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Effects of occlusion of the thoracic aorta on habituation of the flexor withdrawal reflex in the rat Krajina, Vladimir Peter Jan 1972

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EFFECTS OF OCCLUSION OF THE THORACIC AORTA ON HABITUATION OF THE FLEXOR WITHDRAWAL REFLEX IN THE RAT by VLADIMIR PETER JAN KRAJINA B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1969  A THESIS SUBMITTED  IN PARTIAL FULFILMENT OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n the Department of Physiology  We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA August 1972  In p r e s e n t i n g t h i s t h e s i s  in p a r t i a l  f u l f i l m e n t o f the r e q u i r e m e n t s  an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, the L i b r a r y s h a l l make i t  freely available  for  I agree  for  that  r e f e r e n c e and s t u d y .  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s  representatives.  It  of this thesis for financial written  i s understood t h a t copying o r gain shall  permission.  Depa rtment The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Columbia  publication  not be a l l o w e d w i t h o u t my  ii  ABSTRACT Experiments were c a r r i e d out t o i n v e s t i g a t e t h e e x t e n t t o which h a b i t u a t i o n o f t h e f l e x o r r e f l e x depended on mechanisms o p e r a t i n g a t interneurones.  An attempt was made t o cause s e l e c t i v e  interneurones i n the s p i n a l c o r d of the r a t p e r i o d of ischaemia. thoracic aorta. after  occlusion.  spinal  degeneration of  by s u b j e c t i n g t h e c o r d t o a  Ischaemia was produced by temporary o c c l u s i o n o f t h e  The f l e x o r w i t h d r a w a l r e f l e x was t e s t e d 3» 7 o r 1*4- days When compared w i t h d a t a from c o n t r o l a n i m a l s i t  f o u n d t h a t i s c h a e m i a had r e s u l t e d i n both a q u a l i t a t i v e  was  change and a q u a n -  t i t a t i v e d i m i n u t i o n i n t h e amount o f h a b i t u a t i o n which o c c u r r e d d u r i n g t h e p r e s e n t a t i o n o f *K)0 u n i f o r m s t i m u l i .  It  was c o n c l u d e d t h a t t h i s  impair-  ment o f t h e h a b i t u a t i o n p r o c e s s was a consequence o f d e g e n e r a t i o n o f i n t e r n e u r o n e s which n o r m a l l y cause p r o g r e s s i v e i n h i b i t i o n o f t h e f l e x o r r e f l e x pathway.  excitatory  iii TABLE OF CONTENTS Page ABSTRACT  ii  LIST OF FIGURES  iv  LIST OF TABLES  vi  ACKNOWLEDGEMENTS PART I.  PART II.  PART III.  vii  INTRODUCTION  1-10  a)  Habituation  1  b)  The Flexor Withdrawal Reflex  3  c)  Spinal Ischaemia  6  METHODS  11 - 17  a)  Aortic Occlusion  11  b)  Assessment of Habituation of the Flexor Reflex  11  c)  Statistical Analysis of Data  1^  RESULTS  18 - kl  a)  Post-operative Characteristics  18  1.  Control animals  18  2.  Clamped animals  18  b)  Effect of Aortic Occlusion on Habituation  21  PART IV.  DISCUSSION  k2 - 50  PART V.  BIBLIOGRAPHY  51 - 5k  iv  LIST OF FIGURES Figure 1.  Page Diagrammatic representation of the anatomy of biceps femoris muscle of the rat.  15  2.  EMG response of the right biceps femoris muscle.  16  3.  Schematic diagram of the stimulating and recording equipment. Electromyographic discharge (e.m.g.) and simultaneous output from the integrator (i.e.m.g.) recorded under basal conditions and in response to the 2nd, 100th, 200th, 300th, and 400th stimuli in a control rat.  31  Electromyographic discharge (e.m.g.) and simultaneous output from the integrator (i.e.m.g.) recorded under basal conditions and in response to 2nd, 100th, 200th, 300t, and 400th stimuli in a rat which had undergone occlusion of the thoracic aorta for 22 minutes, 7 days earlier.  32  6.  Flexor reflex responses to stimuli presented at 10 sec. intervals to a 7 day control rat.  33  7.  Flexor reflex responses to stimuli presented at 10 sec. intervals to a 7 day clamped rat.  y*  Flexor reflex responses to stimuli presented at 10 sec. intervals to a 3 day clamped rat with hind limb extensor r i g i d i t y which developed following aortic occlusion.  35  Three day rats. Flexor reflex responses to stimuli presented at 10 sec. intervals for control rats in which the thoracic aorta was occluded for 1 min. and rats in which the thoracic aorta was occluded for 22 min.  36  Seven day rats. Flexor reflex responses to stimuli presented at 10 sec. intervals for control rats in which the thoracic aorta was occluded for 1 min. and rats in which the thoracic aorta was occluded for 22 min.  37  Fourteen day rats. Flexor reflex responses to stimuli presented at 10 sec. intervals for control rats in which the thoracic aorta was occluded for 1 min. and rats in which the thoracic aorta was occluded for 22 min.  38  4.  5*  8.  9.  10.  11.  17  V  Figure 12.  13.  14.  Page The r e l a t i o n s h i p between the logarithm of the mean f l e x o r r e f l e x response t o successive groups o f 10 s t i m u l i , and the number of s t i m u l i presented. The groups o f r a t s compared i n the graph are animals examined 3 days a f t e r thoracotomy.  39  The r e l a t i o n s h i p between the logarithm o f the mean f l e x o r r e f l e x response t o successive groups of 10 s t i m u l i , and the number of s t i m u l i presented. The groups o f r a t s compared i n the graph are animals examined ? days a f t e r thoracotomy,  40  The r e l a t i o n s h i p between t h e logarithm o f the mean f l e x o r r e f l e x response t o successive groups of 10 s t i m u l i , and the number o f s t i m u l i presented. The groups o f r a t s compared i n the graph are animals examined 14 days a f t e r thoracotomy.  4l  vi LIST OF TABLES  Student's t test examination of 3 clay control rats compared to 3 clay clamped rats. Student's t test examination of 3 day control rats compared to 3 day clamped rats. Student's t test examination of 7 day control rats compared to 7 day clamped rats. Student's t test examination of 7 day control rats compared to 7 day clamped rats. Student's t test examination of 1^ day control rats compared to Ik day clamped rats. Student's t test examination of lk day control rats compared to 14 day clamped rats. Post-operative characteristics of clamped rats.  vii  ACKNOWLEDGEMENTS  I am indebted to my supervisor, Dr. J . A. Pearson, for his encouragement, advice, assistance, and time.  I thank him for this and for his  friendship. I am grateful to Mr. Kurt Henze for his preparation of the photographic reproductions and assistance with equipment, Mr. Steve Borden for advice on the statistical treatment of the results, Mrs. Gale Butchard, Miss Helen Robertson and Mrs. Jarmila Svoboda for typing the manuscript, and Mr. Mark Fishaut for assistance with surgical manoeuvres.  PART  I.  INTRODUCTION  1 Habituation may be defined as an animal's loss of responsiveness to an inconsequential change in i t s environment, or a gradual quantitative diminution of response to repeated uniform stimuli.  Habituation may result in complete  loss of response but the definition of habituation excludes any qualitative change in the response or any situation where there i s a quantitative change in the stimulus?^ Griffin and Pearson, 1968),  Peckham and Peckham (1887)  lished the f i r s t observation of this phenomenon (habituation), noting  pubthat a  spider stimulated to drop from i t s web by the sounding of tuning fork ceased to respond as the soundings were repeated.  Dodge (1925) introduced the term  "habituation" in preference to the term "adaption" which had been the most common of the names previously applied to describe this type of phenomenon. More recently experiments have been undertaken to investigate the general characteristics of habituation (Prosser and Hunter, 1963; Spencer, Thompson and Neilson, 1966a, 1966b, 1966c; Thompson and Spencer, 1966).  Spencer, Thomp-  son and Neilson (1966a, 1966b, 1966c) specified in great detail the parameters of the overall input - output function of habituation.  Their extensive work  has provided nine general characteristics of habituation: 1)  Given that a particular stimulus e l i c i t s a response, repeated appli-  cations of the stimulus result in decreased response (habituation).  The de-  crease i s usually a negative exponential function of the number of stimulus presentations. 2)  I f the stimulus i s withheld, the response tends to return gradually  to control levels (spontaneous recovery). 3)  If repeated periods of habituation and spontaneous recovery are per-  mitted, habituation becomes progressively more rapid (this might be called potentiation of habituation). 4)  Other parameters being equal, the higher the frequency of stimulation,  the more rapid and/or more pronounced is habituation. 5)  The weaker the stimulus, the more rapid and/or more pronounced  is  2 habituation. 6)  Iterated stimuli may continue to affect the neuronal circuit even  though the response has habituated to zero.  In this case the repeated stimuli  continue to enforce the habituted state. 7)  Habituation of response exhibits "stimulus generalization" between  independent inputs often referred to as "transfer" of habituation (transfer i s a term for the phenomenon which results in habituation to cutaneous stimuli from site "B" when a site "A" had been used as the point of cutaneous stimulus application; "A" and "B" must be close together on the skin or may be points on branches of the same cutaneous sensory nerve). 8)  Presentation of another (usually strong) stimulus results in recovery  of the habituated response 9)  (dishabituation).  Upon repeated application of the dishabituatory.stimulus, the amount  of dishabituation produced habituates (this might be called habituation of dishabituation). These nine common charactersitics now serve as the detailed operational definition of habituation replacing the more general definition given above (characteristic l ) . The study of habituation may yield information which could be of importance to the elucidation of the physiology of some types of behaviour.  Specu-  lation as to the usefulness of habituation as a paradigm of classical conditioning and hence as a simple analogue of learning has been extensively presented by the authors of several reviews (Galambos, 1967; Kandel, 1967). Actual classical conditioning situations, that is those involving the use of natural stimuli and behavioural (effector) responses, bear direct relationship to analogues of classical conditioning situations, that i s those involving a r t i f i c i a l (electrical) stimuli and/or responses (Collier, 1899). There has recently been interest in finding a model neuronal circuit which could be observed in the hope that, l)  the occurrence of change in  3  response to iterated stimuli at the level of a simplified tissue preparation would correspond to a more gross, perhaps behavioural change organism,  in the whole  2 ) the model would already be sufficiently well studied by classi-  cal methods so as to facilitate the study of the model with respect to i t s changing character, and ultimately  3 ) the model would yield knowledge that  might explain the mechanism of the change in response to repeated stimuli. One such model which has been widely studied i s the flexor reflex response in vertebrates. The Flexor Withdrawal Reflex A convenient paradigm of habituation i s provided by the flexor withdrawal reflex in which Sherrington (1897) noted decrementing responses to closely spaced stimuli in the spinal cat. away from a stimulus.  The reflex i s functional in moving a limb  Each flexor motoneurone can be said to have a receptive A wide range of afferents  f i e l d with respect to the flexor reflex stimulus.  can evoke the reflex, including cutaneous, joint, and smaller diameter muscle afferents.  Single motoneurones are subject to both spatial and temporal sum-  mation of stimuli.  The threshold for the reflex i s low in the spinal animal  and higher in the decerebrate.  Habituation of the reflex may be regarded as  a purposeful reaction in the sense that the organism suppresses a spinal withdrawal reflex to a light skin stimulus which has proved to be innocuous. The locus of the process of habituation of the flexor withdrawal reflex has been under investigation.  Because habituation can be obtained by the  stimulation of cutaneous nerves, i t cannot be due to changes at the sensory periphery i . e . receptor adaptation (Spencer et a l . , 1966a; Buchwald, Halas, and Schram, 1965; Groves, Lee, and Thompson, 1969),  Similarly habituation  cannot be due to changes in the threshold of cutaneous afferents for Wickelgren (1967a) has shown that habituation i s not due to a change in the threshold of the peripheral nerve for habituation was greater than might be acccounted for by undetectable changes in the afferent volley.  Decrement then does not occur  4  in either the primary afferent fibers or their terminals because both the afferent volley and the large negative dorsal root potential are unchanged during habituation (Wickelgren, 1967a).  The polysynaptic component of the  glabella reflex has been shown to habituate whereas concomitantly the monosynaptic component has been shown to remain unchanged (Kregelberg, 1962): indeed i t may be potentiated (Wickelgren, 1967a).  Spencer et a l . (1966c)  have shown that excitatory post synaptic potentials (EPSP's) can be evoked in a spinal motoneurone by monosynaptic stimulation, even after EPSP's in the same motoneurone, excited by a polysynaptic route, have undergone habituation. Furthermore, Griffin and Pearson (1968) have shown that the rate of habituation of the polysynaptic flexor reflex i s not influenced by manoeuvers which increase the motoneurone excitability.  Habituation i s not the result of an  increase in presynaptic hyperpolarization as this occurs only following a C fiber volley (Mendell and Wall, 196*0 and habituation occurs when only large fibers are stimulated (Spencer et a l . , 1966a). The possibility remains that habituation may be caused by local dendritic post synaptic changes in the motoneurone membrane which do not affect synapses.  This thesis i s difficult to prove or disprove.  distant  The intracellularly  recorded membrane potential of motoneurones has been measured at the soma (Spencer et a l . , 1966c), and shown to remain unaltered even though the flexor reflex has habituated.  Spencer et a l . (1966c) were able to record habituating  EPSP's in motoneuronal c e l l bodies suggesting that at least some of the habituated motoneurone synapses are quite near the c e l l body.  This implies that  because of the evidence that membrane potential at the c e l l soma remains unaltered, habituated motoneurone synapses possess post synaptic membranes with unaltered membrane potential.  However, relatively large  PSP's w i l l involve  many synapses in a motoneurone and they may l i e up to 0.5 mm from the soma and s t i l l be detectable there.  Hence there is l i t t l e reason to believe that  such distant synapses could not involve a change in local membrane potential  5 even though the membrane potential at the soma appears constant. the evidence i s inconclusive.  In short  Additionally i t could well be that in spite of  constant membrane potential, membrane conductance could vary and thus explain habituation but such evidence has not been investigated. Since habituation does not occur in the primary afferents, in their terminals  or in the motoneurones i t must occur in some population of interneu-  rones.  Evidence suggests that the polysynaptic component (the interneurone  pathway) rather than the monosynaptic component (the sensory afferent neurone and the efferent motoneurone) i s the more likely site of changes responsible for habituation. Two theories have been put forward to explain interneurone action l)  syn-  aptic depression; fatigue at any or a l l synapses between the primary afferent endings and the interneurones, and 2) activity at synapses which inhibit  inhibitory build-up theories; increased  the excitatory pathways to the motoneurones.  A comparison of both low frequency depression and post tetanic potentiation (FTP) with habituation indicates that although the inhibitory mechanism i s more likely to be, correct, synaptic depression cannot be completely ruled out (Wickelgren, 1967b).  Interneurones in the dorsal horn of the rostral l>7 spinal  segment were studied by Wickelgren (1967b). Interneurones were located in the cat rostral L7 spinal segment in laminae IV, V and VI of Rexed (195*0 using the c r i t e r i a for identification of interneurones proposed by Wall (l967)»  Ir-  regular spontaneous activity and very long bursts of spikes (8 or more) f o l lowing a single volley were sufficient to characterize a unit as an interneurone but additional c r i t e r i a proposed by Wall (1967) were applied in doubtful cases.  Wickelgren (1967b), on the basis of several lines of evidence, suggested  that habituating interneurones in the dorsal horn provide input to flexor motoneurones and that habituation of the former i s responsible for the habituation of the latter.  The three supporting lines of evidence are  l ) transfer of  habituation between skin and the superficial peroneal (SP) nerve i s similar in  6 intemeurones and in motoneurones,  2)  the stimulus frequency which produces  the maximum habituation (50-iOO/sec) i s the same for both intemeurones and flexor motoneurones, and 3)  the duration of habituation i s about the same  in both intemeurones and motoneurones, recovery being half complete 3 minutes after cessation of stimulation.  In view of the evidence of habituation of  intemeurones and motoneurones i t i s likely that habituating intemeurones are components of the flexor reflex pathway. Groves, De Marco and Thompson (2969) have, in the cat, located intemeurones of the dorsal horn within L6 to SI in laminae II to IV of Rexed (195*0» which habituated to 2/sec shocks (single pulses 0 , 1 - 0 . 3 msec, duration) applied to the skin of the hind paw at intensities well above threshold.  In  addition Groves et a l . (1969) located 9 cells within layers VII to VIII of Rexed (195*0 which showed a pattern of sensitization (increasing responsiveness) followed by subsequent habituation to cutaneous stimuli.  It would appear that  the role played by intemeurones in the process of habituation demands examination. Spinal Ischaemia There i s a considerable body of evidence which indicates that temporary spinal cord ischaemia can result in degeneration of intemeurones, with l i t t l e concomitant change in the motoneurone population (Davidoff, Graham and Shank, 1967; Murayama and Smith, 1969; Van Harreveld and Shade, 1 9 6 2 ) . Van Harreveld and Schade' (1962) investigated the effect of asphyxiation of the spinal cord of cats upon the nerve c e l l population of L7 segments. Under nembutal narcosis the dura was ligated at Th^Q isolating the caudal part of the dural sac and severing the spinal cord.  The next day a needle was  introduced into the dural cavity between the 6 t h and 7 t h lumbar vertebrae. By introducing Ringer's solution through the needle at a pressure of 20 cm. of mercury the pressure in the dural cavity was increased above blood pressure causing asphyxiation.  The animals were allowed to recover and at the end of  7 a two week period reflex activity in the hind limbs was investigated. spinal cord was then exposed with the animal under ether narcosis.  The  The prepa-  ration was immobilized with Squibb*s Intocostrin and the reflex action potent i a l s were recorded by stimulating a dorsal root and leading off from the ventral root at the same segmental level (usually S i ) .  After the physiological  experiment the cord was fixed for histological investigation using the methods of Schade and Van Harreveld (1962).  A survey of the spinal cords asphyxiated  for 28 to 50 minutes showed a nearly complete destruction of the interneurones in the centrally located gray matter, a very severe loss of nerve cells in the dorsal horn and an extensive destruction of nervous elements in the ventral horn, particularly in i t s central and medial portions.  In the peroneus t i b i a l i s  (PT) neurone pool, which i s situated in the lateral, better preserved region of the ventral horn, most of the nerve cells with a volume larger than 1600 |P survived.  There was almost complete destruction of neurones whose volume was  less than 1600 fu3.  This i s in agreement with previous observations that small  neurones are more susceptible to asphyxlal damage than large ones (Matsushita and Smith, 1970j Prosser and Hunter, 19631 Groves et a l . , 1969} Wickelgren, 1967a, 1967b).  Asphyxiation of the cord for 15 to 20 minutes did not result  in marked alterations of the reflex activity nor did histological examination reveal gross alterations.  Asphyxiation of the cord for 25 to 35 minutes  generally resulted in preparations with considerable extensor tone in the legs and brisk tendon reflexes, often with clonus.  Marked neuronal destruction  had taken place in the PT neurone pool for 93 per cent of the nerve (including motoneurones) cells in the PT neurone pool had been destroyed.  In the cords  of preparations asphyxiated for 50 minutes which did not show tone or reflexes in the legs, 95•5 per cent of the cells in the PT neurone pool had been destroyed. Davidoff et a l . (1967) obtained similar results by occlusion of the thoracic aortae of mature cats just below the origin of the great vessels for  8 periods of 15 to 60 minutes while the animals were under Nembutal anesthesia and a r t i f i c i a l respiration.  After 11 to 35 days 5 P serial sections of L 7  were made and neurones with diameters of less than 20 fx were designated as intemeurones.  This criterion was introduced by Gelfan and Tarlov  (1963).  Atrophy of the spinal cord manifested by dilatation of the central canal and widening of the ventral fissure was noted.  The most severe and statistically  significant losses of intemeurones (comparing control cats with those subjected to Ischaemia) occurred in the central portion of the spinal gray matter. The motoneurones were relatively unaffected.  Cats subjected to longer periods  of aortic occlusion exhibited more severe neurological deficits and more extensive loss of neurones. and Schade (1962),  (1962).  These findings are similar to those of Van Harreveld  As had been noted earlier by Van Harreveld and Schade  Davidoff et a l .  (1967)  have shown that intemeurones are more suscep-  tible to ischaemia than are motoneurones. Van Harreveld and Khattab  (1967)  ligated cat spinal cords at  T10  and  asphyxiated the spinal cord for 50 minutes by increasing the pressure in the dural cavity above the blood pressure.  The spinal cord was fixed by intra-  venous glutaraldehyde infusion from 30 minutes to 2 hours after asphyxiation. Electromicroscopic examination of the cord revealed that the structure of most of the presynaptic terminals was normal.  However, when fixation was under-  taken 2 to 7 hours after asphyxiation, increasingly severe degeneration of terminals, endoplasmic reticulum, and ribosomes became evident.  These changes  in the fine structure of the spinal cord were related to the transient r i g i d i t y (secondary tone) which i s observed after spinal cord asphyxiation of such duration. minutes.  In a separate series of experiments asphyxiation time was 30 to 35 The spinal cords were fixed 2 weeks later when permanent r i g i d i t y  (late tone) has developed.  In these spinal cords the fine structure of the  cytoplasm of the surviving neurones was almost normal.  However, there was  a marked decrease in the number of boutons on the motoneurone bodies.  Glial  9 processes instead of synaptic terminals were the main contacts to the c e l l surfaces.  This i s consistent with the severe destruction of interneurones  which i s found in these preparations.  The micrographs were characterized by  relatively wide spaces, mainly between g l i a l elements. Matsushita and Smith (1970) using the method developed by Gelfan and Tarlov (1963) were able to produce extensor r i g i d i t y of the hind limbs of the rat following the spinal cord ischaemia produced by clamping of the thoracic aorta for 15 to 30 minutes.  The rigidity was investigated with respect to the  nature of the r i g i d i t y produced, and the technique for i t s production. The method of Gelfan and Tarlov (1963) was employed since i t achieved a high i n cidence of r i g i d i t y and a rather selective destruction of interneurones. Reasoning that the inhibition of the monosynaptic reflex response of extensor muscles by stimulation of a sectioned posterior biceps nerve in the rat f o l lows the same mechanism as described for the cat by Eccles, Schmidt and Willis (1962),  Matsushita and Smith (1970) measured the degree of inhibition of the  spinal reflex system.  In the cat (Eccles et a l . , 1962) the mechanisms under-  lying this inhibition are two-foldt  postsynaptic inhibition at a short i n -  terval between posterior biceps stimulation and extensor reflex testing ( t i b i a l nerve stimulation) and presynaptic inhibition lasting for a much longer time. It was shown that the integrity of the postsynaptic inhibition i s more vulnerable to spinal cord ischaemia than are the presynaptic systems. had noticeably less pre- and post-synaptic inhibition.  Rigid rats  Reflexes mediated via  interneurones such as the segmented polysynaptic reflex discharge and inhibition of ankle extensor motoneurones by flexor afferent excitation were reduced in r i g i d rats.  It would appear that there i s good correlation between degree  of ischaemia, degree of r i g i d i t y and degree of interneurone damage. Such findings have suggested a different approach which can be used to study the involvement of interneurones in habituation.  By producing relatively  selective damage of interneurones in the spinal cord, i t should be possible to  10 observe whether this impairment of neural function i s associated with a change in the rate of habituation of the flexor reflex.  If the locus of the habitua-  tion process resides at the interneurone level, ablation  of intemeurones  should alter the habituation. It i s to this end that this study has been undertaken.  PART  II.  METHODS  11  AORTIC OCCLUSION Male rats (of the Long Evans strain, body weight 300-400 g) were anesthetized with ether and spinal cord ischaemia achieved by temporary occlusion of the thoracic aorta at the level of the f i r s t intercostal artery.  Ether anes-  thesia was used exclusively since barbiturate anesthetics tended to produce excessive respiratory depression.  An incision about 2 centimeters long was  made between the hind and fourth ribs on the left side and the upper part of the lung pushed aside with a small cotton b a l l .  The tissue enclosing the  thoracic aorta between the end of the aortic arch and the f i r s t  intercostal  artery was carefully parted by blunt dissection exposing the thoracic aorta. The thoracic aorta was subsequently clamped with a serrafin about 2 centimeters long.  After applying the clip to the aorta, the cotton ball used to retract  the,left lung was removed and the thorax was closed temporarily with haemostats. The animal respired spontaneously during the 22 minute period of aortic acclusion.  The success of the occlusion could be judged to some degree from color  changes in the skin of the feet.  After removal of the aortic clamp, the r i b  cage, musculature and skin were closed separately and the animal was permitted to recover. o  Experiments were carried out at a room temperature which ranged o  from 22 C to 25 C. The rectal temperature of the animals was constantly monitored.  In the test rats the aortic clamp was applied for 22 minutes, while in  control rats the clamp was applied for only 1 minute but in this case the thoracic cavity was not sutured for a further 21 minutes but was closed with haemostats to simulate a clamp period (mock clamping), ASSESSMENT OF HABITUATION OF THE FLEXOR REFLEX The effects of spinal cord ischaemia on habituation of the flexor withdrawal reflex were examined by testing the capability of rats to habituate 3 t 7, or 14 days after clamping of the thoracic aorta.  Control rats were examined  12 3t 7, or Ik days after sham occlusion.  One day before reflexes were assessed,  silver EMG recording electrodes (Johnson Matthey Metals Limited, Grade 5, Diamel, Silver, .007 inches diameter) were implanted in the caudal head of the right biceps femoris muscle and bipolar silver stimulating electrodes were i n serted into the skin of the right ipsilateral hind paw while the rat was under ether anesthesia.  Figure 1 p. 15 illustrates the musculature of the right  hind paw of the r a t .  Figure 2 p. 16 illustrates the EMG discharge during  flexion e l i c i t e d by stimulation of the skin of the ipsilateral hind paw. Recordings made from the rostral head of the biceps femoris (B.F. l) and from the caudal head of the biceps femoris (B.F. 2) are shown.  It can be seen that  the EMG response obtained from electrodes implanted in B.F. 2 (caudal head) i s greater than the response obtained from electrodes implanted in B . F . 1.  In  order that the approach to the problem under investigation could be as consistent as possible, recordings were taken from the caudal head of the biceps femoris with electrodes implanted in a manner that minimized variations from preparation to preparation.  Similarly, stimulating electrodes were always  placed as consistently as possible between the f i r s t and second toes of the hind paw.  After insertion of the electrodes, the rats were placed in Bollman  restraining cages with free access to food and water, and were tested one day later.  Thus, rats were implanted with electrodes 2, 6, or 13 days after aortic  occlusion and tested 3» 7, or lk days after aortic occlusion.  The rat in i t s  Bollman cage was placed in a dark soundproof box, in which i t remained for a 30 minute acclimatization period prior to testing and for the subsequent test period. During the test period ^00 stimuli (30 V, 20 mA, 5 msec^ with an interstimulus interval of 10 sec.were applied to the paw.  Quantitative assessment  of EMG discharge occurring in response to each stimulus was achieved by means of an integration unit.  This unit was programmed to begin integration 10 msec,  after the stimulus, to eliminate the stimulus artefact, and to continue inte-  13 gration for 500 msec.  The integrated response was displayed on a digital  voltmeter and recorded.  Figure 3  used during the experiment.  p. 17 illustrates the electronic system  Prior to the test period, values for background  EMG activity integrated over 5°0 msec, were obtained.  The mean value for  background activity was subtracted from each value obtained in response to the stimuli, to give the net flexor reflex response.  The mean flexor reflex  responses to successive groups of 10 stimuli were calculated. The reflexes of the rats, as assessed  by visual inspection of the  reflexes in response to a pinch, were tested before and after the occlusion of the thoracic aorta.  The degree of muscle tone was noted and a daily record  was kept of any changes in tone and reflex behaviour of the rats. The data accumulated for a l l the rats was examined s t a t i s t i c a l l y .  The  data from rats tested 3 days after occlusion of the thoracic aorta (hereafter referred to as "3 day clamped rats") were compared to data from control rats tested 3 days after mock occlusion of the thoracic aorta (hereafter referred to as "3 day control rats").  Similarly 7 day clamped rats were compared to  7 day control rats and 14 day clamped rats were compared to 14 day control rats. The data were examined in the following manner. The integrated EMG response of the rat was read off the digital voltmeter.  The responses to  the f i r s t 10 stimuli were summed after background EMG activity was subtracted from each response.  Then a mean response was calculated.  This new value  was designated as 100 percent response for i t was the response to the f i r s t 10 stimuli and assumed to be the response of non-habituated reflex.  Subse-  quently, each following mean response to the next 10 stimuli was calculated in the same manner, and then expressed as a percentage of the response of the f i r s t 10 stimuli (percentage of i n i t i a l response). followed for a l l rats from stimuli 1 to 400.  This procedure was  Thus a percentage of the i n i t i a l  response was obtained for each succeeding group of 10 stimuli.  14 Figure 6  33 depicts the graph obtained by testing a control rat.  Percentage of the i n i t i a l response for groups of 10 stimuli are illustrated. Similar graphs were determined for clamped rats as shown in figures 7 and 8 p. 34 and 35.  STATISTICAL ANALYSIS OF DATA Statistical analysis was performed on responses to stimuli 1-10, 41-50, 91-100, 141-150, 191-200, 241-250, 291-300, 341-350, and 391-400, which were obtained for each rat.  These individual response values were pooled within  each of the 3 clamped and 3 control groups.  Thus for a single group of rats  a mean response to stimuli 4i-50 etc. was calculated.  Standard deviation and  standard errors were determined for the mean response.  Using the Student's t  test the mean responses of the control groups were then compared to the mean responses of the clamped group at the same stimuli period. illustrated in figures 9, 10. 11, p. 36, 37, 38.  These data are  As a result of the t test,  t values and standard errors of the combined data were derived.  Tables l a ,  lb, 2a, 2b, 3a, 3b p. 25, 26, 27, 28, 29, 30 present these data as described above.  The calculations were carried but using an IBM 1130 computer.  Finally,  a l l of the data within each group were pooled and a regression line determined using the computer (figures 12, 13, 14 p. 39» 40, 4 l ) .  Determination of wheth-  er a line through the points provided by the responses of a l l rats within a clamped group differed from those of a control group was made.  15  Figure i .  Diagrammatic representation of the anatomy of biceps femoris muscle of the r a t . B.F. 2,  (B.F. 1, r o s t r a l head of the biceps femoris;  caudal head of the biceps femoris; S.T.,  semitendinosis;  Q.F., quadriceps femoris; G.M.,  gluteus maximus; G., gastrocne-  mius; T . A . , t i b i a l i s anterior;)  The gluteus maximus had been  completely removed t o expose the underlying muscles.  100  > u V .  1 0 0 msec.  B.F.  Figure 2. EMG response of the right biceps femoris muscle. B.F. 1 i s a photograph of the oscilloscope tracing of an EMG response from the rostral head of the right biceps femoris muscle while B.F. 2 i s the photograph of the EMG response from the caudal head of the same muscle under identical conditions.  The two recordings were made simultaneously during  the flexor withdrawal reflex.  Stimulus 30V 1msec. duration.  17  Digitimer  stim.  Stimulator  trigger pulse  integrator command pulse  ntegrator  integrated e.m.g.  Rat Digital Voltmeter  e.m.g.  amplified e.m.g.  Figure 3« Schematic diagram of the stimulating and recording equipment.  PART  III.  R E S U L TS  18 POST-OPERATIVE CHARACTERISTICS A.  Control Animals The voluntary control, and reflex characteristics of the musculature of  the hind limbs of control rats were examined before, during, and after thoracotomy, and after habituation and did not appear impaired. Breathing was normal and the rats were able to walk normally immediately after recovery from anesthesia.  The flexor withdrawal response to a pinch was present (often  accompanied by vocalization).  Posture and tone as assessed by resistance  offered to passive movement were normal. or bowel function.  There was no impairment of bladder  Operations were carried out at room temperature (22°C).  The rectal temperature of the control animals dropped from 37°C to approximately 35»5°C by the time the thorax was closed.  The survival rate of control  animals was close to 100%,  Three control animals died during thoracotomy but  none died post operatively.  Thirty control rats are presented in this study,  ten in each category (3, 7, or 14 days after thoracotomy). B,  Clamped Animals The rats subjected to aortic occlusion for 22 minutes exhibited charac-  teristics different from control rats.  Ten rats were examined three days  after aortic occlusion, eleven rats seven days after aortic occlusion and thirteen rats fourteen days after aortic occlusion.  The clamped rats recovered  from anesthesia with greater difficulty than the control rats.  Recovery was  not complete until 45-60 minutes after closure of the chest wound. The rats displayed rapid breathing and/or dyspnea.  It was necessary to apply a r t i f i -  c i a l respiration to many of the clamped rats during the period of their recovery from anesthesia. t i a l recovery of alertness. post-operative mortality.  Dyspnea often persisted for 1 or 2 hours after parThe recovery period was the period of highest In such cases a pink foamy f l u i d was noted draining  from the nostrils and mouth of moribund rats.  A suspected cause of mortality  19 was pulmonary oedema due to an elevated pulmonary capillary pressure caused by aortic - occlusion.  The temperature of the clamped rats which survived,  dropped from 37°C to approximately 34°C by the end of the thoracotomy procedure and rose very slowly again to the 37°C normal temperature over a period od several hours. Clamped rats exhibited flaccid paralysis of the hind limbs for a period of 1 to 2 hours after recovery from anesthesia.  During this period of flac-  cidity, a pinch applied to the paw resulted neither in vocalization nor reflex withdrawal.  After recovery from the flaccidity the hind limb tone  was either  (a) slightly greater in both flexor and extensor muscles or (b) much greater than normal in extensor muscles only. ited difficulty in walking. a rather lateral position. of two or three days.  Rats in category (a) i n i t i a l l y exhib-  Movements were slow and hind limbs were held in This abnormal gait gradually improved over a period  Often the hind limbs appeared swollen and inflamed and  this subsided by the third post-operative day.  Flexor reflex responses could  always be elicited from rats in category (a) but vocalization was usually not present (c.f. table 4 p. 20 ),  This lack of vocalization when testing for  flexor reflex was noted in a l l but one of the 3 day clamped rats, in a l l but five of the 7 day clamped rats, and in a l l but eight of the 14 day clamped rats. Table 4 p.20 notes the characteristics of a l l the clamped rats.  Those  animals which vocalized upon testing for the flexor reflex either developed vocalization several days after the operation or indeed had never lost the a b i l i t y to vocalize.  The rats in category (b) exhibited extensor rigidity  which persisted until the end of the experiment. category.  Three rats f e l l into this  The r i g i d rats had no ability to walk on their hind legs and  dragged their legs "foot-pad" upward. The rats remained r i g i d u n t i l the end of the investigation and in one rat the r i g i d i t y was maintained for 22 days (at which time the rat was sacrificed).  In total two 3 day and one 14 day  RAT NO. 3-lc 3-2C  COMMENTS R curled toes and walks on knuckles, definite r i g i d i t y , slightly in flexor position N - V - walks poorly and slowly, increased sensitivity to touch (painful vocalization), legs held together and up when picked up by t a i l  NV W _ walks poorly and slowly, clumsy, slightly r i g i d in flexor position NV NV w NV w NV w NV w — NV R walks poorly and slowly extensor rigidity, drags hind limbs NV  3-4C 3-5C 3-6C 3-7C 3-8C 3-9C 3-ioc 7 day rats ( l l ) 7-1C NV w 7-2C w w 7-3C N V 7-4C w N V V 7-5C N V 7-6C NV w 7-7C w 7-8C w N V 7-9C w 7-10C w 7-11C w  -  -  loss of bladder function; rat urinated over i t s e l f developed some extensor rigidity post-operatively but lost this if- hrs. later 2 hrs. post-operatively developed some extensor r i g i d i t y , lost this the next day slight vocalization to painful stimulus  14 day rats (13) _ _ walks poorly 14-1C — 14-2C w _ walks poorly, fur i s easy to pull out, walking had improved with time 14-3C NV 14-4C NV w 14-5C w N _V w 14-6C 14-7C w N —V 14-8C w — 14-9C w w 14-10C 14-11C w 14-12C NV R extensor rigidity, drags hind limbs w 14-13C NVi no vocalization upon presentation of painful pinch Wi walks well R« extensor rigidity Table 4. POST OPERATIVE CHARACTERISTICS OF CLAMPED RATS  -  21 r i g i d r a t were i n v e s t i g a t e d .  The incidence of complete r i g i d i t y was very low,  lower than that reported by M a t s u s h i t i and Smith (1970).  A l l the clamped  r a t s except one 7 day clamped r a t had normal bladder and bowel f u n c t i o n .  EFFECT OF AORTIC OCCLUSION ON HABITUATION The r e s u l t s from the habituation experiments are presented i n the f o l lowing 10 f i g u r e s .  Figure 4 p.31 shows t y p i c a l h a b i t u a t i o n obtained i n a  7 day c o n t r o l animal.  I t can be seen from the f i g u r e that the r i g h t f l e x o r  r e f l e x habituates when i t e r a t e d s t i m u l i are presented.  Photographs of the  EMG t r a c e appearing on the osciloscope screen were taken during an unstimul a t e d s t a t e , and a f t e r 2, 100, 200, 300 and 400 s t i m u l i .  The representative  responses t o the i n d i v i d u a l s t i m u l i are 100$, 40.8$, 38.5#, JZ»5% i n i t i a l response. i n t e g r a t e d EMG,  and 28,3$  Beneath each EMG trace i s a r e c o r d of the corresponding Note t h a t both the duration of the EMG discharge and the time  i n t e g r a l of the EMG decrease. Figure 5 P» 32 i l l u s t r a t e s r e s u l t s of a s i m i l a r experiment performed i n a 7'day clamped animal.  I t i s evident that  l ) basal a c t i v i t y during the  p e r i o d p r i o r t o s t i m u l a t i o n i s greater than i n the c o n t r o l ,  2)  the absolute  l e v e l of response i s lower and i t habituates more slowly than i n the c o n t r o l , and  3)  the f l e x o r r e f l e x of the clamped r a t does not habituate t o the same  degree i n terms of asymptotic values as the c o n t r o l r a t . These are general f i n d i n g s i n a l l experiments. Figure 6 p. 33 i l l u s t r a t e s a t y p i c a l experiment on one r a t ,  i n t h i s case  the response t o 400 s t i m u l i i n the 7 day c o n t r o l r a t presented i n f i g u r e 4, p. 31.  Figure 6 i l l u s t r a t e s the i n t e g r a t e d EMG responses expressed as percentages  of the i n i t i a l response recorded as mean responses f o r s t i m u l i 1-10, 41-50, 91-100, 141-150, 191-200, 241-250, 291-300, 3^1-350, 391-400. At s t i m u l i 391-400 the mean response expressed as the percentage of the i n i t i a l response i s  22 30 percent. Figure 7 p. 34 presents a similar experiment, in this case the response to 400 stimuli in the 7 day clamped rat presented in figure 5 p. 32 . sponse to stimuli 391-400 i s 70 percent.  The re-  It i s evident that habituation was  not as marked as that shown in figure 6 p. 33 for the 7 day control animal. Figure 8 p. 35 illustrates the response of a single 3 day clamped rat which exhibited hind limb extensor r i g i d i t y .  These animals did not habituate  and because of the low evidence of rigidity a sample experiment i s presented (figure 8). The data from these 3 r i g i d animals was included in the data of the groups to which the individual animals belonged (one rat belonged to 3 day rats and two belonged to 14 day rats).  The mean response for any group of  stimuli expressed as a percentage of the i n i t i a l response was always well above 100^ of the i n i t i a l response. percent i n i t i a l response.  The response at the end of 400 stimuli was 100  The findings were peculiar to the three rats  that  exhibited hind limb extensor r i g i d i t y . Ten control rats and ten clamped rats were examined three days after aortic occlusion.  Figure 9 p. 36 and tables la + lb p. 25, 26 present the  data obtained for these two groups of rats.  A l l responses in the clamped  group except those to stimuli 91-100 proved to be significantly greater (p<0.05), using the Student's t test, than responses in the control group.  It can be  seen from the graph in figure 9 p. 36 that the responses in the clamped animals remain at approximately 75$ of the i n i t i a l response while in the control animals habituation to 20$ of the i n i t i a l response occurred.  In summary the  3 day control rats habituated to a mean of 20.04$ i n i t i a l response SE * 5.88 at 400 stimuli while the 3 day clamped rats habituated to 53*86$ i n i t i a l response SE i 17.08 after 400 stimuli.  In a l l but one period of stimulation  (91-100) analysed, a significant difference between the two groups of 3 day rats (p<0.05) was obtained. Comparison of the 7 day control and 7 day clamped rats revealed no signif-  23 leant differences between the two groups, for a l l periods of stimulation. The control rats habituated to a mean of 20.3$ i n i t i a l response SE * 3.72 at 400 stimuli while the clamped rats habituated to 41.35% i n i t i a l response SE ± 13.55 at 400 stimuli (fig. 10 p. 37, tables 2a + 2b p. 27. 28). Comparison of the 14 day control rats and 14 day clamped rats revealed a significant difference (p<0.05) between the 10 control animals and 13 experimental animals.  The control rats habituated to a mean of 13.7$ i n i t i a l  response SE ± 3.10 at 400 stimuli while the clamped rats habituated to 62.29$ i n i t i a l response SE * 18.03,  A l l periods of stimulation show a significant  difference between the two groups except the 41-50 and 141-150 stimuli periods (fig..11 p. 38, tables 3a + 3b p. 29, 30). The reflex responses of rats tested  3 or 14 days after aortic occlusion  showed a significantly (p<0.05) lesser degree of habituation than the responses to the corresponding stimuli in control rats.  A statistically significant  impairment of habituation could not be demonstrated when comparison was made between the responses of rats tested 7 days after occlusion and their controls. The greatest impairment of habituation was seen in rats which exhibited exten-. sor r i g i d i t y .  In some cases an increase in response rather than habituation  was seen (figure 8 p. 35). Only 3 rats exhibited extensor r i g i d i t y and none of these rats were in the 7 day group. In the control and in the ischaemic preparation, the duration of EMG discharge in response to the stimulation of the paw, f e l l within the same range (150-250 msec). of the EMG spikes.  The responses did differ in amplitude and frequency  In the control animals there i s an i n i t i a l high frequency  burst followed by a discharge of smaller spikes about 50/sec.  (5O-3OO4UV)  at a frequency of  The responses in animals which had undergone aortic occlusion  did not have the early high frequency component, but consisted solely of a 1  low amplitude, low frequency discharge (figures 4 and 5 p. 31» 32). In the control rats the reflex response habituated rapidly during the presentation  24  of the f i r s t 5° stimuli and more gradually thereafter. In control animals there i s a significant negative linear correlation (p<0.00l) between the logarithm of the reflex response and the number of stimu l i presented.  On the other hand the relationship between log response and  stimulus number in the ischaemic rats was not a linear one (see Fig. 12, 13» 14, p.39 , 40, 41 ). The graph for the clamped animals reveals that for the f i r s t 100 stimuli the response of the clamped rats decreases at a rate similar to the exponent i a l function of the control rats.  However, the decremental relationship  changes at the 100 stimuli point and the line approaches a slope of zero from that point onwards.  This observation led to an examination of the slopes of  the graphs for control rats compared to the slopes of the graphs for clamped rats.  This comparison was undertaken to determine i f indeed there was a  difference between the two lines and thus strengthen the proposal that there i s a difference between clamped and control animals. exponential function in a l l cases.  Control rats f i t an  In a l l cases clamped rats did not have  a slope significantly different from zero and thus did not f i t an exponential function.  The rats were examined statistically by pooling for example a l l  3 day control rats into one group and then performing a linear regression coefficient determination. groups.  The same procedure was executed for a l l other  These data are presented below.  The probability of the slope of the relationship being zero in the case of 3 day control rats, 7 day control rats and 14 day control rats was 0,0050, 0,0003, and 0.0000 respectively.  The probability of zero slope fro the  lines for clamped rats of 3 , 7, or 14 days was 1,0000, 1.0000, and 1.0000 respectively.  Clearly the control animals have a function relationship that  has-a measurable slope and the clamped animals do not. It would appear that both qualitative and quantitative changes in habituation process had been brought about as a result of spinal ischaemia.  3 day rats - control response (% of i n i t i a l response) STIMULUS NUMBER 1-10 3-1 3-2 3-3 3-4 3-5 3-6 3-7 3-8 3-9 3-10  degrees of freedom mean response SD SE log mean  Table l a .  41-50  91-100  141-150  191-200  241-250  291-300  341-350  391-400  26.5 24.1 42.3 41.9 105.9 36.9 27.8 23.1 32.2 36.5  32.3 29.8 20.7 31.0 97.7 36.9 19.5 19.9 16.8 30.3  14.6 23.0 3.5 32.9 103.6 45.8 14.1 9.8 24.6 26.1  23.1 9.6 6.6 29.2 6i.4 30.2 22.4 29.8 16.3 37.3  5.4 23.7 4.8 19.8 68.3 21.8 15.1 2.8 20.2 18.5  9 26.59 15.57 4.92 1.425  9 20.04 18.61 5.88 1.302  100 100 100 100 100 100 100 100 100 100  57.5 35.5 52.4 47.7 72.9 62.2 63.9 80.5 85.3 76.2  40.8 26.9 45.4 68.4 81.2 61.3 51.7 22.0 40.0 41.9  36.7 49.6 30.8 30.2 89.2 50.8 44.4 45.7 23.9 38.8  9 100  9 63.41 15.66 4.95 1.802  9 47.96 18.23 5.76 1.681  9 44.08 18.37 5.81 1.644  2.0  9 39.72 24.27 7.68 1.599  9 33.49 23.54 7.44 1.525  9 29.80 28.61 9.05 1.474  3 DAY RATS. RESPONSE VALUES EXPRESSED AS % OF INITIAL RESPONSE HAVE BEEN EXAMINED BY STUDENT'S t TEST (p<0.05). to  3 day r a t s - clamped response (% o f i n i t i a l  response)  STIMULUS NUMBER 1-10  41-50  91-100  141-150  191-200  241-250  291-300  3^1-350  391-400  Rat 3-10 3-2C 3-30 3-4C 3-50 3-60 3-70 3-80 3-90 3-10C  100 100 100 100 100 100 100 100 100 100  101.0 176.3 102.0 80.0 125.7 60.6 76.5 109.0 69.6 72.1  21.5 63.2 92.2 79.2 108.6 34.4 57.4 100.6 108.7 24.6  63.8 65.9 77.1 100.7 76.6 68.8 46.3 96.9 56.5 64.9  44.5 56.8 77.1 100.7 50.0 104.9 44.6 96.9 86.9 32.9  46.2 39.2 48.6 120.0 49.3 129.5 36.6 122.4 73.9 100.9  72.5 44.0 21.6 135.4 32.4 173.7 34.9 115.4 86.9 47.8  80.6 43.1 33.5 75.3 38.8 201.6 30.9 133.6 82.6 73.6  100.4 49.0 27.3 136.1 28.5 180.3 21.1 137.5 108.7 65.7  degrees o f freedom • mean response SD SE l o g mean  9 100 2.0  9 97.28 34.48 10.90 1.988  9 69.04 33.96 10.74 1.839  combined d a t a ( c o n t r o l compared w i t h clamped) SE 12.58 12.80 t 2.69 1.64 d.f. 18 18 Significance (p 0.05)  Table l b .  S  3 DAY RATS.  N  S  9 71.89 21.22 6.71 I.856  9 69.53 26.80 8.47 1.842  9 76.66 37.76 11.94 I.885  9 76.46 50.69 16.03 1.883  9 79.36 53.08 16.76 1.900  9 75.46 53.86 17.03 I.878  9.32 2.97 18  12.00 2.47 18  14.77 2.92 18  19.33 2.40 18  18.36 2.87 18  18.92 2.93 18  S  CONTROLS COMPARED WITH CLAMPED RATS.  S  S  S  S  S  7 day rats - control response (% of i n i t i a l response) STIMULUS NUMBER 1-10 100 100 100 100 100 100 100 100 100 100  7-1 7-2 7-3 7-4 7-5 7-6 7-7 7-8 7-9 7-10 degrees of freedom mean response SD SE log mean  Table 2a.  41-50  9 100 2.0  9  91-100  141-150  191-200  241-250  67.3 60.8 89.5 51.7 59.3 41.6 28.8 55.4 80.1 73.2  88.0 62.9 61.5 34.3 58.1 63.3 20.5 44.6 44.3 52.3  58.8 73.0 52.4 30.0 46.5 54.6 20.6 15.1 30.0 48.4  52.9 55.9 32.2 34.3 29.5 51.7 13.1 10.5 19.1 34.2  36.4 57.3 21.5 35.1 42.7 34.1 8.2 0.0 28.9 27.0  60.77 17.95 5.68 1.784  9 52.98 18.50 5.85 1.724  9 42.94 18.36 5.80 1.633  9 33.34 16.26 5.14 1.523  9 29.12 16.47 5.21 1.464  291-300  341-350  391-400  28.2 53.0 31.8 33.3 32.4 32.3 8.6 11.5 14.9 28.2  33.4 37.1 22.0 32.2 30.1 30.3 7.8 3.3 22.4 19.4  19.0 37.2 10.3 32.6 26.9 34.0 7.7 3.7 16.4 15.1  9 9 27.42 13.01 4.13 1.438  23.8 11.17 3.53 1.377  9 20.3 11.77 3.72 1.307  7 DAY RATS. RESPONSE VALUES EXPRESSED AS % OF INITIAL RESPONSE HAVE BEEN EXAMINED BY STUDENT'S t TEST (p<0.05).  7 day rats - clamped response {% of i n i t i a l response) STIMULUS NUMBER M-50  91-100  141-150  191-200  100 100 100 100 100 100 100 100 100 100 100  64.4 75.1 40.2 53.9 93.3 57.9 39.7 62.2 51.2 59.2 43.3  55.5 48.4 37.0 33.8 89.0 31.6 44.2 49.1 48.2 47.3 . 44.3  47.6 41.1 30.5 34.2 85.2 31.6 25.9 47.5 54.3 34.2 38.4  44.9 38.0 32.5 54.4 100.0 17.5 17.8 34.4 54.3 25.7 37.5  10 100  10 58.22 15.90 4.80 1.765  10 48.04 15.36 4.63 1.684  10 42.77 16.45 4.96 1.631  10 41.55 23.05 6.95 1.619  7.76 .62  9.22 .88 19  19  NS  NS  1-10 7-1C 7-2G 7-3C  7-4C 7-5C 7-6C 7-7C 7-8C 7-9C 7-10C 7-11C degrees of freedom mean response SD SE log mean combined data SE t d.f. Significance (p<0.05)  Table 2b. 7 DAY RATS.  2.0  19  19  7.96 .02 19  NS  NS  NS  7.76 .32  CONTROLS COMPARED WITH CLAMPED RATS.  241-250 47.2 37.7 26.8 56.6 90.4 29.8 17.4 26.1 52.2 58.5 34.8 10 43.41 20.60 6.21 1.637  8.61 1.64  291-300  341-350  391-400  34.7 19.5 15.9 51.7 129.2 28.1 20.1 25.6 67.0 6.8 41.2  32.4 12.7 17.4 63.1 124.9 31.6 17.4 39.5 59.6 5.4 28.5  50.8 7.5 16.8 54.4 166.0 31.6 10.7 32.3 53.4 10.3 21.0  10 39.98 34.21 10.31 1.602  10 39.32 33.68 10.16 1.595  10 41.35 44.93 13.35 1.616  12.10 1.03 19  11.74 1.29 19  15.40 1.35 19  NS  NS  NS  00  14 day rats - control response (% of i n i t i a l response) STIMULUS NUMBER 1-10  41-50  91-100  141-150  191-200  241-250  Rat 14-1 14-2 14-3 14-4 14-5 14-6 14-7 14-8 14-9 14-10  100 100 100 100 100 100 100 100 100 100  106.0 86.3 71.3 36.7 70.7 54.3 55.1 58.0 44.9 43.9  69.3 42.9 53.3 33.4 50.6 69.5 48.5 47.8 34.4 25.0  70.1 49.4 47.5 45.6 33.8 42.3 33.2 38.5 25.5 26.4  24.8 47.4 33.8 27.9 28.7 36.8 19.4 41.6 37.1 22.5  35.8 26.1 26.1 8.2 22.6 38.7 5.0 39.4 16.7 8.9  18.2 10.4 11.3 10.? 11.0 38.5 12.1 33.0 15.1  degrees of freedom mean response SD SE log mean  9 100  9 47.47 14.54 4.60 1.676  9 41.23 13.14 4.16 1.615  9 32.00 8.88 2.81 1.505  9 22.75 12.82 4.05 1.357  9 17.75 9.96 3.15 1.249  Table 3a.  2.0  9 62.72 21.26 6.72 1.797  291-300  mm  341-350  391-400  6.1 13.1 12.9 6.2 16.9 38.5 3.1 33.6 17.9 14.3  12.2 11.6 5.2 10.4 18.3 31.6 3.9 29.6 8.1 6.3  9 16.26 11.55 3.65 1.211  9 13.72 9.81 3.10 1.138  14 DAY RATS. RESPONSE VALUES EXPRESSED AS % OF INITIAL RESPONSE HAVE BEEN EXAMINED BY STUDENT'S t TEST (p<0.05).  ro vO  14 day r a t s - clamped response (% of i n i t i a l response) STIMULUS NUMBER  Rat  lk-lC lk-2C lk-JQ lk-kC 14-5C 14-6C lk-70 14-8C 14-9C  14-iOC 14-11C 14-12C 14-13C degrees of freedom mean response SD SE l o g mean  1-10  41-50  91-100  141-150  191-200  241-250  291-300  341-350  391-400  100 100 100 100 100 100 100 100 100 100 100 100 100  24.2 148.0 67.0 • 69.3 90.8 57.2 81.3 43.8 90.4 74.1 144.5 128.2 96.3  23.4 124.0 55.1 52.7 62.1 45.6 82.0 27.8 86.9 65.5 200.0 119.9 71.7  21.7 126.0 29.6 87.3 58.3 32.7 72.1 11.7 85.6 36.5 74.3 173.5 65.4  18.4 138.0 30.1 42.9 29.4 27.3 74.1 36.1 90.4 54.8 101.3 110.1 66.3  16.9 100.0 25.6 43.4 24.0 28.0 74.8 23.4 75.0 27.9 243.2 185.2 60.4  12.3 356.0 20.7 52.0 42.5 15.2 71.9 4.8 70.0 17.2 364.8 121.6 58.1  22.7 198.0 3.6 59.1 36.8 15.4 64.7 5.2 65.2 63.4 16.2 111.6 38.7  23.6 254.0 15.7 77.8 18.3 17.0 6O.3 1.6 58.2 54.8 87.8 98.0 42.2  combined data SE t d.f. Significance (p<0.05) Table 3b.  2.0  12 85.82 36.98 10.26 1.939  12 78.21 47.45 13.16 1.893  12 67.44 45.13 12.52 1.829  12 63.02 37.49 10.40 1.799  12 71.37 69.29 19.22 1.853  12 92.85 125.02 34.12 1.968  12 53.89 53.04 14.71 1.732  12 62.29 65.02 18.03 1.79*  -  13.91 1.65 21  16.54 I.85 21  15.66 1.66 21  12.90 2.39 21  23.64 2.04 21  41.56 1.86 21  18.18 2.05 21  22.10 2.19 21  S  S  S  S  S  12 100  NS  14 DAY RATS.  S  NS  CONTROLS COMPARED WITH CLAMPED RATS. V*) o  Stimulus No.  Figure 4,  Electromyographic discharge (e.m.g.) and simultaneous output from the integrator (l.e.m.g.) recorded under basal conditions and in response to the 2nd, 100th, 200th, 300th, and 400th stimuli in a control r a t .  Sham occlusion of the thoracic  aorta had been carried out 7 days earlier.  Habituation of the  reflex i s demonstrated and ttfe responses to the respective stimuli are 100$, 40.8$, 38.5$. 32.5$. and 28.3$ i n i t i a l response.  Stimulus No.  Figure 5.  Electromyographic discharge (e.m.g.) and simultaneous output from the integrator (i.e.m.g.) recorded under basal conditions  and in response to the 2nd, 100th, 200th, 300th, and *K)0th stimuli in a rat which had undergone occlusion of the thoracic aorta for 22 minutes, 7 days earlier.  Note that the basal  activity i s higher but the responses to stimuli are lower than in the control experiment i n F i g . 4. occurred.  Minimal habituation  The responses to the respective stimuli are 100$,  80.5$, 70.5$, 72.0$, and 75.0$ i n i t i a l response.  33  100-x  £  80H  <*—  o  * CD  co c o a. w  60  CD  or X jD  H— CD  DC  40  O •x  20 t  I-  10  Figure 6.  t 1  '  4150  91-  t IN  t I  1  I4|l- 191- 241100 150 200 250 NumJer of Stimuli  1  1  1  291- 341- 391300 350 400  Flexor reflex responses to stimuli presented at 10 sec. intervals to a 7 day control rat. The response i s plotted as a mean response to stimuli 1-10,  41-50, 91-100, i4i-150,  191-200,  241-250, 291-300, 3^1-350, and 391-400. Photographs of EMG discharge shown in Fig. 4 were taken at the points indicated by the arrows.  34  100-  •2  80-  o co  60-  CO  c o o. CO CO  cn X 2. 40C O cu cn  o 20-  r  t — i  10  4150  1  t 1  91- 141100 150  1  t 1  1  1  1  191- 241- 291- 3 4 1 - 391200 250 300 350 400  Number of Stimuli  Figure 7. Flexor reflex responses to stimuli presented at 10 sec. intervals to a 7 day clamped rat.  The response i s plotted as a mean  response to stimuli 1-10, M-50, 91-100, 141-150, 191-200, 241-250, 291-300, 341-350, 391-400. Photographs of EMG discharge shown in F i g . 5 were taken at the points indicated by the arrows.  35  I80-,  .2  160  -  E  o 0  140-  s  120CD W C  o  100-  cr  80-  Q. CO CD  x —  CD  60-  cr  o  X .CD  40-  20-  ~ 10  1  1  41-  50  91100  i  1  1  1  1  141-  191-  241-  291-  341-  150  200  250  300  350  —|  391400  Number of Stimuli  Figure 8,  Flexor reflex responses to stimuli presented at 10 sec, intervals to a 3 day clamped rat with hind limb extensor r i g i d i t y which developed following aortic occlusion. Points plotted are mean responses to consecutive periods of 10 stimuli as noted on the abscissa. Habituation of the reflex did not occur.  36  IOO-«  80  o <D CO  • x  c  o 60 -  Clamped Control  Q. CO <t>  CC X  cu £ 40  o X Ll_  20 -|  I10  1  41-  1  1  91100  141150  1 1 1 1 1 191- 241- 291- 341- 391200 250 300 350 4 0 0  Number of Stimuli  Figure 9 » Three day rats.  Mean ± S.E,  Flexor reflex responses to  stimuli presented at 10 sec. intervals for control rats in which the thoracic aorta was occluded fox 1 min. (crosses) and rats in which the thoracic aorta was occluded for 22 min. ( f i l l e d circles).  The mean response to each successive  group of 10 stimuli i s expressed as a percentage of the mean response to the f i r s t 10 stimuli in each animal.  3?  100-8  80 -  60  40 -  20 -  I10  i 4150  i i 91- 141100 150  Number  Figure 1 0 . Seven day rats.  •  Clamped  x  Control  1 1 1 1 1 191- 241- 291- 341- 3912 0 0 250 3 0 0 350 4 0 0  of Stimuli  Mean ± S.E.  Flexor reflex responses to  stimuli presented at 10 sec. intervals for control rats in which the thoracic aorta was occluded for 1 min. (crosses) and rats in which the thoracic aorta was occluded for 22 min. ( f i l l e d circles).  The mean response to each successive group  of 10 stimuli i s expressed as a percentage of the mean response to the f i r s t 10 stimuli in each animal.  e  38  120-  ~  100-11  80 -  60-  2  Clamped  40-  Control  20I-  10  41-  SO  91100  141150  191200  241250  291300  341350  391400  Number of Stimuli  Figure 11,  Fourteen day rats.  Mean * S,E,  Flexor reflex responses to  stimuli presented at 10 sec, intervals for control rats in which the thoracic aorta was occluded for 1 min, (crosses) and rats in which the thoracic aorta was occluded for 22 min, ( f i l l e d circles).  The mean response to each successive group  of 10 stimuli i s expressed as a percentage of the mean response to the f i r s t 10 stimuli in each animal.  39  r - - 0.986  2.0-H  p<  0.001  m = - 0.0021  c= CD CO  i Regression Oata for C o n t r o l  1.933  Animals  1.8 -  c  o  CL CO CD  CC X  «J  1.6 -  *— CO  cr  o X  £  1.4 H  o  •  Clamped  x  Control  1.2 -  x —i  10  4150  —i  91100  141150  Number  191- 241200 250 of  r-  291- 341- 391300 350 4 0 0  Stimuli  Figure 12. The relationship between the logarithm of the mean flexor reflex response to successive groups of 10 stimuli, and the number of stimuli presented. the case of control rats.  This relationship i s linear in  Regression data i s shown for  control rats only, because the raletionship described in the case of clamped animals i s not exponential.  There i s a  significant negative correlation between these parameters for control rats but not for clamped rats.  The groups of  rats compared in the graph are animals examined 3 clays after thoracotomy.  40  r = - 0.969 "j Regression  2.0-*  p = < 0.001 I Data m = - 0 . 0 0 1 5 for Control c = 1.9033 Animals  CO  x  w o Q. CO CD  cr  1.6  X CD  cr o r  5  1.4  H  •  Clamped  x  Control  o 1.2  1  I10  4150  1—  91100  ~i 141150  191200  Number of  Figure 13.  241250  291300  341350  391400  Stimuli  The relationship between the logarithm of the mean flexor reflex response to successive groups of 10 stimuli, and the number of stimuli presented. the case of control rats.  This relationship i s linear in  Regression data i s shown for  control rats only, because the relationship described in the case of clamped animals i s not exponential.  There i s a  significant negative correlation between these parameters for control rats but not for clamped rats.  The groups of  rats compared in the graph are animals examined 7 days after thoracotomy.  41  r - - 0.968  p • < 0.001  2.0-a  m- - 0.0014 c «= 1.9071  co co c o  Regression Data for Control Animals  1.8  CL  co  0>  CC X  1.6  -  to or o x  i>  1.4  H  •  Clamped  x  Control  o 1.2  I-  10  1 41-  50  I  I  I  1  1  1  1  91- 141- 191- 241- 291- 341- 391100 150 200 250 300 350 400  Number of Stimuli  Figure 14,  The relationship between the logarithm of the mean flexor reflex response to successive groups of 10 stimuli, and the number of stimuli presented. the case of control rats.  This relationship i s linear in  Regression data i s shown for  control rats only, because the relationship described in the case of clamped animals i s not exponential.  There is a  significant negative correlation between these parameters for control rats but not for clamped rats.  The groups of rats  compared i n the graph are animals examined i4 days after thoracotomy.  PART  IV.  DISCUSSION  42 The severity of neurological impairment obtained after aortic occlusion was much less than that observed in an earlier study by Matsushita and Smith (1970). These authors reported that after occlusion of the aorta for 21-23 minutes, 52$ of rats exhibited extensor r i g i d i t y .  In the present work,  marked extensor r i g i d i t y occurred in only 12$ of rats after occlusion for 22 minutes.  This disparity may be, at least partly, due to the fact that a  different strain of rat was used.  It i s possible that subtle differences  exist in the vasculature of the spinal cord between Wistar and Long Evans strains and this could manifest i t s e l f as a difference in susceptibility to asphyxial insult.  It i s possible that variations with the species have  some bearing on the outcome of aortic occlusion.  Murayama and Smith (1969)  report that some cats appeared completely normal postoperatively after occlusion of the thoracic aorta for up to two hundred minutes. Even though ischaemia did not usually result in r i g i d i t y i t did cause significant changes in spinal cord function.  The basal activity in the un-  stimulated muscle was increased,but the magnitude  of the response to an  electrical stimulus to the ipsilateral hind paw was less than in control animals.  This decrease in magnitude of the response i s not likely to be due  to damaged or destroyed afferent fibers.  Van Harreveld (1940) has shown  that afferent and spinal ganglia are not histologically damaged by asphyxiation.  Van Harreveld and Niechaj (1970) have noted that components of the  dorsal root potential (DRP) survived such cord asphyxiation.  In the present  study the responses of r i g i d rats to repeated stimuli either did not habituate or habituated more slowly than the responses of normal rats. Two hypotheses have been advanced to explain the development of r i g i d i t y following spinal ischaemia.  Gelfan and Tarlov (1959) consider that r i g i d i t y  i s due to increased excitability of motoneurones which occurs as a result of their partial denervation by the selective destruction of interneurones which normally innervate them.  It was proposed that the neurones destroyed include  43 those relaying impulses to supraspinal centers as well as to spinal motoneurones, and that this must be the basis for the sensory paralysis in r i g i d preparations.  It was also assumed to be the basis for the motor paralysis.  This chronic r i g i d i t y was not considered to be due to an exaggerated stretch reflex, since i t was neither abolished nor prevented by section of dorsal roots.  The destruction of intemeurones was considered responsible for the  loss of normal regulation of motoneurone activity and consequent muscle responses.  It was proposed that denervation (destruction of intemeurones  also denervates motoneurones) increases the excitability of motoneurones to the point of discharging "spontaneously".  It was also proposed that the  "spontaneous" discharges of such denervated motoneurones are directly responsible for the unremitting and enduring r i g i d i t y . out  on dogs.  This work had been carried  Van Harreveld and Marmont (1939) have suggested that the r i g i d i -  ty produced by ischaemia i s reflex in origin and arises from an increased discharge in fusimotor fibers.  The r i g i d i t y of spinal cats was considered  to be a "high extensor tone" due to the damage of the "tone inhibiting system" by the temporary anoxia  of the lumbosacral cord.  The excitatory component  of spinal cord was proposed to be more resistant to anoxia than the inhibitory component,  Gelfan and Tarlov*s (1959) demonstration of functional failure  of intemeurones relaying reflex excitations as well as inhibitory impulses in r i g i d dogs cannot be f i t t e d into Van Harreveld and Marmont*s (1939) thesis. In contrast to Gelfan and Tarlov, Van Harreveld showed that the strong extensor tone in r i g i d cats i s of reflex origin since transection of the dorsal roots of the lower cord abolishes i t .  Oka and Van Harreveld (1968) stated  that Gelfan and Tarlov (1959) based their postulate mainly on the observation that dorsal root section did not abolish the rigidity permanently.  Yet Oka  and Van Harreveld presented evidence that after section of the relevant dorsal roots of a r i g i d muscle group, nerve endings in contralateral muscles and perhaps in other segments can supply the sensory input for the rigidity  44 developing after radiculotomy.  Only total deafferention and isolation of a  cord segment led to complete and permanent absence of r i g i d i t y .  It should be  pointed out that Van Harreveld and Marmont (1939) using histological preparations noted that only 3 to 75 percent of the normal number of anterior horn cells were present 14 days following asphyxia. with increasing duration of asphyxia.  The number surviving diminished  Both Gelfan and Tarlov (1959) and Van  Harreveld and Marmont (1939) indicated that interneurones were more severely damaged than motoneurones.  Gelfan and Tarlov (1959) revealed that most of  the cells destroyed were interneurones and that a l l large and small motoneurones could survive when 80$ of the interneurones were destroyed. Mucayama and Smith (1969) confirmed the studies of Gelfan and Tarlov (1959) with respect to the spinal origin of the r i g i d i t y , decrease in polysynaptic reflexes,  increased response to repetitive stimulation, presence of  spontaneous alpha and fusimotor neurone activity, and Renshaw c e l l activity. The precise nature of spinal r i g i d i t y however remains difficult to explain. In view of the fact that r i g i d i t y occurred so rarely in the present experiments i t i s not possible to add any information which could help explain i t s origin.  It would seem unlikely that the excitability of the flexor moto-  neurone had increased, as the magnitude of flexor responses was lower than in control rats.  This however could have been due to partial loss of neuronal  elements e.g. afferent fibers and relay pathway.  cells in the excitatory flexor reflex  It i s of interest to note that habituation never occurred in r i g i d  preparations and perhaps related neuroanatomical alterations are common to both characteristics. Rats that have undergone occlusion of the thoracic aorta were pinched on the hind paw to test for the presence of a flexor reflex.  The reflex  could always be e l i c i t e d but compared to the control rats, the rats that have undergone aortic occlusion did not vocalize (in the majority of cases) when a painful pinch was presented.  This finding implies possible impairment of  45 pathways by which the animals identify a painful stimulus applied to the hind paw.  In >view of the paucity of information concerning this finding i t i s  difficult to speculate as to the underlying mechanism. Marayama and Smith (1969) reported that stimulation of the skin by pinching or inserting needles rarely caused any response in acute r i g i d preparations!  there was neither  a withdrawal reflex nor evidence of any sensation of pain by the cat.  No  explanation for this finding was presented. In the present study only three rats exhibited extensor r i g i d i t y and none of these rats were in the 7 day group* The inability to demonstrate a quantitative impairment of habituation in this group may have simply been a consequence of an unequal distribution of r i g i d rats  between the three groups.  The results of this study indicate that aortic occlusion renders an animal incapable of habituation of the hind limb flexor reflex.  It i s proposed  that aortic occlusion caused ischaemia of the spinal cord and this ischaemia resulted in selective destruction of a portion of the spinal neurone pool. In addition to the phenomenon known as habituation a phenomenon opposite to habituation and known as sensitization has been described.  An increased  flexor reflex response to repeated stimuli i s known to occur under ischaemic conditions such as those described by Murayama and Smith (1969),  Sensitization  i s elicited after some procedure i s applied to alter the normal neurophysiological condition of an organism.  Experiments recently carried out on cats  by Groves and Thompson (1970) in which habituation or sensitization of reflexes have been recorded at the same time as changes in the activity of interneurones have led to the so called "dual-process" theory for habituation. A tentative distinction was made between two inferred systems, the "S-R pathway", which i s the most direct route through the central nervous system from stimulus to response, and "state", the collection of pathways, systems, and regions that determines the general level of responsiveness of the organism. Habituation i s assumed to occur  in the S-R pathway and sensitization in the state system.  46  The two processes interact to yield the final common "behavioral outcome. Neurophysiological evidence for this dual-process theory i s strikingly clear. Three categories of interneurones responding to cutaneous stimuli have been found with approximately equal frequencyi  a "nonplastic" type showing no  changes in response, and two types of "plastic" interneurones, one showing habituation (type H) and one showing sensitization (type S),  The nonplastic  types of interneurones do not change with repeated stimulation and do not appear to have any characteristic type of firing pattern.  The type H inter-  neurones exhibit significant habituation even during the period of maximum muscle sensitization as shown by Groves, Be Marco and Thompson ( 1 9 6 9 ) .  Type  H interneurones have a characteristic high-frequency short latency firing pattern and respond within a restricted range of latencies ( 5 - 1 2 . 5 m i l l i seconds).  Type S interneurones exhibit sensitization followed by habituation  to, or below, i n i t i a l control level.  Type S cells characteristically respond  with a longer latency than type H cells (6-180 milliseconds).  The plasticity  of interneurones parallels exactly the two hypothetical processes suggested by Groves and Thompson ( 1 9 7 0 ) . of Rexed ( 1 9 5 2 ) .  Type H interneurones l i e within laminae I-V  Type S interneurones are situated in laminae V-VII.  inferences can be drawn.  Several  F i r s t , the latencies of type H neurones to stimula-  tion of cutaneous nerves are a l l sufficiently short that these neurones could participate in the most direct S-R reflex pathway.  Type S neurones, on the  other hand, usually exhibit latencies which are too long for them to p a r t i c i pate in the most direct reflex paths.  Type S neurones, therefore, might act  from outside the direct S-R reflex pathway onto final interneurones or motoneurones to yield the ultimate behavioral output. Wickelgren (1967b) has completed an analysis of habituation of interneurones in the lumbosacral region of spinal cord in cats.  It was reported  that interneurones habituated and that units with response latencies of more than 6 milliseconds to single shocks were more likely to habituate than units  47 with response latencies of less than 6 milliseconds.  Wall (1967) has described  "novelty detectors" in lamina V of the lumbosacral cord of decerebrate cats. These neurones respond vigorously to the f i r s t few presentations of cutaneous stimuli but soon cease responding i f the stimulus i s repeated.  Mendell (1966)  and Mendell and Wall (1967) described "wind-up" of cells of the spinocervical tract.  This phenomenon was simply a progressive increase in evoked activity  with repetitive stimulation of C (small diameter) fibers, and resembles sensitization very closely.  Prank and Fuortes (1956) described a similar phenomenon  for intemeurones of the lumbosacral cord. These studies point out the basic distinction between sensitization and habituation and suggest that there may be separate populations of intemeurones, the functional properties of which are able to account for the behavioral plasticity observed in the flexor reflex of acute spinal cats. A synaptic hypothesis consistent with the general dual-process theory of response habituation and the known types of intemeurones needs to be developed.  The process as postulated by Groves and Thompson (1970) must occur  at some region of synaptic action on intemeurones, either at the f i r s t synapse formed by afferent fibers or at some subsequent synapse.  The synaptic hypoth-  esis, for convenience i s classified into two categories!  extrinsic and i n -  trinsic.  Postsynaptic and presynaptic inhibition are the two known forms of  extrinsic action that can induce decrements in synaptic transmission.  Addi-  tionally some other as yet unknown form of extrinsic synaptic action may exist.  Two types of intrinsic action have been forwardedt  l)  "Monosynaptic  low frequency depression" (Thompson and Spencer, 1966) which could be a consequence of alterations in mobilization and/or release of transmitter, and 2) "membrane desensitization" (Sharpless, 1964) which would involve decreased responsiveness of the postsynaptic membrane to a transmitter as a result of repeated activation by the transmitter.  Which of the alternatives i s i n fact  the operative process remains to be elucidated.  Indirect evidence for the  48 existence of postsynaptic inhibition as the process on which habituation i s based has been provided by Wickelgren (1967b).  She reports that in three  dorsal horn cells studied, generalization or transfer of habituation occurred in only one direction ( i . e . generalization to stimulus applied to a second site "B" after habituation had been established by stimuli applied to an original site "A" but not vice versa).  Wickelgren (1967b) argues that i f  habituation were due to"synaptic depression", common habituated interneurones ought to show generalization of habituation to both stimuli, whereas postsynaptic inhibition could operate on only one interneurone channel and hence would yield generalization of habituation in only one direction.  The argument  i s indirect and rests entirely on the demonstration of asymmetry of generalization of habituation.  Wickelgren (1967b) has demonstrated three interneurones  which show transfer of habituation i n only one direction,  Glanzmann (1972)  has shown bidirectional generalization of habituation on seven type H interneurones.  It i s significant that at least some interneurones exist which  show unidirectional generalization and hence postsynaptic inhibition could well be the modus operandi.  However the situation remains unsolved.  Evidence against the proposal that inhibition i s the process on which habituation depends i s not strong.  This evidence relies on the report by  Spencer, Thompson and Neilson (1966c) that strychnine and picrotoxin do not prevent habituation.  This work i s confused however by the fact that the  stimulus strength during control periods and during periods of pharmacological intervention with strychnine or picrotoxin was not constant.  Indeed the  stimulus strength was lowered during the later period and this alone could lead to decreased habituation as pointed out by Thompson and Spencer (1966) in an earlier review.  Altered stimulus strength could mask the blocking  effects of the drugs.  Inspection of records, even for periods where the same  stimulus strength was used reveals that with application of strychnine or picrotoxin and combined application of strychnine and picrotoxin there i s a  49  small but real diminution of habituation apparently ignored by Spencer, Thompson and Neilson ( 1 9 6 6 c ) . Habituation of the discharge of intemeurones situated in lamina V of the dorsal horn of the cat has been demonstrated by Wall (1967) and Wickelgren (1967b).  The characteristics of habituation of activity in these inter-  neurones were very similar to those obtained from motoneurones (Wickelgren, 1967a).  Intemeurones in lamina IV, which like those in lamina V respond to  low threshold cutaneous afferents, do not habituate.  Gradual failure of  transmission of activity from neurones in lamina IV to those in lamina V may partly be the cause of habituation (Wickelgren, 1967b).  Wall (1967) has  suggested that habituation at this site may be the result of activity in a side chain of inhibitory intemeurones. afferents may cause  (a)  Thus, stimulation of cutaneous  excitation of lamina V cells, both directly and  via cells in lamina IV, and  (b)  initiate activity in a parallel pathway  which funotions to depress the excitability of neurones in lamina V.  It has  been suggested that the nerve cells of the substantia gelatinosa may be good candidates for such a side chain (Wall, 1 9 6 7 ) . Heimer and Wall (1968) indicated histologically (using the Pink-Heimer technique) that lamina II or the substantia gelatinosa proper, receives a massive termination of dorsal root fibers.  Ralston (1965) has shown that the  marginal cells (15-20 p diameter) which are confined to the surface of dorsal lamina I I , are spindle shaped with their long axes parallel to the surface of the lamina, and appear to receive dorsal root small fiber terminations.  Large  cutaneous fibers were noted to track medially to the dorsal horn to enter lamina IV and synapse with large neurones, also sending collaterals to synapse more dorsally in laminae II and III. substantia gelatinosa  It could well be that cells of the  and possibly also the marginal cells both of which have  been shown to receive cutaneous input (Heimer and Wall, 1968) are components of an inhibitory side chain which may be responsible for producing a decrement  50 of activity in the direct flexor reflex pathway.  The cells of the substantia  gelatinosa are small and small neurones are the most susceptible to asphyxial insult.  It i s possible that the impairment of habituation observed in this  study may have been due to degeneration of substantia gelatinosa cells which had been components of an inhibitory side chain.  It i s l i k e l y that degenera-  tion of interneurones forming the direct excitatory flexor reflex pathway had also occurred, as the magnitude of responses i n the nonhabituating rats was lower than in control rats.  The higher basal activity seen with EMG recordings  of clamped rats prior to testing could be due to the same as yet unsolved, but in these cases attenuated, mechanism responsible for the extensor r i g i d i t y seen in a few preparations.  P A R T  V.  B I B L I O G R A P H Y  51 Buchwald, J . S . , Halas, E.S., and Schram, S.  (1965). Progressive changes in  efferent unit response to repeated cutaneous stimulation in spinal cats. J . Neurophysiol. 28, 200 - 215. Collier, J .  (l899). An investigation upon the plantar reflex with reference  to the significance of i t s variations under pathological conditions, including an enquiry into the retiology of acquired pes cavus.  Brain  22, 71. Davidoff, R . A . , Graham, L . T . , Shank, R . P . , Werman., R., and Aprisin, M.H, (1967). Changes in amino acid concentration associated with loss of spinal interneurones.  J . Neurochem. 14, 1025 - 1031.  Eccles, J . C . , Schmidt, R . F . , and W i l l i s , W.D. (1962). Presynaptic inhibition of the spinal monosynaptic reflex pathway.  J . Physiol. (London), l 6 l ,  282 - 297. Frank, K . , and Fuortes, M.G.F. rones of cats. Galambos, R.  (1956). Unitary activity of spinal interneu-  J . Physiol. 131, 424 - 436.  (1967). Brain Correlates of Learnings  in The Neurosciences,  edited by Quarton, G . L . , Melnechuk, T . , Schmitt, F.0, University Press.  637 - 643.  Gelfan, S. and Tarlov, I.M. origin.  Rockefeller  (1959). Interneurones and r i g i d i t y of spinal  J . Physiol, (london), 146, 594-617.  Gelfan, S. and Tarlov, I.M. (1963). Altered neuron population i n L? segment of dogs with experimental hind limb r i g i d i t y .  Amer. J . Physiol. 205,  606 - 616. Glanzman, D . L . , Groves, P.M., and Thompson, R.F, (1972). Simulus generalization of habituation in spinal interneurones.  Physiology and Behaviour  8, 155 - 158. Griffin, J . P . and Pearson, J . A .  (1968). The effect of bladder distension  on habituation of the flexor withdrawal reflex in the decerebrate spinal cat.  Brain Research 8, 185 - 195.  Groves, P.M., Lee, D . , and Thompson, R.F.  (1969). Effects of stimulus  frequency and intensity on habituation and sensitization in acute spinal cat.  Physiol, and Behav. 4, 383 - 388.  Groves, P.M., De Marco, R., and Thompson, R.F.  (1969).  Habituation and  sensitization of spinal interneurone activity in acute spinal cat. Brain Research 14, 52i - 525. Groves, P.M. and Thompson, R.F.  (1970).  Habituation: A dual-process theory.  Psychological Review 77, 419 - 450. Heimer, L . and Wall, P.  (1968). The dorsal root distribution of the  substantia gelatinosa of the rat with a note on the distribution in the cat.  Exp. Brain Res. 6, 89 - 99.  Hernandez-Peon, R., Jouvet, M., and Scherer, H. at cochlear nucleus  (1957). Auditory potentials  during acoustic habituation.  Acta Neurologica  Latinoamericana 3. 144 - 156. Kandel, E.R.  (1967). Cellular Studies of Learning:  in The Neurosciences,  edited by Quarton, G . L . , Melnechuk, T . , Schmitt, F.O. Rockfeller University Press. Kugelberg, E .  (1962).  666 - 689. Polysynaptic reflexes of c l i n i c a l importance.  Electroenceph. Clin. Neurophysiol. Suppl. 22, 103 - 111. Matsushita, A. and Smith, C M . (1970). r i g i d i t y in the rat.  Brain Research 19, 395 - M l .  Mendell, L.M. and Wall, P.D.  Presynaptic hyperpolarization: a role  (1964),  for fine afferent fibers. Mendell, L.M.  Spinal cord function in post ischemic  J . 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P . and Van Harreveld, A.  (l96l).  The volume distribution of moto-  neurones and interneurones in the peroneus - t i b i a l i s neuron pool. J . Comp. Neurol. 117, 387. Sharpless, S.K.  (1964).  use and disuse. Sherrington, C S .  Reorganization of function in the nervous system Annual Review of Physiology 26, 357 - 388.  (1898).  Experiments in examination of the peripheral  distribution of the fibers of the posterior roots of some spinal nerves.  P h i l . Trans. Roy. Soc.  (London).  Spencer, W.A., Thompson, R . F . , and Neilson, D.R.  Ser. B. 190, 45 - 186. (l966a).  Response decre-  ment of the flexion reflex in the acute spinal cat and transient restoration by strong stimuli.  J . Neurophysiol. 29, 221 - 239.  Spencer, W.A., Thompson, R . F . , and Neilson, D.R.  (1966b).  Alterations in  responsiveness of ascending and reflex pathways activated by iterated cutaneous afferent volleys.  J , Neurophysiol.  Spencer, W.A., Thompson, R . F , , and Neilson, D.R. -  29, 240 - 252.  (1966c).  Decrement of  ventral root electrotomes and intracellularlyrecorded PSP-*-,s produced by iterated cutaneous afferent volleys. - 272.  J . Neurophysiol. 29, 253 -  Thompson, R.F. and Spencer, W.A, (1966),  Habituation:  for the study of neuronal substances of behavior.  A model phenomenon Psychological Review  73, 1, 16 - 43. Van Harreveld, A,  (1940). The effect of asphyxiation of the spinal cord on  pain sensibility,  Amer. J , Physiol. 131, 1, 1 - 9.  Van Harreveld, A. and Marmont, G. (1939). The course of recovery of the spinal cord from asphyxia.  J . Neurophysiol, 2, 101 - 111,  Van Harreveld A. and Khattab, F . I , spinal cords of cats,  (1967).  Electron microscopy of asphyxiated  J . Neuropath. Exper. Neurol. 26, 521 - 536,  Van Harreveld, A, and Schade', J . P .  (1962).  asphyxiation of the spinal cord.  Nerve c e l l destruction by  J , Neuropath. Exp. Neurol. 21, 4l0 -  - 423. Van Harreveld, A. and Spinelli, D. with post asphyxial r i g i d i t y .  (1965).  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