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The role of inhibitory mechanisms in habituation and sensitization of the flexor reflex MacDonald, John Ferguson 1975

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THE ROLE OF INHIBITORY MECHANISMS IN HABITUATION AND SENSITIZATION OF THE FLEXOR REFLEX by JOHN FERGUSON MacDONALD B. Sc., University of Bri t i s h Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the department of Physiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1975 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 i t 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 i s understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Physiology The University of British Columbia Vancouver, B.C., Canada. i ABSTRACT Several authors have suggested that the r e t i c u l a r formation may act as a generator of behavioural i n h i b i t i o n . Furthermore, they have implied that t h i s i n h i b i t i o n may progressively reduce evoked behaviour leading to behavioural habituation,(Hernandez-Peon, 1960; Sokolov, 1963; Stein, 1966). Stein (1966) proposed that recurrent i n h i b i t i o n of the r e t i c u l a r formation might be responsible f o r habituation of behavioural arousal. Wall (1970) presented the hypothesis that PTP of i n h i b i t o r y synapses within the f l e x o r r e f l e x pathway might cause habituation of t h i s r e f l e x . However, experiments performed upon the s p i n a l cat have not indicated a r o l e for i n h i b i t i o n i n habituation of the f l e x o r r e f l e x (Spencer, et a l . , 1966c; Groves and Thompson, 1973). A more reasonable explanation for habituation of t h i s r e f l e x i n the s p i n a l animal i s a reduction i n the e f f i c a c y of e x c i t a t o r y synapses within the d i r e c t r e f l e x pathways (F a r e l , et a l . , 1973). Habituation of the f l e x o r r e f l e x i s not i d e n t i c a l i n the s p i n a l and i n t a c t rat (Pearson and Wenkstern, 1972). Habituation i s more r e a d i l y observed i n the i n t a c t r a t . This thesis has demonstrated that habituation of the f l e x o r r e f l e x i s retarded but not prevented by the i n f u s i o n of drugs which are known to antagonize i n h i b i t i o n (strychnine and b i c u c u l l i n e ) provided the s p i n a l cord i s not transected. This was not the case f o r habituation of t h i s r e f l e x i n the s p i n a l r a t . Indeed, the i n f u s i o n of strychnine a c t u a l l y f a c i l i t a t e d habituation i n the s p i n a l r a t . Spontaneously active s p i n a l interneurones were progressively i n h i b i t e d ( i n h i b i t o r y build-up) by repeated cutaneous stimulation. This build-up of i n h i b i t i o n was greater with intense s t i m u l i than with weak s t i m u l i . However, a s i m i l a r build-up of i n h i b i t i o n was not found a f t e r transection of the s p i n a l cord. I n h i b i t i o n i t s e l f tended to habituate i n the s p i n a l rat regardless of the i n t e n s i t y of the s t i m u l i . The i n j e c t i o n of strychnine eliminated i n h i b i t o r y b u i l d -up i n the i n t a c t rat- i n h a l f of the interneurones tested but was i n e f f e c t i v e i n the remaining interneurones. Decerebration releases a tonic i n h i b i t i o n of the f l e x o r r e f l e x mediated by the r e t i c u l a r formation (Holmqvist and Lundberg, 1961). This thesis has demonstrated that habituation of the f l e x o r r e f l e x i s much more pronounced i n the decerebrate than i n the s p i n a l r a t provided intense s t i m u l i are employed. This evidence suggests that the r e t i c u l a r formation may be responsible for the genesis of i n h i b i t o r y build-up. A s i m i l a r build-up of 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 has been shown following r e p e t i t i v e stimulation of the r e t i c u l a r formation (Abrahams, 1974; Haber and Wagman, 1974). The decrement of the f l e x o r r e f l e x r e l a t e d to a build-up of i n h i b i t i o n was only apparent when the i n t e n s i t y of the s t i m u l i was noxious (possibly painful) and i t may represent a mechanism for adaptation to pain. Serotonergic systems and the raphe^nuclei are associated with an i n h i b i t i o n of behavioural arousal and the i n h i b i t i o n of pain. This thesis has shown that lesions of the nucleus raphe"'' d o r s a l i s and pre-treatment with p-CPA f a c i l i t a t e habituation of the flexor reflex relative to the intact animal (not treated with p-CPA). It i s suggested that the raphe^nuclei inhibit the reticular neurones responsible for the genesis of inhibitory build-up. Habituation of the flexor reflex in the spinal rat i s best explained by a mechanism of reducing synaptic efficacy. However, intersegmental inhibitory mechanisms may be capable of modulating the amplitude of the reflex which in turn may alter the degree of habituation. Furthermore, the decrement of inhibition observed in the spinal rat may be responsible for long term sensitization of the flexor reflex. iv TABLE OF CONTENTS PAGE ABSTRACT i LIST OF TABLES vr LIST OF FIGURES v i : ACKNOWLEDGEMENTS x i i CHAPTER I (INTRODUCTION) Section I Learning and Habituation 1 Section II Integration in the Nervous System 19 Section III Synaptic Plasticity 28 Section IV Inhibition: Habituation and Sensi-tization of the Flexor Reflex 41 CHAPTER II (METHODS) Section I The Flexor Reflex 52 Section II Spinal Interneurones 67 Section III Fixation of the Brain and Spinal Cord 73 CHAPTER III Section I Sta t i s t i c a l Considerations 74 Section II Methological Considerations and Data Presentation 84 CHAPTER IV (RESULTS) Section I The Flexor Reflex 100 Section II Spinal Interneurones (Intact rat)' 137 Section III Spinal Interneurones (Spinal rat) 162 CHAPTER V (DISCUSSION) Section I Inhibition and Habituation of the Flexor Reflex 164 Section II Segmental Inhibition 177 Section III Afferent Inflow to the Spinal Cord 182 Section IV Behaviour of Pain 188 Section V Inhibition and the Theories of Habituation 196 BIBLIOGRAPHY 2 0 9 V LIST OF TABLES Table p a g e I Flexor muscles 44 v i LIST OF FIGURES FIGURE PAGE 1 Conceptualization of conditioning and the effect of motivation/volition. 7 2. Conceptualization of conditioning and stimulus/ response generalization 11 3; Stimulus/response continua and the conceptualization of "associative" learning. 17 4 Schematic diagram of stimulating and recording apparatus 55 5 Location of recording electrodes. Biceps femoris muscle 56 6 Rat i n Bollman restraining cage 58 7 Rat holder for extracellular recording 69 8 Measures of habituation 87 9 Independence of the relative measure of habituation from the response amplitude 88 10 Effect of GDEE on habituation of the FWR 90 11 Sensitivity of the relative measure of habituation to population heterogeneity. 93 12 Action of stimulus intensity on habituation of the FWR in the intact rat. Data expressed as single points 94 13 Action of stimulus intensity on habituation of the FWR in the intact rat. Data expressed as percentages of f i r s t block of 10 responses 95 14 Habituation of the FWR i n the intact and spinal rat. EMG response 97 15 Habituation of the FWR in the intact and spinal rat 99 16 Effect of strychnine on habituation of the FWR in the intact rat. Stimulus intensity 5 v. 101 v i i LIST OF FIGURES (continued) FIGURE PAGE 17 Effect of strychnine on habituation of the FWR in the intact rat. Stimulus intensity 20v. 102 18 Effect of bicuculline on habituation of the FWR in the intact rat. 104 19 Effect of strychnine and bicuculline on habituation of the FWR 105 20 Effect of pre-treatment with p-CPA on habituation of the FWR in the intact rat. Stimulus intensity 5 v. 106 21 Effect of pre-treatment with p-CPA on habituation of the FWR in the intact rat. Stimulus intensity 20 v. 107 22 Effect of lesion in the n.r.d. on habituation of the FWR. 109 23 Extent of n.r.d. lesions. 110 24 Effect of methysergide on habituation of the FWR in the intact rat. 112 25 Habituation of the FWR in the spinal and decerebrate rat. Stimulus intensity 20 v.. 114 26 Habituation of the FWR in the spinal and decerebrate rat. Stimulus intensity 60 v. 116 27 Habituation of the FWR in spinal rats with various levels of transection and decerebrate rats. Single responses 117 28 Effect of asphyxiation on habituation of the FWR in the spinal rat. Stimulus intensity 20 v. 119 29 Effect of asphyxiation on habituation of the FWR in the spinal rat. Stimulus intensity 60 v. and relative measure. 120 30. Effect of asphyxiation on habituation of the FWR in the spinal rat. Stimulus intensity 60 v. and absolute measure 121 v i i i LIST OF FIGURES (continued) FIGURE PAGE 31 Effect of stimulus intensity on habituation of the FWR i n the spinal rat (T5). 123 32 Effect of strychnine on habituation of the FWR in the spinal rat. Stimulus trains, 20 v. 125 33 Effect of strychnine on habituation of the FWR in the spinal rat. Stimulus trains, 60 v. 126 34 Effect of strychnine on habituation of the FWR in the spinal rat. Single pulse, 20 v. 128 35 Effect of stimulus intensity on habituation of the FWR in the spinal rat ( T 1 Q ) . 130 36 Effect of stimulus intensity on habituation of the FWR in the spinal rat (Cy). 131 37 Habituation of the FWR in the intact and anaesthetized rat. EMG response 133 38 Effect of two t r i a l s on habituation of the FWR in the intact and anaesthetized rat. 134 39 Effect of strychnine on habituation of the F/JR in the intact and anaesthetized rat. 135 40 Response patterns of spinal interneurones. High frequency burst and after-discharge 139 41 Habituation of the high frequency burst. 140 42 Effect of stimulus intensity on sensitization of the after-discharge. 142 43 Sensitization of the after-discharge followed by decrement. 143 44 Sensitization of the after-discharge followed by decrement. 144 45. Constant high frequency burst and sensitization of the after-discharge followed by decrement. 145 i x LIST OF FIGURES (continued) FIGURE PAGE 46 Decrement of i n h i b i t i o n and s e n s i t i z a t i o n of ex c i t a t i o n . 147 47 Inhibi t o r y build-up i n response to s i n g l e pulse stimulation. 148 48 Inhibi t o r y build-up i n response to stimulus t r a i n s . 149 49 E f f e c t of stimulus i n t e n s i t y on i n h i b i t o r y b u i l d -up. 151 50 E f f e c t of stimulus frequency on i n h i b i t o r y build-up. 153 51 E f f e c t of a second t r i a l on i n h i b i t o r y build-up. 154 52 Interaction of i p s i l a t e r a l e xcitatory build-up and c o n t r a l a t e r a l i n h i b i t o r y build-up. 156 53 D i s - i n h i b i t i o n of i n h i b i t o r y build-up. 157 54 E f f e c t of strychnine on i n h i b i t o r y build-up. 158 55 E f f e c t of strychnine on i n h i b i t o r y build-up 159 56 Laminar l o c a t i o n of some recorded interneurones. 161 57 Decrement of i n h i b i t i o n i n the cord of the s p i n a l 162 rat . 58 E f f e c t of stimulus s i g n i f i c a n c e on habituation of the t e r r i t o r i a l response of the three-spined 204 stickleback. X LIST OF ABBREVIATIONS C, centrigrade C^, transection at the seventh cervical vertebra cc, cubic cm. cm., centimeter CS, conditioned stimulus CR, conditioned response D. C., direct current dia., diameter DRP, dorsal root potential EJP, excitatory junction potential EMG, electromyographic EPSP, excitatory post-synaptic potential FRA, flexor reflex afferent FRI, flexor reflex interneurone FMN, flexor motorneurones FWR, flexor withdrawal reflex g, gram GDEE, glutamic acid diethyl ester GR, general response GS, general stimulus Hg., mercury hr., hour ILD, inhibition of long duration IPSP, inhibitory post-synaptic potential kg, kilogram LSD, lysergic acid diethyl amide x i LIST OF ABBREVIATIONS (continued) M, motivation mA, milliampere min., minute mg, milligram ml, m i l l i l i t r e mm., millimeter msec. , mil l i s e c o n d MP, megohm n, number n.r.d., nucleus raphe^dorsalis n.r.g., 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 PAD, primary afferent d e p o l a r i z a t i o n PAH, primary afferent hyperpolarization p-CPA, para-Chlorophenylalanine PTD, post-tetanic depression PTP, post-tetanic p o t e n t i a t i o n R, response S, stimulus sec., second T5, T ^ Q, transection at the f i f t h or tenth thoracic vertebrae UR, unconditioned response US, unconditioned stimulus u, micron uA, microampere Ug, microgram v., v o l t x i i ACKNOWLEDGEMENT S The opportunity to a t t a i n a research .'degree i s dependent upon the support and patience of a s c i e n t i s t who perpetuates the expertise of his f i e l d by f o s t e r i n g the education of a student such as myself. In t h i s respect, Dr. J. A. Pearson has made me both indebted and deeply g r a t e f u l . He and the other members of the f a c u l t y of the Department of Physiology have provided an atmosphere condusive to the development of higher education. I also wish to thank Mrs. K. White for her valuable t e c h n i c a l assistance and acknowledgement must also be made to Ms. L. W i l l s , who played a major r o l e i n the performance of experiments based on the study of the nucleus raphe^dorsalis. Furthermore, the many figures and photographs displayed i n t h i s thesis represent many hours of s k i l l e d labour by Mr. K. Henze. However, Mr. Henze has extended the benefit of h i s experience to myself on numerous occasions and i t i s for both of these reasons that I acknowledge Mr. Henze. F i n a l l y , I must thank Ms. A. Redlich and Dr. P. A. Larkin whose encouragement during my undergraduate years was so important. CHAPTER I INTRODUCTION: SECTION I Learning and Habituation A c e n t r a l , i f not i n t r i n s i c , o b j e c t i v e of neurophysiology i s the c o r r e l a t i o n of phy s i o l o g i c a l mechanisms of the ce n t r a l nervous system with the overt and complex behaviour of the organism. Pavlov employed the conditioned r e f l e x as a model from which he could extrapolate the laws of behaviour ( a c q u i s i t i o n , e x t i n c t i o n , and generalization) and inf e r r e d the underlying p h y s i o l o g i c a l mechanisms. Sherrington, however, stressed an understanding of the p h y s i o l o g i c a l mechanisms underlying the behaviour of simple s p i n a l r e f l e x e s . This thesis borrows i t s p h i l o s o p h i c a l approach from both sources and uses a r e f l e x (flexor withdrawal ref l e x ) to determine i f c e r t a i n p h y s i o l o g i c a l mechanisms ( i n h i b i t i o n s ) can account f o r c e r t a i n behavioural c h a r a c t e r i s t i c s of the r e f l e x (habituation and s e n s i t i z a t i o n ) . Russian neurophysiologists, who follow the teachings of Pavlov, suggest that i n h i b i t i o n plays a p a r t i c u l a r i l y s i g n i f i c a n t r o l e i n the manifestation of behaviour. B e r i t o f f (1965:161), f o r example, states that: "Widespread i n h i b i t i o n constitutes the basic factor i n inte g r a t i o n of the cen t r a l nervous system during behavioural reactions.... General i n h i b i t i o n a r i s e s i n the ce n t r a l nervous system and spreads over almost a l l areas of the brain, with concurrent f o c a l states of e x c i t a t i o n i n 2 in only limited neuronal c i r c u i t s . " In contrast, Sherrington and his associates found that inhibition of spinal reflexes was manifest as a contraction of agonist with a concomitant "reciprocal relaxation" of antagonist muscles. Furthermore, cessation of the stimulus resulted in "rebound contraction" of antagonist muscles according to the law of "successive spinal induction". "Reciprocal inhibition" of extensor muscles is blocked by administration of strychnine and this inhibition is followed by excitation ("rebound excitation") once the stimulus is terminated (Graham Brown, 1912; Beritoff, 1965). This inhibition would not seem to bear much resemblence to "general inhibition". There is a basic difference in the definition of the word "inhibition" as employed by the Pavlovian, in comparison with the Sherringtonian, school of thought. The Pavlovian definition of inhibition is less restrictive and refers to the loss or reduction of a response normally elici t e d by the stimulus. A requirement of a dynamic process which intervenes to block or reduce pre-existing activity i s expected by the Sherringtonian school. This difference in definition has lead to considerable confusion (Macintosh, 1975). There i s , however, a point of compromise between "general inhibition" and "reciprocal inhibition." Stimulation of the skin not only leads to an excitation of agonist and inhibition of antagonist muscles but also produces a background (general) inhibition of agonist muscles. Thus, flexors and extensors are inhibited (Graham Brown, 1912; Beritoff, 1965). Furthermore, i t is recognized that stimulation of various brain structures w i l l lead to general inhibition of spinal reflexes (Sherrington, 1906; 3 B e r i t o f f , 1965). Western neurophysiologists have of course extended Sherrington's concept of " r e c i p r o c a l i n h i b i t i o n . " Notable extensions are found i n the development of the new concepts of "feed-back" and "feed-forward" i n h i b i t i o n . In addition an e n t i r e l y new mechanism of i n h i b i t i o n has been recognized i n the form of a depolarization of pre-synaptic terminals (pre-synaptic i n h i b i t i o n ) . However, the basic Sherringtonian conceptuali-zation of the r o l e of i n h i b i t i o n has not been changed. For example, compare t h i s statement made by Eccles (1973:89): "I always think that i n h i b i t i o n i s a sculpturing process. The i n h i b i t i o n , as i t were c h i s e l s away at the d i f f u s e and rather amorphous mass of excitatory performance at every stage of synaptic r e l a y . " , with the e a r l i e r statement made by B e r i t o f f . Behaviourism and Habituation Science has two e s s e n t i a l a s p e c t s — t h e empirical and the explanatory. The empirical aspect i s p r i m a r i l y concerned with the f a c t s of the science as revealed by observation and experiment. The explanatory or t h e o r e t i c a l aspect, on the other hand, consists i n a serious attempt to understand the f a c t s of the science, and to integrate them into a coherent, i . e . , a l o g i c a l , system. (Hull, 1952:1). The study of behaviour has not been r e a d i l y adapted to the s c i e n t i f i c approach. It has suffered from a lack of empirical information and a plethora of unsubstantiated explanations. Central to the study of be-haviour i s the determination of how behaviour i s acquired or how learning takes place. Behaviourists have been forced to consider the a c q u i s i t i o n of behaviour i n terms of the organism's response to experimentally c o n t r o l l e d 4 stimuli but with l i t t l e or no knowledge of the internal workings of the organism. The organism is assumed, largely by necessity, to be a "black box". This has resulted in the adoption of the "empty organism" approach to the study of behaviour. The psychologist can by this means study behaviour by employing theoretical constructs to explain the relationships between the stimulus and the response. Strict empiricism, on the other hand, demands a precise knowledge of these internal mechanisms and how they interact. Thus, behaviourism must be amalgamated with modern neurophysiology. A key link has been and is being sought that would connect empirical neurophysiology with the rudimentary mechanism or mechanisms by which behaviour is acquired. In other words, can the recognizable electrophysio-logical properties of the nerve c e l l and the synapse account for the acquisition of behaviour? The principle of parsimony dictates that the isolation and characterization of a simple system of afferent and efferent connections should be correlated with a simple form of learning. Provided i t is accepted that no two sets of neurones need operate in quite the same manner the process of learning can be empirically defined. Attempts to draw a vinculum between the activity of the nervous system and the acquisition of behaviour, must include definitions of both the overt behaviour and the supposed underlying neuronal mechanism or mechanisms. Defining the process of learning might seem to be a t r i v i a l problem but finding a s c i e n t i f i c a l l y valid definition i s an extremely d i f f i c u l t conundrum. I n i t i a l l y , i t is perhaps of greatest value to use an operational definition. This can be accomplished by imposing limits upon a subjective 5 d e s c r i p t i o n . In t h i s manner, learning can be defined as a change i n per-formance (response) that occurs as a consequence of experience. The change i n performance must be retained for some period of time and i t must not be a t t r i b u t a b l e to a l t e r a t i o n s i n motivation nor to such phenomena as maturation or fatigue. Entelechy can play no r o l e i n t h i s d e f i n i t i o n . Subjective concepts of what constitutes learning can be consolidated by examining several of the t h e o r e t i c a l formulations of H u l l (1943, 1952). Behaviour was defined i n terms of a s e r i e s of postulates which described the mechanisms by which an organism adapts, behaviourally, to a s i g n i f i -can change i n the environment. For example, va r i a b l e s such as amplitude, latency, and p r o b a b i l i t y of response could be f u n c t i o n a l l y r e l a t e d to an i n t e r n a l "excitatory p o t e n t i a l " or "stimulus-response continuum." This "excitatory p o t e n t i a l " ( a c t u a l l y a t h e o r e t i c a l construct i t s e l f ) would be determined by stimulus factors o r i g i n a t i n g both from within and without the organism. The " f i r s t major automatic mechanism", for adapting to the environment i s the r e f l e x , with i t s presumed r i g i d stimulus-response connections. H u l l (1952) stated that no learning takes place at t h i s l e v e l . The stimulus-response pathway was considered to be a device whereby the organism could respond s t e r e o t y p i c a l l y to a r e l a t i v e l y homogenous groups of emergency or defensive s i t u a t i o n s . The latency of response would be small to meet the s u r v i v a l - t h r e a t i n g stimulus. The conditioned r e f l e x was envisaged as the "second major adaptive behaviour mechanism." The capacity to p r o f i t from past experience or simple learning would increase the number of possible response patterns, 6 although at the expense of response time. To a certain extent learning is defined by the exclusion of a number of phenomena, for example fatigue. The distinction between learning and fatigue would seem to be arbitrary. The physiological counterparts of fatigue would include receptor adaptation and muscular fatigue. These processes are clearly excluded because a consensus of opinion would insist that learning is a process of the central nervous system although this may not be true for invertebrates. The central nervous system can modify learning. For example, i t is recognized that volition or the degree of motivation can subvert or f a c i l i t a t e the process of learning. Empirically this distinction would seem to be one of degree of complexity. To be more specific, the learning process is assumed to be located within or at least functionally associated with a specific subset of behavioural units (each unit is equated with a single neurone for the sake of simplicity). The unconditioned stimulus (US) activates a subset of neurones which in turn activate a subset of efferent neurones that produce the unconditioned response (UR). Simi-l a r i l y , the conditioned stimulus (CS) activates a different subset of neurones which in turn activate yet another subset of efferent neurones that produce the conditioned response (CR). The pairing of the US and the CS activate a conjoint subset of efferent neurones that produce the UR and/or the CR (Figure 1). Other non-inclusive subsets of neurones, that are activated by extraneous stimuli (originating from the environment of the organism or from the internal milieu of the organism i t s e l f ) , can modify the activity of the previously mentioned conjoint subsets of 7 ( Figure 1. Subsets of neurones subserving conditioning and modification of conditioning. The c i r c l e s represent the subset of neurones activated by a p a r t i c u l a r stimulus (US, CS, and i n t e r n a l s t i m u l i such as motivation or v o l i t i o n , M). P a i r i n g of the US and CS act i v a t e s and i n t e r s e c t i o n of neurones (URACR) that evokes the CR. An i n t e r s e c t i o n of the neurones activated by the US, CS, and i n t e r n a l s t i m u l i (URHCRAM) can r e s u l t i n f a c i l i t a t i o n or i n h i b i t i o n of the CR. For the sake of s i m p l i c i t y the subsets subserving stimulus elements are not included i n t h i s diagram. 8 neurones. This accounts for the a b i l i t y of neurones subserving v o l i t i o n and motivation to a l t e r the process of learning. Of course there i s not always a s t r i c t demarcation between the conjoint and non-conjoint sub-sets with a resultant p o s s i b i l i t y of g e n e r a l i z a t i o n of the stimulus and/ or response. This type of statement i s made with the fundamental as-sumption that neurones and t h e i r connections form the basis of behaviour and that they are the v e h i c l e by which behaviour i s acquired. The parsimonious approach requires a d e f i n i t i o n of the simplest form of learning. The Pavlovian form of learning ( c l a s s i c a l conditioning paradigm) i s usually recognized as the primary event of learning i n contrast to a more complex Instrumental learning (operant conditioning paradigm). Stated i n b r i e f , conditioning r e f e r s to the transfer of the UR from the US to the CS. The response then evoked by the CS i s c a l l e d the CR. The CS i s "associated" with the US arid/or UR. However, other "non-associational" events may occur simultaneously with conditioning. Repetition of the US, with or without paired presentation of the CS, can evoke an UR of ever decreasing amplitude. This decrement of response following i t e r a t e d US presentation i s r e f e r r e d to as "adaptation" of the UR (Walker, 1967). An augmentation or s e n s i t i z a t i o n of the CR to r e p e t i t i o n of the CS can occur following the conditioning procedure. Neither "adapt-a t i o n " nor s e n s i t i z a t i o n appear to be contingent upon p a i r i n g of the US and CS. The i n t e n s i t y or s i g n i f i c a n c e of the stimulus does have a bearing upon the mutability- of response amplitude. In p a r t i c u l a r , response s e n s i t i z a t i o n i s associated with high i n t e n s i t y stimulation (Hinde, 1966). Presentation of the US can produce a general a c t i v a t i o n of the organism, 9 i n response to any extraneous stimulus, with the r e s u l t that a response which resembles the CR might be evoked following the i n i t i a l presentation of the CS. "Pseudoconditioning" r e f e r s to t h i s generalized s e n s i t i z a t i o n during the conditioning procedure. "Adaptation" and s e n s i t i z a t i o n are considered to be confounding v a r i a b l e s within the conditioning paradigm. It i s c l e a r that these non-a s s o c i a t i o n a l " components are not dependent upon the process of conditioning. "Adaptation" i s s i m i l a r , i f not i d e n t i c a l , to behavioural habituation (Kandel, 1967) and behavioural habituation can be defined as an attenuation of response parameters (amplitude, response duration, p r o b a b i l i t y of response, etc.) following r e p e t i t i o n of the stimulus (Harris, 1943). Peripheral events such as muscular fatigue and receptor adaptation are also excluded from t h i s d e f i n i t i o n as they are from the d e f i n i t i o n of learning. Response s e n s i t i z a t i o n may be defined as an increase i n responsive-ness with r e p e t i t i o n of the stimulus (Groves and Thompson, 1970) and i t i s also considered to be a property of the c e n t r a l nervous system. The c o r r e l a t i o n of s e n s i t i z a t i o n with strong or aversive s t i m u l i has lead to the postulate that s e n s i t i z a t i o n represents a modification of the " c e n t r a l excitatory state" (Sherrington, 1898) or the "excitatory p o t e n t i a l " (Hull, 1952). By t h i s i t i s meant that the presentation of an intense (even p a i n f u l ) stimulus can evoke an a l t e r a t i o n i n the presumed tonic a c t i v i t y of the c e n t r a l nervous system. This influence i s often not s p e c i f i c to the s e n s i t i z i n g stimulus (Hinde, 1966) and a considerable degree of stimulus g e n e r a l i z a t i o n can be expected (Thompson, et a l , 1973). 10 The terminology " c e n t r a l excitatory s t a t e " was used by Sherrington (1906) to describe subliminal f r i n g e phenomena of transient duration but the concept has been extended to imply a " c e n t r a l motivational or drive s t a t e " (Lashley, 1938; S t e l l a r , 1956) or a " c e n t r a l arousal s t a t e " (Lindsley, 1960; Duffy, 1962). Thus, a general a c t i v a t i o n of the organism might predispose the increment of response. S e n s i t i z a t i o n may be represented within the c l a s s i c a l conditioning paradigm (Figure 2). If e i t h e r the US or CS i s of s u f f i c i e n t i n t e n s i t y i t can a l t e r the l e v e l of e x c i t a b i l i t y i n the " s t a t e " system. The con-j o i n t subset of neurones subserving the a s s o c i a t i o n of the US and CS would be f a c i l i t a t e d by an increase i n general e x c i t a b i l i t y through a c t i v a t i o n of an i n c l u s i v e set of neurones (UR CR GR). This f a c i l i t a t i o n would also extend to the generalized stimulus (GG) with a resultant increase i n the generalized response (GR). This concept of an a l t e r i n g l e v e l of e x c i t a b i l i t y (a decrement of e x c i t a b i l i t y ) has also been used to explain behavioural habituation (Jasper, et a l . , 1958). The study of behavioural habituation has brought about a dichotomy of opinion as to which t h e o r e t i c a l explanation best describes the mechanism underlying habituation. These theories can be divided into "dynamic" theories and those proposing some form of progressively r e -ducing " c e n t r a l e f f i c i e n c y " . Proponents of the l a t t e r type of theory suggest that the response occurs l e s s strongly simply because the stimulus no longer possesses the capacity to evoke the response as a r e s u l t of a reduction i n the e f f i c i e n c y of r e f l e x transmission. A l -t e r n a t i v e l y , the stimulus might no longer possess the capacity to evoke 11 * G R » C R Figure 2. Subsets of neurones subserving conditioning and the "state" system. In addition to the subsets activated by the US and CS, a set of "state" neurones is defined (GR). A general stimulus (GS) can activate the "state" neurones which can lead to evocation of a GR which may be different or the same as the CR but that cannot be 'considered a CR. The CS may also cause an activation of the subset GR resulting i n a CR of greater amplitude or of increasing amplitude that is independent of con-ditioning (sensitization). This figure differs from figure 1 in that the subsets US and CS are exclusive of the subset M but are inclusive of the subset GR. 12 the response because some a c t i v e process intervenes. There i s a "dynamic" intervention imposed upon the stimulus-response pathway. The "c e n t r a l e f f i c i e n c y " explanation i s parsimonious whereas the "dynamic" explanation requires the a d d i t i o n a l postulation of an active formof d i s - f a c i l i t a t i o n or i n h i b i t i o n . Thus, an excitatory pathway from stimulus to response i s s u f f i c i e n t f o r the de s c r i p t i o n of a theory'of " c e n t r a l e f f i c i e n c y " but "dynamic" theories must include either a decrease i n tonic f a c i l i t a t i o n of the stimulus-response, pathway or i t must include yet another pathway i n which a progressive i n h i b i t i o n of the stimulus-response pathway can occur. There are numerous examples of "dynamic" theories. The concepts of "conditioned i n h i b i t i o n " (Sokolov, 1965), "reactive i n h i b i t i o n " ( H u l l , 1952), and "afferent neuronal habituation" (Hernandez-Peon, 1960) are t y p i c a l of such theories. These theories lack a cr e d i b l e degree of supportive neuro-p h y s i o l o g i c a l evidence. However, t h i s i s not so with regard to "centr a l e f f i c i e n c y " theories. Many of these theories have been discussed c r i t i c a l l y by Groves and Thompson (1970) and Thompson, et a l . - (1973).; Paramount among the " c e n t r a l e f f i c i e n c y " theories i s the "Dual-Process" theory of habituation (Groves and Thompson, 1970; Thompson, et a l . , 1973) which i s the most prepossessing of the various theories of habituation. This theory i s based upon the culmination of s c i e n t i f i c evidence ranging from studies of the a c t i v i t y of s i n g l e neurones i n the nervous system of simple invertebrates to complex behavioural habituation i n the vertebrate. A b r i e f r a t i o c i n a t i o n of t h i s theory can reduce the problem of defining habituation and s e n s i t i z a t i o n . The "Dual-Process" theory i s stated by Thompson, et a l . (1973:240-241) as follows: 13 1. Every stimulus that evokes a behavioral response has two properties: I t e l i c i t s a response and influences the "stat e " of the organism. The S-R pathway i s the most d i r e c t route through the c e n t r a l nervous system from stimulus to dis c r e t e motor response, however c i r c u i t o u s , redundant, and v a r i a b l e that pathway may be and regardless of whether the response i s learned or unlearned. State i s the general lev^el of e x c i t a t i o n , arousal, a c t i v a t i o n , tendency to respond, etc., of the organism. 2. Repetition of an e f f e c t i v e stimulus r e s u l t s i n an in f e r r e d decremental process i n the S-R pathway which may be termed habituation. (a) During habituation t r a i n i n g , habituation develops exponentially and reaches an asymptotic l e v e l . (b) The rate of development and degree of r e l a t i v e habituation are d i r e c t l y r e l a t e d to stimulus frequency and inversely r e l a t e d to stimulus i n t e n s i t y . Frequency has strong e f f e c t and i n t e n s i t y a weak e f f e c t on habituation. (c) Upon cessation of the habituating stimulus, habituation decays spontaneously (spontaneous recovery). (d) Repeated seri e s of habituation t r a i n i n g and spontaneous recovery r e s u l t i n progressively more habituation. (e) Response habituation w i l l e x h i b i t g e n e r a l i z a t i o n to a test stimulus to the extent that the habituating and test s t i m u l i a c t i v a t e common "habituation" elements. 3. Presentation of an e f f e c t i v e stimulus r e s u l t s i n an in f e r r e d incremental process i n a state of e x c i t a t i o n or tendency to respond of the organism which may be termed s e n s i t i z a t i o n . (a) The process of s e n s i t i z a t i o n occurs i n state system(s) but not i n S-R pathways. (b) During habituation t r a i n i n g , s e n s i t i z a t i o n f i r s t grows and then decays. (c) The amount and duration of s e n s i t i z a t i o n are d i r e c t l y r e l a t e d to stimulus i n t e n s i t y . At higher i n t e n s i t i e s , s e n s i t i z a t i o n i s d i r e c t l y r e l a t e d to stimulus frequency. At low i n t e n s i t i e s there may be l i t t l e or no s e n s i t i z a t i o n . (d) Upon cessation of a stimulus that has produced s e n s i t i z a t i o n , s e n s i t i z a t i o n decays spontaneously. (e) Repeated presentations of a s e n s i t i z i n g stimulus r e s u l t i n progressively l e s s s e n s i t i z a t i o n , that i s , s e n s i -t i z a t i o n decreases or habituates. (f) Response s e n s i t i z a t i o n w i l l e x h i b it g e n e r a l i z a t i o n to a test stimulus to the extent that the s e n s i t i z i n g and test s t i m u l i a c t i v a t e common s e n s i t i z a t i o n elements. (g) Dishabituation, the increase i n a habituated response following presentation of a stimulus other than the habituation stimulus, i s simply an instance of s e n s i t i z a t i o n . 14 (h) Under c e r t a i n circumstances (strong s t i m u l i pre-sented r e g u l a r l y at r e l a t i v e l y slow rate) temporal conditioning of s e n s i t i z a t i o n of state may occur. 4. The two processes of habituation and s e n s i t i z a t i o n occur and develop independently of one another but i n t e r a c t to y i e l d the f i n a l response output function. Habituation may be primarily "phasic" i n i t s a c t i o n on response output, while s e n s i t i z a t i o n may be p r i m a r i l y " t o n i c " Spencer, et a l . (1966a) have defined nine "parametric" c h a r a c t e r i s t i c s of habituation. "Parametric" r e f e r s to various a l t e r a t i o n s of the stimulus parameters used to derive the "parametric" c h a r a c t e r i s t i c s of habituation. Six of these c h a r a c t e r i s t i c s are found i n items 2.a to 2.e, i n c l u s i v e l y . Dis-habituation i s the eighth. The f i n a l c h a r a c t e r i s t i c i s summarized as follows: "The e f f e c t s of habituation t r a i n i n g may proceed beyond the zero or asymptotic l e v e l . " A d d i t i o n a l stimulation a f t e r the response has habituated to the asymptotic l e v e l w i l l r e s u l t i n slower recovery. The question remains as to whether or not habituation and/or s e n s i -t i z a t i o n conform to current c r i t e r i a used to define learning. Thorpe (1963) expressed the opinion that habituation was a form of learning and habituation has been used as a learning paradigm i n the study of the behaviour of neurones i n the invertebrate c e n t r a l nervous system (Kandel and Spencer, 1968). This acceptance of habituation as a form of learning has not been u n i v e r s a l . For example, M i l l e r (1967:644) sets four c r i t e r i a for the acceptance of any behavioural process as an authentic learning event. Learning was defined i n a r e s t r i c t i v e manner: "Learning i s a r e l a t i v e l y permanent increase i n the response strength that i s based upon previous reinforcement and that can be made s p e c i f i c to one out of two or more a r b i t r a r i l y selected stimulus s i t u a t i o n s . " 15 "Relative permanence," M i l l e r defines as a matter of days or months as opposed to minutes or hours. This may exclude s e n s i t i z a t i o n due to i t s short duration (Pearson, 1974) but there are reports that habituation may l a s t f o r long periods (days and months) (Nesmeinanova, 1957; Kozak, et a l . , 1962). Unfortunately the time sequences of a l t e r e d a c t i v i t y i n the nervous system of the vertebrate are usually measured ( e l e c t r o -p h y s i o l o g i c a l l y ) i n orders of magnitude well below those s t i p u l a t e d by t h i s requirement. M i l l e r ' s reference to an "increase i n response strength" and "selected stimulus s i t u a t i o n s " implies several c r i t i c a l aspects of learning. These factors can be i l l u s t r a t e d conceptually i n terms of Hull's "stimulus-response continuum." For example, i f i t i s postulated that there e x i s t s both a stimulus and response continuum for each response and stimulus, r e s p e c t i v e l y , then the process of learning has occurred when a stimulus-response contiguity has been established between the stimulus and the response continuum and between the response and the stimulus continuum (Figure 3). To be more s p e c i f i c , each stimulus has the capacity to evoke a number of response p o t e n t i a l i t i e s ( p o s s i b i l i t y of evoking a . response as opposed to a c t u a l l y evoking a response) on the response con-tinuum and each p o t e n t i a l i t y has an equal p r o b a b i l i t y of being 1strengthened by an ass o c i a t i o n with the stimulus. An " a s s o c i a t i o n " w i l l have been established when the p r o b a b i l i t y of producing any p a r t i c u l a r response p o t e n t i a l i t y on the continuum becomes much greater than that of any other response p o t e n t i a l i t y . In a s i m i l a r manner, a s p e c i f i c response may be produced from a number of stimulus p o t e n t i a l i t i e s on a stimulus continuum. Response Continuum Stimulus Continuum Figure 3. Response (above) and stimulus continua (below) and the development of a stimulus-response contiguity. A stimulus can produce a number of response potentialities (R^...?^) on a response continuum. Prior to conditioning the probability that any "association" w i l l be developed between S and a particular response potentiality (R2) is the same as that of any other potentiality. After conditioning the probability that a S w i l l produce R2 is much greater than that of any other potentiality. A similar arguement holds for a specific response and i t s continuum of stimulus potentialities. 17 When the p r o b a b i l i t y that one stimulus p o t e n t i a l i t y w i l l e l i c i t the response i s greater than that of the others an " a s s o c i a t i o n " w i l l have been established (Figure 3). A super-imposition of the stimulus and r e -sponse continua and an establishment of the stimulus-response c o n t i g u i t y represents the process of learning. The crux of t h i s concept i s the enforcement of an " a s s o c i a t i o n " of the stimulus to the response and/or the response to the stimulus, i n a s p e c i f i c manner. Stimulus and response g e n e r a l i z a t i o n can be defined as a f a i l u r e to develop a s p e c i f i c stimulus-response contiguity. Furthermore, M i l l e r requires that the stimulus-response contiguity take the form of either the c l a s s i c a l or operant conditioning paradigms. M i l l e r excludes habituation, s e n s i t i z a t i o n , and conditioning i n simple organisms from his d e f i n i t i o n . However,- M i l l e r ' s d e f i n i t i o n i s s t i l l based upon the "empty organism" approach with imposed subjective r e s t r i c t i o n s . With no knowledge of the intervening v a r i a b l e s which e s t a b l i s h the stimulus-response contiguity i t i s perhaps too stringent i n i t s exclusion of the phenomena of habituation and s e n s i t i z a t i o n . If the objective i s to analyse the p h y s i o l o g i c a l c o r r e l a t e s of behaviour i t i s more l o g i c a l to adopt a d e f i n i t i o n which allows a component by component examination of the learning process. E i s e n s t e i n (1967:654) has presented an a l t e r n a t i v e d e f i n i t i o n of learning which he states as follows: A system i s said to demonstrate learning when i t s output .(response) to a given test input (stimulus) i s a function of the t o t a l previous input-output pattern of which the t e s t input was a part. That i s , a system can be said to have learned i f i t s output to a given test input i s a 18 function of the s p e c i f i c input-output pattern to which i t has been exposed. Several aspects are often common to the learning process. One of these i s r e p e t i t i o n of the stimulus i n order to re i n f o r c e the as s o c i a t i o n be-tween the stimulus and the response. C r i t i c a l to t h i s i s the temporal a s s o c i a t i o n of more than one set of stimulus-response elements. Habituation and s e n s i t i z a t i o n often occur during the conditioning procedure. They share a r e l a t i o n s h i p with learning i n the sense that the a c q u i s i t i o n of any behaviour requires a r e p e t i t i v e establishment of the stimulus-response pattern (at l e a s t for simple forms of learning) and i n that the response shows a temporal r e l a t i o n s h i p to previous stimulation. They do not nec e s s a r i l y demonstrate an as s o c i a t i o n between sets of stimulus-response elements i n the same way that the US-UR i s associated to the CS-CR. The stereotyped r e f l e x envisaged by H u l l ( " f i r s t major automatic mechanism") can be considered a constant response behaviour. It f a i l s to account for habituation and s e n s i t i z a t i o n of r e f l e x e s , where the r e -sponse i s no longer constant i n quantity, but var i e s as a function of experience (repeated stimulation). However, habituation and s e n s i t i z a t i o n do not a t t a i n the l e v e l of the "second major adaptive behaviour mechanism" or the conditioned r e f l e x i n that the change of behaviour i s not q u a l i -t a t i v e . As a consequence, habituation and s e n s i t i z a t i o n appear to f i t half-way between Hull's " f i r s t major automatic mechanism" and h i s "second major adaptive behaviour mechanism." If learning requires an association between more than one set of stimulus-response elements i t i s doubtful that habituation and s e n s i t i z a t i o n are forms of learning. Perhaps they should be considered sub-learning phenomena. 19 SECTION II Integration i n the Nervous System Acceptance of the "neurone doctrine" has placed ever increasing emphasis upon the neurone as the fun c t i o n a l unit of behaviour. The : cen t r a l problem has been to determine how the coding and storage of information can be represented or circumscribed by such an anatomical structure. Attempts to answer t h i s question have taken several d i r e c t i o n s . One approach has been to a t t r i b u t e information storage to a modification i n e l e c t r o t o n i c p o t e n t i a l s which are integrated across large complex c l u s t e r s or f u n c t i o n a l matrices of c e l l s . Each element of the matrix was assumed to be an i n d i v i d u a l neurone. This conceptual approach had i t s o r i g i n s i n Gestalt psychology, when cognition was envisaged as a coding of b i o - e l e c t r i c f i e l d s , a sort of e l e c t r o n i c hologram of information storage. This hypothesis appeared to be confirmed by the experiments of Lashley. Lesions of the neocortex did not cause s p e c i f i c behavioural d e f i c i t s , but rather, the volume of cortex consumed i n the l e s i o n seemed to be the s i g n i f i c a n t f a c t o r . As a consequence, Lashley (1929:179) r e -jected the hypothesis that behaviour could be represented by s p e c i f i c anatomical l o c i within the neocortex: "Integration cannot be expressed i n terms of connections between s p e c i f i c neurones.... the mechanisms of int e g r a t i o n are to be sought i n the dynamic r e l a t i o n s h i p s among the parts of the nervous system rather than i n d e t a i l s of s t r u c t u r a l d i f f e r e n t i a t i o n . " A lack of knowledge of the topography of the sensory and motor cortex at 20 that time, coupled with p r i m i t i v e s u r g i c a l procedures, are assumed to have l e d to Lashley's spurious conclusion (Kandel and Spencer, 1968). Although the experiments performed by Lashley cannot be used to j u s t i f y h i s conclusion the hypothesis i s not ne c e s s a r i l y i n v a l i d . None the l e s s , Lashley's impact upon the f i e l d of p h y s i o l o g i c a l psychology was s i g n i f i c a n t and many psychologists questioned the p r a c t i c a l i t y of attempts to c o r r e l a t e behaviour with neural structures. Recent evidence has provided some support f o r the concept that neurones might influence each other e l e c t r o t o n i c a l l y . Any sing l e neurone might influence another neurone which i s i n close apposition, e l e c t r o t o n i c a l l y , e i t h e r by means of an e l e c t r o t o n i c synapse or by generating an extra-c e l l u l a r f i e l d p o t e n t i a l of s u f f i c i e n t i n t e n s i t y to a l t e r the a c t i v i t y of the adjacent neurone. The existence of e l e c t r o t o n i c synapses i s now recognized i n invertebrate nervous systems and there i s some evidence that such synapses may also exist i n the c e n t r a l nervous system of the mammal. This type of e l e c t r o t o n i c coupling between neurones seems aptly suited to the synchronization of groups of neurones. I t i s d i f f i c u l t to int e r p r e t such a mechanism of neuronal i n t e r a c t i o n i n terms of complex information processing. However, i t may prove to be a s i g n i f i c a n t means of t r i g g e r i n g c e r t a i n stereotyped behaviours. For example, i n T r i t o n i a , swimming behaviour i s triggered by a group of neurones e l e c t r o t o n i c a l l y coupled together (Getty and Willows, 1974). The recording of e x t r a c e l l u l a r p o t e n t i a l s i n the nervous system has focused, for the most part, upon transient events associated with the propagation of po t e n t i a l s i n the axon, c e l l body, dendrite, or across the synapse. Although i t has not been fashionable to do so, other 21 long term a l t e r a t i o n s i n e x t r a c e l l u l a r f i e l d p o t e n t i a l s have been studied. Some of the e a r l i e s t attempts to record e l e c t r i c a l events i n the nervous system examined f i e l d p o t e n t i a l s (Brazier, 1963; Rowland, 1968). Somjen (1973) uses the term "sustained p o t e n t i a l s " (D. C , steady potentials) to describe these long term p o t e n t i a l s which are recorded from the cortex and sp i n a l cord with respect to a common (ground) electrode. "Sustained p o t e n t i a l s " have also been correlated with behavioural events such as the expectation of response (contingent negative v a r i a t i o n ; Walter, 1968) and the reinforcement of con d i t i o n a l s t i m u l i (Morrell, 1961; Rowland and Goldstone, 1963). There i s increasing evidence that "sustained p o t e n t i a l s " recorded i n the s p i n a l cord, as a consequence of repeated afferent stimulation, are the r e s u l t of a progressive increase i n the concentration of extra-c e l l u l a r potassium (Somjen and Lothman, 1974). Neither "sustained p o t e n t i a l s " nor the simultaneous elevation i n potassium concentration are n e c e s s a r i l y related to dorsal root p o t e n t i a l s (DRP's) (Kriz, et a l . , 1974; Somjen and Lothman, 1974). The duration of "sustained p o t e n t i a l s " i s f a r greater than that of the conventaionally recorded e l e c t r i c a l events such as primary afferent depolarization and hyperpolarization. Increases i n the e x t r a c e l l u l a r concentration of potassium have been correlated with spreading depression (Lothman, et a l . , 1975) which suggests that increases i n potassium concentration might i n h i b i t neuronal d i s -charge (depolarization block) and such a mechanism might account f o r long term i n h i b i t i o n of s p i n a l reflexes (Abrahams, 1974). However, i t i s not yet.kiown i f the changes i n l o c a l e x t r a c e l l u l a r p o t e n t i a l s are of a 22 s u f f i c i e n t magnitude to modulate neuronal a c t i v i t y . "Sustained p o t e n t i a l s " are e l i c i t e d with the discharge of i n h i b i t o r y as w e l l as excitat o r y interneurones suggesting that changes i n potassium concentration may be secondary to the release of i n t r a c e l l u l a r potassium during neuronal discharge (Somjen, 1973).. Changes i n the e x t r a c e l l u l a r concentration have also been reported to a l t e r synaptic transmission (Cooke and Quastel, 1973). In opposition to the hypothesis that behaviour i s represented as b i o e l e c t r o n i c f i e l d s has been the conceptualization of the neurone, and s p e c i f i c a l l y the synapse, as the f u n c t i o n a l unit of behaviour (Eccles, 1964; 1973; Roberts, 1966; Kosower, 1972). The synapse might be c a l l e d the unit of microbehaviour. The quest has been to discover how information processing can occur i n the ce n t r a l nervous system as the r e s u l t of a l t e r a t i o n s i n the function of synapses (synaptic p l a s t i c i t y ) and i n the i n t e r a c t i o n between many synapses. The synapse has not been accessible to d i r e c t examination u n t i l quite recently. T r a d i t i o n a l l y the study of the synapse has been i n d i r e c t and has r e l i e d upon the examination of r e f l e x e s . The concept of the r e f l e x not only l i n k s stereotyped movement to the underlying micro-behaviour of the synapse, but the compounding of reflexes serves to connect physiology to the study of behaviour. Sherrington (1948) c r e d i t s Thomas W i l l i s (1664) as the o r i g i n a t o r of the concept of the r e f l e x . However, Descartes (1677) provided the f i r s t comprehensive d e s c r i p t i o n of the r e f l e x as a means of explaining the agonist-antagonist r e l a t i o n s h i p of the limb muscles. The r e a l i z a t i o n that the behaviour of the simple 23 r e f l e x was the r e s u l t of the a c t i v i t y of synapses must be credited to Sherrington even though he did not have a s p e c i f i c knowledge of the chemical and e l e c t r i c a l events that occur at the synapse. At the time that Sherrington was determining many of the properties of the synapse others were s c r u t i n i z i n g the r e l a t i o n s h i p s between reflexes and complex behaviours (Watson, 1924; Pavlov, 1927). Behaviour has been described as a complexing of simple ref l e x e s but with an important addendum (Pavlov, 1927:14): "The e s s e n t i a l feature of the highest a c t i v i t y of the c e n t r a l nervous system.... consists not i n the f a c t that innumerable s i n g l e s t i m u l i do i n i t i a t e r e f l e x reactions i n the animal, but i n the f a c t that under d i f f e r e n t conditions these same s t i m u l i may i n i t i a t e quite d i f f e r e n t r e f l e x reactions; and conversely the same reaction may be i n i t i a t e d . " The compounding of r e f l e x arcs does not exclude nor does i t explain the development of contiguity between various response-stimulus elements. To be more s p e c i f i c , i f two reflexes are compounded the resultant behaviour i s s y n e r g i s t i c a l l y r e l a t e d to the summated arcs. The elements of the c e n t r a l nervous system p a r t i c i p a t e i n numerous dynamic processes which might be temporarily or permanently rearranged i n order to code and store information. Current knowledge of neuro-p h y s i o l o g i c a l events indicates a number of possible f o c i where modulation of neuronal discharge might take place. These f o c i and the events that occur at these f o c i are summarized below: Afferents 1) blockage 2) d e p o l a r i z a t i o n 3) hyperpolarization ( i n h i b i t i o n or excitation) 24 Synaptic termination 1) location of the terminal with respect to the post-synaptic element (dendrite, soma, axon, etc.) 2) Anatomical pla s t i c i t y a) distance from pre- to post-synaptic membrane (occurrence of synaptic spines) b) number of synaptic contacts and location of contacts upon the target c e l l . c) size and locality of post-synaptic receptive regions d) number and location of trigger zones 3) Chemical transmission a) modulation of synthesis, mobilization, availability for release, transfer to post-synaptic membrane, uptake, enzymatic destruction b) transmitter causes inhibition, excitation, and/or both in the post-synaptic c e l l c) sensitivity of post-synaptic receptors to the transmitter 4) El e c t r i c a l transmission a) pre-synaptic membrane properties b) trans-cellular impedance c) post-synaptic membrane properties 5) Propagation and i n i t i a t i o n a) dendritic (active or passive transmission) b) endogenous pacemaker activity (underlying metabolic processes) c) repetitive discharge of exogenous origin (after-discharge, reverberating circuits) 25 The i n i t i a l event i s the a r r i v a l of the afferent v o l l e y to the b r a i n or s p i n a l cord. Information i s frequency coded within t h i s v o l l e y and d i f f e r e n t groups of afferents carry s p e c i f i c information. The " a l l or none" property of the a f f e r e n t nerves severely l i m i t s the informational content of the v o l l e y . Within the cord the afferent f i b r e s may be subject to blockade (Wall and Johnson, 1958) or to c o l l i s i o n and de-a c t i v a t i o n by antidromically t r a v e l l i n g p o t e n t i a l s (Wall and Gutnick, 1974). The structure and geometric arrangement (fine branching c o l l a t e r a l s ) of the terminal regions of the afferents may contribute f u n c t i o n a l properties to the terminal region (Lloyd, 1971). The terminals are subject to either depolarization (primary a f f e r e n t depolarization (PAD) or to hyperpolarization (primary afferent hyperpolarization . (PAH), and/or both (Hodge, 1972) with concomitant suppression (pre-synaptic i n h i b i t i o n ) or f a c i l i t a t i o n (pre-synaptic f a c i l i t a t i o n ) of a f f e r e n t transmission (Eccles, 1964; Hodge, 1972; Mendell, 1972; Schmidt, 1973). Repetitive interneuronal discharge may account for these changes i n primary afferent e x c i t a b i l i t y (Lloyd, 1971; Schmidt, 1973; Yu and Avery, 19,74) although primary afferent c o l l a t e r a l s may themselves contribute (Wall, 1958; Wall and Johnson, 1958; Lloyd, 1971). The duration of PAD and PAH (200 msec.) i s f a r too b r i e f to account f o r even temporary information storage. However, i t i s becoming apparent that PAD and PAH are a consequence of temporary modulation of a tonic l e v e l of primary afferent depolarization and much longer periods of a l t e r e d e x c i t a b i l i t y may occur with r e p e t i t i v e stimulation (Schmidt, 1973). The release of potassium by discharging afferents has been postulated 26 as a mechanism whereby one afferent terminal can depolarize another. An accumulation of e x t r a c e l l u l a r potassium might account for evoked DRP's (Barron and Mathews, 1938; Krnjevic and Morris, 1972; Vycklicky, et a l . , 1972); however, the time course and magnitude of changes i n the concen-t r a t i o n of e x t r a c e l l u l a r potassium does not c o r r e l a t e with the occurence of DRP's ( L i e b l , et a l . , 1973; K r i z , et a l . , 1974; Somjen and Lothman, 1974). The affe r e n t synapses may terminate upon various elements within the s p i n a l grey matter. The effectiveness of any p a r t i c u l a r synapse w i l l depend upon the l o c a t i o n of the terminal with respect to the post-synaptic neurone ( i . e . , d e n d r i t i c versus somatic) (Jack, et a l . , 1971; M e r r i l l and Wall, 1972). Complex synaptic arrangements have been ob-served such as the glomerular complex found i n the substantia gelatinosa and cl a r k ' s column (Rethelyi and Szentagothai, 1969; Rethelyi, 1970). Afferent terminals p a r t i c i p a t e i n these complexes and they may form the anatomical counterpart for pre-synaptic inhibition(Schmidt, 1973). While gross s t r u c t u r a l r e l a t i o n s h i p s between afferent nerves and target neurones are considered to be g e n e t i c a l l y determined (Sperry, 1951, 1965; Wiesel and Hubel, 1965) more subtle anatomical changes are possible as a consequence of f u n c t i o n a l "use" or "disuse" of pathways. The t o t a l number of synapses, the involvement of synaptic spines, the width of the synaptic c l e f t , and other properties of synapses, may be s t r u c t u r a l l y a l t e r e d by a c t i v i t y or lack of a c t i v i t y i n the pathway (Cragg, 1972, 1974; Lund and Lund, 1972; Eccles, 1973). The s i g n i f i c a n c e of t h i s form of p l a s t i c i t y as a means of s t o r i n g information i s yet to be determined. 27 Informational events occurring at synapses consist of coupled chemical and e l e c t r i c a l processes. Modulation of transmission might occur with a change i n the biochemical reactions of transmission, i n the associated e l e c t r i c a l events, or i n the membrane properties of the pre-or post-synaptic neurones. The a b i l i t y of the chemical synapse to act as an information coder and capacitor may be extensive. The transmitter may hyperpolarize, depolarize or prevent post-synaptic depolarization by increasing conductance with a resultant i n h i b i t i o n or e x c i t a t i o n of the a c t i v i t y of the post-synaptic neurone. 28 SECTION III Synaptic Plasticity (functional) The synapse has long been considered the site of the genesis of habituation and sensitization of spinal reflexes. Sherrington (1898: 140; Sherrington and Sowtown, 1915) described both an i n i t i a l sensi-tization and a later habituation of spinal reflexes: "In most cases a few repetitions tires out the reflex reaction, after increasing some-what for a few repetitions at the beginning of the examination, they begin to fade out, and do so unless a rest i s allowed." Sherrington (1906:218) had no direct experimental access to the synapses mediating spinal reflexes but he postulated, on the basis of indirect evidence, that the origin of habituation was a decrease in the functional efficacy of these synapses: "..'..the seat of fatigue [habituation] is intraspinal and central more than peripheral and cutaneous; and that i t affects the afferent part of the arc inside the cord, probably at the f i r s t synapse." A number of characteristics of habituation (see p. 14, this thesis) were also recognized by Sherrington (1906:218-219): 1) spontaneous recovery The local fatigue of a spinal reflex seems to be recovered from with remarkable speed, to judge by observations on the reflexes of the spinal dog. A few seconds' remission of the stimulus suffices for a marked though incomplete restoration of the reaction. 2) influence of stimulus intensity In my experience, these spinal reflexes fade out sooner under a 29 weak stimulus than under a strong one. 3) stimulus generalization When the scratch r e f l e x e l i c i t e d from a spot of skin i s fatigued, the fatigue holds for that spot but does not implicate the r e f l e x as obtained from the surrounding skin....When the spot stimulated, i s close to the one t i r e d out, the r e f l e x shows some degree of fatigue, but not that degree obtained for the o r i g i n a l spot. The synapses of the c e n t r a l nervous system of the vertebrate are not nearly as accessible to study as those of the c e n t r a l nervous system of invertebrates. As a consequence, synaptic p l a s t i c i t y has been extensively studied i n invertebrates. It can not be stated with c e r t a i n t y that synaptic p l a s t i c i t y occurs by the same mechanism i n vertebrates as i n -vertebrates nor can i t be stated that habituation and s e n s i t i z a t i o n are a consequence of synaptic p l a s t i c i t y . However, habituation and s e n s i t i -zation share many of the c h a r a c t e r i s t i c s of synaptic p l a s t i c i t y and many phenomena demonstrated by both the synapses of vertebrates (peripheral and central) and invertebrates can be placed under the ru b r i c of synaptic p l a s t i c i t y . Synaptic p l a s t i c i t y r e f e r s to an a l t e r a t i o n i n the e f f i c a c y of transmission and a large number of phenomena such as "low frequency depression", post-tetanic potentiation (PTP) and "frequency f a c i l i t a t i o n " may be included i n th i s d e f i n i t i o n . A discussion of synaptic p l a s t i c i t y and habituation i n invertebrates i s j u s t i f i e d i f f o r no other reason than because these phenomena are proposed as l i k e l y mechanisms for habituation and s e n s i t i z a t i o n of s p i n a l r e f l e x e s (Sherrington, 1906; Groves and Thompson, 1970; Thompson, et a l , 1973; F a r e l , et a l . , 1973). Considerable evidence has accrued which associates synaptic p l a s t i c i t y 30 with a number of phenomena which are at l e a s t s u p e r f i c i a l l y s i m i l a r to habituation and s e n s i t i z a t i o n . I t has been found that r e p e t i t i o n of the stimulus can lead e i t h e r to a subsequent post-synaptic (or post- . junctional) p o t e n t i a l of greater or lesser magnitude and duration when compared to the i n i t i a l evoked post-synaptic p o t e n t i a l . The only c e n t r a l synapses r e a d i l y a v a i l a b l e f o r study have been those i n the invertebrates, which demonstrate very s i m i l a r phenomena. If two s t i m u l i are applied with only a b r i e f (several msec.) pause between them the s i z e of the recorded excitatory junction p o t e n t i a l (EJP, i n the case of the neuromuscular junction) or the excitatory post-synaptic p o t e n t i a l (EPSP, c e n t r a l synapses of invertebrates and the post-synaptic response of p e r i p h e r a l gangliq) i n response to the second stimulus w i l l be greater i n magnitude than that evoked by the f i r s t . An increase i n the amount of transmitter released from the pre-synaptic terminal, by the second stimulus, i s usually associated with t h i s "short term f a c i l i t a t i o n " (Del C a s t i l l o and Katz, 1954; Hubbard, et a l . , 1971; Schlapfer, et a l . , 1974). Repetition of the stimulus, at rates below those used to produce "short term f a c i l i t a t i o n " , i s also capable of r e -leasing subsequently larger amounts of transmitter r e s u l t i n g i n "frequency f a c i l i t a t i o n " or "frequency p o t e n t i a t i o n " (Schlapfer, et a l . , 1974). Repetition of the stimulus, at rates below those used to produce "short term f a c i l i t a t i o n " , i s also capable of r e l e a s i n g subsequently larger amounts of transmitter r e s u l t i n g i n "frequency f a c i l i t a t i o n " or "frequency p o t e n t i a t i o n " (Schlapfer, et a l . , 1974) of the synapse examined (Del C a s t i l l o and Katz, 1954; L i l e y , 1956; Dudel and K u f f l e r , 1961; 31 Martin and P i l a r , 1964; M i l e d i and S l a t e r , 1966; Schlapfer, et a l . , 1974). At even lower frequencies of stimulation the s i z e of the post-synaptic p o t e n t i a l may a c t u a l l y be l e s s than that observed a f t e r the i n i t i a l s timulation. This e f f e c t i s c a l l e d "low frequency depression" of the synapse (Del C a s t i l l o and Katz, 1954; L i l e y , 1956; Horn and Wright, 1970; Dudel and K u f f l e r , 1971; Schlapfer, et a l . , 1974). I t may occur due to a decrease i n the amount of transmitter released (Del C a s t i l l o and Katz, 1954; Thies, 1965; M i l e d i and S l a t e r , 1966; Betz, 1970; Horn and Wright, 1970). "Frequency f a c i l i t a t i o n " and "short term f a c i l i t a t i o n " bear some r e l a t i o n s h i p to another phenomenon, post-tetanic p o t e n t i a t i o n (PTP) . While changes i n the post-synaptic p o t e n t i a l may occur during the stimu-l a t i o n , PTP places an emphasis upon changes i n the post-synaptic p o t e n t i a l a f t e r the a p p l i c a t i o n of the t e t a n i z i n g t r a i n s of s t i m u l i . PTP can be defined as a r e l a t i v e l y long term (min. to hrs.) increase i n the magnitude and duration of the post-synaptic p o t e n t i a l following the cessation of a high frequency t r a i n . PTP occurs at the neuromuscular junct i o n , the synapses of peripheral ganglia, and within the c e n t r a l nervous system of both the vertebrate and invertebrates. There i s a general concensus that PTP i s a pre-synaptic event. If divalent calcium ions are added to the bathing medium of the neuromuscular junction, during the period of tetanus (but not afterwards), a f a c i l i t a t i o n of PTP occurs (Rosenthal, 1969; Weinreich, 1971). It has been suggested (Stinnakre and Tauc, 1973) that each spike i n the t r a i n allows greater i n f l u x of calcium into the terminal than previous spikes r e s u l t i n g i n a post-tetanic period of 32 increased transmitter release with subsequent stimulation. In the s p i n a l cord of the mammal, s p i n a l r e f l e x e s undergo PTP (Lloyd, 1949; Eccles and R a i l , 1951; Jefferson and Benson, 1953; Hughes, 1958; Wall and Johnson, 1958; Eccles and Krnjevic, 1959) and the complex EPSP recorded i n the s p i n a l motorneurone undergoes a simultaneous poten-t i a t i o n (Brooks, et a l . , 1950; Curt i s and Eccles, 1960). Spinal reflexes also demonstrate "low frequency depression" (Lloyd, 1949; Wilson, 1958; Eccles, 1964; Kuno, 1964). The EPSP recorded i n s p i n a l motorneurones demonstrates"frequency f a c i l i t a t i o n " as well (Kuno, 1964; Kuno and Weakly, 1972a). Other regions of the vertebrate b r a i n also demonstrate phenomena s i m i l a r to "frequency f a c i l i t a t i o n " (Richards, 1972; B l i s s and L^fmo, 1973; Douglas and Goddard, 1975). The a p p l i c a t i o n of a tentanic t r a i n of s t i m u l i to an a f f e r e n t nerve i s followed by a period of augmentation of r e f l e x e x c i t a b i l i t y that may range from several seconds (Lloyd, 1949; Eccles and R a i l , 1951) to periods of hours (Beswick and Conroy, 1964, 1965; Fentress and Doty, 1966; Spencer and A p r i l , 1970; B l i s s and L^mo, 1973). The duration of the period of PTP i s dependent upon the frequency and duration of the te t a n i z i n g t r a i n s (Lloyd, 1949; Spencer and A p r i l , 1970) but b r i e f intermittent periods of tetanus y i e l d more prolonged periods of PTP (Granit, 1956). If the period of t e t a n i z a t i o n i s increased from a duration of minutes to one of hours the t e t a n i z a t i o n i s not followed by PTP but rather by a period of depression (PTD) which may l a s t up to f i v e hours (Hughes, 1958; Bishop, et a l . , 1959; Fentress and Doty, 1966; Spencer and A p r i l , 1970; 33 Richards, 1972). The previously described phenomena are defined, to a large extent, by the stimulus parameters used to evoke them. If two d i f f e r e n t stimulus parameters are employed to evoke a f a c i l i t a t o r y (or depression) phenomenon there has been a tendency to define two phenomena even though the same mechanism may underlie the observed phenomena. For example, can a decision be made as to what r e l a t i o n s h i p e x i s t s between the mechanism of PTP and that of "frequency f a c i l i t a t i o n " ? Such a question has not yet been ans-wered. It i s l i k e l y that any p a r t i c u l a r system of neural connections displays a number of these phenomena. Richards (1972) provided an example of the complexity of the r e l a t i o n s h i p between the frequency of stimulation and the changing s i z e of the post-synaptic p o t e n t i a l . The system employed by Richards was an i n d i r e c t measure of the complex EPSP's evoked i n the o l f a c t o r y cortex of the guinea pig during r e p e t i t i v e stimulation of the o l f a c t o r y t r a c t . Stimulating t h i s t r a c t at various frequencies resulted i n these a l t e r a t i o n s i n the s i z e of the EPSP: 1) Potentiation of EPSP's occurred following tet a n i c t r a i n s however i f the t r a i n was continued long enough a depression of the EPSP's occurred. 2) A progressive f a c i l i t a t i o n of the s i z e of the evoked EPSP's with stimulus r e p e t i t i o n at frequencies of stimulation from 5 to 40 s t i m u l i per second was found. 3) At a lower frequency of stimulation (0.5 to 2 s t i m u l i per second) the EPSP's were progressively reduced i n s i z e . Therefore, the stimulus parameters determined i f PTP, PTD, frequency f a c i l i t a t i o n , or low frequency depression would occur i n t h i s p a r t i c u l a r pathway. 34 Kandel and Tauc (1965a, b) described a form of f a c i l i t a t i o n i n the abdominal ganglion of Ap l y s i a that occurred when two consecutive s t i m u l i were applied to heteronymous pathways. This f a c i l i t a t i o n was not de-pendent upon properties of the post-synaptic neurones suggesting that t h i s f a c i l i t a t i o n was also pre-synaptic i n o r i g i n . However, processes such as "frequency f a c i l i t a t i o n " and PTP are homosynaptic, that i s they can be demonstrated even f o r a s i n g l e pre-synaptic terminal. The "heterosynaptic" f a c i l i t a t i o n described by Kandel and Tauc (1965a, b) i s believed to occur as a consequence of a synaptic a c t i o n upon the pre-synaptic terminal. Pre-synaptic i n h i b i t i o n (heterosynaptic i n h i b i t i o n ) was also observed i n the ganglion (Tauc, 1965). "Heterosynaptic f a c i -l i t a t i o n " develops a f t e r fewer stimulus r e p e t i t i o n s than PTP and i t has a much longer time course than PTP i n the "homosynaptic" pathway (Kandel and Tauc, 1965b; Tauc and Epstein, 1967). "Heterosynaptic f a c i l i t a t i o n and i n h i b i t i o n " both demonstrated "low frequency depression" (they became les s e f f e c t i v e with r e p e t i t i o n of the stimulus). Many of the previous forms of "synaptic p l a s t i c i t y " bear a resemblance to habituation and s e n s i t i z a t i o n . However, i t i s not s u f f i c i e n t to assume that "low frequency depression" i s the underlying mechanism of habituation and that s e n s i t i z a t i o n i s the r e s u l t of "frequency f a c i l i t a t i o n " or "heterosynaptic f a c i l i t a t i o n " . These properties of synapses must be shown to occur i n the r e f l e x arc during simultaneous habituation and s e n s i t i z a t i o n of that r e f l e x . A r e f l e x i d e a l l y suited f o r such an exami-nation would be a monosynaptic r e f l e x with large and ac c e s s i b l e pre- and post-synaptic neurones. The monosynaptic EPSP recorded from the post-35 synaptic neurone should demonstrate the parametric c h a r a c t e r i s t i c s of habituation and s e n s i t i z a t i o n . If t h i s were found to be the case, then i t might be assumed that habituation and s e n s i t i z a t i o n occurred by some form of synaptic depression or f a c i l i t a t i o n , r e s p e c t i v e l y . A degree of caution must be exercised, however, because no extrapolation can be made about the mechanisms of habituation and s e n s i t i z a t i o n i n t h i s r e f l e x to the same behavioural phenomena i n more complex systems. Furthermore, i f synaptic p l a s t i c i t y i s a general c h a r a c t e r i s t i c of chemical synapses i t i s s e l f - e v i d e n t that habituation and s e n s i t i z a t i o n of a monosynaptic r e f l e x w i l l be the r e s u l t of synaptic p l a s t i c i t y (assuming the synapse i s the s i t e of habituation and s e n s i t i z a t i o n ) . Therefore, the next question i s whether or not synaptic p l a s t i c i t y underlies habituation and s e n s i t i z a t i o n i n more complex systems of neurones. In p a r t i c u l a r , can i n h i b i t i o n play a r o l e i n habituation? Invertebrates are Obvious candidates f o r such studies of habituation and s e n s i t i z a t i o n as the a c c e s s i b i l i t y of t h e i r neurones i s f a r greater than that of the vertebrate c e n t r a l nervous system. Habituation and s e n s i t i z a t i o n have been extensively studied i n invertebrates (Eisenstein and Peretz, 1973; Pakula and Sokolov, 1973; Wyers, et a l . , 1973). The most complete study of habituation and s e n s i t i z a t i o n has been performed using the g i l l withdrawal r e f l e x of A p l y s i a . Mechanical stimulation of the siphon (mantle, shelf, purple gland, and siphon) caused a r e f l e x i v e withdrawal of the g i l l and siphon. Repeated e l i c i t a t i o n of t h i s with-drawal resulted i n habituation of the response which demonstrated eight of the nine parametric c h a r a c t e r i s t i c s of habituation (there was a lack 3 6 of g e n e r a l i z a t i o n which i s to be expected i n a monosynaptic r e f l e x ) . Habituation of t h i s r e f l e x lasted for periods of several minutes to periods of three weeks (Carew, et a l . , 1971; Carew, et a l . , 1972; Carew and Kandel, 1973). The a c t i v a t i o n of t a c t i l e receptor neurones i n the skin of the siphon evoked putative monosynaptic EPSP's i n the appropriate motor-neurones, and although some interneurones were activated during stimu-l a t i o n of the r e f l e x the monosynaptic component i s l i k e l y the most s i g -n i f i c a n t component contributing to habituation of t h i s r e f l e x . During habituation of the relex these EPSP's underwent a decrement i n s i z e which also demonstrated eight of the c h a r a c t e r i s t i c s of habituation. Further-more, these EPSP's were l i k e l y monosynaptic as they were con s i s t e n t l y short i n latency, persisted i n bathing solutions high i n divalent ions (known to block the a c t i v i t y of interneurones), and the i n j e c t i o n of t e t r a - e t h y l ammonium into the pre-synaptic neurone increased t h e i r magni-tude and duration ( t h i s drug when in j e c t e d i n t r a c e l l u l a r l y increases the duration of the pre-synaptic spike and presumably increases the amount of transmitter released) (Bryne, et a l . , 1974). The post-synaptic membrane showed no long term change i n i t s con-ductance which might have accounted f o r the decrement of the EPSP ( C a s t e l l u c c i , et a l . , 1970). and there was a one to one r e l a t i o n s h i p between the spikes i n the sensory neurone and the EPSP's i n the motorneurones. Repeated i n t r a c e l l u l a r stimulation of the sensory neurone evoked constant amplitude spikes, but the EPSP underwent a simultaneous decrement (Bryne, et a l . , 1974). Therefore, i t has been proposed that habituation of t h i s 37 r e f l e x i s a consequence of a form of "low frequency depression" or "synaptic depression" (Carew and Kandel, 1973; C a s t e l l u c c i and Kandel, 1974). I t was found that dishabituation and s e n s i t i z a t i o n of t;he g i l l -withdrawal r e f l e x occurred by a mechanism independent of habituation. Dishabituation appeared to be i d e n t i c a l with "heterosynaptic f a c i l i t a t i o n " , and a strong stimulus presented to a heteronymous pathway potentiated (dishabituated) the EPSP's evoked i n the motorneurones by stimulation of a habituated pathway, and the r e f l e x i t s e l f (Carew, et a l . , 1971). E l i c i t a t i o n of t h i s r e f l e x with noxious stimulation induced periods of response s e n s i t i z a t i o n which la s t e d up to three weeks (Pinsker, et a l . , 1973). There i s some evidence to suggest that the g i l l pinnule of A p l y s i a i s capable of performing a r e f l e x i v e withdrawal independently of the c e n t r a l nervous system. For example, neurones which may be the sensory and motor components of the r e f l e x are located within the pinnule i t s e l f (Peretz and Estes, 1974). Peretz and Moller (1974) demonstrated that the amplitude or general e x c i t a b i l i t y of the pinnule withdrawal was dependent, i n part, upon excitat o r y a c t i v i t y o r i g i n a t i n g from the anterior g i l l ganglion (a p e r i p h e r a l ganglion). In ganglionectomized preparations. (abdominal ganglion removed) the further removal of the a n t e r i o r g i l l ganglion resulted i n a decrease i n response amplitude, a reduced rate of habituation, and prevented dishabituation. This implies that t h i s ganglion may play some ro l e i n the establishment of habituation of the pinnule withdrawal response. 38 I n t r a c e l l u l a r recording from neurones of the a n t e r i o r g i l l ganglion (during habituation of the response) revealed a long latency and poly-phasic p o t e n t i a l ( i n i t i a l d e p olarization followed by hyperpolarization) evoked by t a c t i l e stimulation of the pinnule. The hyperpolarization phase became progressively greater i n duration with successive s t i m u l i . This r e s u l t e d i n a gradual reduction i n the tonic a c t i v i t y of some of the neurones i n the ganglion. Stimulation of the connective, from ganglion to pinnules, did not cause an observable muscular response. On t h i s basis, Peretz and Moller (1974) postulated that the anterior g i l l ganglion contributed to pinnule withdrawal response amplitude by a mechanism of "heterosynaptic f a c i l i t a t i o n " . As w e l l , they suggested that a progressive i n h i b i t i o n of the tonic a c t i v i t y of the g i l l ganglion leads to a progressive d i s - f a c i l i t a t i o n of the pinnule response which contributed to habituation of t h i s response. The concept that habituation might occur as a consequence of build-up of i n h i b i t i o n was also proposed by Holmgren and Frenk (1961). They recorded a progressive increase IPSP's recorded from neurones i n the p a r i e t a l ganglion of Helix following r e p e t i t i v e stimulation. Inhibitory synapses are also capable of demonstrating f a c i l i t a t o r y phenomena. Tauc (1969) reported that hyperpolarization evoked i n c e r t a i n ganglionic.(neurones underwent a progressive increase i n amplitude and duration with stimulus r e p e t i t i o n . There was a d i r e c t r e l a t i o n s h i p between the extent of t h i s build-up of i n h i b i t i o n (ILD, i n h i b i t i o n of long duration) and the s i z e of the i n i t i a l stimulus. A more extensive examination of the f a c i l i t a t i o n of i n h i b i t o r y synapses was performed on 39 neurones i n the abdominal ganglion of A p l y s i a by Waziri, et a l . (1969), and i t was shown that i n h i b i t o r y synapses were capable of "frequency f a c i l i t a t i o n " and PTP. Although t h i s mechanism was considered to be a possible contributory factor to habituation i t was rejected because strong s t i m u l i were more e f f e c t i v e than weak s t i m u l i i n producing the build-up of i n h i b i t i o n and because i t was not possible to demonstrate d i s -i n h i b i t i o n following a strong shock to a heteronomous pathway. Employing the parametric c h a r a c t e r i s t i c s of habituation i t would seem that i f i n -h i b i t i o n were to account for habituation, the build-up of i n h i b i t i o n should be greater with weak than strong s t i m u l i and dis-habituation should occur by d i s - i n h i b i t i o n . However, there i s some l a t i t u d e i n the i n t e r -p r e t a t i o n of the r e l a t i o n s h i p between stimulus i n t e n s i t y and degree of habituation (see p. 92, t h i s t h e s i s ) . Furthermore, i f dishabituation i s i n f a c t a super-imposed "heterosynaptic f a c i l i t a t i o n " or s e n s i t i z a t i o n of the decremented pathway i t i s not necessary for d i s - i n h i b i t i o n to occur. In f a c t , i t might be argued that t h i s i s why the rate of habituation i s greater following a dishabituatory stimulus. Recently, F a r e l , et a l . (1974) have demonstrated that r e p e t i t i v e stimulation of the l a t e r a l column of the s p i n a l cord (frog) evoked a mono-synaptic EPSP which demonstrated eight of the parametric c h a r a c t e r i s t i c s of habituation. This decrement of the EPSP suggests that "synaptic depression" occurs i n a s i m i l a r manner i n the vertebrate s p i n a l cord. Spencer, et a l . (1966c) found that monosynaptic EPSP's evoked i n f l e x o r motorneurones of the s p i n a l cord (cat) did not demonstrate decremental behaviour when the f l e x o r r e f l e x was undergoing simultaneous habituation. 40 They concluded that the polysynaptic component of the f l e x o r r e f l e x habituates but monosynaptic EPSP's evoked i n f l e x o r motorneurones do not. However, habituation of monosynaptic s p i n a l r e f l e x e s has been reported i n man ( D i m i t r i j e v i c and Nathan, 1973). It seems rather s u r p r i s i n g that monosynaptic EPSP's evoked i n f l e x o r motorneurones did not display some form of synaptic p l a s t i c i t y . This may be a t t r i b u t a b l e to the stimulus parameters employed by Spencer, et a l (1966c) or perhaps i t suggests that p l a s t i c i t y i s confined to s e l e c t synapses. E l e c t r o t o n i c synapses ap-parently do not demonstrate synaptic p l a s t i c i t y (Martin and P i l a r , 1964), and Bennett(1968) has suggested that synaptic p l a s t i c i t y i s a d i s t i n g u i s h i n g property of the chemical synapse. In conclusion, i t must be stressed that the properties of invertebrate synapses may not be the same as those of vertebrate synapses. However, the many s i m i l a r i t i e s between synapses i n invertebrates and vertebrates must j u s t i f y at l e a s t a discussion of these properties. Furthermore, the s i m i l a r i t i e s between habituation of simple refl e x e s i n invertebrates and habituation of s p i n a l r e f l e x e s i n the vertebrate suggest that a s i m i l a r mechanism may underlie both forms of habituation (Farel, et a l . , 1974). 41 SECTION IV I n h i b i t i o n : Habituation and S e n s i t i z a t i o n of the Flexor Reflex Habituation and s e n s i t i z a t i o n have been demonstrated i n numerous sensory systems (Buchwald and Humphrey, 1973), but the f l e x o r withdrawal r e f l e x has been used as a model i n order to develop the parametric c h a r a c t e r i s t i c s of habituation and s e n s i t i z a t i o n . A f t e r Sherrington's o r i g i n a l d e s c r i p t i o n of habituation and s e n s i t i z a t i o n , s p i n a l reflexes continued to be the preferred models by which to study behavioural habituation (Prosser and Hunter, 1936; Spencer et a l . , 1966 a, b, c; Wickelgren, 1967 a, b). The c e n t r a l o r i g i n of habituation was confirmed either by stimulating a f f e r e n t nerves d i r e c t l y (Wickelgren, 1967b) or by recording motor responses from the v e n t r a l r o o t l e t s (Buchwald, et a l . , 1965), thereby demonstrating that i n the mammal neither peripheral r e -ceptors nor the neuromuscular junction are c r i t i c a l f o c i f o r habituation. Few studies have u t i l i z e d the i n t a c t preparation when examining habituation of the f l e x o r r e f l e x . The almost exclusive use of the s p i n a l preparation was encouraged by a desire to reduce the r e f l e x to i t s simplest anatomical components. The objective was to i s o l a t e a s i n g l e s i t e of the o r i g i n of habituation (presumably synaptic). The f l e x o r r e -f l e x i s not an i d e a l r e f l e x for such a study. For example, studies of the monosynaptic gill-withdrawal r e f l e x of A p l y s i a or of the disynaptic plantar r e f l e x (Egger and Wall, 1971) seem f a r more suited to such an approach. The f l e x o r r e f l e x undergoes habituation i n the i n t a c t rat ( G r i f f i n 42 and Pearson, 1968b), and man ( D i m i t r i j e v l c , et a l . , 1972). Habituation of t h i s r e f l e x d i f f e r s i n the i n t a c t animal when compared to the s p i n a l animal. In p a r t i c u l a r , the amplitude of the f l e x o r r e f l e x i s i n i t i a l l y greater i n the i n t a c t animal than i n the s p i n a l with a concomitant a l t e r -a t i o n i n the i n i t i a l rate of s e n s i t i z a t i o n ( D i m i t r i j e v i c , et a l . , 1972; Pearson and Wenkstern, 1972). This aspect w i l l be discussed subsequently (this t h e s i s , p. 96). Habituation of the f l e x o r r e f l e x of the rat i s also strongly c o n t r o l l e d by supraspinal structures such as the f r o n t a l cortex ( G r i f f i n and Pearson, 1968b) and the dorsomedial thalamus ( G r i f f i n , 1970). While the f l e x o r r e f l e x may not be i d e a l f or the study of synaptic p l a s t i c i t y i t provides a model of r e l a t i v e complexity s u f f i c i e n t to de-termine the mechanisms by which habituation and s e n s i t i z a t i o n are con-t r o l l e d . Furthermore, i f multisynaptic mechanism contribute to habituation i n more complex systems than the monosynaptic r e f l e x , the f l e x o r r e f l e x w i l l allow an examination of these processes. The f l e x o r withdrawal r e f l e x (FWR) was defined as a r e f l e x i v e with-drawal of either the hind or forelimb evoked by stimulation of the skin of the limb or i t s afferent nerves (Sherrington, 1910). The hindlimb displays a f l e x i o n of the hip, knee, and ankle; the forelimb a f l e x i o n of the elbow, shoulder, and w r i s t . The FWR may be e l i c i t e d i n the i n t a c t , decerebrate, or s p i n a l animal, although the response d i f f e r s somewhat i n each of the preparations (Graham Brown, 1912). Muscles of the limbs which undergo contraction during the FWR are defined as f l e x o r muscles (Sherrington, 1910), and regardless of the strength of stimulation only a f i x e d subset or group of muscles w i l l contract. Spread of contraction to 43 other muscles w i l l not occur. The f l e x o r muscles are l i s t e d i n Table I (Sherrington, 1910:31). Reflexive r e l a x a t i o n of muscles also occurs during f l e x i o n and these muscles are defined as extensor muscles. This f u n c t i o n a l d i s t i n c t i o n between f l e x o r and extensor muscles does not always correspond to c l a s -s i c a l anatomical nomenclature. The receptive f i e l d of the FWR re f e r s to that area of the skin of the limb wherein applied s t i m u l i w i l l evoke withdrawal. Noxious s t i m u l i are by f a r the most e f f e c t i v e s t i m u l i i n e l i c i t i n g the r e f l e x . Within the receptive f i e l d there e x i s t s a hierarchy with regard to the threshold of f l e x o r e l i c i t a t i o n , and the threshold i s lowest i n the skin of the foot and i t increases proximally along the limb. It i s greatest i n the sk i n of the thigh. Evocation of the r e f l e x from any point w i t h i n the receptive f i e l d produces a stereotyped withdrawal. This f l e x i o n i s com-posed of a "group of refle x e s almost i d e n t i c a l i n form which when con-current combine i n harmonious action on the same common paths." (Sherrington, 1910). Stimulation of the dorsal roots also evokes FWR of the muscles of the segment activated. In t h i s regard, f l e x i o n of the ankle occurs most strongly with stimulation of caudal dorsal roots while hip f l e x i o n occurs most strongly with stimulation of more cephalic roots. However, stimu-l a t i o n of any dorsal root w i l l evoke a f u l l FWR but with the greatest strength of contraction prevalent i n the muscles of the segment d i r e c t l y innervated by the stimulated root. This tendency extends to i n d i v i d u a l afferent nerves, and i f a stimulus of increasing i n t e n s i t y i s applied to 44 Table I Flexor Muscles Ilio-psoas Pectineus ( s l i g h t ) Sartorius (part inserted into Tensor fas c i a e femoris Rectus femoris G r a c i l i s Semitendinous Posterior biceps femoris p a t e l l a ) Tenirissmus T i b i a l i s anticus Peroneous longus Extensor longus digitorum 45 to the skin of the foot (or to the a f f e r e n t nerve supplying the foot) f l e x i o n w i l l occur i n i t i a l l y at the ankle, then at the knee, and f i n a l l y at the hip. Sherrington (1910) makes a d i s t i n c t i o n between two types of flexor r e f l e x e s . Withdrawal of the limb i n response to a noxious (painful) stimulus r e s u l t s i n a r e l a t i v e l y long l a s t i n g response. This i s the FWR as a p r o t e c t i v e or "nociceptive" r e f l e x . This r e f l e x i s e l i c i t a b l e from a large surface area of the limb and also from the underlying muscles and f a s c i a . The flexor r e f l e x can also be evoked from subcutaneous structures of the limb i n response to non-noxious s t i m u l i . Thus, a d i s t i n c t i o n i s made between a nociceptive f l e x i o n and a f l e x i o n evoked by weak s t i m u l i . The l a t t e r f l e x i o n , i t was suggested, i s a component of r e f l e x stepping while the former i s a protective r e f l e x a l l i e d to the response of the animal to p a i n f u l stimulation. The "nociceptive" FWR was dominant as described by Sherrington (1910:73): The p e c u l i a r character, i n f a c t the "adequacy" of i t s stimulus l i e s i n i n t e n s i t y . It i s prepotent when p i t t e d against the locomotor r e f l e x : and t h i s i s a further mark of i t s nociceptive character, since nociceptive r e -flexes l i k e p a i n f u l sensations are h a b i t u a l l y "dominant" i n competition against others. Lloyd (1943) c l a s s i f i e d a number of subcomponents of the FWR on the basis of the type of afferent activated to e l i c i t the r e f l e x . A c t i v a t i o n of group I a f f e r e n t s (either by d i r e c t stimulation of the muscles nerves or by tendon tap) caused a contraction i n f l e x o r muscles i f the nerve originated from that f l e x o r muscle. This was not the FWR described by Sherrington (1910). Stretching of a f l e x o r muscle caused contraction of that muscle due to a c t i v a t i o n of group II muscle nerves. Stimulation of 46 group II and I I I nerves from muscle, skin, and j o i n t s evoked a general f l e x o r r e f l e x . However, the a c t i v a t i o n of group III cutaneous afferents (Delta f l e x i o n ) and group IV afferents leads to a FWR comparable to that observed by Sherrington (1910) that i s , the "nociceptive" FWR. Stim u l i that a c t i v a t e high threshold afferents also maximally a c t i v a t e low threshold afferents unless an attempt i s made to s e l e c t i v e l y block them. Mendell and Wall (1964) reported that s e l e c t i v e stimulation of group IV afferents did not e l i c i t any r e f l e x discharge or the so c a l l e d "C (group IV) r e f l e x . " It was suggested that group IV afferents merely f a c i l i t a t e d the influence of low threshold afferents by a mechanism of PAH. In d i r e c t c o n t r a d i c t i o n to these r e s u l t s many others have found that group IV afferents were capable of e l i c i t i n g a d i s c r e t e FWR and/or PAD (Franz and Iggo, 1968; Clark, et a l . , 1935; Franz, et a l . , 1966; Laporte and Bessou, 1958; Alderson and Dowman, 1960; Janig and Zimmerman, 1971). More recent evidence suggests that PAD and PAH can be evoked i n the terminals of any p a r t i c u l a r group of afferents provided that group afifaf-y,. .. ferents i s i t s e l f stimulated (Mendell, 1972; Whitehorn and Burgess, 1973). The "C fl e x o r r e f l e x " was r a p i d l y blocked by anaesthetic doses of ether, ethyl c h l o r i d e , or sodium pentobarbital, but i t was les s l i k e l y to be reduced i n animals anaesthetized with chloralose (Franz and Iggo, 1968). This may account for the f a i l u r e of Mendell and Wall (1964) to record r e f l e x discharge following stimulation of group IV afferents as t h e i r animals were anesthetized with sodium pentobarbital. I t seems l i k e l y that a d i s t i n c t "C f l e x o r r e f l e x " does e x i s t even i n the absence of low threshold afferent a c t i v i t y . This d i s t i n c t i o n may be somewhat a r b i t r a r y 47 as i t has been shown that nociceptors are capable of evoking a powerful and independent discharge of dorsal horn interneurones (Iggo, 1974). The FWR i n the int a c t animal may include c e r t a i n "long r e f l e x e s " such as the "spino-bulbo-spinal" f l e x i o n r e f l e x . Apparently a c t i v a t i o n of afferents capable of e l i c i t i n g the FWR also activates a pathway to the brain stem r e t i c u l a r formation that i n turn can eit h e r excite or i n h i b i t f l e x o r motorneurones and the polysynaptic f l e x o r pathway (Shimamura and Aoki, 1969). Spinal transection and i n j e c t i o n of sodium pen t o b a r i t a l blocked "spino-bulbo-spinal" r e f l e x e s . Groves and Thompson (1970) described two t h e o r e t i c a l (inferred constructs) processes of behavioural and r e f l e x decrement which occur as a consequence of regular stimulus r e p e t i t i o n . The f i r s t i s "habituation" to r e l a t i v e l y weak s t i m u l i and the other i s "habituation of s e n s i t i z a t i o n " which occurs with r e p e t i t i o n of r e l a t i v e l y intense s t i m u l i . The many systems that demonstrate habituation as a consequence of "synaptic de-pression" tend to suggest that "habituation" occurs by a s i m i l a r mechanism. It i s not clear i f "habituation of s e n s i t i z a t i o n " i s merely a super-imposition of s e n s i t i z a t i o n and "habituation" or whether i t represents an altogether d i f f e r e n t process (Groves and Thompson, 1970). Habituation of the FWR of the s p i n a l cat has been employed as a model i n which to determine whether or not i n h i b i t i o n might be responsible for t h i s observed response decrement (Spencer, et a l . , 1966c). The de-crement of the polysynaptic EPSP's evoked i n fl e x o r motorneurones was associated with habituation of the f l e x o r r e f l e x , but the i n j e c t i o n of strychnine or p i c r o t o x i n was found to be i n e f f e c t i v e i n disrupting 48 habituation of the v e n t r a l root electrotonus. This was tested i n both decerebrate-spinal preparations and s p i n a l animals anaesthetized with pentobarbital. The "nociceptive" FWR described by Sherrington (1910) i s not l i k e l y to be that observed by Spencer et a l . , (1966 a, b, c) . For example, Spencer et a l . , (1966a) indicated that group II and some group III afferents were involved, but i t was never c l e a r which afferents were activat e d at any p a r t i c u l a r stage of the experiments. A f a i l u r e by various authors to i n d i c a t e either the i n t e n s i t y of stimulation or the afferents activated by that stimulation of the skin (Spencer et a l . , 1966a, b, c; Wickelgren, 1967a; Groves and Thompson, 1973) have made i t impossible to determine the contribution of high threshold afferents to habituation and s e n s i t i z a t i o n of the FWR-. The exclusive use of the sp i n a l preparation has also negated the p o s s i b i l i t y of determining i f any contribution i s made by supraspinal structures and/or "spino-bulbo-s p i n a l " f l e x o r r e f l e x e s . The i n j e c t i o n of strychnine or p i c r o t o x i n resulted i n an elevation of the response amplitude of the recorded v e n t r a l root electrotonus. In compensation, Spencer et a l . (1966c) lowered the stimulus i n t e n s i t y for those animals which received an i n j e c t i o n of strychnine or p i c r o -toxin but not for control animals. One of the parametric c h a r a c t e r i s t i c s of habituation (see t h i s t h e s i s , p. 14) i s that a reduction i n stimulus i n t e n s i t y w i l l r e s u l t i n a greater rate of habituation. This r a i s e s the question of whether any impairment of habituation caused by the drugs might have been counteracted by the opposite influence of reducing stimulus i n t e n s i t y . The j u s t i f i c a t i o n f o r such a manoeuver can also be questioned 49 on the basis that an a r t i f i c a l l y induced increase i n response amplitude does not n e c e s s a r i l y a l t e r the rate of habituation of the FWR ( G r i f f i n and Pearson, 1968a; Pearson and Vickars, 1974) which negates the l o g i c of reducing stimulus i n t e n s i t y for treated animals. While t h i s study demonstrated that habituation can continue to take place following the i n j e c t i o n of strychnine or p i c r o t o x i n the p o s s i b i l i t y remained that i n h i b i t o r y mechanisms might contribute at l e a s t to some forms of response decrement. Wall (1970) postulated that habituation of the FWR might be due to PTP of i n h i b i t o r y synapses i n the f l e x o r r e f l e x pathway. PTP of both pre-synaptic and post-synaptic i n h i b i t i o n has been reported i n the mammalian s p i n a l cord (Spencer and A p r i l , 1970; Schmidt, 1973). It does not seem l i k e l y , however, that i n h i b i t o r y synapses should undergo PTP with stimulus parameters which do not produce PTP of excitatory synapses. This i s c e r t a i n l y the case i n invertebrates. Therefore i t seems more probable that habituation to low i n t e n s i t y stimulation does not involve the build-up of i n h i b i t o r y a c t i v i t y . Strong s t i m u l i that cause s e n s i -t i z a t i o n might, on the other hand, provoke a build-up of i n h i b i t o r y and excitatory a c t i v i t y . This proposed build-up of i n h i b i t i o n might l i m i t s e n s i t i z a t i o n and determine the rate of i t s decrement with r e p e t i t i o n of the stimulation. A process of i n h i b i t o r y build-up might be found at a v a r i e t y of l e v e l s of the c e n t r a l nervous system. These i n h i b i t o r y mechanisms might be inherent to the s p i n a l segments d i r e c t l y involved with the f l e x o r r e f l e x , however, intersegmental i n h i b i t i o n o r i g i n a t i n g from outside these 50 segments might also contribute. Supraspinal i n h i b i t i o n might be manifest through modulation of "spino-bulbo-spinal" r e f l e x e s or perhaps more d i r e c t l y through an a l t e r a t i o n i n the tonic influence of i n h i b i t i o n o r i g i n a t i n g from the bulbar r e t i c u l a r formation ( o r i g i n a l l y described by Magoun and Rhines, 1946). The bulbar r e t i c u l a r formation (and for that matter the s p i n a l cord) i s also under the c o n t r o l of suprabulbar i n h i b i t o r y systems (Pompeiano, 1973). In summary i n h i b i t i o n of the FWR could o r i g i n a t e from various l e v e l s of the nervous system such as: 1) intrasegmental 2) intersegmental 3) supraspinal 4) suprabulbar. This study was designed to explore the c o n t r i b u t i o n of i n h i b i t o r y mechanisms to habituation and s e n s i t i z a t i o n of the FWR. This was ac-complished by studying the r e f l e x i n the i n t a c t , decerebrate, and s p i n a l rat i n order to i s o l a t e the c o n t r i b u t i o n of various l e v e l s of the c e n t r a l nervous system. Pharmacological i s o l a t i o n was achieved by eliminating post-synaptic i n h i b i t i o n ( i n f u s i o n , of strychnine), pre-synaptic i n -h i b i t i o n and some forms of post-synaptic i n h i b i t i o n ( i n f u s i o n of b i c u c u l l i n e ) , and bulbo-spinal i n h i b i t i o n mediated by serotonergic systems (pre-treat-ment with para-chlorophenylalanine (p-CPA) which i s a s e l e c t i v e depletor of brain serotonin, i n f u s i o n of the drug methysergide which antagonizes serotonergic transmission, and by l e s i o n i n g the nucleus raphe^dorsalis (n.r.d.) one of the brain stem n u c l e i p a r t i c u l a r l y high i n content of serotonin and which when stimulated causes i n h i b i t i o n of the f l e x o r r e f l e x ) . Segmental mechanisms were also examined by comparing the e f f e c t of various l e v e l s of s p i n a l transection and by subjecting the s p i n a l cord to a temporary period of asphyxiation with the objective of causing 51 r e l a t i v e l y s e l e c t i v e destruction of i n h i b i t o r y s p i n a l interneurones. CHAPTER II 52 METHODS: SECTION I The Flexor Reflex Animal Subjects Albino rats (Wistar s t r a i n ) were employed as experimental preparations i n a l l cases. Male rats (200 to 700 g) were used e x c l u s i v e l y i n order to avoid any inhomogeneity i n sample populations due to sexual d i f f e r e n c e s . An attempt was made to avoid rats that showed any i n d i c a t i o n of re s p i r a t o r y disease. This was c r i t i c a l , as the use of ether as an anaesthetic greatly accentuates r e s p i r a t o r y d i s t r e s s . Conscious rats had to be restrained following the implantation of chronic stimulating and recording electrodes and during t e s t i n g of f l e x o r r e f l e x e s . Restraint of animals during the period a f t e r electrode implantation, and p r i o r to t e s t i n g , required that some s p e c i f i c attention be paid to the care of the rats (p.57, t h i s t h e s i s ) . Reflex Response E l e c t r i c a l s t i m u l i were delivered, subcutaneously, to the hindpaw of the rat i n order to evoke a defensive withdrawal of the limb. The representative measure of f l e x o r r e f l e x response used i n these experiments was the integrated electromyographic (EMG) discharge recorded from the posterior head of the biceps femoris muscle. The anterior head of t h i s muscle i s extensor i n function, but the posterior head p a r t i c i p a t e s i n func t i o n a l f l e x i o n at the knee (Sherrington, 1910). The integrated EMG may correspond e i t h e r to the tension developed i n the muscle or the a c t i v i t y i n the motor nerve (electroneurogram) both of which are considered adequate measures of r e f l e x response (Steiner, 53 Thexton, and Weber, 1973). The integrated EMG of the pos t e r i o r head of the biceps femoris has previously been correlated, with a high degree of s i g n i f i c a n c e , to the isometric tension developed i n t h i s muscle. Habituation of the isometric tension and the integrated EMG, following r e p e t i t i v e stimulation, was also very s i m i l a r ( G r i f f i n and Pearson, 1967). I t can be concluded that the integrated EMG of the posterior head of the biceps femoris i s an acceptable measure of the amplitude of the f l e x o r r e f l e x as described by Sherrington (1910). Stimulus Delivery and Response Recording Uniform, si n g l e p u l s e . s t i m u l i (5 msec, duration), were delivered by means of a Devices stimulator which was triggered at regular i n t e r v a l s by a Devices Digitimer. An in t e g r a t i o n unit was also appropriately triggered by the Digitimer. The electromyographic signals were amplified (Tektronix preamplifier type 122) and quantified by full-wave r e c t i f i c a t i o n followed by e l e c t r o n i c i n t e g r a t i o n . The i n t e g r a l values of the EMG were displayed on a d i g i t a l voltmeter and were recorded by the experimenter. EMG a c t i v i t y was integrated f o r a period of 250 msec. (10 to 260 msec, a f t e r i n i t i a t i o n of the stimulus pulse) subsequent to each stimulus pulse. Evoked a c t i v i t y was simultaneously monitored on a Tektronix 565 o s c i l l o s c o p e . Just p r i o r to t e s t i n g , and immediately following t e s t i n g , a reading of background a c t i v i t y was recorded. The average background value was always subtracted from the evoked value of the EMG. The corrected values of the EMG were then used f or an assessment of the amplitude of the f l e x o r r e f l e x . 54 Stimulus t r a i n s (0.5 msec, pulse duration, 50 p u l s e s / s e c , 0.5 sec. t r a i n duration) were given i n a manner s i m i l a r to the d e l i v e r y of s i n g l e pulses. However, a Devices Frequency Generator was added to the c i r c u i t i n order to t r i g g e r uniform stimulus t r a i n s rather than si n g l e pulses. The Devices generator was triggered by the Digitimer which i n turn triggered the Devices stimulator. The EMG a c t i v i t y was integrated for a 500 msec, period (10 to 510 msec.) a f t e r termination of the stimulus t r a i n . This i n t e g r a l i s a measure of "after-discharge" as described by Sherrington (1906). I t was not possible to record evoked a c t i v i t y during the stimulus t r a i n due to the presence of large stimulus a r t i f a c t s . A schematic diagram i s given i n f i g u r e 4. Stimulating Electrodes Chronic stimulating electrodes consisted of two lengths of diamel coated s i l v e r wire (0.007 i n . d i a . ) . The ends of these were bared for about 0.5 cm. and s u r g i c a l l y inserted into the skin of the hind-paw, always i n the same l o c a t i o n , i . e . , one was placed between the t h i r d and fourth d i g i t and the other was inserted on the l a t e r a l aspect of the f i f t h d i g i t . Recording Electrodes Recording electrodes were constructed of diamel coated s i l v e r wire (0.010 i n . dia.) bared for approximately 0.5 cm. at the ends. The bared ends were inserted, s u p e r f i c i a l l y , into the posterior head of the biceps femoris muscle (see f i g u r e 5). Care was taken to ensure that 55 stim. Rat e.m.g. Pu l se Generator Digitimer Stimulator trigger pulse amplified e.m.g. integrator command pulse Integrator integrated e.m.g. Digital Voltmeter .Speaker Figure 4. Schematic diagram of the stimulating and recording equipment. 56 Figure 5. Anatomical l o c a t i o n of the biceps femoris muscle of the r a t i n r e l a t i o n s h i p to other muscles. (B.F. 1, anterior head of the biceps femoris: B.F.2, pos t e r i o r head of the biceps femoris: S;T., semitendinosis: Q.F., quadriceps femoris: G.M., gluteus maximus: G., gastrocnemius: T.A., t i b i a l i s a n t e r i or (antcus). The gluteus maximus had been completely removed to expose the underlying muscles. The l o c a t i o n of EMG electrodes i s indicated by the E. the electrodes did not penetrate through the biceps femoris into underlying extensor muscles. The ends of the wires were positioned about 3 mm. from each other. Recording (and stimulating) electrodes were always implanted one day p r i o r to t e s t i n g of r e f l e x e s . Restraint and Care Following the implantation of stimulating and recording electrodes i t was necessary to r e s t r a i n rats i n order to prevent them from damaging or p u l l i n g out the electrodes. Rats were placed i n Bollman r e s t r a i n i n g cages (see f i g u r e 6) with free access to food and water. The maximum period of r e s t r a i n t was two days. P r i o r to te s t i n g of the r e f l e x rats were placed into a dark and soundproof box for an a c c l i m a t i z a t i o n period of 20 min. Testing of f l e x o r reflexes took place with the rats i n s i d e t h i s box. Special care was taken with s p i n a l rats to ensure that the urinary bladder of each rat was emptied at le a s t twice each day and immediately p r i o r to t e s t i n g of the r e f l e x . Anaesthetics In a l l experiments i t was necessary to anaesthetize rats with ether - (Mallinckrodt Chemical Works). Ether anaesthesia was employed fo r a l l acute s u r g i c a l procedures such as electrode implantation, decerebration, s p i n a l l i g a t i o n , and s p i n a l asphyxiation. The depressant action of ether upon the centralnervous system i s w e l l known. Therefore, at l e a s t 24 hours were permitted to elapse following termination of the ether anaesthesia and the beginning of t e s t i n g . The only exception was Figure 6. A rat i n a Bollman r e s t r a i n i n g cage. Flexor reflexes were tested while the rat was restrained i n t h i s manner. 59 i n experiments i n which a comparison was made between acute s p i n a l r a t s and •.decerebrate rats when only 90 minutes separated the termination of the a p p l i c a t i o n of ether and t e s t i n g of the r e f l e x . An i n t a c t r at usua l l y recovered from ether anaesthesia within t h i s time period (90 min.) but r e s i d u a l anaesthesia may have been present during t e s t i n g of the r e f l e x . Special care had to be taken to give the ether for short and frequent periods during the induction of s p i n a l asphyxiation. Often the rats had to remain anaesthetized f o r periods of up to 1 hour. The only other anaesthetic employed was urethane (ethyl carbamate, 25% s o l u t i o n i n 0.9% s a l i n e ) . Barbituates were avoided because of t h e i r antagonistic actions upon "C fl e x o r r e f l e x e s " , "after-discharge", and spontaneous a c t i v i t y within the s p i n a l cord (Beecher, et a l . , 1939; Wall, 1967a; Franz and Iggo, 1968). As i n h i b i t i o n was examined i n th i s study, chloralose was not used because of i t s tendency to prolong primary a f -ferent depolarization (Schmidt, 1963, 1973). It must be admitted that l i t t l e i s known of the actions of urethane upon the c e n t r a l nervous system although i t may have a considerable depressant a c t i o n upon s p i n a l ac-t i v i t y (Schmidt, 1963). This anaesthetic was not given i n the conventional manner ( I n t r a p e r i t o n e a l l y ) . Rats were i n i t i a l l y anaesthetized with ether i n order to permit the implantation of tracheal and jugular cannulae. Urethane was then given intravenously to a t o t a l dosage of 0.25 g/kg or l e s s , and anaesthesia was not maintained f o r long periods a f t e r t e s t i n g . This l i g h t l e v e l of anaesthesia was necessary so that motor responses (EMG) were not t o t a l l y suppressed. 60 Surg i c a l Procedures Spinal L i g a t i o n (Transection) A laminectomy r e f e r s to the s u r g i c a l exposure of the s p i n a l cord by removal of a v e r t e b r a l p o s t e r i o r arch. In the rat t h i s Is a r e l a -t i v e l y simple, although somewhat d e l i c a t e procedure. An i n c i s i o n of the skin (5 to 10 cm.) was made over the appropriate region of the sp i n a l cord. The muscles and fasciae of the back were then grossly separated from the v e r t e b r a l column by blunt d i s s e c t i o n . L i t t l e or no bleeding occurred. A pair of r e t r a c t o r s was used to hold the large muscles away from the s p i n a l column, while the muscle attachments to the column were scraped away using the back of a s c a l p e l blade. At lea s t 2 vertebrae were cleared of a l l soft t i s s u e on t h e i r dorsal surfaces. The points of a p a i r of bone forceps were then inserted i n t e r v e r t e b r a l l y , and the dorsal (posterior arch) of the appropriate vertebra was gradually clipped away. A previously blunted surgical., needle (1/2 i n . , h a l f c i r c l e ) , with attached dental f l o s s , was slipped under the cord without causing v i s i b l e damage to the cord or dura. The dental f l o s s was then drawn t i g h t and knotted several times, c o n s t r i c -t i n g and e f f e c t i v e l y transecting the cord. The dura remained i n t a c t but the region of the cord under the l i g a t i o n i s forced caudal and cephalad to the l i g a t i o n . No doubt can e x i s t that the cord was transected. This method of transection reduced vascular d i s r u p t i o n i n comparison to a c t u a l l y c u t t i n g the cord. In addition, the cord caudal to the l i g a t i o n was l e f t i n an enclosed dural sac. This was necessary i n experiments 61 i n which asphyxiation of the cord was to be induced. I f the rat was to be kept c h r o n i c a l l y , a small amount of a n t i b i o t i c was applied to the wound before the various layers of muscle and skin were sutured closed. Asphyxiation Both c o n t r o l and experimental r a t s had t h e i r cords l i g a t e d at the l e v e l of the second thoracic vertebra. A hypodermic needle (26 gauge) was inserted into the dural sac formed caudal to the l i g a t i o n . Ex-perimental rats were subjected to 22 min. of asphyxiation. With experi-mental rats the needle was connected, by means of polyethylene tubing, to a manometer b o t t l e containing i s o t o n i c s a l i n e heated to 40° C. The pressure i n the b o t t l e was gradually increased, f o r c i n g s a l i n e into the dural sac, r a i s i n g the i n t r a - d u r a l pressure, and causing occlusion of the s p i n a l vasculature (Van Harreveld, 1943). The pressure was increased to 240 mm. Hg. over a period of 1 min. and was maintained at t h i s l e v e l . Tremor and extension of the hindlimbs occurred i n most rats when the pressure reached 90 to 120 mm. Hg. I f . t h i s did not occur, the rat was not included i n t h i s study. Decerebrati'on The s k u l l of the anaesthetised rat was placed i n a Student's stereotaxic holder (David Kopff) and an i n c i s i o n (2 to 4 cm.) was made across the skin of the dorsal surface of the head. The f a s c i a e and underlying layers of ti s s u e were cleared o f f the top of the s k u l l i n a region between the p a r a s a g i t t a l ridges. Lambda and bregma were i d e n t i f i e d . At a point mid-way between lambda and bregma the muscles attached to the p a r a s a g i t t a l ridges were cut free from the s k u l l . Using 62 a high speed dental d r i l l s l i t s were cut into the bone extending from the base of the skull to i t s dorsal surface, from both sides. The bone was not cut in the region directly overlying';: the parasagittal sinus. The dura was exposed undamaged and a blunted surgical needle, with dental floss, was passed, under the ventral surface of the brain. DecerebEati'onwas accomplished by tightly knotting-, the dental floss to the remaining dorsal piece of bone. Provided this procedure was care-f u l l y performed l i t t l e blood loss resulted as the major vessels and sinuses were ligated rather than cut. Some rats failed to breath spon-taneously after decerebratio'n and these rats were not used in this study. In decerebrate rats the carotid blood pressure was monitored using a Statham pressure transducer and a transducer convertor (S. E. Laboratories, Ltd.). Blood pressure was recorded on a Gilson pen recorder. . Experimental Procedures Strychnine Sulphate Strychnine sulphate (Sigma Chemical Co.) is soluble in physio-logical saline, and the drug was merely dissolved in the required volume of saline. The pH was adjusted to 7.4 i f required. Bicuculline Bicuculline (K and K Laboratories, Inc.) was dissolved in saline by adding several drops of HCl (5N) to acidify the solution. The solution was then neutralized with NaOH (5N), prior to infusion. Methysergide Methysergide (Sandoz Pharmaceuticals) was acidified to f a c i l i t a t e 63 dissolution in saline. This drug was neuralized prior to infusion. Infusion of Strychnine, bicuculline, and methysergide Intact rats had jugular cannulae implanted one day prior to testing. Single pulse stimuli of 5 or 20 v stimulus strength were presented as series of 300 stimuli with an inter-stimulus interval of 5 sec. The infusion of a drug (or saline) was begun 20 min. prior to testing of the reflex (3 ml/hr.) and continued un t i l the end of testing 45 min. later. Control rats received an infusion of saline but experimental rats received an infusion of: strychnine sulphate (30 yg/kg/min.) bicuculline (60 yg/kg/min.) strychnine sulphate (15 yg/kg/min.) and bicuculline (30 yg/kg/min.) methysergide (56 yg/kg/min.). Pre-treatment with-p?GPA P-CPA (Pfizer, Inc.) suspended in saline (30 mg/ml) was injected intraperitoneally twice prior to testing. The i n i t i a l injection was given 4 days prior to testing of the reflex and was followed by a second injection 2 days prior to testing. Each injection contained a dosage of 300 mg/kg. The procedure for preparation of this drug for injection was taken from Koe and Weissman (1966). Control rats received injections of saline. Rats received 300 stimulations with an inter-stimulus interval of 5 sec. (stimulus strength 5 or 20 v). Rats with Lesions of the h.f.d. (nucelus raphe""dorsalis) The lesion electrode was lowered into the region of the n.r.d. Using a Students stereotaxic rat holder and attached electrode holder. 64 The position of the electrode tip was determined as follows: A (frontal plane), 160; L(sagittal plane), 0; H(horizontal plane), 800 (Ko'nig and Klippel, 1963). Current was applied using a Wyss coagulator in the case of experimental rats but not,for control rats. Three days f o l -lowing this procedure, recording and stimulating electrodes were im-planted. Testing of the flexor reflex was carried out one day later. Rats were stimulated 300 times (inter-stimulus interval 5 sec.) with a stimulus strength of 5 v. The rats were perfused with fixative and sections of the midbrain were cut on a freezing microtome and stained with cresyl violet to verify the location of lesions. Comparison between Decerebrate^and Spinal Rats Spinal ligation (T,.) was performed upon control rats and decerebration upon experimental rats as earlier described. Ninety min. later flexor reflexes were tested using stimulus trains (20 v or 60 v, inter-stimulus interval 10 s e c ) . Prior to d'ecgr^ ebrntLbril polyethylene cannulae (PE 60) were inserted into the right carotid artery of each rat. Arterial blood pressure was monitored and recorded during testing of flexor reflexes. The range of mean blood pressure was 80 to 150 mm. Hg. After testing rats were perfused with fixative and their brains were removed to deter-mine the level and completeness of decerfebarati.on, Spinal Rats and Asphyxiation of the Cord Control rats and experimental rats had their cords ligated at the level of the second thoracic vertebra Cl^). Experimental rats were sub-jected to 22 min. of asphyxiation of the spinal cord. Two days after spinal ligation the flexor reflex was tested using stimulus trains 65 ( 20 v or 60 v, inter-stimulus i n t e r v a l 10 s e c ) . Infusion of Strychnine A group of s p i n a l rats (T,. l i g a t i o n ) was tested using stimulus t r a i n s . Control r a t s received an i n f u s i o n of s a l i n e whereas experimental rats received an i n f u s i o n of strychnine sulphate (30 yg/kg/min.) ad-ministered i n the same manner as i t was for i n t a c t r a t s . Two days a f t e r s p i n a l l i g a t i o n the f l e x o r r e f l e x was tested (20 or 60 v, inter-stimulus i n t e r v a l 10 s e c ) . Infusion of Strychnine or B i c u c u l l i n e A group of s p i n a l r a t s (T^) was tested using s i n g l e pulse stimu-l a t i o n . The day following s p i n a l l i g a t i o n jugular cannulae were im-planted. Two days following s p i n a l l i g a t i o n the f l e x o r r e f l e x was tested (20 v, inter-stimulus i n t e r v a l 5 s e c ) . Each rat received two s e r i e s of 300 s t i m u l i separated by 2 hours. During the second s e r i e s of s t i m u l i control rats received an i n f u s i o n of s a l i n e while experimental rats received an i n f u s i o n of strychnine sulphate (30 yg/kg/min.) or b i c u c u l l i n e (60 yg/kg/min.). A further group of rats received s a l i n e during both the f i r s t and second seri e s of s t i m u l i . Level of Spinal Transection One group of rats had t h e i r cords l i g a t e d at the l e v e l of the seventh c e r v i c a l vertebra whereas another group was l i g a t e d at the l e v e l of the tenth thoracic vertebra. The following day the f l e x o r r e f l e x was tested using stimulus t r a i n s (20 v or 60 v, intern-stimulus i n t e r v a l 10 sec.) . 66 In j e c t i o n of Strychnine i n Anaesthetized Rats Recording and stimulating electrodes were implanted as previously described. One day l a t e r , rats were anaesthetized with ether while jugular cannulae were inserted. Ether anaesthesia was discontinued and replaced by urethane anaesthesia-. (Oi 25 g/kg) . Single pulse stimu-l a t i o n (60 v, interrstimulus i n t e r v a l 1.5 sec.) was employed and EMG a c t i v i t y was integrated f o r 550 msec, a f t e r each stimulus. Two serie s of 120 s t i m u l i , separated by 2 min., were applied to both c o n t r o l and experimental r a t s . One min. p r i o r to the second s e r i e s of s t i m u l i , the co n t r o l r a t s received an intravenous i n j e c t i o n of s a l i n e (1 ml), whereas experimentals received an i n j e c t i o n of strychnine sulphate (0.4 mg/kg, 1 ml.) . 67 SECTION II Spinal Interneurones Flexor Reflex Interneurones The f l e x o r r e f l e x i s a polysynaptic r e f l e x . However, there i s v i r t u a l l y no evidence, either anatomical or p h y s i o l o g i c a l , that s p e c i f i e s which interneurones are d i r e c t l y involved i n the f l e x o r r e f l e x (Wall, 1970, 1973). The response of s p i n a l interneurones i n the lumbar region of the cord has been correlated with the a c t i v i t y of the f l e x o r r e f l e x (Groves and Thompson, 1973). The d e f i n i n g c h a r a c t e r i s t i c of f l e x o r r e f l e x interneurones i s not t h e i r capacity to evoke a c t i v i t y (or i n h i b i -tion) i n f l e x o r motorneurones, but t h e i r property of response to f l e x o r r e f l e x a f f e r e n t s . Therefore, the s p i n a l interneurones that respond to f l e x o r r e f l e x afferents may not project to motorneurones and may con-t r i b u t e to ascending afferent t r a c t s , etc. Dorsal horn interneurones, i n p a r t i c u l a r , have many c o l l a t e r a l axons which branch extensively and which may terminate at the motor n u c l e i or at supraspinal structures (Matsushita, 1969). Flexore r e f l e x interneurones may also project to various structures i n the c e n t r a l nervous system. Apparatus and Procedures The use of microelectrodes to record s p i n a l a c t i v i t y requires sub-s t a n t i a l r e s t r a i n t of the s p i n a l column. This was accomplished using a s p i n a l column holder designed s p e c i f i c a l l y for the r a t . I t i s composed 68 of a heavy metal base with two adjustable holders. One holder is used to f i x the position of the rat's hips. Attached to the second holder is a towel clamp (Irex, Co. Ltd.) which can be used to grasp a dorsal vertebral spine or process. The towel clamp i s connected to, and can be adjusted by a Narishige micromanipulator. Another such manipulator, also mounted on the second holder, serves to hold the microelectrode (Figure 7). The anterior end of the rat was suspended from a plexiglass ring which was sutured to the skin of the back in order to form an o i l bath over the exposed cord. To this ring was attached a metal rod which was in turn clamped to a standard ring stand. The snout of the rat was also tied to this support rod. Rats were anaesthetized with ether in order to permit the implantation of tracheal and jugular cannulae. Intravenous doses of 0.5 ml (25% solution) of urethane were then given repeatedly over a period of 30 min. The rats remained anaesthetized for the remaindert of the ex-periment usually without subsequent injections of urethane. The total' dosage was 0.5 g./kg. Rats were also paralysed with gallamine triethio-dide (Flaxedil, 10 mg./ml) to a total dosage of 60 mg./kg. No reflex responses could be eli c i t e d from these animals even with very intense stimuli. A laminectomy was performed to expose the lumbar enlargement. The rat was then placed in the spinal cord holder, the dural was removed, and the exposed cord was immediately covered with o i l . The temperature of this o i l bath and rectal temperature were monitored and kept at 37° C. Following paralysis with Flaxedil the rats were maintained on 69 Figure 7. Photograph showing the rat holder that was used to hold animals during the recording of interneurones i n the s p i n a l cord. 70 a rodent r e s p i r a t o r (300 cc/min., p o s i t i v e pressure 0.5 to 1.0 cm. water). A pneumothorax.was then performed i n order to prevent r e s p i r a t o r y move-ments from being transferred to the cord. Stimulating electrodes (21 gauge, hypodermic needles) were inserted i n t o the skin of the hind-paw on the same side as the recording s i t e . Occasionally electrodes were also inserted into the c o n t r a l a t e r a l hind-paw i n a l o c a t i o n homologous to that of the i p s i l a t e r a l stimulating electrodes. A microelectrode was placed into the micromanipulator and connected to a cathode follower by means of s i l v e r wire. The cathode follower was grounded to the r a t . The r a t , s p i n a l cord holder, lamp, and cathode follower were located i n s i d e a double-lined, copper wire cage f u l l y surrounded on a l l sides except for an opening at the front of the toajge which allowed the experimenter access to the preparation. The s i g n a l from the cathode follower was fed into a Tektronics 3A9 d i f f e r e n t i a l amplifier.and a Tektronics 565A o s c i l l o s c o p e from which s i n g l e unit r e -sponses could be monitored. The o s c i l l o s c o p e could be triggered simul-taneously with the cutaneous stimulation. Single pulse s t i m u l i or t r a i n s of s t i m u l i were delivered as previously described. Spikes were counted using an EKG Ltd. rate meter which had the capacity to s e l e c t the highest amplitude spikes. A Tennelec d i g i t a l rate meter was triggered following the termination of the stimulus a r t i f a c t and t h i s rate meter could be set to count spikes for a pre-selected i n t e r v a l a f t e r the stimulus. The number of s p i k e s / i n t e r v a l and the unit response i t s e l f were displayed on a B e l l and Howell u l t r a v i o l e t recorder. The power l i n e was supplied with a voltage transformer i n order to eliminate l i n e i nterference. 71 The depth of penetration of the microelectrode was noted whenever a unit was recorded. Some recording locations were marked by iontophor-e s i s of the dye pontamine sky blue (4 to 10 yA, 1 to 2 min.). F o l -lowing termination of the experiment the rat was perfused with f i x a t i v e and the cord was removed for h i s t o l o g i c a l v e r i f i c a t i o n of the recording s i t e s . Microelectrodes Hard glass tubing (Pyrex Lab. glassware: tubing lab. std. wall -2 mm.), previously cleaned i n chromic a c i d , was pul l e d and broken back, to t i p diameters ranging from 1 to 2 y with resistances ranging from 2 to 7 These electrodes were then f i l l e d with NaCI (4M) or a 2% so l u t i o n of pontamine sky blue i n sodium acetate (0.5M). F i l l i n g was accomplished by i n j e c t i n g the.desired s o l u t i o n i n t o the b a r r e l of the electrode. The t i p was then broken to the required t i p diameter and the electrode t i p f i l l e d by c a p a i l l a r y a c t i o n . Small fcubMes. could often be teased out of the t i p with lengths of very f i n e glass. E l e c -trodes which were to be f i l l e d with pontamine sky blue often f i l l e d more r e a d i l y i f the t i p was broken p r i o r to i n j e c t i o n of the s o l u t i o n into the b a r r e l of the electrode. Saline electrodes tended to have lower t i p resistances (2 - 5 M fi) than electrodes f i l l e d with the dye s o l u t i o n (4 - 7 M Q). Tip resistances were measured using a f u l l wave generator. Preparation of Pontamine Sky Blue Solution (from Hellon, 1971) 1) To a 0.5 M s o l u t i o n of sodium acetate a quantity of pontamine sky blue (ESBE Laboratories), s u f f i c i e n t to make a 2% s o l u t i o n , 72 . was*-added. The dye i s r e a d i l y soluable. 2) This s o l u t i o n was then f i l t e r e d (0.4 u m i l l i p o r e f i l t e r ) at l e a s t three times. I t was then centrifuged (Beckman micro-sample centrifuge) f o r 5 min. The supernate was the f i n a l s o l u t i o n used for f i l l i n g electrodes. Cleaning: of Glass Tubing Tubing used to construct electrodes had to be absolutely clean- i n order to permit f i l l i n g of the electrode. This was accomplished by completely immersing the glass i n a s o l u t i o n of chromic acid (potassium dichromate i n sulphuric acid) for a period of 2% hr. The tubing was rinsed at l e a s t 3 times with d i s t i l l e d water and oven dr i e d . The tubing was l e f t i n a sealed container u n t i l required. 73 SECTION III Fi x a t i o n of the Brain and Spinal Cord F i x a t i o n of the c e n t r a l nervous system for v e r i f i c a t i o n of l e s i o n and electrode t r a c t s , l e v e l of decerebration, and l o c a t i o n of pontamine sky blue markers was accomplished by in f u s i n g f i x a t i v e into the heavily-anaesthetized r a t . A thoracostomy was quickly performed and the heart freed from surrounding t i s s u e . A length of polyethylene tubing (PE 60) connected to a pressure b o t t l e containing s a l i n e , was inserted through a s l i t made i n the l e f t v e n t r i c l e and ultim a t e l y i n t o the aorta. A small s l i t was made i n the r i g h t v e n t r i c l e to allow f l u i d to escape from the vascular system. A pressure of 200 to 300 mm. Hg. was applied to the pressure b o t t l e f o r a period of 1 to 2 min. producing a s a l i n e f l u s h of the vascular system. Without removing the polyethylene tubing i t was reconnected to a second pressure b o t t l e containing a s o l u t i o n of 10% formol-saline. Pressure was applied to the second b o t t l e and the rat was perfused with f i x a t i v e f o r 20 min. The desired section of the bra i n or cord was removed and placed i n 10% formol-saline f o r 24 hrs. Sections were then cut on a freezing microtome and stained with c r e s y l v i o l e t (or safranin-0 for pontamine sky blue markers). 74 CHAPTER I I I SECTION I S t a t i s t i c a l Considerations Current research ethics demand the use of conventional s t a t i s t i c a l t e s t s . The v a l i d i t y of any s t a t i s t i c a l procedure i s d i r e c t l y dependent upon whether or not the assumptions of that s t a t i s t i c a l test are v a l i d f or the prescribed experimental conditions. S t a t i s t i c s can be a power-f u l a i d i n determining differences between the experimental and c o n t r o l populations. However, i f s t a t i s t i c a l tests are applied without a knowledge of the t e s t ' s inherent assumptions a t o t a l l y erroneous con-c l u s i o n may be derived. The l e v e l of mathematical comprehension r e -quired to determine i f the assumptions of a t e s t are upheld i s often formidable, e s p e c i a l l y when the test s t a t i s t i c i s based upon a compari-son of a sample population to a hypothetical "normal" d i s t r i b u t i o n . For t h i s reason a short discussion of the c r i t e r i a by which decisions of s t a t i s t i c a l appropriateness are derived i s appropriate. S t a t i s t i c s i s not simply the study of p r o b a b i l i t i e s . S t a t i s t i c s i s the mathematical comparison of a characterized p r o b a b i l i t y model with sampled data i n order to make inferences about the p r o b a b i l i t y of occurrence of any p a r t i c u l a r event. Seldom can the a c t u a l or " r e a l " population d i s t r i b u t i o n be described i n d e t a i l , nor can any s i n g l e proba-b i l i t y model describe a l l possible population d i s t r i b u t i o n s . The best 0 75 known probability model is the "normal" or "'Gaussian" distribution. It is not the only model nor i s i t necessarily the most appropriate model. Laplace and Gauss both described the normal distribution in relationship to the observed distribution of errors of astronomical measurement. These errors intuitively seemed to occur as frequently below the true value (a theoretical and by definition unmeasureable quantity approxi-mated by the sample mean) as above and the larger the magnitude of any error the less frequently i t would occur. Thus, the error appears to be distributed symmetrically and unimodally about the true value. The frequency of occurrance of these errors decreases moriotohdeally: as:.the error deviates in magnitude from this true value. If the magnitude of the errors is of the size (x) and i f the relative frequency of occurrence of an error of magnitude (x) is (y) then such a distribution can be described as follows: 1) dy/y = -C x dx 2) In y = -Cx2/2 + k 3) or y = e-C(*2/2) +k 4) or y = Ke~ C x 2/ 2 Equation (4) gives the probability density of a normal distribution arrived at by the method used by Laplace and Gauss (C + k, constants). Astronomical observations were found to correspond with considerable regularity to this distribution. Quetelet, the Belgian astronomer, found that variations in anthropological measures of military personnel :.als'6 corresponded to the normal distribution. For example, the variations in the heights of men were found to be distributed normally about a 76 hypothetical true population mean. From t h i s correspondence of d i s -t r i b u t i o n s with widely separated experimental measures there was a tendency to elevate the normal d i s t r i b u t i o n to the status of a law. Seemingly choatic events could be described by the order of the normal d i s t r i b u t i o n . Fisher described t h i s attempt to dogmatize the normal d i s t r i b u t i o n as follows (1923:181): A l l sorts of measurements were taken and the r a p i d l y growing c o l l e c t i o n s of s t a t i s t i c a l data r e l a t i n g to economic and s o c i a l conditions as recorded by various government s t a t i s -t i c a l bureaus furnished material for further i n v e s t i g a t i o n s . But unfortunately in. a l l these in v e s t i g a t i o n s the Gaussian error law came to act as a v e r i t a b l e Procrustean bed to which a l l possible measurements should be made to f i t . . . . S t a t i s -t i c i a n s could not c o n c i l i a t e themselves with the thought of the possible presence of "skew" frequency curves, although numerous data offered complete defiance to the Gaussian dogma and exhibited.a marked skew frequency d i s t r i b u t i o n . Sup-posedly great a u t h o r i t i e s argued naively that the reason the data did not f i t the curve of Gauss was that the observations were not numerous enough to eliminate the presence of skew-ness. In other words, skewness was regarded as a by-product of sampling and was believed could be made to^disappear com-p l e t e l y i f we would take an i n f i n i t e number of observations. Against t h i s background of the universdM-ty->* of the normal d i s -t r i b u t i o n i t became apparent that while a g r i c u l t u r e , astronomy, and economics often contained data f i t t i n g the normal d i s t r i b u t i o n the behavioural sciences were l i k e l y to deviate from the expected normality. I n t u i t i v e l y i t was also apparent that Laplacian error law contradicted the objectives of experimental science. The error law implies that as errors become i n f i n i t e t h e i r d i s t r i b u t i o n approaches, that of the normal d i s t r i b u t i o n . Thus, as the experimenter s t r i v e s f o r greater and greater c o n t r o l of h i s experimental parameters h i s data w i l l l e s s 1 77 l i k e l y resemble an approximation of the normal d i s t r i b u t i o n . I r o n i c a l l y , the power of a s t a t i s t i c a l test based upon normality i s i n d i r e c t l y dependant upon the f a i l u r e to a t t a i n experimental c o n t r o l . When the objective i s the comparison of two independant populations i t was evident that by s e t t i n g the population variances equal to each other the d e r i v a t i o n of the resultant test s t a t i s t i c could be con-siderably s i m p l i f i e d . As a consequence, parametric tests (based on normality) require an a d d i t i o n a l pre-condition of homoscedastic (equal) variances. A test of equal variances then becomes a test which s p e c i f i e s whether or not two populations are. drawn from i d e n t i c a l normal popu-l a t i o n s . I f the normality of the population d i s t r i b u t i o n s and the equality of t h e i r variances can be assumed then the means of the popu-l a t i o n s and the difference:;.between them can be s p e c i f i e d . Seldom can these conditions be met absolutely; however, they are^.usually considered to be approximately normal. This has resulted i n a b e l i e f i n the q u a s i - u n i v e r s a l i t y of the normal d i s t r i b u t i o n a conclusion which i s a t t r a c t i v e to the mathematician but which leaves an experimenter i n a quandry. Once making the assumptions, the mathematics i s simple and exact and f a s c i n a t i n g l y b e a u t i f u l : and Mathe-maticians w i l l frankly say that i t i s our concern as researchers, not t h e i r s , whether the assumptions are l e g i -timate i n the p a r t i c u l a r research s i t u a t i o n s with which we work. I t happens that i n most of the research i n our f i e l d (behaviour) the assumptions are so far fetched as to abort the r e s u l t s of c a r e f u l work. (Peters, 1943) In p a r t i c u l a r the behavioural s c i e n t i s t found that he was using a parametric test based upon i n f i n i t e sample s i z e , homoscedasticity, and normality of d i s t r i b u t i o n when he had no idea of the shape of the 78 underlying d i s t r i b u t i o n and .when he r e g u l a r l y employed very small sample s i z e s . In answer to t h i s problem s t a t i s t i c s has been returned to the approach current i n the time of B e r n o u l l i . P r o b a b i l i t i e s were expressed as the r a t i o of the number of successful outcomes of an event to a f i n i t e number of possible outcomes i n opposition to the i n f i n i t e or asymptotic d i s t r i b u t i o n where p r o b a b i l i t i e s were obtained by i n t e g r a t i o n over mathematical d e n s i t i e s . Therefore, tests which were independent of d i s t r i b u t i o n were developed so that non-normality would not o b l i t e r a t e test v a l i d i t y . " D i s t r i b u t i o n - f r e e " tests possess t h i s property. In other words, a " d i s t r i b u t i o n - f r e e " test i s independent of the d i s t r i b u t i o n of the sampled population. As a consequence, sample populations do not have to demonstrate either normality nor homoscedasticity i n order to employ such t e s t s . Tests which do require normality and homoscedas-t i c i t y of the sample populations are c a l l e d "parametric" te s t s because they depend upon these parameters of the sample populations. On the other hand, tests which are independent of these parameters are c a l l e d "non-parametric" t e s t s . Thus, they are also " d i s t r i b u t i o n - f r e e " t e s t s . This thesis makes use of parametric and^rionrparametric s t a t i s t i c a l t e s t s . Both types of tests have inherent assumptions. Non-parametric tests assume: 1) Sample observations are independent. 2) The v a r i a b l e under study has underlying continuity. 3) The v a r i a b l e under study can be measured at l e a s t on the o r d i n a l scale (some tests v a l i d f o r measurements made of the nominal s c a l e ) . 79 Parametric tests assume a l l of the above except (3) and also assume: 4) Sample populations are normally d i s t r i b u t e d . 5) Sample variances are equal (homoscedastic). 6) Measurements are made at least on the i n t e r v a l scale. Most of the above assumptions can only be assumed to be true or at le a s t almost true (Siegel, 1956). However, the homoscedasticity of variances can be tested using Snedecor's F test (or Variance Ratio t e s t ) . Non-parametric t e s t s have some advantages i n c e r t a i n s i t u a t i o n s , whereas parametric te s t s have advantages over non-parametric te s t s i n other s i t u a t i o n s . The advantages of non-parametric tests are summarized as follows: 1) Non-parametric te s t s make few assumptions about the population d i s t r i b u t i o n . 2) I f small sample si z e s are used (n = 6) then there i s no a l t e r n a t i v e but to use a non-parametric t e s t . In f a c t the " s t a t i s t i c a l e f f i c i e n c y " (Bradley, 1968) of non-parametric tests i s greatest with small sample sizes and l e a s t with i n f i n i t e sample s i z e s . 3) Non-parametric tests are derived with minimal mathematics. 4) They are e a s i l y applied and require l i t t l e time i n t h e i r a p p l i c a t i o n . 5) Even i f a l l the assumptions of parametric tests are upheld non-parametric tests are only s l i g h t l y l e s s powerful than parametric t e s t s . The disadvantages of non-parametric tests are summarized as follows: 1) Provided a l l the assumptions of the parametric test are met the non-parametric test i s wasteful of information. This can be overcome simply by increasing sample s i z e , however. 80 2) Non-parametric tests cannot be adapted to study i n t e r a c t i o n s i n the analysis of variance model without making s p e c i a l assumptions about a d d i t i v i t y . In t h i s regard, the an a l y s i s of variance model requires one a d d i t i o n a l assumption. This assumption i s that the means of normal and homoscedastic populations must be l i n e a r combinations of e f f e c t s due to columns and/or rows. The a p p l i c a t i o n of s t a t i s t i c a l tests involves several steps and these steps are l i s t e d as follows: 1) The n u l l hypothesis (H Q) i s stated. 2) The appropriate s t a t i s t i c a l t e s t i s chosen depending on whether or not the experimental data are v a l i d f o r the assumptions underlying the chosen t e s t . 3) A sample s i z e i s chosen (N) and a l e v e l of s i g n i f i c a n c e i s s p e c i f i e d . 4) The sampling d i s t r i b u t i o n i s determined or i t i s assumed. 5) A region of r e j e c t i o n of H D i s chosen. 6) The value of the s t a t i s t i c a l test i s calculated f o r the experimental data. If th i s value l i e s within the region of r e j e c t i o n the H o may be rejected. The a p p l i c a t i o n of s t a t i s t i c a l tests and the required assumptions are summarized and presented as follows: 1) The analysis of the data from t h i s thesis employs a common H Q. The f l e x o r response of the co n t r o l group i s assumed to be i d e n t i c a l to that of the experimental group. In other words, the mean (u) responses from each group are assumed to be equal (H Q:u(control) = u (experimental)). 81 The al t e r n a t e hypothesis or the research hypothesis i s that the experimental group has a response greater (or less than) that of the co n t r o l group. Thus, there are two research hypotheses (H-^ : y(experimental) > y (control) and H2: y (experimental) < y ( c o n t r o l ) ) . 2) The l e v e l of s i g n i f i c a n c e (p) i s the p r o b a b i l i t y of r e j e c t i n g H 0 when i t i s i n fact true (type I e r r o r ) . The lowest value of (p) accepted i n t h i s thesis was 0.050 a value which i s commonly accepted i n the behavioural sciences (Siegel, 1956). Thus, i f the value of a s t a t i s -t i c a l test was found to be equal to or less than 0.050 the H Q was r e -jected i n favour of the research hypothesis (a s i g n i f i c a n c e l e v e l of 0.052 was accepted f o r the Mann Whitney U tes t s when ^ - 7). 3) The sample d i s t r i b u t i o n was assumed to be normal. 4) The region of r e j e c t i o n was determined from H-^  or and there-fore one-tailed regions of r e j e c t i o n were employed. 5) The f l e x o r response i s assumed to be measured on a r a t i o scale. 6) Individual observations (responses) are assumed to be independent. 7) Experimental and control groups were assumed to be independent. Therefore, the s t a t i s t i c a l tests employed were Student's t - t e s t , two-way analysis of variance, Snedecor's F t e s t , and Mann-Whitney U test (Bailey, 1959; S i e g e l , 1956; Snedecor, 1956). There were two exceptions to t h i s independence of experimental and cont r o l groups. In experiments 2D and 3A c o n t r o l and experimental groups were rel a t e d or dependent (animals serve as t h e i r own controls) and a Wilcoxon Matched-Pairs Signed-Ranks test was employed (Siegel, 1956). At no time can data from co n t r o l and experimental groups be pooled unless the groups are dependent. 82 8) P r i o r to the a p p l i c a t i o n of any parametric test (Student's t - t e s t , two-way ana l y s i s of variance) Snedecor's F t e s t was applied i n order to determine i f the assumption of equal variances would be upheld. The only occasion when the variances were found to be s i g n i f i c a n t l y d i f f e r e n t was f o r experiments 2B and 2E. Cochran's modification of the t - t e s t (Snedecor, 1956) was then applied because i t corrects for unequal variances. However, non-parametric tests do not make t h i s assumption and a p p l i c a t i o n of the Mann-Whitney U tests was more appropriate (and indeed more powerful). 9) The data was presented i n many cases, g r a p h i c a l l y . Such a presentation usually includes a measure of dispersion and often standard errors are drawn on the graphs. Standard errors are not measures of dispersion f o r non-parametric t e s t s and should not be included for data tested i n t h i s manner ( i n t e r q u a r t i l e ranges are the non-parametric counterpart but this measure i s seldom included i n a graphic presentation). When parametric tests were employed standard errors were not included i n the graphic presentation f o r a number of reasons which are as follows: a) The value of the s t a t i s t i c a l test gives the exact answer to the question of whether or not the H Q should be rejected whereas standard errors give only a crude approximate. b) The use of standard errors on the graphs of t h i s thesis often confused rather than c l a r i f i e d the data presented. 10. The choice of s t a t i s t i c a l tests was based upon three f a c t o r s : 1) the appropriateness of the test (for example do the data support the assumptions of the test) 2) the power of the test (The power of a test i s defined as the p r o b a b i l i t y of r e j e c t i n g the H G when i t i s i n fa c t f a l s e , 83 i . e . , the power i s equal to one minus the type II error. The type II error i s the p r o b a b i l i t y of accepting the H q when i t i s f a l s e . ) 3) the power-efficiency of the te s t . The power-efficiency of a tes t i s defined as the increase i n sample sizenecessary to make one s t a t i s t i c a l test as powerful as another s t a t i s t i c a l t e s t . For example the power-e f f i c i e n c y of the Mann-Whitney U test i s approximately 95% for moderate sample sizes (N = 9 to 20) when compared to the most powerful parametric test (Student's t-test) (Siegel, 1956). 84 SECTION II Methological Considerations and Data Presentation Habituation of behavioural responses cannot be assumed to be the r e s u l t of the operation of a s i n g l e mechanism and i t i s l i k e l y that at l e a s t two or more processes are responsible for determining the degree of response decrement (Hinde, 1970; Groves and Thompson, 1970; Peeke and Peeke, 1973). This complication of i n t e r a c t i o n of a number of processes i s further aggravated by a lack of uniformity i n the method used to measure the degree (or rate) of habituation. The degree of habituation i s measured i n three ways which are l i s t e d below: 1) The degree of habituation may be measured as the number of stimulations required to reach a s p e c i f i e d response l e v e l . For example, the number of stimulations required to extinguish the response i s often • taken as the s p e c i f i e d c r i t e r i o n . This may require as few as 20 stimu-l a t i o n s i n the case of the galvanic skin response (Jackson, 1974) or i t may take many hundreds of stimulations as i n the case of the f l e x o r r e f l e x ( G r i f f i n and Pearson, 1967). This i s not a p a r t i c u l a r l y u seful measure when the f l e x o r r e f l e x i s employed as the r e f l e x may never reach zero response (dependent upon stimulus i n t e n s i t y ) . The response does tend to approach an asymptotic l e v e l but i f t h i s l e v e l i s taken as the c r i t e r i o n l e v e l i t must be recognized that i t i s only an approximate value. Further-more, t h i s measure i s only a p a r t i c u l a r case of the absolute decrement of response (described below (2)) measure. 85 2) The degree of habituation may be measured as the absolute change i n response during the habituation procedure. This absolute measure i s often found to be d i r e c t l y r e l a t e d to the amplitude of the i n i t i a l response (Peeke, 1969; Groves and Thompson, 1970). 3) The degree of habituation may be measured as the r e l a t i v e decre-ment of response during the habituation procedure. There are three methods of measuring r e l a t i v e habituation. One technique widely used when measuring habituation has been to give a small number of s t i m u l i at widely spaced i n t e r v a l s p r i o r to the habituation procedure (testing of habituation) Groves, et a l . , 1969; Thompson, et a l . , 1973). The average of these responses i s then used as a reference to which test responses (recorded during the habituation procedure) are compared. A s i m i l a r technique was used by Wickelgren (1967a) but a further ser i e s of widely spaced s t i m u l i were given following the t e s t i n g of habituation. The degree of habituation was then considered to be the percentage decrease i n response of the post-test stimuli, to the pre-test s t i m u l i . These methods have the disadvantage that the pre- and post-test s t i m u l i may themselves a l t e r habituation (Farel, 1971; Davis, i n press). This t h e s i s avoids using pre- or post-test s t i m u l i and uses the i n i t i a l response of the f l e x o r r e f l e x as a reference. A l l other responses were then expressed as percentages of t h i s reference. This method of data presentation was recommended by F i g l e r (1970) and has been employed previously i n order to study habituation and s e n s i t i z a t i o n of the f l e x o r r e f l e x ( G r i f f i n and Pearson, 1968a; Pearson and Krajina, 1972; Pearson, 1974). The r e l a t i v e measure of habituation i s independent of the amplitude of the f l e x o r r e f l e x . 86 To i l l u s t r a t e how these various measures of habituation can be used to a r r i v e at c o n f l i c t i n g conclusions three hypothetical response curves are presented. These curves are i l l u s t r a t e d i n Figure 8 and are labeled a, b, and c. From t h i s f i g u r e i t can be shown that the degree of habituation depends upon the type of measure employed. If the time (number of sti m u l i ) to reach c r i t e r i o n i s taken as a measure of habituation the greatest habituation occurred with curve c(c > b > a). If the absolute change i n response i s taken as a measure of the degree of habituation the greatest habituation occurred with curve a (a > c > b). On the other hand, the greatest r e l a t i v e decrement of response occurred with curve c (c > a > b). This thesis employs a r e l a t i v e measure of habituation because r e l a t i v e measures of habituation are independent of the amplitude of the r e f l e x and because a r e l a t i v e measure can be used to answer t h i s important question: What i s the r e l a t i o n s h i p of the f i n a l response l e v e l to that of the i n i t i a l response level? The responses of each animal were converted to the r e l a t i v e measure p r i o r to averaging the data for a group of animals i n order to maintain an independence from response amplitude. The im-portance of t h i s technique can be i l l u s t r a t e d by giving several simple (hypothetical) response curves. Figure 9 i l l u s t r a t e s the response curves for a sample population of two animals (a and b). Curve c represents the average (absolute) response curve for the sample population and curve d i s the average ( r e l a t i v e ) response curve derived by the method employed i n t h i s t h e s i s . Curves a and b demonstrated i d e n t i c a l degrees of r e l a t i v e habituation and the sample variance i s equal to zero for curve d. The 0 4 T 6 ~r 8 1 0 Stimulus Number Figure 8. The degree of habituation i s shown to be dependent upon the type of measure used to determine habituation. Three hypothetical response curves are presented (a, b, and c ) . Figure 9. Demonstration of the independence of the r e l a t i v e measure of habituation from response amplitude. Two hypothetical response curves are presented (a and b above) and the average of these two curves i s also shown (c). The r e l a t i v e response curve i s shown below (d). The variance of the r e l a t i v e curve i s not effected by differences i n response amplitude but the average (absolute) response curve has a variance dependent upon response amplitude. 89 absolute measure of response decrement was 3 units for curve a and 1.5 units for curve b. The sample variance (maximum) for curve c i s 2.25 u n i t s . This serves to i l l u s t r a t e that the absolute measure of habituation i s dependent upon response amplitude while the r e l a t i v e measure i s independent. Furthermore, i f we compare two sample populations i t would not be s u r p r i s i n g to f i n d that the absolute measure i s i n s e n s i t i v e to differences i n habituation (because of dependence upon response amplitude) while the r e l a t i v e measure i s more s e n s i t i v e to group differences i n the degree of habituation. However, i t can not be e m p i r i c a l l y assumed that an a l t e r a t i o n i n response amplitude w i l l not modify the degree of habituation. To be more s p e c i f i c , an experimental manipulation may a l t e r the amplitude of the r e f l e x and also a l t e r the r e l a t i v e measure of habituation. For example, i t has been suggested that glutamate may be the transmitter released from primary afferent terminals i n the s p i n a l cord. The action of glutamate and of primary afferent terminals upon sp i n a l interneurones i s blocked or reduced by the glutamate anatagonist glutamic a c i d d i e t h y l ester (Haldeman and McLennan, 1972). Infusion of t h i s drug might therefore reduce the sensory input to the s p i n a l cord and reduce the amplitude (contribution of polysnaptic pathways) of the f l e x o r r e f l e x . Figure 10 i l l u s t r a t e s the habituation curves for r a t s r e c e i v i n g an i n f u s i o n of s a l i n e (controls) or glutamic a c i d d i e t h y l ester. This drug caused a s i g n i f i c a n t reduction i n the i n i t i a l response amplitude of the f l e x o r r e f l e x (p f 0.010, Mann-Whitney U t e s t ) , and the r e l a t i v e degree of habituation was s i g n i f i c a n t l y l e s s i n these r a t s compared to those r e c e i v i n g s a l i n e (p f 0.010, Mann-Whitney U t e s t ) . 90 z °~\ 1 r 1 , , , 21-25 46-50 71-75 96-100 121-125 146-150 Stimulus Number Figure 10. The e f f e c t of glutamate acid d i e t h y l ester (GDEE) on habituation of the f l e x o r r e f l e x ( i n t a c t r a t s ) . Infusion of s a l i n e (controls, 8 rats) or GDEE (8 rats) was begun 1 min. p r i o r to t e s t i n g of the r e f l e x and was continued u n t i l i t s termination. GDEE reduced the r e l a t i v e degree of habituation (p 5 0.010, Mann-Whitney U test) by reducing the i n i t i a l response amplitude (p 5 0.010). r 91 It i s u n l i k e l y , however, that t h i s a l t e r a t i o n i n r e l a t i v e habituation i s due to any factor other than a reduction i n the number of s p i n a l i n t e r -neurones p a r t i c i p a t i n g i n the r e f l e x . Often the r e l a t i v e measure of habituation has been presented without any i n d i c a t i o n of the i n i t i a l response amplitude. This i s an important question as the amplitude of the f l e x o r r e f l e x i s d i r e c t l y r e l a t e d to stimulus i n t e n s i t y and because the parametric c h a r a c t e r i s t i c s of habituation are dependent upon the type of measure employed. In f a c t , Groves and Thompson (1970) have proposed that the r e l a t i v e degree of habituation i s inversely r e l a t e d to stimulus i n t e n s i t y but that the absolute degree of habituation i s d i r e c t l y r e l a t e d to stimulus i n t e n s i t y . Therefore, i n t h i s thesis the average absolute response curves for each sample population are presented as w e l l as the r e l a t i v e measures of habituation. The usual method of presenting the absolute response i s to average the absolute responses of the animals i n each group. While t h i s allows a determination of whether or not the amplitude of the f l e x o r r e f l e x i s greater i n one group than another i t i s a r e l a t i v e l y poor measure of the degree of habituation. For example curve c i n Figure 9 i s an example of t h i s method of presenting the data. Although t h i s curve indicates a 50% decrement of response the variance (maximum) of t h i s curve i s equal to 2.25 units. In comparison the average r e l a t i v e response curve (d) has zero variance. Admittedly t h i s example i s an extreme case but i t serves to i l l u s t r a t e that the variance of the absolute curve increases with dispersion of values of r e f l e x amplitude whereas the variance of 92 the r e l a t i v e response curve does not. However, the r e l a t i v e response curve i s p a r t i c u l a r l y , s e n s i t i v e to sample population heterogeneity, with regard to habituation and s e n s i t i z a t i o n . Figure 11 gives a n i ' ! extreme example of t h i s type of population heterogeneity. Curve a demonstrates habituation (absolute or r e l a t i v e ) . The average absolute response curve (c) shows neither habituation nor s e n s i t i z a t i o n but the maximum variance of the curve i s s t i l l 2.25 u n i t s . The average r e l a t i v e response curve (d) indicates a s l i g h t s e n s i t i z a t i o n but the maximum variance of t h i s curve i s enormous (variance = 5,625%). Thus, i t i s absolutely c r i t i c a l to r e t a i n some form of absolute response measure to guard against the e f f e c t s of population heterogeneity. . c -Another factor i n the presentation of data, also of some importance i s the representation of data points (response values) i n blocks (averages of small numbers of responses). If data i s presented i n t h i s manner an early s e n s i t i z a t i o n of the f l e x o r r e f l e x (spinal animal) i s overlooked (Groves, et a l . , 1969). In the i n t a c t animal an i n i t i a l response i n c r e -ment may also be overlooked. For example Figure 12 shows f l e x o r responses of two groups of i n t a c t r a t s tested with a stimulus i n t e n s i t y of 5 or 20 v. Rats tested with a 5 v. stimulus showed an i n i t i a l increment of response while rats tested with a 20 v. stimulus showed a r e l a t i v e l y rapid and greater degree (absolute and r e l a t i v e ) habituation. If the data i s now presented as blocks of responses (averages of 10 responses) the i n i t i a l increment of response (rats tested with 5 v. st i m u l i ) i s not apparent (Figure 13). Furthermore, over the course of 300 stimulus presentations the r e l a t i v e degree of habituation was s i g n i f i c a n t l y Figure 11. Demonstration of the s e n s i t i v i t y of the r e l a t i v e measure of habituation to sample population heterogeneity. Two hypothetical response curves are presented (a and b, above) and the average of the two curves i s shown ( c ) . The r e l a t i v e response curve i s shown below (d). The variance of the average response curve (c) i s r e l a t i v e l y small compared to that of the r e l a t i v e response curve (d). This type of population heterogeneity r e s u l t s i n very large dispersion of the r e l a t i v e measures. 94 • • 20 Volts o o 5 » Stimulus Number Figure 12. Response of the flexor r e f l e x to repeated stimulation with a stimulus strength of 5 v. or 20 v. The greatest degree of habituation (absolute or r e l a t i v e ) occurred with the group tested with 20 v. s t i m u l i . The data are presented as averages of i n d i v i d u a l responses. These data were taken from control rats used i n the comparison with rats which received an in f u s i o n of strychnine. CO <D CO c o Q. CO 0> a: t5 c o CD H— O <S a) co c o CL CO CD o: o X CD 100 20 V o—o 5 V 95 100 200 Stimulus Number 300 Figure 13. Response of the f l e x o r r e f l e x to repeated stimulation with a stimulus strength of 5 v. or 20 v. The greatest r e l a t i v e decrement of response occurred with the group tested with 5 v. s t i m u l i . The responses are blocked (averaged i n groups of 10) and presented as percentages of the f i r s t block of responses. This i s the same data that were shown i n Figure 12. 96 greater for rats tested at 5 v. compared to those tested at 20 v. (Figure 13, p 1 0.010; Mann-Whitney U t e s t ) . Thompson, et a l . (1973) have recently suggested that stimulus i n t e n s i t y has only a weak e f f e c t upon the r e l a t i v e degree of habituation. This parametric c h a r a c t e r i s t i c of habituation i s open to i n t e r p r e t a t i o n . On the basis of t h i s r e s u l t two decremental components of the f l e x o r r e f l e x may be defined as follows: 1) a decrement of response apparent early i n the test s e r i e s provided the i n t e n s i t y of stimulation i s r e l a t i v e l y great 2) a decrement of response apparent l a t e i n the test s e r i e s which i s only weakly rel a t e d to stimulus i n t e n s i t y . A number of the experimental manipulations employed i n t h i s thesis are dependent upon or are performed upon the i n t a c t , decerebrate, or s p i n a l r a t . However, decerebration and s p i n a l transection can cause a s u b s t a n t i a l modification of the f l e x o r r e f l e x . For. example, the f l e x o r . r e f l e x i s t o n i c a l l y i n h i b i t e d following decerebration (Holmqvist and Lundberg, 1961). In a d d i t i o n , s p i n a l transection can cause a tem-porary suppression of the f l e x o r reflex.due to the phenomenon of s p i n a l shock, however, s p i n a l shock i s e i t h e r very short l i v e d or non-existent i n the s p i n a l r a t . There i s some i n d i c a t i o n that even i n primates s p i n a l shock may be due to vascular d i s r u p t i o n . Intra-dural section of the cord may prevent s p i n a l shock i n the monkey f o r example (Denny- . Brown, et a l . , 1973). The a p p l i c a t i o n of repeated s t i m u l i c h a r a c t e r i s t i c a l l y e l i c i t s a smaller amplitude f l e x o r r e f l e x (biceps femoris) i n the s p i n a l com-pared with the i n t a c t rat (Figure 14). This diffe r e n c e i n response 97 Intact Stimulus Number Spinal If*'1 ' It 5 0 1 5 0 5 0 / J V 100 msec 5 0 0 Figure 14. EMG discharge evoked i n response to stimulation of the i p s i l a t e r a l hind paw. In the l e f t column are shown the responses obtained from a r a t with an i n t a c t s p i n a l cord. In the ri g h t column are shown the responses from a s p i n a l r a t . Habituation i n the in t a c t rat and s e n s i t i z a t i o n i n the s p i n a l r at are i l l u s t r a t e d . This fi g u r e i s taken from Pearson and Wenkstern (1972:110). 98 amplitude i s diminished with many stimulus r e p e t i t i o n s . As a consequence the i n t a c t rat shows an i n i t i a l decrement of response to a l e v e l cor-responding to that attained i n an incremental manner by the s p i n a l r at (Figure 15). This d i s t i n c t i o n i n response amplitude may be re l a t e d to the depression of r e f l e x "after-discharge" that occurs following s p i n a l transection (Forbes, et a l . , 1923). In order to e l i c i t an "after-discharge" of the f l e x o r r e f l e x i n the s p i n a l animal Sherrington (1906) used a stimulus t r a i n as opposed to a s i n g l e pulse stimulation. Sherrington (1906) also found that the s i z e of the "after-discharge" was d i r e c t l y r e l a t e d to stimulus i n t e n s i t y and that the maximum "after-discharge" occurred following ces-sat i o n of the stimulus t r a i n . Therefore, i n order to evoke a discharge of s u f f i c i e n t s i z e to demonstrate habituation of the f l e x o r r e f l e x i n the s p i n a l and decerebrate rat i t was necessary to use high i n t e n s i t y stimulus t r a i n s . 99 Figure 15. Mean f l e x o r r e f l e x responses to s t i m u l i applied at 10 sec. i n t e r v a l s (top) and 1 sec. i n t e r v a l s (bottom). Habituation i n i n t a c t rats i s shown on the l e f t , and s e n s i t i z a t i o n i n s p i n a l rats i s shown on the r i g h t . This f i g u r e i s taken from Pearson and Wenkstern (1972:113). 100 CHAPTER IV RESULTS: SECTION I The Flexor Reflex Experiments on Unanaesthetized Rats with Intact Spinal Cords IA. Infusion of Strychnine The e f f e c t of strychnine of f l e x o r r e f l e x responses to 300 s t i m u l i , at an i n t e n s i t y of 5 v., i s i l l u s t r a t e d i n Figure 16,(above). The responses to each successive group of 10 s t i m u l i were averaged and ex-pressed i n absolute units (volts per in t e g r a t i o n period: 250 msec.) or as percentages of the average of the f i r s t 10 responses ( r e l a t i v e measure). The amplitudes of responses were con s i s t e n t l y and s i g n i f i c a n t l y greater i n those rats to which strychnine had been administered than they were i n control r a t s . The r e l a t i v e degree (and rate) of habituation was unaffected by strychnine i n f u s i o n (Figure 16, below). For experiments i n which a stimulus strength of 20 v. was used administration of strychnine resulted i n an impairment of r e l a t i v e habituation (Figure 17, below), but did not cause a s i g n i f i c a n t e levation of the l e v e l of r e f l e x response to the i n i t i a l , s t i m u l i (Figure 17, above). The 5 v. stimulus has been found to be ju s t suprathreshold ,for r e f l e x ac-t i v a t i o n of the biceps femoris muscle (unpublished observation) and the 20 v. stimulus has been found to be j u s t suprathreshold f o r group IV afferents i n the s u r a l nerve (unpublished observation). IB. Infusion of Bicu c u l l i n e The e f f e c t of b i c u c u l l i n e on habituation of the r e f l e x (20 v. stim u l i ) 101 — I 1 I I I 100 200 300 Stimulus Number Figure 16. The e f f e c t of strychnine on habituation of the f l e x o r r e f l e x . The con t r o l group received an i n f u s i o n of s a l i n e (15 rats) and experimental rats received an i n f u s i o n of strychnine (10 r a t s ) . The stimulus i n t e n s i t y was 5 v. The absolute response of experimental rats was greater than that of the controls (p - 0.010, Student's t - t e s t ) . The r e l a t i v e degree of habituation was the same i n both groups (below). Figure 17. The e f f e c t of strychnine on habituation of the f l e x o r r e f l e x . The con t r o l group received an i n f u s i o n of s a l i n e (15 rats) and experimental rats received an i n f u s i o n of strychnine (12 r a t s ) . The stimulus i n t e n s i t y was 20 v. The absolute response of experimental rats was s i g n i f i c a n t l y greater than that of controls (p S 0.010, Student's t - t e s t ) . The r e l a t i v e degree of habituation was greater i n control animals (p - 0.010, Student's t - t e s t ) . 103 i s shown i n Figure 18. As was the case with strychnine , t h i s drug also reduced the r e l a t i v e degree of habituation (Figure 18, below). However, b i c u c u l l i n e had no s i g n i f i c a n t action on the absolute response amplitude of the r e f l e x . This i s an example where the r e l a t i v e measure of habi-tuation i s s e n s i t i v e to an a l t e r a t i o n i n habituation that i s obscured i n the absolute response curve due to i t s dependence upon response amplitude (see p. 91 , t h i s t h e s i s ) . IC. Infusion of both Strychnine and B i c u c u l l i n e The responses from experiments i n which a combination of strychnine and b i c u c u l l i n e were given (20 v. stimuli) were compared to those i n which strychnine or b i c u c u l l i n e were given alone (Figure 19). Whereas the drugs given together did not r e s u l t i n an impairment of the r e l a t i v e degree of habituation a f t e r 300 s t i m u l i , there was a greater impairment of decrement ( i n fact an increment occurred) during the early stages of the experiment than was apparent for e i t h e r strychnine or b i c u c u l l i n e alone. The combined drug i n f u s i o n also reduced the r e l a t i v e degree of habituation with regard to animals receiving an i n f u s i o n of s a l i n e . ID. Pre-treatment with p-CPA Pre-treatment of rats with p-CPA caused an a l t e r a t i o n i n e x c i t a -b i l i t y of the f l e x o r r e f l e x i n rats tested at e i t h e r 5 or 20 v. Figures 20 and 21 (above) i l l u s t r a t e the changes that occurred i n absolute response. Regardless of the i n t e n s i t y of stimulation responses to the i n i t i a l s t i m u l i i n the p-CPA treated animals were greater than those i n t h e i r respective controls. Following the presentation of 300 s t i m u l i t h i s r e l a t i o n s h i p was reversed and therefore the absolute degree of 104 Stimulus Number Figure 18. The e f f e c t df b i c u c u l l i n e on habituation of the f l e x o r r e f l e x . . The co n t r o l group received an i n f u s i o n of s a l i n e (10 rats) and the experimental groups re-ceived an i n f u s i o n of b i c u c u l l i n e (10 r a t s ) . The ab-solute measure indic a t e d no s i g n i f i c a n t differences between control and experimental rats (above). The r e l a t i v e degree of habituation was greatest i n con-t r o l animals (p - 0.010, Student's t - t e s t ) . 105 ure 19. The e f f e c t of a combination of strychnine and b i c u c u l l i n e (9 rats) on habituation of the f l e x o r r e f l e x i s compared to that of strychnine and b i c u c u l l i n e alone. The a s t e r i s k (*) indicates a s i g n i f i c a n t difference (Student's t - t e s t , p f 0.050). Further control groups (s a l i n e infusion) are shown i n the two previous f i g u r e s . 106 o 1-5 96-100 196-200 296-300 Stimulus Number ure 20. The e f f e c t of pre-treatment with p-CPA on habituation of the f l e x o r r e f l e x using a stimulus strength of 5 v. Con-t r o l rats (11 rats) received sham implants whereas experi-mental rats (10 rats) received p-CPA implants. The r e l a t i v e degree of habituation was s i g n i f i c a n t l y greater f o r experi-mental rats (below). Furthermore, the i n i t i a l responses f o r experimental rats were s i g n i f i c a n t l y greater than those f o r controls, and f i n a l responses were s i g n i f i c a n t l y lower for experimental rats (above). See the.text f o r discussion of s i g n i f i c a n c e l e v e l s . 107 o 1-5 96-100 196- 200 296-300 Stimulus Number ure 21. The effect of pre-treatment with p-CPA on habituation of the flexor reflex using a stimulus strength of 20 v. Each group consisted of 9 rats. Pre-treatment with p-CPA had the same effect upon absolute and relative response;" curves as i t did when the rats were tested with a stimulus strength of 5 v. See the text for discussion of s i g n i f i -cance levels. 108 habituation was greater i n p-CPA treated animals. A two-way analysis of variance (Bailey, 1959) was used to assess the i n i t i a l arid f i n a l differences i n response amplitude. The i n i t i a l responses i n treated rats; were s i g n i f i c a n t l y greater (p - 0.010) than those i n the control group and the f i n a l responses i n treated rats were s i g n i f i c a n t l y lower (p - 0.010). than those of t h e i r appropriate controls. The p r o b a b i l i t y l e v e l s r e f e r only to the i n i t i a l and f i n a l 20 responses. <• Treated rats demonstrated a s i g n f i c a n t l y greater degree of r e l a t i v e habituation (p < 0.025 for rats tested at 20 v. and p < 0.001 f o r rats tested at 5 v) as assessed using the Mann-Whitney U t e s t . In accordance with the parametric c h a r a c t e r i s t i c s of habituation the greatest degree of r e l a t i v e habituation occurred with the group of c o n t r o l rats tested with the 5 v. s t i m u l i i n comparison with the c o n t r o l group tested with 20 v. s t i m u l i (Mann-Whitney U t e s t , p - 0.050). IE. Rats with le s i o n s of the n.r.d. (nucleus raphe''dorsalis) The e f f e c t o f - l e s i o n s of the n.r.d. on f l e x o r r e f l e x amplitude i s shown i n Figure 22 (above). The stimulus strength was 5 v. Confirmation of the locations of these le s i o n s of the n.r.d. was demonstrated i n a l l rats included i n the experimental group and the extent of these le s i o n s i s i l l u s t r a t e d i n Figure 23. Two-way analysis of variance was applied to the i n i t i a l and f i n a l 20 responses. In lesioned rats the i n i t i a l amplitude of the r e f l e x was s i g n f i c a n t l y greater than that of controls and the f i n a l response amplitude was s i g n i f i c a n t l y lower (p < 0.001). The r e l a t i v e degree of habituation was s i g n i f i c a n t l y greater i n lesioned rats than i n controls (p - 0.001; Mann-Whitney U t e s t ) . 109 1-5 96-100 196-200 296-300 Stimulus Number ure 22. The effect of a lesion in the n.r.d. on habituation of the flexor reflex using a stimulus strength of i'Sjv. Control rats were sham lesioned whereas experimental rats were lesioned in the n.r.d. The greatest relative degree of habituation occurred with experimental rats (below). The i n i t i a l response amplitude was greater in experimental rats and the f i n a l response amplitude was greater in the control rats (above). See;the text for discussion of significance levels. 110 Figure 23. The extent of the lesions of the n.r.d. i s indicated by the shaded area. This diagram was been redrawn from the at l a s of Konig and K l i p p e l (1963). I l l Two-way analysis of variance was performed on the i n i t i a l and f i n a l 20 responses of the treated (and lesioned) rats and t h e i r appropriate controls. Such an analysis i n d i c a t e d a s i g n i f i c a n t e f f e c t of t r i a l s (p 5 0.010) f o r a l l comparisons. This simply means that there was a s i g n i f i c a n t l y d i f f e r e n t e f f e c t of repeating the stimulus on the experi-mental as opposed to the c o n t r o l group. There was no s i g n i f i c a n t e f f e c t of groups which i s not s u r p r i s i n g as the experimental and c o n t r o l r e -sponse curves i n v a r i a b l y crossed over each other (the group analysis simply asks i f the experimental group tended to be greater or l e s s than the control group without regard to differences occurring at d i f f e r e n t places on the curves). A s i g n i f i c a n t t r i a l s versus group i n t e r a c t i o n was demonstrated with animals pre-treated with p-CPA and tested with 5 v. s t i m u l i (p ^ 0.050) and f o r lesioned rats (p - 0.010), therefore demonstrating a difference i n the rate of habituation between experi-mental and control animals. IF. Infusion of Methysergide This dosage of methysergide d i d not have a s i g n i f i c a n t e f f e c t upon the habituation of the f l e x o r r e f l e x . However, the i n i t i a l few re-sponses were s i g n i f i c a n t l y , l a r g e r than comparable co n t r o l responses (Student's t - t e s t , p 1 0.050) (Figure 24, above). This resulted i n a s l i g h t increase i n the i n i t i a l r e l a t i v e s e n s i t i z a t i o n of the r e f l e x (not s t a t i s t i c a l l y s i g n i f i c a n t ) (Figure 24, below). 112 Figure 24. The e f f e c t of methysergide on habituation of the f l e x o r r e f l e x using a stimulus i n t e n s i t y of 20 v. Each group consisted of 9 r a t s . The asterisks^*) i n d i c a t e that the response during i n f u s i o n of the drug was s i g n i f i c a n t l y greater than the corresponding control responses (above). The r e l a t i v e degree of s e n s i t i z a t i o n i s shown below. 113 Experiments on Unanaesthetized Rats with Transected •"" Spinal Cords (or Decerebration) 2k. Comparison between Decerebrate and Spinal Rats Rats which had undergone s p i n a l transection exhibited f l a c c i d p a r a l y s i s of the hind limbs, but the muscular tone of decerebrate r a t s was not obviously d i f f e r e n t from that of i n t a c t r a t s . These decerebrate rats displayed exaggerated s t a r t l e response to strong s t i m u l i , and the ap p l i c a t i o n of a strong pinch to a hind-paw resulted i n a powerful r e f l e x withdrawal of the limb. The magnitude of t h i s response quickly diminished with repeated stimulation. S i m i l a r i l y , "pseudaffective r e f l e x e s " such as v o c a l i z a t i o n and o r i e n t a t i o n of the head towards the s i t e of stimulation, when present, also gradually diminished. These findings are s i m i l a r to those described by Sherrington (1906). Dis-habituation of the FWR could be brought about by pinching the contra-l a t e r a l hind-paw. For decerebrate rats the r e l a t i v e degree of habituation of the fl e x o r r e f l e x was not s i g n i f i c a n t l y d i f f e r e n t to that of s p i n a l rats provided the i n t e n s i t y of the stimulus t r a i n s was 20 v. (Figure 25, below). The amplitude of the f l e x o r r e f l e x was not s i g n i f i c a n t l y d i f f e r e n t i n decerebrate rats when they were compared to equivalent responses i n s p i n a l rats (p < 0.052), Mann-Whitney U t e s t ) . When a stimulus strength of 60 v. was employed the f l e x o r r e f l e x responses of decerebrate rats d i f f e r e d .from those of s p i n a l r a t s both i n terms of the amplitude of the responses and with regard the the r e l a t i v e degree of habituation. 114 i—i—i—i—i—i—i—r 1-5 21-25 Stimulus Number 46-50 Figure 25. Habituation of the f l e x o r r e f l e x to repeatedly applied s t i m u l i (20 v.) i n s p i n a l and decerebrate r a t s . There were 8 rats i n each group. Decerebration had no s i g n i -f i c a n t e f f e c t on the absolute (above) or r e l a t i v e degree of habituation (below). 115 The responses to the i n i t i a l 5 s t i m u l i were s i g n i f i c a n t l y greater (p 1 0.003, Mann-Whitney U test) i n decerebrate rats than the corresponding responses i n s p i n a l rats (Figure 26, above). In the case of the decere-brate rats the r e f l e x magnitude decreased r a p i d l y , whereas the opposite occurred i n s p i n a l r a t s , so that a f t e r 50 stimulations the responses from decerebrate rats were s i g n i f i c a n t l y lower (p f 0.005, Mann-Whitney U test) than those from rats which had undergone s p i n a l transection. Thus, decerebrate rats demonstrated r e l a t i v e habituation while s p i n a l rats showed a long term s e n s i t i z a t i o n (Figure 26, below); p = 0.000, Mann-Whitney U t e s t ) . In order to emphasize the r a p i d i t y of response habituation i n decerebrate rats Figure 27 i s included. In t h i s f i g u r e i n d i v i d u a l re-sponses f o r decerebrate rats (tested with 60 v. t r a i n s ) and f o r s p i n a l rats (60 v.) with various l e v e l s of transection are shown. Regardless of the l e v e l of s p i n a l transection s p i n a l rats demonstrated an i n i t i a l increment of response while decerebrate rats i n v a r i a b l y showed decrement (p =; 0.000, Mann-Whitney U t e s t ) . 2B. Spinal Rats and Asphyxiation of the Cord F l a c c i d p a r a l y s i s , the normal immediate consequence of s p i n a l tran-section, was seen i n both co n t r o l rats and rats which had undergone asphyxiation f o r 22 min. The control rats had strong withdrawal re-flexes i n response to'i a pinch of the hind-paw within 30 min. a f t e r l i g a t i o n of the cord but no r e f l e x could be e l i c i t e d i n the rats which had under-gone asphyxiation u n t i l 4 to 5 bouis a f t e r the cords were l i g a t e d . Groves , et a l . (1969) described both an early and a l a t e s e n s i t i z a t i o n 116 j i — i — i 1—I 1 1—I 1—i .1-5 2 1 - 2 5 4 6 - 5 0 Stimulus Number ure 26. Changes in flexor reflex response,to repeated application of intense stimuli (60 v.) in spinal and decerebrate rats. The i n i t i a l response amplitude was.greater in decerebrate rats but this response f e l l u n t i l i t was significantly less in decerebrate rats (above). Decerebrate rats showed relative habituation while spinal rats showed a long term sensitization (below). There were 8 rats in each group. 117 Figure 27. A comparison of habituation of the flexor reflex between spinal and decerebrate rats a l l tested with a stimulus intensity of 60 v. Each response is ex-pressed as a change in amplitude from the f i r s t res-ponse (volts). Decerebrate rats showed only decrement of response while spinal rats invariably showed an increment of response.. 118 of the f l e x o r r e f l e x i n the s p i n a l cat. A s i m i l a r observation was made i n t h i s study but the early component tended to be obscured by pre-senting the data i n blocks (see p. 92 , t h i s t h e s i s ) . For t h i s reason • the i n i t i a l 20 responses are occasionally p l o t t e d as i n d i v i d u a l data points. Figures 28 and 29 (above) i l l u s t r a t e t h i s early response s e n s i t i z a t i o n . Using 20 v. stimulus t r a i n s the early s e n s i t i z a t i o n and the l a t e r habituation were not s i g n i f i c a n t l y d i f f e r e n t i n asphyxiated rats com-pared to controls (Figure 28). However, rat s tested with 60 v. stimulus t r a i n s demonstrated an a l t e r a t i o n i n the early phase of s e n s i t i z a t i o n and i n the l a t e r r e l a t i v e habituation (Figure 29). S t a t i s t i c a l s i g n i f i -cance was indicated using Snedecor's F test (p ^ 0.050). This indicates a s i g n i f i c a n t difference between groups with regard to variances. The Student's t - t e s t cannot therefore be employed without a c o r r e c t i o n f o r unequal variances. Such a c o r r e c t i o n was accomplished using Cochran's modification of the t - t e s t (see p. 82, t h i s t h e s i s ) . This test indicated a s i g n i f i c a n t difference between experimental and c o n t r o l groups (p 1 0.050). The Mann-Whitney U t e s t was. also employed (p - 0.010) and i t i s i n f a c t superior to Cochran's modification as i t makes no as-sumptions about equal variances. The large variances that were calculated f o r the group of rats that underwent asphyxiation (demonstrated by a s i g n i f i c a n t F test) suggest sample population heterogeneity with regard to habituation and s e n s i t i z a t i o n (see p. 91', t h i s t h e s i s ) . Therefore, i t i s necessary to c a r e f u l l y consider the absolute response data. Figure 30 i l l u s t r a t e s these 119 A Figure 28. The e f f e c t of asphyxiation on habituation of the f l e x o r r e f l e x i n the s p i n a l r a t . Stimulus t r a i n s (20 v.) were applied at 10 sec. i n t e r v a l s . The r e l a t i v e degree of s e n s i t i z a t i o n (above) and habituation (below) were s i m i l a r f o r control (14 rats) and asphyxiated rats (14 r a t s ) . 120 A Figure 29. The e f f e c t of asphyxiation on habituation of the f l e x o r r e f l e x i n the s p i n a l r a t . Stimulus tr a i n s (60 v.) were applied at 10 sec. i n t e r v a l s . The r e l a t i v e degree of s e n s i t i z a t i o n was s i g n i f i c a n t l y greater i n asphyxiated rats (above) while the degree of habituation was s i g n i f i -cantly less that that of controls (7 r a t s , 11 rats asphyxiated). /Zo8 4 0 0 -, Stimulus Number .121 Figure 30. Flexor responses, i n absolute units ( v o l t s ) , of control and asphyxiated rats tested with a stimulus i n t e n s i t y of 60 v. 122 r e s u l t s f o r control and asphyxiated rats tested at 60 v., i n absolute response u n i t s . No s t a t i s t i c a l difference was apparent between these two curves using the Mann-Whitney U test (at the beginning and end of curves, p > 0.050). The groups did show a s i g n i f i c a n t d i f f e r e n c e i n terms of variances (F test) during the i n i t i a l and f i n a l stimulations which negates the use of a parametric t e s t , but t h i s s i g n i f i c a n c e can i t s e l f be used to i n d i c a t e that the rats were drawn from two d i f f e r e n t and independent populations. Regardless of s t a t i s t i c a l s i g n i f i c a n c e , two aspects are apparent from t h i s graph and they are as follows: 1) asphyxiation reduced the i n i t i a l response amplitude and 2) asphyxiation increased the f i n a l response amplitude, i n r e l a t i o n s h i p to c o n t r o l responses. Expressing the data as percentages indi c a t e d an i n i t i a l increase i n early s e n s i t i z a t i o n ( r e l a t i v e ) but t h i s r e l a t i o n s h i p holds only because of a reduction i n r e f l e x amplitude. There was a tendency, however, for asphyxiation to reduce a l a t e r r e f l e x decrement. Figure 31 i l l u s t r a t e s a comparison between controls ( s p i n a l rats) tested with 20 or 60 v. t r a i n s i n which the data are expressed as r e l a t i v e measures. The r e l a t i v e degree of habituation was s i g n i f i c a n t l y greater i n rats tested with 60 v. t r a i n s (p ^ 0.010, Mann-Whitney U test) and the degree of s e n s i t i z a t i o n was s i g n i f i c a n t l y greater i n rats tested with 20 v. t r a i n s (p - 0.010, Mann-Whitney U t e s t ) . These data contradict the parametric c h a r a c t e r i s t i c of habituation that states the r e l a t i v e degree of habituation i s inversely r e l a t e d to stimulus i n t e n s i t y . 123 A Figure.31. The effect of varying stimulus intensity on habituation of the flexor reflex in the spinal rat. A greater relative degree of habituation occurred with high i n -tensity stimulation than with low arid a.greater degree of sensitization occurred with low rather than high i n -tensity stimulation. 4 0 0 -, CO 10 c O Q. CO co cr 3 0 0 c o Q. 10 CU CC CO rr 200 H 100 o 20 V -• 60 V Stimulus Number _ 100 -o 20 V • 60 V 51-55 Stimulus Number 96-100 124 2C. Infusion of strychnine For s p i n a l rats transected at the l e v e l of the f i f t h thoracic vertebra, and tested with a stimulus strength of 20 v. ( t r a i n s ) , strychnine c o n s i s t e n t l y elevated the amplitude of response (Figure 32, above). However there was no a l t e r a t i o n i n the r e l a t i v e rate and degree of habituation (Figure 32, below). Stimulation with 60 v. t r a i n s resulted i n a s e n s i t i z a t i o n of con t r o l responses, but the i n f u s i o n of strychnine prevented t h i s c h a r a c t e r i s t i c response pattern (Figure 33,. below). Rats which received an i n f u s i o n of strychnine demonstrated a • s i g n i f i c a n t l y greater response amplitude (p - 0.001, Mann-Whitney U test) but following 25 stimulus presentations both groups responded at the same l e v e l of response. Therefore, i t can be concluded that strychnine prevented a long term s e n s i t i z a t i o n and revealed habituation. 2D. Infusion of strychnine and b i c u c u l l i n e (single pulse s t i m u l i ) It was d i f f i c u l t to demonstrate habituation of the f l e x o r r e f l e x i n the s p i n a l r a t using s i n g l e pulse s t i m u l i (see p. 98, t h i s thesis) and as a consequence i t was decided to take the largest amplitude block of 10 responses as a reference and to express a l l subsequent responses as a percentage of t h i s reference, provided t h i s block oc-curred within the f i r s t 5 response blocks. It was also necessary to increase the stimulus frequency to 1 per second i n order to accentuate habituation. The experimental procedure used for s p i n a l rats (tested with s i n g l e pulse stimuli) was d i f f e r e n t than that used i n other experiments i n t h i s t h e s i s . Two serie s of 300 s t i m u l i were given to each r a t . During the 125 1-5 21-25 41-45 61-65 81-85 95-100 Stimulus Number Figure 32. The effect of strychnine on habituation of the flexor reflex in the spinal rat. The stimuli were trains (20 v.). Intravenous infusion of saline (9 rats) ^ r strychnine (12 rats) was commenced 20 min. prior to the f i r s t stimulus and continued until the end of the experi-ment. Strychnine significantly elevated response amplitude but had no effect upon the relative degree of habituation. 126 1-5 21-25 41-45 61-65 81-85 95-100 Stimulus Number j ure 33. The effect of strychnine on habituation of the flexor reflex in the spinal rat. The stimuli were trains (60 v). Strychnine significantly elevated i n i t i a l response am-plitude and the controls showed a long term sensitization whereas rats receiving strychnine demonstrated habituation (15 control rats and 10 experimentals). 127 f i r s t s e r i e s s a l i n e was administered. This was followed two hours l a t e r by the second ser i e s during which an i n f u s i o n of strychnine or b i c u c u l l i n e was given. Habituation to the f i r s t s eries of s t i m u l i thus served as a con t r o l to which the e f f e c t of a drug on habituation, during the second s e r i e s , could be compared. A separate c o n t r o l group of rats was tested using t h i s regime but s a l i n e was given during both s e r i e s . The purpose of t h i s group was to ensure that the degree of habituation during the second ser i e s of s t i m u l i was the same as that of the f i r s t and t h i s proved to be the case. In Figure 34 the r e l a t i v e degree of habituation during the f i r s t s e r i e s of s t i m u l i (saline) and during a second ser i e s (strychnine) i s shown. Strychnine caused a s i g n i f i c a n t reduction i n the f i n a l degree of habituation. B i c u c u l l i n e had no s i g n i f i c a n t e f f e c t upon habituation of the FWR. Because experimental and con t r o l groups are dependent (each r a t serves as i t s own control) s t a t i s t i c a l s i g n i f i c a n c e was assessed using the Wilcoxon Matched-Pairs Sign-Ranks test ( S i e g e l , 1956). 2E. Level of Spinal Transection In order to determine i f the l e v e l of transection might have some influence upon habituation and s e n s i t i z a t i o n of the FWR a serie s of rats had t h e i r cords transected at the l e v e l of the l a s t c e r v i c a l vertebra (C7) and were compared to a group of rats with transection at the tenth thoracic vertebra (T^g). The most r o s t r a l s e ction was chosen rather than the section used by Wickelgren (1967a) so that the supraspinal drive to the motor neurones of the diaphragm remained i n t a c t whereas the most caudal section was chosen rather than the T^2 Figure 34. The e f f e c t of strychnine on habituation of the f l e x o r r e f l e x using si n g l e pulse s t i m u l i of 20 v. stimulus strength. Experimental rats served as t h e i r own controls <£8 r a t s ) . Strychnine caused a s i g n i f i c a n t reduction i n the r e l a t i v e degree of habituation (p 1 0.010, Wilcoxon-Matched Pairs Signed-Ranks t e s t ) . 129 section used by Groves, et a l . (1969) to ensure that the d i r e c t FWR pathway was not damaged. Rats with e i t h e r l e v e l of transection were tested with 20 v. and 60 v. stimulus t r a i n s . Rats with the caudal section demonstrated no s i g n i f i c a n t d i f f e r e n c e i n response amplitude when tested with 20 or 60 v. stimulus t r a i n s (p > 0.052, Mann-Whitney U test) (Figure 35, labove) . The greatest r e l a t i v e degree of habituation occurred with the 20 v. stimulus t r a i n s (p 1 0.025, Mann-Whitney U test) (Figure 35, below). In contrast, rats with the r o s t r a l transection demonstrated s i g n i f i -cantly greater response amplitude when tested with 60 v. t r a i n s i n comparison to s i m i l a r r a t s tested with 20 v. t r a i n s (Figure 36, p -0.025, Mann-Whitney U t e s t ) . Tested at a stimulus strength of 20 v., rats with the r o s t r a l transection were also s i g n i f i c a n t l y lower i n response amplitude than with the caudal transection, regardless of the stimulus i n t e n s i t y (p f 0.052, Mann-Whitney U t e s t ) . The group of r a t s , with r o s t r a l transection, tested with 60 v. t r a i n s demonstrated sample population heterogeneity with regard to r e l a t i v e degree of habituation and s e n s i t i z a t i o n (see p. 91, t h i s t h e s i s ) . To be s p e c i f i c 2 rats showed low response amplitude with s e n s i t i z a t i o n while 6 rats showed high amplitude responses and marked habituation. As a consequence, the r e l a t i v e measure of habituation had a variance of thousands of percent and i t f a i l e d to be a reasonable measure. To avoid t h i s p a r t i c u l a r d i s t o r t i o n the data was presented as differences i n absolute response from the i n i t i a l block of responses (Figure 36, below). The greatest absolute degree of habituation occurred with the 60 v. stimulus 130 Figure 35. The effect of stimulus intensity on habituation of the flexor reflex in the spinal rat. There was no significant difference between rats tested with 20 v. (8 rats) and those tested with 60 v. (8 rats) in absolute response; however, the relative degree of habituation was greatest for rats tested with the lower intensity. These rats were transected at the level of the tenth thoracic vertebra. 131 <D co . c o CL co CD or X 0> H— 0 ce _ 4.0 o cu CO 2 3.0 5 c 3 2.0 H ^ 1.0 H 0.0 1-5 2-25 41-45 61-65 81-85 95-100 Stimulus Number gure 36. The effect of stimulus intensity on habituation of the flexor reflex in the spinal rat. Rats testedswith -20 v. trains (6 rats) had responses significantly lower than those rats tested with 60 v. trains (8 rats). As a consequence, rats tested with the highest intensity had a greater absolute decrement of responses. These rats were transected at the level of the seventh cervical vertebra. 132 , t r a i n s p r i m a r i l y because of the higher i n i t i a l response amplitude. Flexor Reflex i n Anaesthetized Rats 3A. Inj e c t i o n of Strychnine The use of urethane anaesthesia greatly changed the response pattern of the FWR i n the i n t a c t r a t . The response of an unaesthetized r a t (EMG) i s shown i n Figure 14 (p. 9 7 ) . Following i n j e c t i o n of urethane the r e f l e x was manifest as an e a r l y and l a t e r e f l e x (Figure 37). Repetition of the stimulus produced a fusion of these components u n t i l the duration of the response outlasted the inter-stimulus i n t e r v a l . Continued stimulation resulted i n a decrement of the response usually due to a dropping out of i n d i v i d u a l units and a reduction i n the amplitude and duration of the EMG discharge. The integrated EMG recorded during two consecutive series of stimulation i s shown i n Figure 38. There was no s i g n i f i c a n t difference between the f i r s t and second s e r i e s of s t i m u l i . A second group of rats was tested i n .-the same manner with the exception that rats received an i n j e c t i o n of strychnine 1 min. p r i o r to the second serie s of s t i m u l i . These r e s u l t s are i l l u s t r a t e d i n Figure 39 i n ab-solute response ( v o l t s ) . The response amplitude at the end of the second run was s i g n i f i c a n t l y greater than at the end of the f i r s t s e r i e s of s t i m u l i (p f 0.050, Wilcoxon Matched-Pairs Signed-Ranks t e s t ) . The f i r s t run for controls and those ra t s that received strychnine were not s i g n i f i c a n t l y d i f f e r e n t (p 1 0.050, Wilcoxon Matched-Pairs Signed-Ranks t e s t ) . In order to determine the influence which strychnine had upon the 133 Figure 37. Responses of the f l e x o r r e f l e x (EMG, biceps femoris) to repeated stimulation (60 v., 22.5 mA, inter-stimulus i n t e r v a l 1.5 sec.) i n the anaesthetized, i n t a c t r a t . The EMG i s shown above and the integrated EMG (550 msec.) i s respresented below. The i n i t i a l stimulus evoked a short latency response (10 to 20 msec.) followed by a long latency (500 msec.) response. On presentation of the second stimulus the early and l a t e responses fused. 134 o—o I st. Run 0 5 1 0 1 5 110 115 1 2 0 Stimulus Number Figure 38. S e n s i t i z a t i o n and "habituation of s e n s i t i z a t i o n " of the fle x o r r e f l e x i n the anaesthetized, i n t a c t rat (60 v. 22.5 mA, inter-stimulus i n t e r v a l 1.5 s e c ) . The graph i l l u s -s trates the mean responses of rats (7 rats) r e c e i v i n g two series of s t i m u l i separated by 2 min. One min. p r i o r to the second seri e s an i n j e c t i o n of s a l i n e was given. The f i r s t s e r i e s had no s i g n i f i c a n t e f f e c t on the second s e r i e s . 135 3.0 -, o> in i 10 I CD CO c o CL CO CL) or CD or 2.0-4 \ \ \ \ -0-\ \ \ Strychnine Control o.o J no - 1 — 115 120 Stimulus No. Figure 39. The e f f e c t of strychnine on "habituation of s e n s i t i z a t i o n " of the f l e x o r r e f l e x i n the i n t a c t r a t . Control and strychnine s e r i e s were separated by two min. One min. p r i o r to the second serie s of s t i m u l i an i n j e c t i o n of strychnine was given. Strychnine s i g n i f i c a n t l y impaired the absolute degree of "habituation of s e n s i t i z a t i o n . " (8 r a t s , 60 v., 22.5 mA, inter-stimulus i n t e r v a l 1.5 sec.). 136 decrement of the response, a s p e c i a l measure was synthesized. Responses to the 110th to 120th s t i m u l i were averaged and expressed as a percentage of the means of the 6th to 15th responses, for both the f i r s t and second serie s of s t i m u l i . This percentage decrement was then expressed as a differ e n c e between the f i r s t and second runs for both controls and ex-perimentals. The percentage decrement of the second run was always sub-tracted from that of the f i r s t run which meant that t h i s d i f f e r e n c e could be either p o s i t i v e or negative. A negative d i f f e r e n c e would i n -dicate that l e s s decrement had occurred i n the second run as compared to the f i r s t run. This measure gives an i n d i c a t i o n of the influence of strychnine on response decrement which i s l a r g e l y independent of the influence of the f i r s t run on the second. Comparison of experimentals with controls, using the Mann-Whitney U t e s t , demonstrated a s i g n i f i c a n t impairment of response decrement as a consequence of strychnine (p f 0.005, con t r o l +8.8%, strychnine + 65.2%) 137 SECTION II Spinal Interneurones The•microelectrode was lowered v e r t i c a l l y through the dorsal surface of the cord u n t i l either spontaneous or evoked a c t i v i t y was encountered. The d i s t r i b u t i o n of spontaneously a c t i v e units was not uniform, and the more v e n t r a l the l o c a t i o n of the electrode t i p the more l i k e l y that spontaneous a c t i v i t y would be detected. These unit discharges f u l f i l l e d c r i t e r i a that would define them as neuronal discharge (Dafny and Gilman, 1973). Spontaneously a c t i v e interneurones had r e s t i n g discharge rates which ranged from about 5 to 200 spikes/sec. These units usually had a r e l a t i v e l y stable discharge rates. Often i n preparations that had been used f o r long periods the discharge was o s c i l l a t o r y . Such units were not studied. Orthodromic stimulation evokes a number of c h a r a c t e r i s t i c d i s -charge patterns: 1) A short duration high frequency burst (100 to 1000/sec.) with a r e l a t i v e l y short latency (2 to 5 m s e c ) . Increasing the i n t e n s i t y of stimulation decreases the latency of the response and increases the number of spikes i n the burst (5 to 20 spikes). 2) A long duration and moderate frequency t r a i n (10 to 200/sec.) of discharges. These neurones have greater latencies (5 to 500 m s e c ) . This type of response has been given several names incl u d i n g l a t e discharge, sustained discharge, r e p e t i t i v e discharge, and after-discharge. This d i s t i n c t i o n i n response patterns i s not absolute and many 138 intermediate patterns are encountered. This c l a s s i f i c a t i o n of e x c i t a t o r y response patterns has been established i n the s p i n a l cord by numerous authors (Frank and Fuortes, 1956; Wall, 1959; P r i c e and Wagman, 1970; Groves and Thompson, 1973). The term after-discharge i s adopted i n t h i s t hesis because of the s i m i l a r i t y of t h i s neuronal response pattern and "after-discharge" of the f l e x o r r e f l e x . Figure 40 i l l u s t r a t e s an example of each of the response patterns. These interneurones were not spontaneously a c t i v e which served to f a c i l i t a t e observation of the r e -sponse patterns. Repetitive stimulation has d i f f e r e n t e f f e c t s upon each of the d i s -charge patterns. Frank and Fuortes (1956) o r i g i n a l l y reported that the after-discharge b u i l t up with stimulus r e p e t i t i o n (increased i n duration and frequency). This build-up was confirmed by a number of authors i n -cluding Groves and Thompson (1973) who equated i t with s e n s i t i z a t i o n of the FWR and labeled these interneurones "S" c e l l s . Certain of these interneurones demonstrated a l a t e r decrement of discharge following the i n i t i a l build-up and these were c a l l e d "S-H" c e l l s . On the other hand, Groves and Thompson (1973) found that interneurones which responded with a high frequency burst underwent only decrement (reduction i n number of evoked spikes) following stimulus r e p e t i t i o n ("H" c e l l s ) . In these respects, the r e s u l t s of the present study did not d i f f e r from previous studies. Interneurones that responded with a high frequency burst often had r e l a t i v e l y low thresholds to cutaneous stimulation. Habituation of such an interneurone i s i l l u s t r a t e d i n Figure 41. The threshold for e x c i t a t i o n 139 Figure 40. Interneuronal responses typical of a high frequency burst (above) and after-discharge (below). 140 I i Stimulus Number Figure 41. An example of habituation of a high frequency burst i l l u s t r a t e d g r a p h i c a l l y . The response to 500 uniform s t i m u l i (1 mA) was a gradual decrement of response. The response to the 1st and 500th s t i m u l i are also shown. 141 (0-6mA) of t h i s interneurone was l e s s than that of the FWR. Increasing the stimulus strength greatly reduced the rate of decrement of t h i s type of response and at stimulus i n t e n s i t i e s ranging from 10 v. (3.8 mA) to 60 v. (22.5 mA) these units showed l i t t l e i n the way of decrement (see Figure 45). Interneurones which demonstrated after-discharge were examined i n considerably more d e t a i l than those showing a high frequency burst. As the i n t e n s i t y of the stimulation was increased, the degree of s e n s i t i z a t i o n of after-discharge increased (Figure 42), and increasing stimulus frequency had a s i m i l a r e f f e c t . Other interneurones demonstrated a l a t e r phase of response decrement (Figures 43 and 44). Groves and Thompson (1973:187) found that c e r t a i n interneurones r e -sponded with both a high frequency burst and a l a t e r after-discharge. These p a r t i c u l a r interneurones demonstrated decrement of the high frequency burst and build-up of the after-discharge. In t h i s study a number of interneurones was encountered that responded with both patterns of discharge but i t often happened that the high frequency burst had a .... lower threshold to cutaneous stimulation. Interneurones which responded with a high frequency burst were often found to possess a l a t e r a f t e r -discharge provided the i n t e n s i t y of stimulation was increased s u f f i c i e n t l y . Records obtained from such an interneurone i s shown i n Figure 45. The high frequency burst had a threshold well below 1 mA but the a f t e r -discharge was not evoked u n t i l the stimulus strength was rai s e d to 8 mA. The upper part of the f i g u r e demonstrates the response of the high freqeuncy burst to r e p e t i t i v e stimulation (20 mA, 58 v . ) . 142 70 -, 0 10 20 30 40 50 90 Stimulus Number Figure 42. S e n s i t i z a t i o n of after-discharge to various i n t e n s i t i e s of stimulation i l l u s t r a t e d g r a p h i c a l l y . Increasing the i n t e n s i t y increased the degree of s e n s i t i z a t i o n . 143 2 sec . Figure 43. S e n s i t i z a t i o n of after-discharge and a l a t e r decrement of response following r e p e t i t i v e stimulation (20 mA). This interneurone was not spontaneously a c t i v e . The decrement of s e n s i t i z a t i o n was gradual and was si m i l a r to that shown i n Figure 44. 144 4 0 -i 30 H o CO CO \ CO <u CL CO 2 0 ' 10' o-» - 3 -I +1 I 30 I 60 — I 9 0 Stimulus No. Figure 44. S e n s i t i z a t i o n of after-discharge and a l a t e r decrement of response following r e p e t i t i v e stimulation. This i n t e r -neurone was spontaneously a c t i v e . The decrement of s e n s i t i z a t i o n was gradual. 145 Stimulus Number Figure 45. Response of an interneurone demonstrating both a high frequency burst and a l a t e r afters-discharge. The high frequency burst did not change with repeated stimulation (above) whereas the after-discharge showed a marked b u i l d -up of response followed by a l a t e r decrement (below). This interneurone was not spontaneously a c t i v e . 146 L i t t l e or no change occurred i n evoked response. However, when the period of counting was increased to include the after-discharge (the high frequency burst was not counted) there was a considerable b u i l d -up of response followed by a decrement. I t i s only an assumption that t h i s represents the a c t i v i t y of a s i n g l e interneurone. Interneurones were both excited and i n h i b i t e d by the stimulation, but both the i n h i b i t o r y and excitatory phases were capable of under-going progressive changes following r e p e t i t i v e stimulation. The factor which determined whether excitatory or i n h i b i t o r y influences would dominate appeared to be the frequency of stimulation. This factor was o r i g i n a l l y described by Frank and Fuortes (1956:429): "Apparently the afferent v o l l e y produced short periods of i n h i b i t i o n which fused and predominated over more enduring phases of e x c i t a t i o n when the frequency of stimulation was s u f f i c i e n t l y high." Figure 46 i l l u s t r a t e s the response of an interneurone that was i n h i b i t e d by the stimulus. This i n h i b i t i o n was reduced with repeated stimulation due to build-up of e x c i t a t i o n . The duration of t h i s i n h i b i t i o n was greater with higher stimulus i n t e n s i t i e s and a p a r t i c u l a r l y strong stimulus was used to accentuate the i n h i b i t i o n . One of the objectives of t h i s t hesis was the demonstration of the build-up of i n h i b i t o r y a c t i v i t y . A number (28) of spontaneously a c t i v e interneurones was progressively i n h i b i t e d by the r e p e t i t i o n of cutaneous stimulation, and t h i s occurred with s i n g l e pulse stimulation (Figure 47) and with a p p l i c a t i o n of stimulus t r a i n s (Figure 48). Maximal build-up of i n h i b i t i o n was demonstrated with stimulus t r a i n s . The cessation of 147 A. B. C. 1 2 3 30 32 I sec Figure 46. I n h i b i t i o n of a spontaneously a c t i v e interneurone. A t y p i c a l record of pre-stimulus a c t i v i t y i s shown i n (A). Repeated stimulation (30 mA) every 0.5 sec. i n i t i a l l y i n h i b i t e d t h i s a c t i v i t y (B) but the i n h i b i t i o n gradually wore off and was replaced by a build-up of a c t i v i t y (C) reaching a l e v e l greater than i t s pre-stimulus condition. The decrement of i n h i b i t i o n may therefore be the consequence of a competing build-up of e x c i t a t i o n . 148 5 6 Figure 47. Inhibitory build-up of a spontaneously a c t i v e interneurone i n response to s i n g l e pulse stimulation (20 mA, i n t e r -stimulus i n t e r v a l 1.5 s e c ) . Repeated stimulation was associated with a gradual dropping out of spontaneous ac-t i v i t y u n t i l no a c t i v i t y was present. No recovery occurred u n t i l stimulation was recommenced 20 sec. a f t e r the l a s t stimulus (40). An increase i n the background a c t i v i t y occurred during the stimulation and t h i s might represent the a c t i v i t y of nearby interneurones, perhaps the inhibitory" interneurone dr i v i n g the spontaneously active interneurone. 149 B. D. Figure 48. Inhibitory build-up of a spontaneously a c t i v e interneurone i n response to stimulus t r a i n s (10 mA, 0.5 msec, pulses, 0.5 sec. t r a i n duration, inter-stimulus i n t e r v a l 10.0 s e c ) . Each stimulus t r a i n i s shown as an open box and the responses to the f i r s t (A), f i f t h (B), and f i f t e e n t h (C) are shown. F u l l recovery i s i l l u s t r a t e d i n (E). The time bar indicates 1.5 s e c with regard to (A,B,C and E), however, i t in d i c a t e s 4.5 s e c f o r (D). (D) i l l u s t r a t e s the gradual recovery of a c t i v i t y . No a c t i v i t y was present u n t i l 3.5 min. following the l a s t stimulus (25 tra i n s ) and a second s e r i e s of t r a i n s given a f t e r f u l l recovery i n h i b i t e d the interneurone f o r a period of 7.0 min. following the cessation of the s t i m u l i . Single pulse s t i m u l i also produced i n h i b i t o r y build-up s i m i l a r to that seen i n Figure 47. 150 spontaneous a c t i v i t y outlasted the period of stimulation ( a f t e r -i n h i b i t i o n ) from periods of 0.5 sec. to 7 min. An interneurone was considered to exhibit i n h i b i t o r y build-up only i f i t f u l f i l l e d c e r t a i n c r i t e r i a : 1) the pre-stimulus a c t i v i t y had to be r e l a t i v e l y constant 2) the stimulus had to c l e a r l y evoke inhibition of the spontaneous a c t i v i t y 3) the period of i n h i b i t i o n (duration of halted a c t i v i t y ) had to increase progressively with repeated stimulation 4) the interneurone had to c l e a r l y demonstrate recovery a f t e r cessation of the stimulation 5) the build-up of i n h i b i t i o n had to be demonstrated at l e a s t twice for each interneurone. This build-up of i n h i b i t i o n demonstrated a number of c h a r a c t e r i s t i c s as follows: 1) During stimulus repetition the period of i n h i b i t i o n developed gradually (Figure 49). This i n h i b i t i o n often appeared to reach asym-p t o t i c l e v e l . A t o t a l of 28 interneurones demonstrated .inhibitory build-up but another 13 showed a build-up of i n h i b i t i o n followed by a decay of t h i s i n h i b i t i o n during the stimulation period. The maximal duration of i n h i b i t i o n i n the l a t t e r c e l l s was usually r e l a t i v e l y short:: ( < 500 msec.) i n comparison to interneurones showing i n h i b i t o r y build-up, and the decay of i n h i b i t i o n was associated with a simultaneous build-up of e x c i t a t i o n (Figure 46). 2) The rate of build-up and the duration of the i n h i b i t i o n were 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 of stimulation (confirmed i n 16 interneurones). In t h i s respect, most i n h i b i t o r y build-up was observed at i n t e n s i t i e s that activated L.high ..threshold afferents (groups I I I and IV), 151 16.0 - i 7.5 m A ( 20 V ) 20 mA (58 V ) 10 ~i 1 r 20 30 40 Stimulus Number 1 50 15 -i o CD CO N ro cu CO to CO cu CE c D CD 2 During Stimulation Pre=and Post= Stimulation 10 H 5 H 1-5 5.0 2.5 Time (sec.)before I st. Stimulus " i 1 r 21-25 41-45 61-65 91-95 Stimulus Number 12 3.7 6.2 8.7 Time (sec.)after last Stimulus Figure 49. The e f f e c t of stimulus i n t e n s i t y on i n h i b i t o r y build-up. Above i s shown the progressive increase i n the duration of i n h i b i t i o n for two r e l a t i v e l y strong stimulus strengths (7.5 and 20 mA). With a lower stimulus strength (1.9 mA) the frequency of spontaneous a c t i v i t y was gradually r e -duced (below). The rate and duration of i n h i b i t o r y b u i l d -up i s proportional to the i n t e n s i t y of stimulation. 152 although i n h i b i t o r y build-up was observed at i n t e n s i t i e s w ell below that of group IV afferents ( > 6 mA). Figure 49 i l l u s t r a t e s the influence of three d i f f e r e n t stimulus strengths on i n h i b i t i o n of the same i n t e r -neurone .' Although a c l e a r period of i n h i b i t i o n was not apparent with r e l a t i v e l y low stimulus i n t e n s i t y (5 v., 1.9 mA), r e p e t i t i o n of the stimulus caused a gradual reduction i n spontaneous a c t i v i t y . There was a r e l a t i o n s h i p between the frequency of the spontaneous a c t i v i t y and the i n t e n s i t y of stimulation capable of h a l t i n g the a c t i v i t y . The higher the frequency of the spontaneous a c t i v i t y the greater was the i n t e n s i t y of stimulation required to h a l t the a c t i v i t y . Several i n t e r -neurones with r e l a t i v e l y low spontaneous rates were strongly i n h i b i t e d by weak s t i m u l i (5 v., 1.9 mA). 3) The rate of i n h i b i t o r y build-up was d i r e c t l y r e l a t e d to the frequency of stimulation (confirmed i n 9 interneurones) (Figure 50). A build-up of i n h i b i t i o n was observed i n some interneurones with f r e -quencies as low as 0.2/sec. when using s i n g l e pulse stimulation and at frequencies as low as 0.1/sec. when stimulus t r a i n s were employed. 4) A f t e r cessation of the stimulation, the i n h i b i t i o n appeared to decay spontaneously and the spontaneous^ a c t i v i t y was often depressed f o r periods a f t e r the interneurones re-commenced discharging. 5) Repeated seri e s of s t i m u l i often resulted i n a more rapid development of i n h i b i t i o n and longer periods of i n h i b i t i o n a f t e r the cessation of stimulation (Figure 51). Confirmed i n 4 c e l l s . 153 2 . 0 - , 0 . 6 7 / s e c 0.4 / s e c 5 15 2 5 S t i m u l u s N u m b e r Figure 50. The e f f e c t of stimulus frequency on i n h i b i t o r y build-up. The build-up was proportional to the frequency of stimu-l a t i o n . 154 Stimulus Number Figure 51. The e f f e c t of repeated s e r i e s of s t i m u l i on i n h i b i t o r y build-up. Two s e r i e s of s t i m u l i (10 mA, inter-stimulus i n t e r v a l 1.5 sec.) were separated b y l min. The build-up of i n h i b i t i o n was greatest i n the second s e r i e s . This may indi c a t e a r e s i d u a l i n h i b i t i o n was c a r r i e d over from the f i r s t to second seri e s of s t i m u l i , although the i n t e r -neurone recovered i t s spontaneous discharge rate between the f i r s t and second s e r i e s . 155 6) In several interneurones an i n t e r a c t i o n between the build-up of excitatory a c t i v i t y by repeated ( i p s i l a t e r a l ) stimulation and the b u i l d -up of i n h i b i t i o n by repeated ( c o n t r a l a t e r a l ) ' stimulation was observed (Figure 52). Repetition of the i p s i l a t e r a l stimulus evoked an a f t e r -discharge which gradually doubled (the number of spikes evoked by each stimulus doubled) u n t i l a plateau of response was reached. The i p s i -l a t e r a l stimulation was continued, but a simultaneous stimulation of the c o n t r a l a t e r a l hind paw was begun. This resulted i n a gradual reduction of the evoked a c t i v i t y . Following cessation of the c o n t r a l a t e r a l but not the i p s i l a t e r a l , stimulation there was a gradual build-up of response u n t i l the plateau l e v e l was again reached. 7) The interneurone i l l u s t r a t e d i n Figure 47 was stimulated during the period of i n h i b i t i o n that occurred following the i n i t i a l s e r i e s of s t i m u l i . The interneurone was d i s - i n h i b i t e d by re-implementation of the s t i m u l i , presumably due to an e x c i t a t i o n o r i g i n a l l y masked by the build-up of i n h i b i t i o n (Figure 53). Continued r e p e t i t i o n of the d i s - i n h i b i t i n g s t i m u l i lead, again, to a build-up of i n h i b i t i o n . This e f f e c t was confirmed i n 3 other interneurones. 8) In 6 r a t s , when an interneurone was encountered that demonstrated i n h i b i t o r y build-up, an i n j e c t i o n of strychnine was given (0.1 mg/Kg). '.. . In h a l f of these r a t s the build-up of i n h i b i t i o n was blocked by strychnine (Figure 54) and without elevating the presstimulus discharge of the interneurone (Figure 55), or at l e a s t by no great extent. Strychnine blocked t h i s i n h i b i t i o n i n 3 of the r a t s (3 interneurones), however, i n 3 others i t e i t h e r had l i t t l e e f f e c t on the i n h i b i t i o n or i t reduced the build-up i n a s s o c i a t i o n with a large increase i n the spontaneous discharge 156 Stimulus No. (Contralateral) 0 5 10 15 20 25 Stimulus No. (Ipsilateral) Figure 52. The i n t e r a c t i o n of excitatory and i n h i b i t o r y build-up. This interneurone showed a build-up of response to i p s i -l a t e r a l stimulation (20 mA, inter-stimulus i n t e r v a l 1.5 s e c ) . C o n t r a l a t e r a l stimulation (same parameters) pro-gres s i v e l y i n h i b i t e d the i p s i l a t e r a l evoked response. On cessation of the c o n t r a l t e r a l stimulation the response b u i l t -up to i t s o r i g i n a l response l e v e l . 157 I s e c Figure 53. D i s - i n h i b i t i o n of i n h i b i t o r y build-up. Inhibitory b u i l d -up of th i s interneurone i s shown i n Figure 47. Twenty sec. af t e r cessation of the stimulation the stimulus i s r e -commenced (1) and then turned o f f a f t e r a second stimulus (2). The c e l l was f u l l y recovered by these two s t i m u l i . If the stimulation was begun again and continued i n h i b i t o r y build-up also occurred again. I i 158 i i Figure 54. The e f f e c t of strychnine on i n h i b i t o r y build-up. Inh i b i t o r y build-up was demonstrated before i n j e c t i o n of strychnine (above), however, a f t e r strychnine the i n h i b i t i o n was a l -most eliminated and no build-up occurred. 159 2.0 - i - I.5H i.o H a> CL 0.5 H 0.0 Control C O a. cn r 45 h 40 1-35 £ -3 -I + 1 I 10 1~ 15 20 25 Stimulus No. gure 55. The e f f e c t of strychnine on i n h i b i t o r y build-up i l l u s -trated g r a p h i c a l l y . This i s the same interneurone shown i n Figure 54. Pre-stimulus discharge rates are indicated and strychnine did not elevate spontaneous a c t i v i t y . A l -though some i n h i b i t i o n remained a f t e r i n j e c t i o n of strychnine repeated stimulation d i d not r e s u l t i n a build-up of i n h i b i t i o n . 160 rate. This suggests that i n h i b i t i o n , other than strychnine s e n s i t i v e i n h i b i t i o n , may be p a r t i a l l y responsible f o r i n h i b i t o r y build-up. A number of interneurones was anatomically located i n the cord by iontophoresis of the dye pontamine sky blue. These interneurones (recording s i t e s ) were then placed i n the appropriate Rexed laminae (Figure 56) according to Steiner and Turner (1972) who examined lami-nation i n the cord of the r a t . Depths of recorded interneurones were correlated with the depths of marked interneurones i n order to determine the approximate l o c a t i o n of non-marked interneurones. Interneurones demonstrating i n h i b i t o r y build-up were located i n laminae V to VII and two were recorded i n the v i c i n i t y of lamina I. A t o t a l of 154 interneurones were recorded i n the s p i n a l cords of i n t a c t rats ( t o t a l of 41 r a t s ) . Of these interneurones 101 were exc l u s i v e l y excited by the stimulus, 13 demonstrated both e x c i t a t i o n and i n h i b i t i o n , 12 were i n h i b i t e d but repeated stimulation did not a l t e r the duration of the i n h i b i t i o n , and 28 demonstrated i n h i b i t o r y build-up. The majority of the interneurones excited by the stimulus demonstrated after-discharge, although many interneurones responded with a high f r e -quency burst and an after-discharge. 161 Figure 56. The laminary l o c a t i o n of various interneurones. Re-cording s i t e s were marked with pontamine sky blue and lamination was determined as indicated by Steiner and Turner (1972). The l o c a t i o n of 9 i n h i b i t o r y build-up responses i s indicated above. 162 SECTION I I I Interneurones Recorded i n the Cord of Spinal Rats An attempt was made to locate interneurones i n the s p i n a l r a t (6 r a t s , transection T5) that demonstrated i n h i b i t o r y build-up. C h a r a c t e r i s t i c a l l y interneurones that demonstrated r e l a t i v e l y long periods of i n h i b i t i o n to a s i n g l e stimulus ( > 100 msec.) also demonstrated i n h i b i t o r y build-up, i n the i n t a c t r a t . I t has not been possible to locate any interneurones demonstrating i n h i b i t o r y build-up i n the s p i n a l r a t ; however, the sample of interneurones i s small (10 interneurones). On the contrary, the r e l a t i v e l y long duration i n h i b i t i o n ( a f t e r - i n h i b i t i o n ) which was observed i n the s p i n a l r a t , decayed with stimulus r e p e t i t i o n regardless of the stimulus strength or frequency. This decrement of the a f t e r - i n h i b i t i o n occurred even though these interneurones did not appear to be excited by the stimulus. Figure 57 i l l u s t r a t e s the t y p i c a l i n h i b i t o r y response to s i n g l e pulse stimulation and to stimulus t r a i n s . Figure 57. The decrement (habituation?) of inhibition found in interneurones in the spinal rat. Single pulse stimuli evoked an inhibition of decreasing duration (20 mA, inter-stimulus interval 1.5 s e c ) . Only one inter-neurone is shown in this figure. 164 CHAPTER V DISCUSSION: SECTION I I n h i b i t i o n and the Flexor Reflex (FWR) This study has been based upon the supposition that i n h i b i t o r y processes might contribute s i g n i f i c a n t l y to the establishment and i n t e -gration of behavioural events which manifest themselves as habituation and s e n s i t i z a t i o n of the f l e x o r r e f l e x and, r e s i s t i n g a current trend towards reductionism, i t was thought important to consider the cybernetic r o l e of i n h i b i t i o n . Therefore, i t was not the objective of t h i s study to propose a general theory of habituation, but rather i t was designed to examine the possible r o l e that i n h i b i t i o n might play i n modifying flexorc responses to repeated stimulation. I s o l a t i o n of various components of the r e f l e x has been accomplished both s u r g i c a l l y and pharmaco-l o g i c a l l y . A number of preparations were studied (decerebrate, s p i n a l , and i n t a c t ) instead of r e l y i n g s o l e l y upon the use of the s p i n a l pre-paration, and the j u s t i f i c a t i o n f o r t h i s appraoch becomes apparent from an understanding of the composition of the f l e x o r r e f l e x . In order for some form of i n h i b i t o r y process to contribute to habituation of the f l e x o r r e f l e x a c t i v a t i o n of f l e x o r r e f l e x afferents must cause either i n h i b i t i o n or a d i s - f a c i l i t a t i o n of the f l e x o r r e f l e x . D e f i n i t i o n of Flexor Reflex Afferents (FRA's) A s i n g l e shock to the skin or nerve of a limb evokes an i n d i r e c t (multisynaptic) f a c i l i t a t i o n of f l e x o r motorneurones and likewise 165 r e c i p r o c a l i n h i b i t i o n of extensor motorneurones, located i p s i l a t e r a l to the s i t e of stimulation. This i s the f l e x o r r e f l e x defined by Sherrington (1910). Recent e l e c t r o p h y s i o l o g i c a l experiments have described c e r t a i n afferents as f l e x o r r e f l e x afferents (FRA's) by v i r t u e of t h e i r actions upon f l e x o r and extensor motorneurones. Stimulation of group II and III muscle afferents (Lloyd, 1943, 1946; Brock et a l . , 1951; Laporte and Lloyd, 1952; Laporte and Bessou, 1959; 2 Eccles and Lundberg, 1959 a,b; P a i n t a l , 1961) and group I I and I I I cutaneous afferents (Lloyd, 1943; Hagbarth, 1952; Laporte and Bessou, 1958; Eccles and Lundberg, 1959 a,b) evoke e x c i t a t i o n of i p s i l a t e r a l f l e x o r motorneurones and i n h i b i t i o n of i p s i l a t e r a l extensor motorneurones. Cutaneous group IV afferents may also be considered FRA's (Franz .and Iggo, 1968). This d e f i n i t i o n of FRA's was extended by Wall (1970:180-182) i n r e l a t i o n s h i p to t h e i r action tin f l e x o r motorneurones: Motorneurones which supply axons to f l e x o r muscles are driven into a c t i v i t y by a v a r i e t y of peripheral s t i m u l i . A class of these s t i m u l i sets o ff the f l e x o r r e f l e x , which seems designed to move the limb away from the stimulus point. Which muscles contract or which motorneurones f i r e depends therefore not only on the type of affe r e n t f i b r e s stimulated but also on t h e i r s p a t i a l o r i g i n . In other words, each motorneurone can be s a i d to have a receptive f i e l d with respect to the f l e x o r r e f l e x stimulus... As a stimulus at one point i s increased i n strength, more and more muscles take part i n the response. In terms of the sing l e motorneurone, t h i s means that threshold v a r i e s within i t s receptive f i e l d . Increase i n stimulus strength not only r e c r u i t s more neurones but also produces r e p e t i t i v e f i r i n g of a c t i v e motorneurones. Repetitive stimulation produces r e p e t i t i v e response. The motorneurones are there-fore subject to both s p a t i a l and temporal summation...The threshold f o r the r e f l e x i s affected by the existence of other peripheral s t i m u l i and c e n t r a l a c t i v i t y . 166 However, fl e x o r and extensor motorneurones are not the exclusive targets of ERA'sandOscarsson (1967, 1970) defined ascending t r a c t s , carrying information which was not r i g h t l y c l a s s i f i e d as eit h e r proprioceptive or exteroceptive, as FRA t r a c t s . These t r a c t s project to the cerebellum and with many components i n the medial lemniscal system, to the cerebral sensorimotor cortex (Grampp and Oscarsson, 1968). The ascending FRA pathways have the following features i n common (Evarts, 1971:100): 1) The information from the periphery i s without modality s p e c i -f i c i t y , because e x c i t a t i o n and/or i n h i b i t i o n i s evoked by a l l components of the FRA. 2) The receptive f i e l d s are large and may include one or several limbs. They sometimes consist of excitatory and i n h i b i t o r y areas, but they permit only crude s p a t i a l d i s c r i m i n a t i o n . 3) The FRA e f f e c t s to ascending pathways are mediated by pools of interneurones i n the s p i n a l cord and/or brainstem. 4) These interneurones are strongly excited and i n h i b i t e d by descending t r a c t s . The actions of FRA's are mediated by s p i n a l and b r a i n stem interneurones which can be denoted " f l e x o r r e f l e x interneurones" (FRI's). This conundrum i s presented as follows: Can these FRI's be regarded pr i m a r i l y as f l e x o r r e f l e x centres, under the control of descending influences, or do they represent a focus by which descending motor a c t i v i t y i s modulated by peripheral input? However, such a d i s t i n c t i o n may be meaningless as considerable evidence suggests that motor performance depends, at le a s t i n part, on the i n i t i a t i o n of e x c i t a t i o n and i n h i b i t i o n 167 of r e f l e x arcs by higher centres (Lundberg, 1966; Hongo, et a l . , 1966a, b). Oscarsson (1970) has suggested that ascending FRA's function to provide a continuous feedback as to the state of the FRI's i n r e l a t i o n s h i p to motor commands from higher centers. Exceptions to Reciprocal I n h i b i t i o n Exceptions to the r u l e of i p s i l a t e r a l e x c i t a t i o n of f l e x o r motor-neurones and r e c i p r o c a l i n h i b i t i o n of extensor motor neurones have long been recognized. For example, Sherrington (1906) described "extensor thrust" whereby pressing the skin underneath the toe pads of the hindfoot (dogs) e l i c i t e d extension of that limb. S i m i l a r i l y i t was found that stimulation of the appropriate p e r i p h e r a l nerve trunks could provoke i p s i l a t e r a l extension (Sherrington and Sowton, 1911). Thus, i t was concluded that i p s i l a t e r a l stimulation sometimes y e i l d s r e f l e x con-t r a c t i o n of extensor muscles (Graham Brown and Sherrington, 1912). Contradictions to r e c i p r o c a l e x c i t a t i o n and i n h i b i t i o n were further emphasized i n the experiments of Graham Brown (1912). Flexion of the ankle was e l i c i t e d by e l e c t r i c a l stimulation of a cutaneous nerve of the limb. The v a r i a b i l i t y of response l e d to the suggestion that i p s i l a t e r a l stimulation not only activates f l e x o r and i n h i b i t s extensor muscles but also i n h i b i t s f l e x o r and excites extensor muscles. (Ranson and Hinsey, 1930). A s i m i l a r c o n t r a d i c i t o n was found with stimulation of the forelimbs (Denny-Brown and L i d d e l l , 1928). These r e f l e x actions did not correspond to "extensor thrust", however, because a c t i v a t i o n of t h i s r e f l e x i s r e s t r i c t e d to the plantar nerves (Sherrington, 1906) whereas the aff e r e n t sources o f . f l e x o r i n h i b i t i o n and extensor 168 e x c i t a t i o n were widely d i s t r i b u t e d to stimulation of various nerves. Hagbarth (1952) described i n h i b i t i o n of f l e x o r monosynaptic re -sponses (myographic and v e n t r a l root electrotonus) and e x c i t a t i o n of ex-tensor responses with i p s i l a t e r a l cutaneous stimulation. Pinching the s k i n of the limb could e i t h e r f a c i l i t a t e or i n h i b i t f l e x o r responses depending upon the l o c a t i o n of the stimulus on the surface of the skin. I t was recognized that f o r any f l e x o r or extensor muscle both e x c i t a -tory and i n h i b i t o r y f i e l d s could be mapped out on the skin of the limb. I n h i b i t i o n of Flexor Motorneurones by FRA's Eccles and Lundberg (1959a) f i r s t reported that FRA's not only evoked EPSP's i n f l e x o r motorneurones but also but also IPSP's. I t was suggested that FRA's might ac t i v a t e two pathways to FMN one e x c i t a t o r y and one i n h i b i t o r y . Both of these pathways are themselves subject to supraspinal i n h i b i t o r y c o n t r o l . That i s FRI's are i n h i b i t e d by supra-s p i n a l centres, a d i s t i n c t i o n which became apparent when the decerebrate preparation was compared to the s p i n a l animal (Job, 1953; Eccles and Lundberg, 1959b). Holmqvist and Lundberg (1961) described the transmission from FRA's to f l e x o r motorneurones i n a v a r i e t y of preparations. In the s p i n a l cat a c t i v a t i o n of FRA's evoked, overwhelmingly, e x c i t a t i o n of f l e x o r motorneurones and i n h i b i t i o n of extensor motorneurones. The only ex-ception has been shown to occur with stimulation of the skin overlying an; extensor muscle which excites that extensor muscle and i n h i b i t s f l e x o r s (Hagbarth, 1952). In the decerebrate cat both the ex c i t a t o r y and i n h i b i t o r y pathways to f l e x o r motorneurones were t o n i c a l l y i n h i b i t e d . 169 A "low pontine l e s i o n " s e l e c t i v e l y released, from descending i n h i b i t i o n , the i n h i b i t o r y pathways from FRA's to f l e x o r and extensor motorneurones. A "caudal medullary l e s i o n " s e l e c t i v e l y released the excitatory pathways from FRA's to f l e x o r and extensor motorneurones. In the i n t a c t (anaes-thetized) animal both pathways were patent although the excitatory pathway to f l e x o r motorneurones was predominant. The preceding d e s c r i p t i o n serves to I l l u s t r a t e that FRA's evoke i n -h i b i t i o n of i p s i l a t e r a l motorneurones, i n very d i f f e r e n t degrees, de-pending upon the preparation examined. This i n h i b i t i o n of f l e x o r motor-neurones may be present i n the d i r e c t f l e x o r r e f l e x pathway or i t may be manifest i n d i r e c t l y as a descending i n h i b i t i o n of FRI's. I n h i b i t i o n . of f l e x o r motorneurones was shown to occur i n a number of motor n u c l e i including that of the biceps femoris (Holmqvist and Lundberg, 1961). I t was not c l e a r i f group IV afferents were also capable of i n h i b i t i n g f l e x o r motorneurones but pinching the skin and thermal stimulation of these afferents produced i n h i b i t i o n of f l e x o r motor responses i n the s p i n a l cat (Hagbarth, 1952). Strychnine Sensitive I n h i b i t i o n , Habituation, and I n h i b i t o r y Build-up Eccles (1964) and others have demonstrated a close r e l a t i o n s h i p between strychnine s e n s i t i v e i n h i b i t i o n and the i n t r a c e l l u l a r l y recorded IPSP's of motorneurones. The short duration of the i n d i v i d u a l IPSP's makes i t u n l i k e l y that they represent a basic mechanism of habituation. However, IPSP's e l i c i t e d by stimulation of j o i n t and cutaneous afferents can be prolonged due to "....temporal dispersion of the a c t i v a t i o n of the i n h i b i t o r y synapses rather than by the slower action of i n d i v i d u a l synapses" (Eccles, 1964;160). In other words, prolonged IPSP's can be 170 evoked i n motorneurones as a consequence of high i n t e n s i t y stimulation of cutaneous nerves and, i n addition, r e p e t i t i v e interneuronal discharge of an i n h i b i t o r y pathway has been suggested as a reasonable explanation f o r t h i s phenomenon (Eccles, 1964). A. Spinal Animal The complex IPSP evoked i n f l e x o r motorneurones by a c t i v a t i o n of FRA's undergoes a progressive decrement (as does the EPSP) during habitua-t i o n of the f l e x o r r e f l e x i n the s p i n a l cat (Spencer, et a l . , 1966c). Furthermore, Renshaw c e l l i n h i b i t i o n does not cause habituation of the f l e x o r r e f l e x i n the s p i n a l cat (Spencer, et a l . , 1966a). S i m i l a r l y , Buchwald, et a l . (1965) found that the i n h i b i t i o n of t o n i c a l l y active units i n the v e n t r a l root of the s p i n a l cat underwent a progressive decrement during habituation of phasic (excited) u n i t s . These r e s u l t s would seem to agree with the f i n d i n g of t h i s thesis wherein the i n h i b i t i o n of t o n i c a l l y a c t i v e interneurones i n the s p i n a l cord of the s p i n a l rat also underwent a progressive decrement during repeated stimulation. Administration of strychnine f a i l e d to prevent habituation of the v e n t r a l root electrotonus i n the s p i n a l cat (Spertcer, et a l . , 1966c). In contrast, t h i s thesis did demonstrate an impairment of the r e l a t i v e degree of habituation of the f l e x o r r e f l e x (see p. 128) i n the s p i n a l rat (when s i n g l e pulse stimulation was employed). This impairment of habituation was probably not due to an action on the underlying mechanism of habituation. For example, i t was d i f f i c u l t to demonstrate habituation i n the s p i n a l rat us ing s i n g l e pulse stimulation and there was always an i n i t i a l s e n s i t i z a t i o n of response.. Furthermore, Pearson and Richardson 171 (personal communication) have demonstrated that the i n f u s i o n of strychnine ( s p i n a l r a t , si n g l e pulse stimuli) caused a gradual p o t e n t i a t i o n of s e n s i t i z a t i o n . The impairment of decrement observed i n the s p i n a l rat i s l i k e l y due to a p o t e n t i a t i o n of s e n s i t i z a t i o n and not due to an ac t i o n on habituation. When stimulus t r a i n s were employed, strychnine not only f a i l e d to retard habituation but a c t u a l l y f a c i l i t a t e d decrement (or at l e a s t prevented a long term s e n s i t i z a t i o n ) (see p. 126 , t h i s t h e s i s ) . Spencer, et a l . (1966c) also found that strychnine occasionally f a c i l i t a t e d decrement of the v e n t r a l root electrotonus. The progressive decrement of i n h i b i t i o n of t o n i c a l l y a c t i v e interneurones may account for long term s e n s i t i z a t i o n of the f l e x o r r e f l e x i n the s p i n a l r a t . Therefore, i t i s u n l i k e l y that strychnine s e n s i t i v e i n h i b i t i o n c o n t r i -buted to habituation of the f l e x o r r e f l e x i n the s p i n a l r a t . B. Intact Animal This thesis has demonstrated an impairment of the r e l a t i v e and absolute degree of habituation of the f l e x o r r e f l e x i n the i n t a c t r a t . The i n t e n s i t y of the stimulation had to be r e l a t i v e l y great (20 v ) . This impairment of the r e l a t i v e degree of habituation, unlike the r e s u l t s f o r the s p i n a l r a t , was greatest during the i n i t i a l period of the t e s t (see p. 102> t h i s t h e s i s ) ; however, a reduced habituation during the i n f u s i o n of strychnine may have occurred f or several reasons: 1) Strychnine may have eliminated a tonic i n h i b i t i o n of interneurones so that new interneurones now p a r t i c i p a t e i n the f l e x o r r e f l e x . These interneurones may have l i t t l e or no tendency to demonstrate habituation with a resultant reduction i n the degree of response: (motor) habituation. 172 Thus, the impairment by strychnine would simply be the consequence of an a l t e r a t i o n i n the number of interneurones p a r t i c i p a t i n g i n the r e f l e x . 2) Strychnine may have changed the frequency of a c t i v i t y within the r e f l e x arc. I f i t i s assumed that habituation i s a r e s u l t of synaptic depression then an increase i n a c t i v i t y should reduce the degree of habituation. Furthermore, strychnine may have had an e f f e c t upon the process underlying habituation that i s independent of strychnine's actions upon post-synaptic i n h i b i t i o n . 3) The impairment of habituation might have been due to a blockage of a build-up of i n h i b i t i o n e i t h e r i n the d i r e c t (spinal) pathway from FRA's to f l e x o r motorneurones or i n d i r e c t l y as a blockage of a b u i l d -up of inhibition o r i g i n a t i n g from supraspinal structures. The former p o s s i b i l i t y i s u n l i k e l y but the l a t t e r p o s s i b i l i t y seems to be supported by the demonstration of i n h i b i t o r y build-up of s p i n a l interneurones i n the i n t a c t r a t . I t i s u n l i k e l y that t h i s build-up of i n h i b i t i o n occurs i n the i n h i b i t o r y pathway from FRA's to extensor motorneurones because there i s no such build-up i n the s p i n a l r a t even though t h i s pathway i s predominant i n the s p i n a l animal (Holmqvist and Lundberg, 1961). Therefore, the r e s u l t s of t h i s thesis suggest that a build-up of supraspinal i n h i b i t i o n may contribute to habituation of the f l e x o r r e f l e x i n the i n t a c t r a t . The f l e x o r r e f l e x i s i n h i b i t e d t o n i c a l l y by the r e t i c u l a r formation. I f t h i s i n h i b i t i o n were to represent a build-up of supraspinal i n h i b i t i o n the decerebrate rat should demonstrate a more pronounced habituation than the s p i n a l r a t . This thesis has shown that the decerebrate rat 173 demonstrated a rapid decrement of response to repeated stimulation (stimulus t r a i n s 60 v., see p. 116 , t h i s t h e s i s ) . This rapid decrement was best i l l u s t r a t e d i n Figure '27 (p. 117>, t h i s t h e s i s ) . No group of s p i n a l r a t s , regardless of the l e v e l of s p i n a l transection, ever f a i l e d to show some degree of s e n s i t i z a t i o n during the i n i t i a l 10 s t i m u l i , but decerebrate rats uniformly demonstrated a rapid decrement. The threshold for the e l i c i t a t i o n of the FWR i s greater i n the decerebrate than i n the s p i n a l animal (Sherrington and Sowton, 1915) and t h i s d i f f e r e n c e i n threshold may be a t t r i b u t a b l e to the tonic i n h i b i t i o n of FRA transmission by the b r a i n stem. I t was found i n t h i s thesis that the response of the FWR was i n i t i a l l y greater than that of s p i n a l rats but the amplitude of the r e f l e x was r a p i d l y reduced to l e v e l s below that observed i n s p i n a l rats provided the stimulus was repeated (see p. 116 , t h i s t h e s i s ) . Even tested at a strength of 20 v. decerebrate rats demonstrated a margihally- greater amplitude of response f o r the f i r s t 5 responses. Perhaps tonic b r a i n stem i n h i b i t i o n of FRA trans-mission requires stimulus r e p e t i t i o n before i t develops f u l l y . Thus, there would appear to be an a s s o c i a t i o n of rapid response decrement with tonic b r a i n stem i n h i b i t i o n of the FWR. This seems to be a re-statement of an observation made by Graham Brown (1912:262) with re-gard to the f l e x o r r e f l e x i n the decerebrate as compared to the s p i n a l animal: "....the greater e x c i t a b i l i t y of the factor of i n h i b i t i o n ["refers to decerebrate preparation^; the greater the l i a b i l i t y of the f a c t o r of contraction to " f a t i g u e " [^habituation]...." 174 B i c u c u l l i n e Sensitive I n h i b i t i o n , Habituation and In h i b i t o r y Build-up The actions of b i c u c u l l i n e are highly complex. This drug blocks the a c t i o n of gamma-amino-butyric acid (GABA) whether i t i s the post-synaptic hyperpolarization of interneurones ( C u r t i s , et a l . , 1970; C u r t i s , et a l . , 1971b) i n the br a i n and s p i n a l cord or i f i t i s the depolarization of primary aff e r e n t terminals i n the s p i n a l cord (Levy, 1974). There-fore, the administration of b i c u c u l l i n e i s associated with the antagonism of some forms of post- and pre-synaptic i n h i b i t i o n ( C u r t i s , et a l . , 1971a, b. c; Schmidt, 1973; Levy, i974; Levy and Anderson, 1974). The complexity of the actions of b i c u c u l l i n e are demonstrated by i t s actions upon dorsal root p o t e n t i a l s (DRP's). This drug eliminates DRP's (both negative and p o s i t i v e ) and pre-synaptic i n h i b i t i o n induced by stimulation of s p i n a l afferents (segmental DRP's) (Levy and Anderson, 1974), but DRP's and presumably pre-rsynaptic i n h i b i t i o n of primary afferents ( s p i n a l cord) induced by a c t i v a t i o n of supraspinal structures (supraspinal DRP's) are f a c i l i t a t e d by intravenous i n j e c t i o n of b i -c u c u l l i n e (Benoist, et a l . , 1972). However, a p p l i c a t i o n of b i c u c u l l i n e d i r e c t l y to the surface of the s p i n a l cord eliminates both segmental and supraspinal DRP's. On the other hand, a p p l i c a t i o n ,of b i c u c u l l i n e to the surface of the cortex f a c i l i t a t e d supraspinal DRP's induced by stimulation of the cortex. This suggests that the f a c i l i t a t i o n of supraspinal DRP's i s the r e s u l t of d i s - i n h i b i t i o n at the c o r t i c a l l e v e l (Benoist, et a l . , 1974). The Flexor Reflex I t has been demonstrated that a l t e r a t i o n s xn ttis 6xcit3.bility (PAD) 175 of cutaneous afferent terminals (located in the region of lamina IV) were not related to habituation of the flexor reflex in the spinal cat (Groves, et a l . , 1970). Furthermore, picrotoxin failed to prevent habituation of the ventral root electrotonus in the spinal cat (Spencer, et a l . , 1966c). Also, bicuculline failed to impair habituation of the flexor reflex in the spinal rat (this thesis). However, these result do not eliminate the possibility that pre-synaptic.inhibition or bicucul-line ..sensitive inhibition might contribute to habituation of the flexor reflex in the intact animal. This thesis has demonstrated that bicu-culline impairs the relative degree of habituation of the flexor reflex in the intact rat.(see p..'104, this thesis). Because bicuculline had no effect in the spinal rat i t might be suggested that i t s actions in the intact animal are primarily at the supraspinal level. For example the frontal cortex exerts an inhibitory action upon the reticular formation and lesions.of the frontal cortex remove this inhibition and also impair habituation of the,flexor reflex (Grifffin and Pearson, 1968b) Bicuculline might have had a similar action. Recent evidence has stressed the importance of descending brain stem Inhibition in the control of spinal reflexes. Afferent input to the spinal cord can be inhibited by repetitive stimulation of various brain stem structures (Hagbarth and Kerr, 1954) and long term (hrs.) inhibition of spinal reflexes may occur (Abrahams, 1974). Furthermore, brain stem stimulation may cause either post-or pre-synaptic inhibition of FRA transmission (Pomeiano, 1973). This inhibition i s exerted at the 176 l e v e l C . b f ^ t h e FRI's and i t i s c a r r i e d to the cord as part of the dorsal r e t i c u l o - s p i n a l system (Pompeiano, 1973), and i t originates from regions located i n the medial part of the lower b r a i n stem (HoMqvLst and Lundberg, 1961). In the decerebrate animal t h i s i n h i b i t i o n i s t o n i c a l l y a c t i v e . This system i s believed to be independent of another system of des-cending i n h i b i t i o n which originates i n the medullary j raphe''nuclei and which sends axons to the s p i n a l cord within the d o r s o - l a t e r a l funiculus (Brodal, >. et a l . , 1960). Destruction of the medullary ; raphe n u c l e i or i n f u s i o n of serotonergic antagonists was shown to give a p a r t i a l release from the tonic descending i n h i b i t i o n of transmission of the FRA's (Engberg, et a l . , 1968). 177 SECTION II Segmental I n h i b i t i o n The hindlimbs are subject to i n h i b i t i o n 'during a c t i v a t i o n of the forelimbs (Schiff-Sherrington e f f e c t ) , and repeated stimulation of afferent nerves has been reported to depress s p i n a l reflexes by increasing "general i n h i b i t i o n " ( B e r i t o f f , 1965). This "general i n h i b i t i o n " i s not r e s t r i c t e d to those pathways d i r e c t l y involved with the a c t i v a t i n g stimulus and cannot be accounted for e n t i r e l y on the basis of PTD or a s i m i l a r phenomenon. Furthermore "general i n h i b i t i o n " can also be induced by repeated stimulation of supraspinal structures. I t has been postu-l a t e d ( B e r i t o f f , 1965) that "general i n h i b i t i o n " i s mediated by the substantia gelatinosa and i t s segmentally arranged connections^ c a r r i e d i n the t r a c t of Lissauer. U n t i l recently l i t t l e experimental evidence was a v a i l a b l e to support such an hypothesis; however, a recent study of the cutaneous recpetive f i e l d s of the Macaque monkey has provided s u b s t a n t i a l evidence for the existence of segmentally mediated i n h i b i t i o n (and to a l e s s e r extent excitation) that i s t o n i c a l l y a c t i v e and mediated by the substantia gelatinosa and the t r a c t of Lissauer (Denny-Brown, et a l . , 1973). I t was found ( s p i n a l animal) that' the receptive;-field for an i s o l a t e d dorsal root preparation was under a tonic i n h i b i t o r y influence of the c e l l s of the substantia gelatinosa located both caudad and r o s t r a l to the "entry zone of the i s o l a t e d root by as many as four segments. This i n h i b i t i o n was c a r r i e d i n the t r a c t of Lissauer and i t was eliminated following i n j e c t i o n of strychnine. 178 Stimulation of the substantia gelatinosa i n h i b i t s f l e x o r responses ( I s o l l i a n i , 1958) to cutaneous stimulation and t h i s suggests that the l e v e l of f l e x o r r e f l e x e x c i t a b i l i t y might be under the control of the substantia gelatinosa. In the present study a caudal transection (T^rj) resulted i n a f l e x o r response amplitude that was s i g n i f i c a n t l y greater than that of the rats with r o s t r a l transection (C^). This i s based upon a comparison of rats tested at 20 v. (C^) with rats tested at 20 v. and 60 v. ( T 1 0 ) (see pp. 130-132,? t h i s t h e s i s ) . This depression of r e f l e x e x c i t a b i l i t y , obviously could not have been due to a damaging of the d i r e c t f l e x o r r e f l e x pathways and,, as i t appeared to be overcome i n rats tested at the higher i n t e n s i t y (C,, 60 v.), a tonic depression of r e f l e x e x c i t a b i l i t y i s implied. One possible explanation i s that the reduced r e f l e x e x c i t a b i l i t y i s the r e s u l t of a tonic i n h i b i t i o n by the substantia gelatinosa which extends from the region of the caudal transection to the r o s t r a l transection. During the i n f u s i o n of strychnine (20 v., t r a i n s ) there was no impairment of f l e x o r response decrement,.A With higher i n t e n s i t y t r a i n s (60 v.) strychnine appeared to favour response decrement by preventing a long term s e n s i t i z a t i o n of the r e f l e x . This s e n s i t i z a t i o n was c h a r a c t e r i s t i c of rats with s p i n a l transection at T5. From Figure 30 (p. 126 ) i t can be seen that strychnine released an i n i t i a l period.of high r e f l e x e x c i t a b i l i t y , and therefore i t might be suggested that, i n the s p i n a l r a t (T5) without strychnine, f l e x o r i n h i b i t i o n i s gradually reduced i n effectiveness r e s u l t i n g i n a long term s e n s i t i z a t i o n . To a 179 c e r t a i n extent such an imp l i c a t i o n seems to co r r e l a t e with the decrement of i n h i b i t i o n of s p i n a l interneurones i n the s p i n a l r a t . Temporary asphyxiation of the s p i n a l cord leads to the s e l e c t i v e destruction of small interneurones ( < 20 u dia.) p a r t i c u l a r l y within the dorsal horn (Davidoff, et a l . , 1967). The onset of asphyxiation i s associated with a reduction i n the amplitude of dorsal root p o t e n t i a l s (DRP'.s). One p a r t i c u l a r component of the DRP (DRP nomenclature of Lloyd and Mclntrye, 1949), DRPV was susceptible to asphyxiation. Periods of asphyxiation, s u f f i c e n t to cause destruction of small s p i n a l i n t e r -neurones, resulted i n a • permanent.loss" of DRPV but not other components of DRP*s (Van Harreveld and N i e c h a j i , 1970). Lloyd (1971) postulated that DRPV most l i k e l y represents the a c t i v i t y of interneurones and Wall (1962, 1964) has postulated that the o r i g i n of t h i s a c t i v i t y is: the substantia gelatinosa. The close s t r u c t u r a l r e l a t i o n s h i p of substantia gelatinosa neurones with g l i a l elements (Ralston, 1968; Scheibel and Scheibel, 1969), coupled with t h e i r uniquely small s i z e (5 to 20 ]l d i a . ) , has lead to the suggestion that they have a p a r t i c u l a r l y high metabolic rate and requirement f o r oxygen. I t would therefore seem l i k e l y that the c e l l s of the substantia^, gelatinosa are susceptible to asphyxiation. Davidoff, et a l . (1967) reported that temporary ischaemia of the s p i n a l cord caused a s e l e c t i v e decrease i n the concentrations of glycine, aspartate, and glutamate, and there was a c o r r e l a t i o n between the decrease i n glycine and aspartate and the destruction of small 180 interneurones. The concentrations of GABA and glutamine were unaltered by ischaemia of the cord. I t was assumed that decreases i n the concentra-tions of glycine and aspartate were the consequence of the destruction of s p i n a l interneurones. This was further supported by a loss of i n h i b i t i o n and polysynaptic r e f l e x e s . The loss of glutamate, however, was not d i r e c t l y r e l a t e d to the loss of interneurones. The authors suggested that a de-crease i n the glutamate concentration of dorsal root terminals may have occurred as a consequence of destruction of the interneurones upon which they normally synapse. Pearson and K r a j i n a (1972) reported that s p i n a l ischaemia (22 min.) resul t e d i n an impairment of habituation of the FWR i n the i n t a c t r a t . O r i g i n a l l y , i t was decided to use asphyxiation to cause s e l e c t i v e destruction of s p i n a l interneurones i n 1 t h e s p i n a l r a t . The e f f e c t of destruction of FRI's ( f l e x o r r e f l e x interneurones) would be two f o l d : 1) impairment of FRA transmission of e x c i t a t i o n to FMN. 2) impairment of FRA transmission of i n h i b i t i o n to FMN. I t was the objective of t h i s experiment to cause a s e l e c t i v e disruption of the l a t t e r pathway and therefore asphyxiation would present an i n d i r e c t means to impair a hypothetical build-up of i n h i b i t i o n . However, t h i s hypothesis i s no longer tenable. Asphyxiation d i d impair f l e x o r response decrement i n the s p i n a l r a t . However, i t must also be recognized that i t did so by a decrease i n the i n i t i a l l e v e l of r e f l e x e x c i t a b i l i t y ( t h i s t h e s i s , p. 121). At the end of the stimulus s e r i e s the r e f l e x e x c i t a b i l i t y was a c t u a l l y greater i n asphyxiated than c o n t r o l r a t s . This was perhaps the consequence of removal of segmental i n h i b i t i o n . Davidoff, et a l . , (1967) postulated that depletion of glutamate was responsible f o r the loss of polysnaptic reflexes following long 181 periods of s p i n a l ischaemia, possibly due to depletion of primary affer e n t terminals. Infusion of the glutamate antagonist glutamic a c i d d i e t h y l ester (Haldeman/ and McLennan, 1972) can also impair habituation* of the FWR but pr i m a r i l y because of a reduction i n the i n i t i a l l e v e l of r e f l e x e x c i t a b i l i t y ( t h i s t h e s i s , p 90 ). The i n i t i a l reduction i n r e f l e x e x c i t a b i l i t y which occurred following asphyxiation may have been the r e s u l t of the loss of glutamate}, (or destruction of interneurones) and therefore the loss of excitatory pathways to f l e x o r motorneurones. On the other hand, the l a t e r increased r e l f e x e x c i t a b i l i t y may have been the consequence of a loss of i n h i b i t o r y interneurones. 182 SECTION III Afferent' Inflow to the Spinal Cord The afferent nerves and t h e i r r e l a t e d sensory apparatus have never been conclusively eliminated as contributors to habituation and/ or s e n s i t i z a t i o n of the FWR, although habituation of the r e f l e x can continue i f d i r e c t stimulation of a f f e r e n t nerves i s substituted f o r cutaneous stimulation (Wickelgren, 1976b). Repeated cutaneous stimu-l a t i o n at frequences below 1/sec. can lead to " f a t i g u e " of group IV aff e r e n t v o l l e y (Torebjork and H a l l i n , 1974) whereas s l i g h t l y higher frequencies of stimulation can lead to " f a t i g u e " of lower threshold afferents (Campbell and Taub, 1973; Torebjork and H a l l i n , 1974). There i s also evidence that long duration hyperpolarizations (sec.) occur i n i s o l a t e d p e r i p h e r a l nerves ( p a r t i c u l a r l y non-myelihated afferents) following r e p e t i t i v e stimulation of the nerves (Ritchie and Straub, 1956; 1957). This hyperpolarization served to increase the ma-gnitude and duration of the pre-synaptic spike and thus an increase i n the amount of transmitter:: released by the primary afferent terminals might be expected. "Wind-up" ( s e n s i t i z a t i o n ) might be a consequence of such an e f f e c t as i t has been reported to be a property of repeated a c t i v a t i o n of group IV a f f e r e n t s . However, hyperpolarization of non-myelinated nerves did not occur at frequencies of stimulation below 6/sec. whereas "wind-up" occurs at frequencies as low as 0.3/sec. Spencer, Thompson, and Neilson (1966a) concluded that habituation 183 of the f l e x o r r e f l e x does not depend upon the type of a f f e r e n t a c t i v a t e d but rather upon the number of afferents activated. C l e a r l y , there i s a r e l a t i o n s h i p between high threshold afferents (group I I I and IV) and s e n s i t i z a t i o n . There i s some evidence that group I I I afferents are themselves capable of e l i c i t i n g s e n s i t i z a t i o n of the FWR i n the s p i n a l . ra t (Richardson and Pearson, personal communication); however, i t should always be .remembered that s t i m u l i s u f f i c i e n t to a c t i v a t e group IV afferents are also supramaximal for lower threshold a f f e r e n t s . In addition, the e l e c t r i c a l s t i m u l i employed i n t h i s study may have lead to a c t i v a t i o n of .deep affer e n t nerves (muscle,, j o i n t , e t c . ) . . . • Stimulus electrodes form an epicentre of.current i n t e n s i t y with a current f i e l d of decreasing density r a d i a t i n g away from t h i s centre. Even with low i n t e n s i t y stimulation high threshold afferents may be activated at the elctrodes. S i m i l a r l y with high i n t e n s i t y stimulation only low threshold afferents may be activated at the edge of the current f i e l d and the number of low threshold afferents activated may be much larger than that activated by low I n t e n s i t y s t i m u l i . The stimulus strength, and therefore the s i z e of the current f i e l d , w i l l also determine the s i z e of the sensory f i e l d a ctivated f o r any i n d i v i d u a l FRI or f l e x o r motorneurone. As a consequence, the stimulus strength may also determine whether or not the f i e l d a ctivated i s excitatory, i n h i b i t o r y o f both. These factors make i t d i f f i c u l t to determine i f i n h i b i t o r y build-up i s dependent (or for that matter independent) of the type of a f f e r e n t activated. Nociceptors are e s p e c i a l l y influenced by l o c a l chemical changes 184 where various substances such as histamine, serotonin, bradykinin, and prostaglandins are implicated. Although nociceptors (mechanical and thermal) respond with la t e n c i e s too short to be accounted for by chemical intermediates r e p e t i t i v e stimulation may lead to a s i g n i f i c a n t accumulation of such substances. These chemical factors might have s i g n i f i c a n t actions i n terms of receptor s e n s i t i v i t y and r e f l e x s e n s i t i z a t i o n . Further-more, vasomotor responses to noxious stimulation of the skin (galvanic s k i n response, GSR) are subject to habituation and might a l t e r receptor s e n s i t i v i t y and s e n s i t i z a t i o n of the f l e x o r r e f l e x . Le Blanc and Gapiton (1974) have reported i n h i b i t o r y build-up i n response to strong e l e c t r i c a l s t i m u l i and also to strong mechanical stimulation of the s k i n which suggests that i n h i b i t o r y build-up i s not dependent upon some c h a r a c t e r i s t i c of the e l e c t r i c a l stimulation. The r o l e of sensory mechanisms i n behjavioural p l a s t i c i t y of the FWR . i s beyond the scope df t h i s thesis but i t i s recognized that these mechanisms may have played a s i g n i f i c a n t r o l e . The f a i l u r e to demonstrate i n h i b i t o r y build-up i n the s p i n a l r a t does seem to i n d i c a t e that the build-up of i n h i b i t i o n does not depend upon peri p h e r a l mechanisms. Furthermore, preliminary experiments have shown i n h i b i t o r y build-up i n response to r e p e t i t i v e stimulation of the s u r a l nerve.. Stimulation of the skin w i l l cause ei t h e r e x c i t a t i o n or i n h i b i t i o n (or both) of the FRI's depending upon: 1) the l o c a t i o n of the stimulus with respect to the receptive"' f i e l d (excitatory or i n h i b i t o r y ) of the FRI. 2) the type of a f f e r e n t activated 3) the number of afferents activated 4) the i n t e g r a t i v e control of access to the FRI exerted by higher centres 185 of the ce n t r a l nervous system. Schmidt (1973) has suggested that the afferent input to the s p i n a l cord i s for the most part "surplus", that i s any stimulus evokes more af-ferent a c t i v i t y than the s p i n a l cord can reasonably process. As a con-sequence, i n h i b i t i o n i s required to reduce the affere n t input. This process of i n h i b i t i o n may occur at the receptor l e v e l ( t h i s mechanism i s found i n i n t e r t e b r a t e s ) , the l e v e l of the primary afferent terminal (pre-synaptic i n h i b i t i o n ) or at the l e v e l of the second order sensory neurones. Furthermore, Schmidt proposed that the greater the af f e r e n t inflow to the cord the greater would be the resultant i n h i b i t i o n . This woj^ld serve two purposes: 1) to adjust the s e n s i t i v i t y ( i n t e n s i t y ) of the inflow 2) to focus afferent signals into s p e c i f i c i n t e g r a t i v e components of the nervous system (In a sense to "focus a t t e n t i o n " on to s p e c i f i c elements of the massed afferent input. This would i n f a c t increase the information content of the afferent inflow). This type of i n h i b i t o r y control would have s p a t i a l and temporal aspects as w e l l (Melzack, 1973;82): I t i s apparent, then, that a c e n t r a l c e l l normally has large skin area that can drive i t (the receptive f i e l d s of many f i b r e s that project on to a ce n t r a l c e l l ) , but that only a portion of the f i b r e s i s capable of doing so at any time. Because receptive f i e l d s may vary i n s i z e and s e n s i t i v i t y from moment to moment, any transient input- such as a stimulus - pro-duces a c t i v i t y that must be selected by the b r a i n from a con t i n u a l l y changing background. The r e s u l t s of t h i s thesis suggest that both the i n c l u s i o n ( i f the stimulus l i e s within an excitat o r y f i e l d ) and exclusion ( i f the stimulus l i e s with an i n h i b i t o r y f i e l d ) of afferent a c t i v i t y are capable of undergoing progressive changes i n response to repeated stimulation. For example, the more intense the stimulus (and thus the greater the afferent 186 inflow) the greater w i l l be the resultant i n h i b i t i o n ( i n h i b i t o r y build-up). This i n h i b i t i o n i s dependent upon c e n t r i f u g a l * pathways. In t h i s regard, Haber and Wagman (1974) have reported i n h i b i t o r y build-up of s p i n a l interneurones i n response to r e p e t i t i v e stimulation of 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 (n.r.g ). The n.r.g. demonst-rated both excitatory and i n h i b i t o r y build-up to repeated cutaneous stimulation of the limbs (Le Blanc and Gapiton, 1974). On the other hand, r e p e t i t i v e stimulation of t h i s nucleus lead to i n h i b i t o r y build-up of spontaneously a c t i v e s p i n a l interneurones d i s t r i b u t e d i n laminae I and IV to VII. Inhibitory build-up of evoked a c t i v i t y e l i c i t e d by intense p e r i p h e r a l stimulation was also demonstrated (Haber and Wagman, 1974). The n.r.g. therefore seems to be a possible source of the i n h i b i t o r y build-up observed i n the experiments of t h i s t h e s i s . I n h i b i t o r y build-up may be secondary to excitatory build-up of i n t e r -neurones d r i v i n g i n h i b i t o r y interneurones; however, i n h i b i t o r y synapses are also capable of p l a s t i c changes. For example, the c h a r a c t e r i s t i c s of i n h i b i t o r y build-up, with regard to stimulus i n t e n s i t y , are s i m i l a r to the frequency f a c i l i t a t i o n of i n h i b i t o r y synapses observed i n Aplysia (Waziri, et a l . , 1969) and the mammalian s p i n a l cord (Kuno and Weakly, 1972b). Most l i k e l y , i n h i b i t o r y build-up i s the r e s u l t of a recruitment of the influence of interneurones demonstrating some form of frequency f a c i l i t a t i o n or PTP but requ i r i n g the p a r t i c i p a t i o n of supraspinal structures. Complex i n t e r a c t i o n s such as progressive d i s - i n h i b i t i o n and d i s - f a c i l i t a t i o n may also be involved. B r i e f periods (sec.) of noxious stimulation of the skin have also 187 been shown to a l t e r a c t i v i t y at various l e v e l s of the nervous system such as the s p i n a l cord, the r e t i c u l a r formation and the thalamus, either i n h i b i t i n g or f a c i l i t a t i n g a c t i v i t y f o r 5 to 10 min. Longer periods of stimulation resulted i n longer periods of a l t e r e d a c t i v i t y . These e f f e c t s were highly dependent upon the depth of anaesthesia and were observed only during moderate and not l i g h t or heavy anaesthesia (Melzack, et a l . , 1968, 1969). The i n h i b i t i o n of a c t i v i t y recorded i n t h i s study i s assumed to be due to e x c i t a t i o n of i n h i b i t o r y interneurones that exert t h e i r e f f e c t s upon spontaneously a c t i v e interneurones. The blockage of i n h i b i t o r y build-up following an i n j e c t i o n of strychnine tends to support t h i s assumption. I t i s d i f f i c u l t to determine the longest period of i n h i b i t i o n which might be induced. This i s d i f f i c u l t to do because a f t e r - i n h i b i t i o n i s measured i n terms of the absence of a c t i v i t y . If the spontaneously a c t i v e interneurone i s " l o s t " during the period of i n h i b i t i o n the ex-perimenter may be l e f t i n the ludicrous s i t u a t i o n of recording a lack of a c t i v i t y where no a c t i v i t y e x i s t s . Therefore,, only l i m i t e d periods of i n h i b i t i o n are s a f e l y recorded (due to movement of the electrode) and the longest possible duration of a f t e r - i n h i b i t i o n may be much greater than that reported i n t h i s t h e s i s . In t h i s regard, the i n h i b i t i o n of the plantar r e f l e x described by Abrahams (1974) developed progressively with repeated stimulation of the brain, l a s t e d a number of hours a f t e r termination of the stimulation, and remained even a f t e r subsequent transection of the s p i n a l cord. 188 SECTION IV The Behaviour of Pain The r e s u l t s of t h i s thesis indicated that i n h i b i t o r y build-up and habituation of the f l e x o r r e f l e x to repeated noxious (painful) s t i m u l i bear some r e l a t i o n s h i p to the perception of pain because: 1) Strychnine impaired habituation of the f l e x o r r e f l e x only i f the i n t e n s i t y of stimulation was s u f f i c i e n t l y strong to a c t i v a t e group IV af f e r e n t s . This stimulus (20 v., 7.5 mA) was s u f f i c i e n t to cause pain when tested on t h i s experimenter (electrodes inserted into the s k i n ) . 2) Inhi b i t o r y build-up 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 of stimulation. This build-up of i n h i b i t i o n was p a r t i c u l a r l y apparent with s t i m u l i of an i n t e n s i t y s u f f i c i e n t to cause pain. 3) The f l e x o r r e f l e x has been c l o s e l y associated with the perception of pain by a number of authors. Sherrington (1910), i n f a c t , defined the f l e x o r r e f l e x as a "nociceptive r e f l e x " . The sudden a p p l i c a t i o n of a noxious (painful) stimulus i s followed by a number of stereotyped responses or "pseudaffective r e f l e x e s " as named by Sherrington (1906). Melzack and Wall (1965:976) described these "psuedaffective r e f l e x e s " (now shortened to " a f f e c t i v e r e f l e x e s " ) : Sudden,' unexpected damage to the skin i s followed by 1) a s t a r t l e response; 2) a f l e x i o n r e f l e x ; 3) postural readjustment; 4) v o c a l i z a t i o n ; 5) o r i e n t a t i o n of the head and eyes to examine the damaged area; 6) autonomic responses; 7) evocation of past experience i n a s i m i l a r s i t u a t i o n and pr e d i c t i o n of the consequence of the stimulation; 8) many other patterns of behavior aimed at diminishing the sensory and a f f e c t i v e components of the whole experience, such as rubbing the damaged area, avoidance behavior, and so f o r t h . 189 The decerebrate dog displays such "pseudaffective r e f l e x e s , " i n response to noxious stimulation, which are presumably divorced from any higher perception of pain. Sherrington (1906) observed that i n the decerebrate dog these responses underwent a rapid decrement of response amplitude following repeated e l i c i t a t i o n : * "The movement, even when most vigorous and prompt, dies away r a p i d l y , to be succeeded i n some cases by a few weaker r e p e t i t i o n s , each successively weaker and more transient thanrthe l a s t . " Sherrington did not. i n d i c a t e i f t h i s rapid response decrement extended to the f l e x o r r e f l e x . The r e s u l t s of t h i s thesis have confirmed Sherrington's r e s u l t s and have extended them to show that the f l e x o r r e f l e x also undergoes a rapid decrement i n the decerebrate animal. The FWR shares a number of c h a r a c t e r i s t i c s with the perception of pain. The sudden a p p l i c a t i o n of a p a i n f u l stimulus to the s k i n i s per-ceived ( i n man), almost immediately as a sharp pain or " f i r s t pain" which i s then followed, approximately 0.5 to 1.0 sec l a t e r , by a more d i f f u s e , burning, or "second pain". This perceptual separation of pain into an early and a l a t e component has been a t t r i b u t e d to d i f f e r i n g conduction v e l o c i t i e s of cutaneous a f f e r e n t s : " f i r s t pain" with group III (A) afferents and "second pain" with group IV (C) afferents (Lewis and Pochin, 1938 a,b; Noordenbos, 1959; S i n c l a i r and Stokes, 1964; S i n c l a i r , 1967). Phylogenetically, " f i r s t pain" i s believed to cor-respond to a r e l a t i v e l y new and s p e c i f i c discriminatory or " e p i c r i t i c system", and the "second pain" i s believed to be representative of an old and d i f f u s e or "protopathic system" (Head, 1920; Landau and Bishop, 1953; C o l l i n s , et a l . , 1960). In a s i m i l a r i f not i d e n t i c a l manner, 190 the f l e x o r r e f l e x i s also composed of an early and a l a t e r e f l e x component (Kugelburg, 1948; D i m i t r i j v e c and Nathan, 1970; Pr i c e , 1972; t h i s t h e s i s , p. 133). The early f l e x o r r e f l e x has been associated with " f i r s t pain" and the l a t e f l e x o r r e f l e x i s associated with "second pain" ^ P r i c e , 1972). The threshold for the perception of pain and that of the FWR, i n man, are i d e n t i c a l (Hardy, 1953). Price (1972) has suggested that FRI's also function as the interneurones transmitting pain. Furthermore, the perception of pain may habituate (Glaser and G r i f f i n , 1962; Le Blanc and Potvin, 1966; Campbell and Taub, 1973; Torebjork and H a l l i n , 1973; Pearson, personal communication) or s e n s i t i z e (Noordenbos, 1959; P r i c e , 1972) following r e p e t i t i v e stimulation. The discharge of sp i n a l interneurones may also be correlated with the early f l e x o r r e f l e x (high frequency burst) and the l a t e f l e x o r r e f l e x (after-discharge). Repetitive stimulation r e s u l t s i n a s e n s i -t i z a t i o n of the a f t e r discharge or "wind-up" (Mendell and Wall* 1965; •" Mendell, 1966; Wagman and P r i c e , 1969; P r i c e and Wagman, 1970, 1971; thi s thesis p. 143). The i n h i b i t i o n of neuronal after-discharge may be re l a t e d to the induction of analgesia and the decrement of after-discharge may r e s u l t i n a reduction i n the perception of pain. Therefore, i n t e r -a ction might be expected between the s e n s i t i z a t i o n of after-discharge and the build-up of i n h i b i t i o n . Intense stimulation of the skin of a limb i s known to induce, analgesia of the c o n t r a l a t e r a l limb by "c o u n t e r - i r r i t a t i o n " . ( M e n d e l l , 1973) . Strong stimulation of the skin of a limb i s also associated with i n h i b i t i o n of c o n t r a l a t e r a l s p i n a l interneurones believed to transmit 191 noxious information (Taub, 1964; Mendell, 1966; Brown and Franz, 1969). Therefore, r e p e t i t i v e stimulation of the skin might be expected to cause a progressive i n h i b i t i o n of after-discharge of c o n t r a l a t e r a l s p i n a l interneurones. This hypothesis was supported by the r e s u l t s of t h i s thesis where such an i n t e r a c t i o n was demonstrated (p.156, t h i s t h e s i s ) . Stimulation of the c e n t r a l grey of the br a i n stem i n the v i c i n i t y of the n.r.d. induces a profound i n h i b i t i o n of the FWR, the i n t e r -neurones of lamina V of the s p i n a l cord, and produces a strong analgesia i n the skin of the hindlimbs (Reynolds, 1969; Liebeskind, et a l . , 1974; Mayer and Liebeskind, 1974; Melzack and Melinkoff, 1974). Wall (1970) proposed that lamina V interneruones were l i k e l y to be FRI's; however, no anatomical connexion.has been established between lamina V interneurones and f l e x o r motorneurones. There i s evidence which suggests that lamina VI interneurones send c o l l a t e r a l s to the motorneuronal pools (Matsushita, 1969). Considerable attention has been focused upon the r o l e of lamina V interneurones i n the transmission of pain. These interneurones respond to noxious cutaneous stimulation either e l e c t r i c a l , chemical, or natural (pinch) (Liebeskind, et a l . , 1974). Some lamina V interneurones con-t r i b u t e to the s p i n o c e r v i c a l and spinothalamic t r a c t s , and they are also the s i t e for convergence of v i s c e r a l and somatic group IV affere n t input to the s p i n a l cord (Pomeranz:,. et a l . , 1968). In response to peripheral s t i m u l i extensive excitatory and i n h i b i t o r y f i e l d s can be mapped for any p a r t i c u l a r lamina V interneurone. In ad d i t i o n s t i m u l i applied to the i n h i b i t o r y f i e l d can i n h i b i t the response of such a neurone to a^noxious 192 stimulus applied to i t s excitatory f i e l d (Wall, 1967; Pomeranz, et a l . , 1968; Hillman and Wall, 1969; Selzer and Spencer, 1969; Wagman and Pri c e , 1969; P r i c e , et a l . , 1971; Besson, et a l . , 1974). The r e s u l t s of t h i s t hesis have shown that lamina V interneurones demonstrate i n h i b i t o r y build-up (p. 161 , t h i s t h e s i s ) . The i n h i b i t o r y f i e l d s of lamina V interneurones are under strong i n h i b i t o r y c o n t r o l by supraspinal mechanisms and the s i z e of the receptive f i e l d s are t o n i c a l l y reduced i n the decerebrate animal (Wall, 1967). The i n h i b i t i o n of lamina V interneurones, induced by stimulation of the n.r.d., was blocked by the i n j e c t i o n of l y s e r g i c a c i d d i e t h y l amide (LSD) (Liebeskind, et a l . , 1974), and the i n h i b i t i o n of s p i n a l reflexes that occurs following stimulation of the ventro.-medial region of the medulla (bulbospinal i n h i b i t i o n ) was also blocked by i n f u s i o n of LSD and methysergide (Clineschmidt and Anderson, 1970). The administration of LSD?caused a s p e c i f i c i n h i b i t i o n of the a c t i v i t y of the neurones i n the raphe^nuclei (Haigler and Aghajanian, 1974). Furthermore, the cutaneous analgesia induced by stimulation of the n.r.d. i s blocked by p r i o r administration of p-CPA ( A k i l and Mayer, 1972) presumably due to the s p e c i f i c depletion of serotonin (Koe and Weissman, 1966; Sheard, 1973). In addition, l e s i o n s of the raphe^nuclei (dorsal and median), pre-treatment with p-CPA, and i n f u s i o n of serotonin antagonists such as LSD are associated with a p o t e n t i a t i o n of s e n s i t i z a t i o n of various behavioural responses (Carlton and Advokat, 1973; Davis and Sheard, 1974; Davis and Sheard, i n press); or increases i n dishabituatory responses (Aghajanian and Sheard, 1968; Conner, et a l . , 1970); or decreased thresholds 193 to noxious s t i m u l i (Tehen, 1967; Harvey and L i n t s , 1971). In d e r i v a t i o n from these various r e s u l t s , blockage of descending serotonergic i n h i b i t i o n would be expected to produce a greater degree of f l e x o r response s e n s i t i z a t i o n and perhaps hyperalgesia. In t h i s study the i n i t i a l l e v e l of r e f l e x e x c i t a b i l i t y (absolute s e n s i t i z a t i o n ) was increased by pre-treatment with p-CPA, by l e s i o n s of the n.r.d., and to a l e s s e r extent by i n f u s i o n of methysergide. These r e s u l t s imply that the removal of bulbospinal i n h i b i t i o n and/or the influence of the n.r.d. eliminates a tonic l e v e l of i n h i b i t i o n of the FWR. Continued r e p e t i t i o n of the s t i m u l i eventually lead to a FWR of an amplitude below that of controls, i n p-CPA treated and lesioned (n.r.d.) r a t s . This p a r t i c u l a r f i n d i n g i s d i f f i c u l t to r e c o n c i l e i n terms of the removal of a tonic i n h i b i t i o n . It appears, however, that the raphe n u c l e i exert an i n h i b i t o r y influence upon behavioural arousal. The arousal mechanism seems to exert both excitatory and i n h i b i t o r y influences upon s p i n a l re f l e x e s ( t h i s t h e s i s , p. 200 ) which suggests that both components may be released by removal of the influence of the n.r.d. and the serotonergic system. Thus a greater expression of i n h i b i t o r y build-up might be predicted with a resultant increase i n response decrement. These findings further support a c o r r e l a t i o n between a pot e n t i a t i o n of s e n s i t i z a t i o n of the FWR and cutaneous hyperalgesia. Cutaneous analgesia induced by repeated stimulation of the b r a i n stem does not commence immediately following the i n i t i a t i o n of the stimulus (Melzack and Melinkoff, 1974). Furthermore, the period of analgesia may outlast the stimulation by periods of up to 5 min. (Reynolds, 1969). 194 Repetitive peripheral stimulation can also induce an analgesia which develops gradually and which outl a s t s the period of stimulation (Wall and Sweet, 1967; Meyer and F i e l d s , 1972; Andersson, et a l . , 1973; Campbell and Taub, 1973). Electro-analgesia induced i n t h i s way can occur i f the i n t e n s i t y of stimulation i s non-noxious, although noxious stimulation may be required to evoke pericutaneous analgesia. I t has been postulated that some forms of analgesia may occur as the r e s u l t of i n h i b i t i o n of the sensory transmission of pain, and the gradual induction of e l e c t r o -analgesia has been a t t r i b u t e d to a progressive build-up or recruitment of i n h i b i t i o n within the c e n t r a l nervous system. I t has also been suggested that the balance between e x c i t a t i o n and i n h i b i t i o n of s p i n a l interneurones might determine the effectiveness of pain transmission (Melzack, 1973). Weak s t i m u l i , below group IV afferent thresholds, have been shown to i n h i b i t the resonse of dorsal horn Interneurones to noxious s t i m u l i (Wall, 1967). The s t i m u l i employed i n t h i s thesis were usually of an i n t e n s i t y above the threshold of group IV afferents (6 mA) i n order to produce a marked i n h i b i t o r y build-up of s p i n a l interneurones; however, some interneurones demonstrated a reduction i n spontaneous a c t i v i t y with r e p e t i t i o n of r e l a t i v e l y weak s t i m u l i . The induction of acupuncture analgesia usually involves r e p e t i t i o n of s t i m u l i at much greater frequences and for much longer periods than used i n t h i s study. It i s possible that increasing the period of stimulation and the frequency of stimulation would produce a s i g n i f i c a n t build-up of i n h i b i t i o n of the neurones sub-serving the transmission of pain. Considerable i n t e r e s t has developed as to the possible r e l a t i o n s h i p 195 between the transmission of pain and c e n t r a l i n h i b i t i o n (Melzack, 1973). Dusser de Barenne (1910) presented evidence that the t o p i c a l a p p l i c a t i o n of strychnine to the dorsal surface of the s p i n a l cord produced hyper-a l g e s i a and h y p e r - r e f l e x i a i n the dermatomes supplied by that region of the cord. Of course h y p e r - r e f l e x i a was to be expected; however, Dusser de Barenne also reports behaviour that must be associated with spontaneous pain and hyper-algesia: A few seconds a f t e r the repeated contact of the poison [strychnine] with the s p i n a l cord at t h i s spot, the dog that mean-while has nearly awakened from the narcosis, begins to l i c k the skin of the r i g h t h a l f of the trunk over a region, extending l i k e a band of moderate breadth from the mid-dorsal to the mid-ventral l i n e , passing over the most caudal ribs....The hyper-reflectory symptoms are: 1) wrinkling of the skin, 2) curving of the verte-b r a l column, the concavity turned to the r i g h t , 3) with i n t e r v a l s scratching movements of the r i g h t hind-limb, resembling c l o s e l y those of the well known Sherrington's "scratch r e f l e x " . . . . Con-t i n u a l l y repeating t h i s gentle, mechanical i r r i t a t i o n , I gradually approached that region, and as soon as I have passed i t s boundary, the hyper-reflectory symptoms described above are aroused or be-come much more intense, whilst i n most cases, the animal shows at the same time by howling and b i t i n g that the subjective symp-toms are likewise aroused or t h e i r i n t e n s i t y increased. These r e s u l t s indicated a tonic i n h i b i t i o n of pain transmission at the s p i n a l l e v e l . I nhibitory build-up, decrement of the FWR to p a i n f u l s t i m u l i , and acupuncture analgesia (McLennan and G i l f i l l a n , personal communication) are antagonized by the i n f u s i o n of strychnine which im-p l i e s a common mechanism for i n h i b i t o r y build-up and some forms of analgesia. Furthermore, acupuncture analgesia i s also blocked or reduced following the i n j e c t i o n of b i c u c u l l i n e and methysergide (McLennan and G i l f i l l a n , per-sonal communication) and acupuncture analgesia has been associated with an increase i n the brain content of serotonin (Acupuncture Anaesthesia Research Group, 1973). 196 SECTION V I n h i b i t i o n and the Theories of Habituation The major theories of habituation are as follows: 1) Dual-Process theory (Groves and Thompson, 1970) 2) Conditioned I n h i b i t i o n (Sokolov, 1965; Stein, 1966) 3) Afferent Neuronal Habituation (Hernandez-Peon, 1961) and each theory recognizes two separate sensory systems. One, a s p e c i f i c sensory system, which ref e r s to the c l a s s i c a l paucisynaptic affe r e n t pathways that carry modally, temporally, and s p a t i a l l y s p e c i f i c information from the receptors to the corresponding sensory-motor c o r t i c a l p r o j e c t i o n areas or to the motorneurones. This i s the "S-R" pathway described by Groves and Thompson (1970). The other system i s the non-s p e c i f i c sensory system ("state" system) which i s associated with phenomena such as arousal, attention, and " a f f e c t i v e " responses. The information c a r r i e d by t h i s system i s not temporally or s p a t i a l l y coded nor i s i t modality s p e c i f i c . In other words, i t has c h a r a c t e r i s t i c s s i m i l a r to the information transmitted by FRA's. The non-specific system i s character-ized by s e n s i t i z a t i o n of r e f l e x responses whereas the s p e c i f i c sensory system demonstrates habituation (Webster, 1971; 1974). Response decrements do occur i n the non-specific sensory system (Sharpless and Jasper, 1956; Hernandez-Peon, 1960; Knispel and Siegel, 1973; Deutsch and Dennis, 1975), however. The " s t a t e " system may be synonymous with the brain stem r e t i c u l a r mechanism that controls the l e v e l of behavioural e x c i t a b i l i t y or arousal. This c e n t r a l excitatory state i s conceptually i d e n t i c a l to the " c e n t r a l arousal s t a t e " (Lindsley, 1960; Duffy, 1962). The r e t i c u l a r formation i s 197 usually accepted as the major component of the arousal system (Moruzzi, 1964) and stimulation of the r e t i c u l a r formation w i l l cause EEG arousal and dishabituation (or s e n s i t i z a t i o n ) of habituation i n the s p e c i f i c sensory system (Mancia, et a l . , 1959). Hernandez-Peon,rs (I960) hypothesis that "afferent neuronal habituation" r e s u l t s from a " r e t i c u l a r c e n t r i f u g a l i n h i b i t i o n " upon the" s p e c i f i c sensory pathways has not been confirmed (Buchwald and Humphrey, 1973). The Dual-Process theory i s based almost e n t i r e l y on habituation of the f l e x o r r e f l e x i n the s p i n a l cat. As a consequence, the flexor r e f l e x was i s o l a t e d from the r e t i c u l a r formation and other c e n t r a l nervous system structures c r i t i c a l to the expression of behavioural arousal. In essence the Dual-Process theory presents two i n f e r r e d constructs, one a process of response decrement i n the S-R pathway and the other a process of response increment i n the " s t a t e " system (see t h i s t h e s i s , p. 13 ), which when summated, are responsible for the f i n a l motor response of the f l e x o r r e f l e x . Confirmation of t h i s hypothesis was found at the interneuronal l e v e l . The process of i n f e r r e d decrement was represented by habituation of the high frequency burst discharge and s e n s i t i z a t i o n of the after-discharge was representative of the i n f e r r e d incremental process. Weak (touch, h a i r movement, etc.) stimulation of the skin tends to evoke a high frequency burst i n s p i n a l interneurones (Price and Browe, 1973; t h i s thesis) which undergoes a decrement of response (Groves and Thompson, 1973; t h i s thesis) when the stimulus i s repeated. The a f t e r -discharge of s p i n a l interneurones i s evoked,, f o r the most part, by high i n t e n s i t y (pinch) stimulation of the skin (Price and Browe, 1973; t h i s thesis) 198 and t h i s response builds-up with repeated stimulation. This s e n s i t i z a t i o n of after-discharge i s often, but not always, followed by a decrement of response i n both the s p i n a l (Groves and Thompson, 1973) and i n t a c t animal (t h i s t h e s i s ) . It i s assumed that the decrement of the high frequency burst i s l i k e l y to be a form of "synaptic depression" and that the s e n s i t i z a t i o n of after-discharge i s perhaps a consequence of "frequency f a c i l i t a t i o n " . Even the laminar l o c a t i o n of interneurones d i s p l a y i n g the high frequency burst i s s i m i l a r to those associated with the trans-mission of r e l a t i v e l y s p e c i f i c sensory information (Price and Browe, 1973). In contrast, s p i n a l interneurones d i s p l a y i n g after-discharge are located i n laminae which contain interneurones contributing to s p i n o - r e t i c u l a r t r a c t s and which receive a large- convergence of a c t i v i t y from s p i n a l and supraspinal structures (Matsushita, 1969; Bowsher, 1972). Thus, a f t e r -discharge of s p i n a l interneurones may be i t s e l f considered a property of the " s t a t e " system whereas the high frequency burst response i s l i k e l y c h a r a c t e r i s t i c of the s p e c i f i c sensory system. The r e s u l t s of t h i s thesis and the r e s u l t s presented by Spencer, et a l . (1966c) demonstrate that drugs such as strychnine, p i c r o t o x i n , and b i c u c u l l i n e do not stop habituation of the f l e x o r r e f l e x i n the s p i n a l and i n t a c t animal. Neither does the i n h i b i t i o n of s p i n a l interneurones i n the s p i n a l r a t ind i c a t e any s i g n i f i c a n t p o t e n t i a t i o n of i n h i b i t i o n (Groves and Thompson, 1973; t h i s thesis) during repeated cutaneous stimulation. In the s p i n a l rat i n h i b i t o r y mechanisms, possibly short chained intersegmental systems, may modify FWR amplitude but do not appear to play a c a u s i t i v e r o l e i n response decrements. In fa c t they may prevent response decrements under c e r t a i n circumstances ( t h i s t h e s i s , p.178)-199 Thus, there i s no evidence to i n d i c a t e that habituation i s p r i m a r i l y a r e s u l t of "conditioned i n h i b i t i o n . " If chemical synapses c h a r a c t e r i s -t i c a l l y display various forms of synaptic p l a s t i c i t y i t i s not u n l i k e l y that habituation and s e n s i t i z a t i o n are the consequence of the properties of the synapses of the c e n t r a l nervous system. Habituation of the f l e x o r r e f l e x i n the i n t a c t r a t i s not i d e n t i c a l to habituation of t h i s r e f l e x i n the s p i n a l rat (p. .97 , t h i s t h e s i s ) . Habituation i s r e a d i l y apparent i n the i n t a c t animal with an i n t e n s i t y of stimulation which produces almost no habituation i n the s p i n a l animal. This d i f f e r e n c e i s probably r e l a t e d to the elimination of spino-bulbo-s p i n a l r e f l e x e s (Shimamura and Aoki, 1969) and the reduction of f l e x o r r e f l e x "after-discharge" (Forbes, et a l . , 1923). Therefore, supraspinal structures contribute a component of e x c i t a t i o n to the s p i n a l f l e x o r r e f l e x , and i n t a c t r a t s may show a maximal response to strong s t i m u l i without a p r i o r build-up of a c t i v i t y ( p . 97, t h i s t h e s i s ) . Habituation of t h i s supraspinal component i s greatest with r e l a t i v e l y strong s t i m u l i and least with weak s t i m u l i (p. 94). It i s impaired by the i n -fusion of strychnine and b i c u c u l l i n e and i t i s f a c i l i t a t e d by pre-treatment with p-CPA and lesions of the n.r.d. The r e t i c u l a r formation has been proposed as the s i t e of generation of behaviour that i s associated with reactions to environmental s t i m u l i of novel, aversive, and p a i n f u l perceptual c h a r a c t e r i s t i c s . Stimuli of these types produce arousal, increased v i g i l a n c e , escape behaviour, and a f f e c t i v e responses. Some of these reactions such as behavioural arousal, EEG arousal, and the o r i e n t i n g r e f l e x are assumed to be mediated by the 200 the r e t i c u l a r formation. Somatic and autonomic reflexes may also be the v e h i c l e s of such behaviours. For example, the f l e x o r r e f l e x has a nociceptive component and a low threshold component associated with r e f l e x stepping (Sherrington, 1910). Presumably the nociceptive com-ponent i s associated with r e t i c u l a r a c t i v a t i o n , arousal, etc. In other words, the f l e x o r r e f l e x i s both an a f f e c t i v e r e f l e x and a simple s p i n a l r e f l e x . I t i s proposed that cutaneous s t i m u l i are capable of a c t i v a t i n g r e t i c u l a r interneruones which i n turn have the capacity to i n h i b i t tonic a c t i v i t y i n the f l e x o r r e f l e x pathway. Repeated stimulation then r e s u l t s i n a build-up of t h i s i n h i b i t o n . This i n h i b i t i o n i s p a r t i a l l y blocked by strychnine which may i n d i c a t e the involvement of glycine and g l y c i n e -l i k e transmitters. However, other forms of i n h i b i t i o n are also involved (perhaps GABA mediated i n h i b i t i o n ) . The decrement of the FWR r e l a t e d to a build-up of i n h i b i t i o n i s only apparent when the stimulus i s of an i n t e n s i t y which i s noxious (possibly p a i n f u l ) and i t may represent a mechanism for adaptation to p a i n f u l s t i m u l i repeated at regular i n t e r v a l s . Serotonergic systems are known to i n h i b i t r e t i c u l a r a c t i v i t y (Simon, et a l . , 1973) and behavioural arousal (Fibiger and Campbell, 1971; Mabry and Campbell, 1972) presumably by an a c t i o n upon the " l i m b i c - f o r e -b r a i n " system which i t s e l f exerts an i n h i b i t o r y a c t i o n upon the r e t i c u l a r formation (Morgane and Stern, 1973). Thus, lesions of the nucleus raphe''' d o r s a l i s and pre-treatment with p-CPA may eliminate an i n h i b i t i o n of the r e t i c u l a r neurones responsible for the build-up of i n h i b i t i o n observed 201 i n the s p i n a l cord. This proposed mechanism of habituation i s s i m i l a r to a " r e t i c u l a r c e n t r i f u g a l i n h i b i t i o n " of the non-specific sensory system suggested by Hernandez-Peon (1960). Voronin and Sokolov (1960) came to the conclusion that habituation of the o r i e n t i n g r e f l e x was the r e s u l t of "conditioned i n h i b i t i o n " . This concept was based i n part upon a study of the p l a s t i c responses evoked by repeated stimulation ( l i g h t ) of neurones i n the v i s u a l system (Sokolov, 1965). In t h i s s i t u a t i o n , repeated stimulation evoked s e n s i t i z a t i o n of neurones i n the r e t i n a , superior c o l l i c u l u s , and the geniculate body but interneurones of the v i s u a l cortex (pyramidal neurones) and e s p e c i a l l y the interneurones of the hippocampus, underwent rapid response decrements. Inhibitory build-up of spontaneously a c t i v e interneurones was also demonstrated (Sokolov, 1969:699) i n the v i s u a l cortex. Such r e s u l t s prompted Sokolov (1965) to suggest that PTP of the v i s u a l pathways resulted i n s e n s i t i z a t i o n of a c t i v i t y i n some interneurones and an "elaboration of i n h i b i t i o n " i n others. Furthermore Sokolov (1965:342) stated that: "The observation of complete i n h i b i t i o n of spike discharges during habituation leads to the conclusion that interneurones are involved not as negative feed-back loops of pyramidal neurones, but as " p a r a l l e l i n h i b i t i o n chains," summating p a r a l l e l inputs of excitatory and i n h i b i t o r y pathways to pyramidal neurones." Sokolov (1965) i n f e r r e d that the mechanism of t h i s "elaboration of i n h i b i t i o n " might be PTP of i n h i b i t o r y synapses. There i s an obvious s i m i l a r i t y between Sokolov's r e s u l t s and the r e s u l t s of t h i s t h e s i s . Arousal induced by stimulation of the r e t i c u l a r formation appears 202 to be under the i n h i b i t o r y c o n t r o l of the f r o n t a l cortex (Clemente and Sternum, 1967; Lynch, et a l . , 1969; Lynch, 1970) i n the r a t . Therefore, the observation that l e s i o n s of the f r o n t a l cortex prevent habituation of the FWR ( G r i f f i n and Pearson, 1968b) i s probably r e l a t e d to an increase i n behavioural arousal. A stimulus which o r i g i n a l l y did not a c t i v a t e the arousal system might now demonstrate a continuous d i s h a b i -tuation (or s e n s i t i z a t i o n ) of the f l e x o r r e f l e x . Decerebration should release r e t i c u l a r arousal from the i n h i b i t o r y influence of the f r o n t a l cortex and thus cause an impairment of f l e x o r response habituation. How-ever, i n comparison to the f l e x o r r e f l e x i s o l a t e d from the r e t i c u l a r formation by s p i n a l transection, decerebration seems capable of f a c i l i t a t i n g i n i t i a l response decrements ( t h i s t h e s i s , p. 117 ). Thus, with high i n t e n s i t y stimulation the r e t i c u l a r formation contributes to habituation of the arousal component of the f l e x o r r e f l e x . Lesions of the n.r.d. and pre-treatment with p-CPA also f a c i l i t a t e d f l e x o r habituation i n comparison to the i n t a c t animal which suggests such manipulations increase arousal and habituation of arousal. Thompson, et a l . (1973 :214) state that: "Under c e r t a i n circumstances (strong s t i m u l i presented r e g u l a r l y at r e l a t i v e l y slow rate) temporal conditioning of s e n s i t i z a t i o n of state may occur." This thesis presents no s u b s t a n t i a l evidence for temporal conditioning of i n h i b i t i o n , but i f the excitatory component i s capable of such conditioning i t i s not un-reasonable to expect a s i m i l a r conditioning of the i n h i b i t o r y component of the "state" system. According to Sokolov (1960, 1969) the o r i e n t i n g r e f l e x i s a complex 203 of responses to novel s t i m u l i which serves to increase s e n s i t i v i t y to periph e r a l s t i m u l i . As such, Sokolov would p r e d i c t a resistance to habi-tuation with s t i m u l i of a stengtb close to threshold f o r the r e f l e x and less resistance with strong s t i m u l i . This hypothesis contradicts the Dual-Process theory which would predict that low i n t e n s i t y stimulation w i l l produce the greatest habituation. A number of studies, examining a v a r i e t y of r e f l e x and behavioural responses, has indicated a contradiction to t h i s aspect of the Dual-Process theory. Two of these studies (Wickel-gren, 1967a; Pearson and MacDonald, 1973) may have produced contradictory r e s u l t s simply due to a secondary a l t e r a t i o n i n FWR e x c i t a b i l i t y as-sociated with the l e v e l of s p i n a l transection. On the other hand, experiments performed on the i n t a c t animal have also produced contradic-tory r e s u l t s (Harper, 1968; Peeke, 1969; Davis and Wagman, 1968; Jackson, 1974; t h i s t h e s i s , p. 94 ). An example i s i l l u s t r a t e d i n Figure 58 taken from Peeke (1969). These r e s u l t s r e f e r to the b i t i n g behaviour ( t e r -r i t o r i a l ) of male three-spined sticklebacks i n response to s t i m u l i of two d i f f e r i n g strengths. One curve (RM) represents the response to another and l i v e male stickleback whereas the other (MM) respresents the response to a crude model of a male stickleback. Assuming that the natural stimulus ( l i v e stickleback) i s perceived as being of greater s i g n i f i c a n c e or i n t e n s i t y these r e s u l t s are s u p r i s i n g l y s i m i l a r to those shown i n Figure 11 (th i s t h e s i s , p.94 ). Similar r e l a t i o n s h i p s have been shown for the galvanic skin response (Harper, 1968; Jackson, 1974) and for the s t a r t l e response of the ra t (Davis and Wagman, 1968). In these various studies the absolute and r e l a t i v e habituation were greatest with 204 I •-• RM 1 o—o MM ! I 2 3 4 5 6 7 8 9 10 DAYS i \ Figure 58. Mean number of b i t e s per minute delivered at the stimulus by a male three-spined stickleback Gasterosteus aculeatus, for a group presented with a r e a l , male con s p e c i f i c con-fined to a glass tube (RM) and a group presented with a crude wood model male (MM) moved i n a c i r c u l a r path i n the t e r r i t o r y of the subject f i s h . Each point represents the average of 3 successive minutes (Peeke, 1969). 205 the highest i n t e n s i t y of stimulation. In the case of the FWR t h i s r e l a t i o n s h i p held only for the i n t a c t r a t and, furthermore, i f the stimu-l a t i o n was repeated a s u f f i c i e n t number of times i t was obvious that the r e l a t i v e degree of habituation was greatest with the lower i n t e n s i t y of stimulation. Thompson, et a l . , (1973:245) account for the c o n t r a d i c t i o n i n the r e s u l t s presented by Peeke (1969) as follows: "....the periods of r e -sponse measurement are c r i t i c a l — too large time blocks [analogous to response blocks used i n t h i s thesis] wash out i n i t i a l response s e n s i t i z a t i o n " (see p. 92, t h i s t h e s i s ) . Thus, the response decrement i s . c l a s s i f i e d as "habituation of s e n s i t i z a t i o n " by Groves and Thompson, (1970). Unfortunately, "habituation of s e n s i t i z a t i o n " i s not s t r i c t l y distinguished from that occurring i n the s p i n a l animal as opposed to that i n the i n t a c t animal. The terminology "habituation of s e n s i t i z a t i o n " also implies the necessity for a build-up or increment of response and as a consequence I propose the term "arousal habituation" which r e f e r s to a decrement of that component of r e f l e x response a t t r i b u t a b l e to a c t i v a t i o n of the " s t a t e " system. S p e c i f i c a l l y , "arousal habituation" i s the gradual withdrawal of the " s t a t e " system from the r e f l e x l e v e l . Both the s i z e of the arousal component and the degree of arousal habi-tuation are d i r e c t l y r e l a t e d to stimulus i n t e n s i t y . The c o n t r i b u t i o n a of the " s t a t e " system i s both excitatory and i n h i b i t o r y provided the r e t i c u l a r formation i s not i s o l a t e d from the r e f l e x arc under study. With t h i s preface an addendum to the Dual-Process theory i s offered. Presentation of a strong stimulus w i l l a c t i v a t e the " s t a t e " system 206 with a resultant r e t i c u l a r a c t i v a t i o n ( t h i s r e s u l t s i n the i n t e r p o l a t i o n of e x c i t a t i o n into the r e f l e x a r c ) . Repeated stimulation may cause s e n s i t i z a t i o n of the arousal (excitation) but a maximal response may be triggered by the f i r s t stimulus presentation. The withdrawal of arousal ("arousal habituation") from the r e f l e x arc i s due to a build-up of i n h i b i t i o n of the more tonic components of the r e f l e x . This i n h i b i t i o n i s probably manifest at a l l l e v e l s of the " s t a t e " system and i t may be analogous to .the "general i n h i b i t i o n " of the s p i n a l r e f l e x e s described by B e r i t o f f (1965). The build-up of i n h i b i t i o n demonstrates these c h a r a c t e r i s t i c s : a) The process of i n h i b i t o r y build-up occurs i n the " s t a t e " system (requires the r e t i c u l a r formation) but not i n the S-R pathway. It i s manifest as a progressive reduction i n the tonic a c t i v i t y of the r e f l e x ("after-discharge"). b) During repeated stimulation the build-up of i n h i b i t i o n i s progressive and therefore arousal decays or habituates.- The i n h i b i t i o n may l a t e r decay but other decremental mechanisms (synaptic depression?) w i l l have become predominant. c) The rate of i n h i b i t o r y build-up and the duration of i n h i b i t i o n following cessation of the stimulation ( a f t e r - i n h i b i t i o n ) are 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 of stimulation. At low i n t e n s i t i e s l i t t l e or no a c t i v a t i o n of the arousal system occurs. d) The rate of i n h i b i t o r y build-up i s d i r e c t l y r e l a t e d to stimulus frequency. e) The i n h i b i t i o n outlasts the duration of stimulation ( a f t e r -i n h i b i t i o n ) and then decays spontaneously. 207 f) Repeated s e r i e s of strong s t i m u l i r e s u l t i n progressively longer periods of a f t e r - i n h i b i t i o n . g) I n h i b i t o r y build-up may demonstrate genera l i z a t i o n to the ex-tent that the generalized stimulus l i e s within an i n h i b i t o r y f i e l d . h) D i s - i n h i b i t i o n of a f t e r - i n h i b i t i o n may occur i f the d i s -i n h i b i t o r y stimulus l i e s within an excitatory f i e l d . i ) There i s a p o s s i b i l i t y that temporal conditioning of a f t e r -i n h i b i t i o n w i l l occur with repetittonsof strong s t i m u l i at r e l a t i v e l y low rates. The r e s u l t s of t h i s thesis have presented evidence for one further i n f e r r e d construct which might underlie habituation of a p a r t i c u l a r component of the f l e x o r r e f l e x . This process i s one of a s e n s i t i z a t i o n of c e n t r i f u g a l i n h i b i t i o n of the more tonic elements of the FWR. To t h i s extent t h i s thesis d i f f e r s from the Dual-Process theory and supports Sokolov's theory of Conditioned I n h i b i t i o n . However, t h i s thesis adds to and does not contradict the Dual-Process theory. Furthermore, i t supports the concept that stimulation of sensory afferents e l i c i t s both s i g n i f i c a n t i n h i b i t i o n and e x c i t a t i o n which co-exist w i t h i n the c e n t r a l nervous system. The i n t e r a c t i o n of t h i s developing e x c i t a t i o n and i n h i b i t i o n presents a unique way i n which the c e n t r a l nervous system can control sensory inflow to the s p i n a l cord and r e f l e x behaviour. Pavlovian i n h i b i t i o n does not draw a d i s t i n c t i o n between the phenomena of "synaptic depression" and i n h i b i t o r y build-up ( i n h i b i t i o n i s used i n the Sherringtonian sense) (This t h e s i s , p. 2 ). Either mechanism might be c l a s s i f i e d as being a manifestation of i n h i b i t i o n 208 by Beritoff; however, a mechanism such as "synaptic depression" seems at least superficially, to be analogous to Sherrington's concept of central "fatigue". Sherrington's concept of inhibition f a i l s to account for the long term inhibition of the plantar reflex (Abrahams, 1974) and the long term analgesia which is related to inhibition (Liebeskind, et a l . , 1974), both of which are induced by repeated stimulation of the brain and which have the capacity to last for periods of hours after cessation of the stimulus. This type of inhibition is a confirmation of the Russians' hypothesis of "general inhibition", but is this a form of Sherringtonian inhibition? It i s readily admitted that the inhibition of spontaneously active spinal interneurones (after-inhibition) is of a duration well below that described for the inhibition of the plantar reflex; however, f a c i l i t a t i o n of the action of excitatory synapses can last for many hours in vertebrates and i t i s not unreasonable to expect similar phenomena to occur for inhibitory synapses. 209 REFERENCES Abrahams, V. C. 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