures in the brain stem with reqard to thei r possible i n h i b i t o r y e f f e c t s on spinal interneurones. For a nucleus to be considered as a candidate i t would be expected to: (1) Respond to noxious s t i m u l i (It must be emphasized here that the peripheral stimulation used to e l l i c i t the f l e x o r 5 1 withdrawal r e f l e x i s noxious.); (2) Have i n p u t from the s p i n a l c o r d ; (3) Have output to the s p i n a l c o r d ; and (4) T h i s decendinq output to the s p i n a l c o r d must be i n h i b i t o r y . There are two groups of n u c l e i i n t h i s area whose c e l l s have p r o p e r i t i e s which suggest that they may be i n v o l v e d i n , or r e s p o n s i b l e f o r i n c r e a s i n g s p i n a l i n h i b i t i o n . These are the r e t i c u l a r f o r m a t i o n and the raphe n u c l e i . In the next few paragraphs the known r e l e v a n t c h a r a c t e r i s t i c s of these two systems w i l l be d i s c u s s e d . fiJTICJJLAJ FORMATION Anatomical s t u d i e s of s e v e r a l v e r t e b r a t e s p e c i e s show that a l a r g e p r o p o r t i o n of the ascending axons a r i s i n g from the s p i n a l cord terminate i n the medial r e t i c u l a r f ormation of the medulla and pons (Anderson and Berry, 1959; Bowsher, 1957; B r c d a l and S o s s i , 1955; S o s s i , 1957; F i e l d s , P a r t r i d g e , and Winter, 1970; F i e l d s , wagner and Anderson, 1975; B o s s i and Brodal, 1957). There are complex l o c a l c o n n e c t i o n s w i t h i n the medial pontcmedullary r e t i c u l a r formation, ( S c h e i b e l and S c h e i b e l , 1958) and i t has convergence of i n p u t s from m u l t i p l e sensory m o d a l i t i e s (French, Verzeano and Morgan, 1953; Nanta and Kuypers; Pomeiano, 1973). Although p h y s i o l o g i c a l s t u d i e s have i n d i c a t e d l i t t l e modality s p e c i f i c i t y or topographic o r g n a i z a t i o n of i n p u t ( B e l l , 52 S i e r r a , Buendia, and Segundo, 1961; Bowsher, Mallat, P e t i t , and Albe-Fessard 1968; Casey, 1969; Segundo, Takenaka, and Ecabo, 1967; Wolstencroft, 1964), a role for t h i s region i n pain transmission i s suggested by evoked responses to noxious stimulation of the periphery and to selective a c t i v a t i o n of small diameter peripheral f i b r e s (Bowsher, Mallart, P e t i t , and Albe-Fessard, 1968; Casey, 1969; Goldman, C o l l i n s , Taub and Fitzman, 1972). Futhermore, there i s a marked decrease i n responsiveness to noxious stimulation after b i l a t e r a l lesions of the medial r e t i c u l a r formation in the caudal brainstem lesions (Anderson and Pearl, 1975) . Peripherally applied noxious s t i m u l i have been shown to evoke both increases and decreases i n background f i r i n g rate of single units of the r e t i c u l a r formation (LeBlanc and Gapiton, 1974; Arutzumov, N a r i k a s v i l l i and Tatevosyan, 1872; Guilfcaud, Besscn, Oliveras and Wyon-Maillard, 1973; F i e l d s , Clanton, and Anderson, 1976). In the mesencephalic r e t i c u l a r n u c l e i , 11 out of 105 c e l l s showed a proqressive increase with repeated stimulation and of these, 5 did not respond to the f i r s t stimulus ( B e l l , S i e r r a , Buendia and Segundo, 1964). The existance of r e t i c u l o s p i n a l neurones has been shown i n the cat (Kohnstramm, 1899; Magni and W i l l i s , 1963; and Wolstencroft, 1964) and rat (Fox, 1970; and i n the present study). F i e l d s , Wagner and Anderson (1975) have demonstrated that a c t i v i t y of s p i n a l neurons can be greatly reduced ty weak e l e c t r i c a l stimulation of the nucleus r e t i c u l a r i s giqanto-53 c e l l u l a r i s . Furthermore Haber and Waqman (1974) described t h i s i n h i b i t i o n as becoming progressively greater with repeated stimulation. I t has also been demonstrated that the s p i n a l projection from the medial brain stem r e t i c u l a r formation i n h i b i t s nociceptive information (Fields, Clantcn and Anderson, 1977) confirming that t h i s nucleus i s c r u c i a l for processing information concerning pain. A low pontine transection reveals a descending i n h i b i t o r y pathway to flexor motor-neurons and t h i s i n h i b i t i o n i s abolished by spinal transection (Hclmquist and Lindberg, 1961) i n d i c a t i n g that i t originated in the medulla. Similar conclusions were made by Besson, Guilbaud and LeBars (1975) on the basis of experiments using decerebrate cats. Thus the r e t i c u l a r nuclei have connections to and from the spinal cord, respond to noxious stimulation and have in h i b i t o r y e f f e c t s on spinal a c t i v i t y . RAPJHJ NUCLEI The dorsal raphe nucleus recieves a compliment of somato-sensory input including that conveying information about pain (Becker, Gluck, Neilsen and darae, 1968; Bowsher, 1957; Liebeskind and Mayer, 1971 Mehler, 1969). These studies showed that noxious peripheral stimulation enhanced or i n i t i a t e d unitary a c t i v i t y i n the dorsal raphe n u l e i . It has also been shown by Liebeskind, Guilbaud, Besson and Oliveras (1973) that 54 s t i a u l a t i c n of the d o r s a l raphe nucleus i n h i b i t s the responses of s p i n a l i n t e r n e u r o n s i n v o l v e d i n the sensory pathways. Many s t u d i e s i n r a t s (Bowsher, 1957; Melzack and M e l i n k o f f , 1S74; Meyer, Wolfe, A k i l , Carder and L i e b e s k i n d , 1971; and Reynolds, 1969) c a t s ( L i e b e s k i n d et a l . , 1973) and man (Adams, 1976) have shown t h a t s t i m u l a t i o n of t h i s nucleus i n h i b i t s responses to p a i n . Axons a r i s i n g from the d o r s a l raphe nucleus do not p r e l e c t i n a caudal d i r e c t i o n ( D a h l s t r o i , Fuxe, L a r s s o n , O l s i n and Unqerstedt, 1966) and thus the connection with the s p i n a l cord must be v i a an i n d i r e c t route. T h i s route c o u l d be v i a the r e t i c u l a r n u c l e i . Briqgs (1976) has shown t h a t neurons i n the r a t bulbar r e t i c u l a r formation are e x c i t e d by s t i m u l a t i o n i n the nucleus raphe maqnus, nucleus raphe p a l l i d u s or nucleus raphe ohscurus. Sakamura (1975) on the other hand, demonstrated an i n h i b i t o r y i n f l u e n c e of the midbrain raphe nucleus upon neurons of the r e t i c u l a r f o r m a t i o n of the midbrain and pons. For the raphe n u c l e i o f the more caudal p a r t s of the b r a i n stem i t i s not necessary to devise such a complex r e l a y system. D a h l s t r c n and Fuxe (1969) have shown t h a t these n u c l e i have a d i r e c t p r o j e c t i o n to the s p i n a l cord. O l i v e r a s , Redjerai, Guilfcaud and Besson ( 1975) have demonstrated t h a t e l e c t r i c a l s t i m u l a t i o n of the i n f e r i o r c e n t r a l nucleus of the raphe i n the medulla t o t a l l y suppresses b e h a v i o u r a l r e a c t i o n s to limb pinches. Moreover s t i m u l a t i o n of t h i s area i n h i b i t s the a c t i v i t y of s p i n a l neurones t o noxious i n p u t s ( F i e l d s , Eausbaum, 55 Clanton and Anderson, 1977). Thus, neurones of both raphe and r e t i c u l a r n u clei receive ascending information concerninq noxious s t i a u l i and have descending i n h i b i t o r y influences. I f the development of i n h i b i t i o n of spinal neuronal a c t i v i t y i s dependent upon a particular supraspinal nucleus then i t miqht be expected that repeated stimulation, both of that nucleus and of the periphery would have the same e f f e c t on a given s p i n a l interneurone. In the present study, stimulation of nucleus r e t i c u l a r i s pcntis caudalis has been demonstrated to have such an e f f e c t . fiaber and laqman have, as mentioned e a r l i e r , shown a build-up of inhib i t o r y influences in the spinal cord to repeated stimulation of r q i . Neither of these works have investiqated the e f f e c t s of stimulation of raphe nuclei. With reqard to the i n h i b i t o r y e f f e c t of stimulation of the nucleus raphe magnus on s p i n a l interneurones, the only published data a v a i l a l a b l e are those of Fields, Eausbaum, Clanton and Anderson (1977). Repeated t r a i n s of s t i m u l i were shown to have a powerful i n h i b i t o r y e f f e c t , but the duration of the i n h i b i t i o n did not appear to show any consistent trend. I t should be pointed out however, that these workers were not primarily concerned with, nor were their experiments desiqned to adequately investiqate any possible change tc repeated stimulation. 56 I t would appear t h a t nucleus r e t i c u l a r i s - g i q a n t o c e l l u l a r i s and nucleus r e t i c u l a r i s p o n t i s - c a u d a l i s are i n v o l v e d i n the development of a p r o q r e s s i v e i n h i b i t i o n of s p i n a l i n t e r n e u r o n e s . 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