Physiology and Behavior, Vol. 14, pp. 297-304. Brain Research Publications Inc., 1975. Printed in the U.S.A.
Classical Conditioning, Sensitization and Habituation in the Spinal Cat I RUSSELL G. DURKOVIC
Department o f Physiology, Upstate Medical Center, Syracuse, New York 13210
(Received 23 August 1974) DURKOVIC, R. G. Classical conditioning, sensitization and habituation in the spinal cat. PHYSIOL. BEHAV. 14(3) 297-304, 1975. - Changes in the flexion reflex of the tibialis anterior muscle of acute spinal cats were examined during conditioning, sensitization and habituation paradigms. Experimental animals were classically conditioned by pairing electrical stimulation of the saphenous nerve (CS) with stimulation of the superficial peroneal nerve (US). Recordings from these nerves assured known and constant stimulus inputs. The response observed was an increase in the magnitude of the reflex response to the CS over training. Habituation (CS only) and sensitization (CS and US presentations, unpaired) control animals exhibited no such reflex facilitation. The results of post-tetanic potentiation studies indicated that the intertrial intervals used were not a factor in the differences observed between experimental and control groups. The results give positive support to the concept of spinal conditioning and emphasize the potential of this model system for the study of neural correlates of learning. Spinal conditioning
Sensitization
Habituation
Flexion reflex
Spinal cord
can be recorded throughout the duration of the experiment to monitor their constancy. Third, the stimulus inputs are confined to specific cutaneous nerves. This is particularly important in limiting the complexity of the neural model. Finally, the specific types of nerve fibers activated for the CS and US are known and can be varied. This technique is proving useful in further studies designed to elucidate the role of different fiber groups in the elicitation of the conditioned response (R. G. Durkovic and A. R. Light, manuscript in preparation). The present paper describes the results of conditioning, sensitization and habituation experiments which were based in part on a preparation which Fitzgerald and Thompson [7] used for pilot studies on spinal conditioning. Their preparation appeared to have several advantages for conducting neurophysiological analyses of the phenomenon such as rapid development of the conditioned response and an experimental animal that is well restrained. In the acute spinal cat Fitzgerald and Thompson utilized electrical stimulation of the skin of the thigh as the CS and shock to the foot pad as the US. They found that paired CS-US trials resulted in conditioning of a hind limb flexion reflex, while unpaired CS and US presentations did not. The conditioned response was an increase in the magnitude of contraction of the tibialis anterior muscle in response to the CS. Recently, an additional study of conditioning in the spinal cat has
THE history of attempts to develop simplified neural preparations for analyses of learning mechanisms includes many successful [1, 2, 4, 6, 7, 10, 17, 21] as well as unsuccessful [3, 8, 12, 13, 15, 18] attempts to demonstrate conditioned responding of the spinal cord. As a result of the contradictory results of these experiments as well as the emphasis that has been placed upon the role of higher neural structures in learning, the existence of conditioning at the spinal level has not gained general acceptance. Nevertheless, relative to higher neural centers, the simplicity of the spinal preparation could offer very important advantages for the investigation of the neurophysiological mechanisms of conditioning. Therefore, the present classical conditioning experiments were undertaken to examine the suitability of the spinal preparation as a model system for such investigations. These experiments differ from previous spinal conditioning studies primarily with regard to stimulus control. Stimulation of individual exposed cutaneous nerves was used for the CS (conditioned stimulus) and the US (unconditioned stimulus), and recordings from these nerves were made for each animal. There are several notable advantages to this method. First, possible artifacts which might arise from receptor adaptation or fatigue are avoided. Second, the compound action potentials representing the conditioned and unconditioned stimulus inputs to the spinal cord
1This research was supported by Joint Awards Council and the University Awards Committee, S.U.N.Y. Research Foundation Grant No. ND 67226. The author would like to express his appreciation to Mr. Alan Light and Dr. Ernest Damianopoulos for their helpful comments regarding the manuscript and to Dr. Paul Sheehe for his help with the statistics. 297
298
DURKOVIfl
been completed by Thompson and co-workers [ 17 ] and has yielded even more impressive evidence for the occurrence of conditioned flexion reflexes in this preparation. In this latter study the animals were paralyzed with a neuromuscular blocking agent and the conditioned responses measured were recordings of reflex volleys from the deep peroneal nerve. The US was shock to the skin of the ankle and the CS was electrical stimulation of the superficial peroneal cutaneous nerve. In two sets of experiments using different intertrial intervals, paired CS-US presentations produced significant differences over unpaired CS-US sensitization control groups during acquisition. The present study supports the positive results of this earlier work and in addition supplies important information regarding the control and magnitude of the conditioned and unconditioned stimuli. METHOD Animals Results are described from experiments on 40 adult, male and female cats ranging in weight 3 - 5 . 2 kg. Animals were assigned randomly to conditioning, sensitization or habituation groups with ten animals in each group. An additional group of 10 cats was used to determine the time course of post-tetanic potentiation of the flexion reflex. Procedure Animals were anesthetized with ether, intubated and artificially respired with a mixture of room air and 1-2% Halothane. The common carotid arteries were isolated and a cannula inserted into one of them for monitoring blood pressure. The other artery was ligated. Following spinal cord transection at the T-10 level, animals were made decapitate by occluding the vertebral arteries with a clamp. Further anesthesia was discontinued at this point. End tidal CO2 levels ( 3 - 4 % was considered acceptable) and arterial pressure (mean levels of 65 to 110 mmHg were considered acceptable) were continuously monitored. Rectal temperature was maintained at 37°C with the aid of a heating pad. For administering fluids (lactated Ringer's solution, approximately 10 cc per hour) a small cannula was inserted into a forelimb vein. The hind quarters were stabilized with hip pins and the left hind leg was rigidly fixed with a femur pin and a foot clamp. The distal tendon of the tibialis anterior muscle was severed and attached to a Grass force displacement transducer (resting tension approximately 50 g). The output of the transducer was connected to high and low gain amplifiers of a two-channel polygraph. The internal saphenous and superficial peroneal nerves (both pure cutaneous nerves) were freed from surrounding tissue for placement of platinum wire stimulating and recording electrodes leaving both central and peripheral connections intact. All exposed nervous tissue was covered with warm mineral oil in pools formed from skin flaps. A Grass $88 stimulator was used to deliver current to the s t i m u l a t i n g electrodes. Electrical stimulation of the saphenous nerve (1 V, 0.2 msec pulses at 10/sec for 1.5 sec) served as the CS. The US was electrical stimulation of the superficial peroneal nerve (1 V, 0.2 msec pulses at 40/sec for 0.5 sec). For animals used in conditioning, sensitization and habituation studies a minimum of 2 hr passed between
decapitation and initiation of conditioning procedures in order to allow the preparation some time to recover from the effects of spinalization and anesthetic. At this time each animal received a test CS followed 1 rain later by a test US. This was to assure the correct functioning of stimulating and recording electrodes. Five minutes after the test US presentation all animals received 5 CS alone trials separated from one another by 1 rain intervals. One min after the last CS alone presentation animals received one of three sets of stimulus presentations. Conditioning animals received 30 CS-US presentations, the US overlapping the last 0.5 sec of the CS. The intertrial interval (ITI) was 1 rain. Sensitization animals received 30 unpaired CS and US presentations, the CS alternated with the US every 30 sec. Thus, over this 30-rain period, conditioning and sensitization animals received the same number of CS and US presentations, only the stimuli were paired for conditioning animals and unp a i r e d for sensitization animals. Habituation animals received 30 CS alone trials with a I min Iq-I. One minute after this initial 30-min period all animals received 30 additional CS alone presentations with a 1 min ITI. For each trial the maximum tension developed by the tibialis anterior muscle during the first second of the CS presentation (Fig. 1) was the primary response measured. For most animals this peak tension usually occurred in response to the second, third or fourth pulse of the CS. The mean of the responses to the first 5 CS alone persentations established the initial control level for a given animal. Subsequent 5 trial mean responses were converted to a percentage of this initial control response. The tension measurements were due exclusively to contraction of the anterior tibial muscle since (i)section of the nerve to the anterior tibial muscle abolished all recorded responses to the CS and the US, even though the surrounding muscles continued to respond, and (ii) the CS, US or stimulation of the common peroneal nerve produced the same tension responses before and after the anterior tibial muscle was isolated from surrounding tissues, leaving its attachments to bone intact. The three experimental groups were compared by analysis of variance and Duncan's New Multiple Range Test [5 ]. Unless otherwise stated, significance was tested for c~ = 0.05. Comparisons were made of the 30 trial mean percent changes from baseline for conditioning and extinction periods. As a measure of the trend of the responses over trials, regression coefficients were calculated for each animal from the 5 trial mean percentage data for both the conditioning and the extinction periods. Because of the exponential shape of these curves, the regression coefficients were calculated from the logarithms of these data. These coefficients were compared in the same manner as the mean percent data. In order to obtain a measure of the time course of the effect of the US on the flexion response to the CS, i0 additional cats were prepared in the same manner as the cats used in the conditioning studies. Following the usual surgical and anesthetic recovery period animals were given 5 series of saphenous and superficial peroneal nerve stimulus presentations at the same intensities as those used in the conditioning studies. A series consisted of 15 single shocks to the saphenous nerve at 30 sec intervals. Five seconds before the sixth shock of each series the superficial peroneal nerve was stimulated for 0.5 sec at 40/sec. The interseries interval was 10 min. For each cat the peak tension developed by the tibialis anterior muscle in
SPINAL C O N D I T I O N I N G
299 response to each pulse to the saphenous nerve was measured and converted to a percentage of the m e a n response to the first 5 of these stimuli. A D u n n e t t ' s t test [5] was used to compare this control mean percentage with subseq u e n t interval m e a n percentages. Oscilloscope displays of c o m p o u n d action potentials from the saphenous and superficial peroneal nerves of all 40 cats were m o n i t o r e d visually to assure that the neural inputs to the spinal cord were constant in magnitude over the course of each experiment. In some cases these recordings were filmed using a Grass camera. A c t i o n potentials such as those pictured in Fig. 2B were considered acceptable. Such recordings indicated that the stimuli were near maximal for Ao~ and A6 fiber groups [ 11 ]. Nine cats were tested in addition to the 40 animals comprising this study, but their results could not be used. In 5 of these, the recordings of the cutaneous nerve c o m p o u n d action potentials declined m a r k e d l y in amplitude over the course of the experiment, possibly because of injury or lack of blood supply to the nerve. In four o t h e r cats there was no initial reflex response to saphenous nerve stimulation. This appeared to be a result of depressed spinal reflexes in these animals.
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alone were 284 + 104 S.E., 306 + 74 S.E., and 338 * 86 S.E. grams for conditioning, sensitization and habituation groups respectively, and these did not differ significantly from one another (Duncan’s New Multiple Range Test). Subsequent 5 trial mean responses to the CS are shown for each group as a percentage of the control level responses. In the conditioning group (paired CS-US presentations) the response to the CS increased rapidly in magnitude during the first 10 trials (e.g., see Fig. 2A), and this level was maintained throughout subsequent conditioning trials. Beginning with Trial Block 4 the variability in response of the conditioning group began to increase (Fig. 3). This was because the responses of 4 animals began to decline toward the control level, while that of the other 6 either remained near the plateau level or increased in magnitude. The sensitization group (unpaired CS and US presentations) ex-
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hibited no reflex facilitation in response to the CS, the reflex remaining near control level throughout the first 30 CS presentations. With regard to the responses of individual sensitization animals, 3 of these exhibited small flexion increases in response to the CS over the initial 30 trials. The responses of these 3 sensitization animals did not appear to differ from those of the two lowest responders in the conditioning group. Repeated presentations of the CS alone (habituation group) resulted in a rapid decline from control level during the first and second 5 trial blocks, the reflex declining more gradually during subsequent trials. The responses of every animal in this group declined from control levels during the initial thirty trials. Significant differences were found among the mean responses of the different experimental groups during conditioning, F(2,27) = 24.91, p0.6). Tests for correlations between mean CR (conditioned response) scores and UR magnitude and changes over conditioning and sensitization were not significant. There were no significant correlations between an animal's 30 trial mean CR and: ( i ) t h e magnitude of the UR, (ii) the ratio of the control magnitude to the UR magnitude or (iii) the change in UR magnitude between the first and sixth five trial blocks. Statistical analyses of the extinction data in Fig. 3
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MINUTES FIG. 5. Group data (n = 10) from post-tetanic potentiation experiments. Mean responses of tibialis anterior muscle to single shocks to the saphenous nerve (S.N.) before and at intervals following a 40/sec, 0.5 sec stimulus to superficial peroneal nerve (SPN stim.) plotted as percent of pre-SPN stimulus responses. Vertical lines represent standard deviations of the mean. i n d i c a t e d t h a t significant d i f f e r e n c e s existed a m o n g m e a n responses of the t h r e e groups, F ( 2 , 2 7 ) = 4.39, p < 0 . 0 2 5 . T h e m u l t i p l e range test s h o w e d t h a t t h e 30 trial m e a n r e s p o n s e s of t h e c o n d i t i o n i n g g r o u p were significantly greater t h a n t h o s e of t h e s e n s i t i z a t i o n and h a b i t u a t i o n groups. O t h e r c o m p a r i s o n s o f g r o u p m e a n d a t a and regression c o e f f i c i e n t s were n o t significant. Several animals were r u n o n m o r e t h a n o n e b e h a v i o r a l paradigm and t h e single trial data f r o m o n e of t h e s e are p r e s e n t e d in Fig. 4. This a n i m a l was initially tested as an h a b i t u a t i o n a n i m a l a n d t h e d a t a f r o m t h e first 65 CS alone (Fig. 4, o p e n circles) p r e s e n t a t i o n s were used along w i t h similar d a t a f r o m o t h e r cats t o f o r m t h e h a b i t u a t i o n g r o u p curve in Fig. 3. S u b s e q u e n t tests o n t h e s e n s i t i z a t i o n and c o n d i t i o n i n g paradigms were carried o u t in this a n i m a l t o c o m p a r e t h e results of d i f f e r e n t m o d e s o f s t i m u l u s present a t i o n o n flexor reflex e x c i t a b i l i t y in a single animal. T h e h a b i t u a t i o n d a t a in Fig. 4 b e a r a close r e s e m b l a n c e t o t h e h a b i t u a t i o n g r o u p d a t a p i c t u r e d in Fig. 3. T h e r e was a rapid decline in t h e r e s p o n s e to t h e CS b e t w e e n t h e s i x t h a n d t w e l f t h CS p r e s e n t a t i o n s and this data is similar t o t h e g r o u p d a t a in Fig. 3. S u b s e q u e n t decline in t h e r e s p o n s e was m o r e gradual.
F o l l o w i n g a 1 h r rest period t h e a n i m a l was tested o n t h e sensitization p a r a d i g m exactly as n o r m a l s e n s i t i z a t i o n animals had b e e n tested. However, t h e d a t a f r o m this and s u b s e q u e n t tests are n o t i n c l u d e d in Fig. 3. T h e r e s p o n s e to the initial CS o f this series was similar in m a g n i t u d e to t h a t observed in the first series o f CS p r e s e n t a t i o n s over 2 h r before. This was followed in t h e s u b s e q u e n t 4 CS presentat i o n s b y a decreased r e s p o n s e to levels near t h o s e r e a c h e d at the end of t h e h a b i t u a t i o n series. With t h e i n t r o d u c t i o n of a l t e r n a t e CS and US p r e s e n t a t i o n s (Fig. 4, b l a c k triangles) t h e r e was an increase in t h e r e s p o n s e to t h e CS to magnitudes n o t r e a c h e d since t h e initial 5 CS's were p r e s e n t e d . This was followed b y a decrease in t h e r e s p o n s e over t h e last h a l f o f the s e n s i t i z a t i o n series and t h e s u b s e q u e n t 30 CS alone p r e s e n t a t i o n s . N o t e t h e similarities o f these individual a n i m a l d a t a w i t h t h e curves o f t h e s e n s i t i z a t i o n g r o u p in Fig. 3. A n o t h e r 1 h r rest period was followed b y t h e same series of stimuli t h a t t h e a n i m a l s o f t h e c o n d i t i o n i n g g r o u p had received. T h e responses t o t h e initial CS alone trials again showed evidence of some s p o n t a n e o u s r e c o v e r y f r o m t h e previous CS alone r e s p o n s e levels o f the p r e c e d i n g series. Pairing of CS w i t h US (Fig. 4, solid circles) resulted in
SPINAL CONDITIONING evidence of flexor reflex facilitation beginning at trial four with a continuous increase through trial fourteen. The CS alone (extinction) trials which followed produced a rapid decrement in the response but the response did not decline to the levels reached in previous CS alone series. This last set of data corresponds closely to the conditioning group data of Fig. 3. The behavioral data show that a facilitation of the flexor reflex response to the CS takes place when the CS is paired closely in time with the US (conditioning). However, if the CS is presented alone (habituation) or alternated in a series with the US (sensitization) such reflex facilitation does not occur. One explanation of the difference between conditioning and sensitization animals in this experiment could be that the US produces a post-tetanic potentiation of the response to the CS which is minimal 30 seconds after delivery of the US, but which increases significantly after one minute. The post-tetanic potentiation experiments shown in Fig. 5 were designed to test this possibility. It was found that following presentation of the US (40/sec stimulation of superficial peroneal nerve for 0.5 sec) there was a potentiation of the response to single saphenous nerve stimuli. However, the time course of the post-tetanic potentiation decayed monotonically. Furthermore, the only point found to be significantly different from pre-US control levels was the response observed five seconds after the US presentations (t = 8.633, d f = 108, p
Classical Conditioning, Sensitization and Habituation in the Spinal Cat I RUSSELL G. DURKOVIC
Department o f Physiology, Upstate Medical Center, Syracuse, New York 13210
(Received 23 August 1974) DURKOVIC, R. G. Classical conditioning, sensitization and habituation in the spinal cat. PHYSIOL. BEHAV. 14(3) 297-304, 1975. - Changes in the flexion reflex of the tibialis anterior muscle of acute spinal cats were examined during conditioning, sensitization and habituation paradigms. Experimental animals were classically conditioned by pairing electrical stimulation of the saphenous nerve (CS) with stimulation of the superficial peroneal nerve (US). Recordings from these nerves assured known and constant stimulus inputs. The response observed was an increase in the magnitude of the reflex response to the CS over training. Habituation (CS only) and sensitization (CS and US presentations, unpaired) control animals exhibited no such reflex facilitation. The results of post-tetanic potentiation studies indicated that the intertrial intervals used were not a factor in the differences observed between experimental and control groups. The results give positive support to the concept of spinal conditioning and emphasize the potential of this model system for the study of neural correlates of learning. Spinal conditioning
Sensitization
Habituation
Flexion reflex
Spinal cord
can be recorded throughout the duration of the experiment to monitor their constancy. Third, the stimulus inputs are confined to specific cutaneous nerves. This is particularly important in limiting the complexity of the neural model. Finally, the specific types of nerve fibers activated for the CS and US are known and can be varied. This technique is proving useful in further studies designed to elucidate the role of different fiber groups in the elicitation of the conditioned response (R. G. Durkovic and A. R. Light, manuscript in preparation). The present paper describes the results of conditioning, sensitization and habituation experiments which were based in part on a preparation which Fitzgerald and Thompson [7] used for pilot studies on spinal conditioning. Their preparation appeared to have several advantages for conducting neurophysiological analyses of the phenomenon such as rapid development of the conditioned response and an experimental animal that is well restrained. In the acute spinal cat Fitzgerald and Thompson utilized electrical stimulation of the skin of the thigh as the CS and shock to the foot pad as the US. They found that paired CS-US trials resulted in conditioning of a hind limb flexion reflex, while unpaired CS and US presentations did not. The conditioned response was an increase in the magnitude of contraction of the tibialis anterior muscle in response to the CS. Recently, an additional study of conditioning in the spinal cat has
THE history of attempts to develop simplified neural preparations for analyses of learning mechanisms includes many successful [1, 2, 4, 6, 7, 10, 17, 21] as well as unsuccessful [3, 8, 12, 13, 15, 18] attempts to demonstrate conditioned responding of the spinal cord. As a result of the contradictory results of these experiments as well as the emphasis that has been placed upon the role of higher neural structures in learning, the existence of conditioning at the spinal level has not gained general acceptance. Nevertheless, relative to higher neural centers, the simplicity of the spinal preparation could offer very important advantages for the investigation of the neurophysiological mechanisms of conditioning. Therefore, the present classical conditioning experiments were undertaken to examine the suitability of the spinal preparation as a model system for such investigations. These experiments differ from previous spinal conditioning studies primarily with regard to stimulus control. Stimulation of individual exposed cutaneous nerves was used for the CS (conditioned stimulus) and the US (unconditioned stimulus), and recordings from these nerves were made for each animal. There are several notable advantages to this method. First, possible artifacts which might arise from receptor adaptation or fatigue are avoided. Second, the compound action potentials representing the conditioned and unconditioned stimulus inputs to the spinal cord
1This research was supported by Joint Awards Council and the University Awards Committee, S.U.N.Y. Research Foundation Grant No. ND 67226. The author would like to express his appreciation to Mr. Alan Light and Dr. Ernest Damianopoulos for their helpful comments regarding the manuscript and to Dr. Paul Sheehe for his help with the statistics. 297
298
DURKOVIfl
been completed by Thompson and co-workers [ 17 ] and has yielded even more impressive evidence for the occurrence of conditioned flexion reflexes in this preparation. In this latter study the animals were paralyzed with a neuromuscular blocking agent and the conditioned responses measured were recordings of reflex volleys from the deep peroneal nerve. The US was shock to the skin of the ankle and the CS was electrical stimulation of the superficial peroneal cutaneous nerve. In two sets of experiments using different intertrial intervals, paired CS-US presentations produced significant differences over unpaired CS-US sensitization control groups during acquisition. The present study supports the positive results of this earlier work and in addition supplies important information regarding the control and magnitude of the conditioned and unconditioned stimuli. METHOD Animals Results are described from experiments on 40 adult, male and female cats ranging in weight 3 - 5 . 2 kg. Animals were assigned randomly to conditioning, sensitization or habituation groups with ten animals in each group. An additional group of 10 cats was used to determine the time course of post-tetanic potentiation of the flexion reflex. Procedure Animals were anesthetized with ether, intubated and artificially respired with a mixture of room air and 1-2% Halothane. The common carotid arteries were isolated and a cannula inserted into one of them for monitoring blood pressure. The other artery was ligated. Following spinal cord transection at the T-10 level, animals were made decapitate by occluding the vertebral arteries with a clamp. Further anesthesia was discontinued at this point. End tidal CO2 levels ( 3 - 4 % was considered acceptable) and arterial pressure (mean levels of 65 to 110 mmHg were considered acceptable) were continuously monitored. Rectal temperature was maintained at 37°C with the aid of a heating pad. For administering fluids (lactated Ringer's solution, approximately 10 cc per hour) a small cannula was inserted into a forelimb vein. The hind quarters were stabilized with hip pins and the left hind leg was rigidly fixed with a femur pin and a foot clamp. The distal tendon of the tibialis anterior muscle was severed and attached to a Grass force displacement transducer (resting tension approximately 50 g). The output of the transducer was connected to high and low gain amplifiers of a two-channel polygraph. The internal saphenous and superficial peroneal nerves (both pure cutaneous nerves) were freed from surrounding tissue for placement of platinum wire stimulating and recording electrodes leaving both central and peripheral connections intact. All exposed nervous tissue was covered with warm mineral oil in pools formed from skin flaps. A Grass $88 stimulator was used to deliver current to the s t i m u l a t i n g electrodes. Electrical stimulation of the saphenous nerve (1 V, 0.2 msec pulses at 10/sec for 1.5 sec) served as the CS. The US was electrical stimulation of the superficial peroneal nerve (1 V, 0.2 msec pulses at 40/sec for 0.5 sec). For animals used in conditioning, sensitization and habituation studies a minimum of 2 hr passed between
decapitation and initiation of conditioning procedures in order to allow the preparation some time to recover from the effects of spinalization and anesthetic. At this time each animal received a test CS followed 1 rain later by a test US. This was to assure the correct functioning of stimulating and recording electrodes. Five minutes after the test US presentation all animals received 5 CS alone trials separated from one another by 1 rain intervals. One min after the last CS alone presentation animals received one of three sets of stimulus presentations. Conditioning animals received 30 CS-US presentations, the US overlapping the last 0.5 sec of the CS. The intertrial interval (ITI) was 1 rain. Sensitization animals received 30 unpaired CS and US presentations, the CS alternated with the US every 30 sec. Thus, over this 30-rain period, conditioning and sensitization animals received the same number of CS and US presentations, only the stimuli were paired for conditioning animals and unp a i r e d for sensitization animals. Habituation animals received 30 CS alone trials with a I min Iq-I. One minute after this initial 30-min period all animals received 30 additional CS alone presentations with a 1 min ITI. For each trial the maximum tension developed by the tibialis anterior muscle during the first second of the CS presentation (Fig. 1) was the primary response measured. For most animals this peak tension usually occurred in response to the second, third or fourth pulse of the CS. The mean of the responses to the first 5 CS alone persentations established the initial control level for a given animal. Subsequent 5 trial mean responses were converted to a percentage of this initial control response. The tension measurements were due exclusively to contraction of the anterior tibial muscle since (i)section of the nerve to the anterior tibial muscle abolished all recorded responses to the CS and the US, even though the surrounding muscles continued to respond, and (ii) the CS, US or stimulation of the common peroneal nerve produced the same tension responses before and after the anterior tibial muscle was isolated from surrounding tissues, leaving its attachments to bone intact. The three experimental groups were compared by analysis of variance and Duncan's New Multiple Range Test [5 ]. Unless otherwise stated, significance was tested for c~ = 0.05. Comparisons were made of the 30 trial mean percent changes from baseline for conditioning and extinction periods. As a measure of the trend of the responses over trials, regression coefficients were calculated for each animal from the 5 trial mean percentage data for both the conditioning and the extinction periods. Because of the exponential shape of these curves, the regression coefficients were calculated from the logarithms of these data. These coefficients were compared in the same manner as the mean percent data. In order to obtain a measure of the time course of the effect of the US on the flexion response to the CS, i0 additional cats were prepared in the same manner as the cats used in the conditioning studies. Following the usual surgical and anesthetic recovery period animals were given 5 series of saphenous and superficial peroneal nerve stimulus presentations at the same intensities as those used in the conditioning studies. A series consisted of 15 single shocks to the saphenous nerve at 30 sec intervals. Five seconds before the sixth shock of each series the superficial peroneal nerve was stimulated for 0.5 sec at 40/sec. The interseries interval was 10 min. For each cat the peak tension developed by the tibialis anterior muscle in
SPINAL C O N D I T I O N I N G
299 response to each pulse to the saphenous nerve was measured and converted to a percentage of the m e a n response to the first 5 of these stimuli. A D u n n e t t ' s t test [5] was used to compare this control mean percentage with subseq u e n t interval m e a n percentages. Oscilloscope displays of c o m p o u n d action potentials from the saphenous and superficial peroneal nerves of all 40 cats were m o n i t o r e d visually to assure that the neural inputs to the spinal cord were constant in magnitude over the course of each experiment. In some cases these recordings were filmed using a Grass camera. A c t i o n potentials such as those pictured in Fig. 2B were considered acceptable. Such recordings indicated that the stimuli were near maximal for Ao~ and A6 fiber groups [ 11 ]. Nine cats were tested in addition to the 40 animals comprising this study, but their results could not be used. In 5 of these, the recordings of the cutaneous nerve c o m p o u n d action potentials declined m a r k e d l y in amplitude over the course of the experiment, possibly because of injury or lack of blood supply to the nerve. In four o t h e r cats there was no initial reflex response to saphenous nerve stimulation. This appeared to be a result of depressed spinal reflexes in these animals.
1 sec 1200
600 .{
no
Z
9
o~
z
uJ P
20[ J 1
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-4 usi,FIG. 1. Reproduction of polygraph record from a typical conditioning trial showing the tension of the tibialis anterior muscle. Upper record (low sensitivity) used for unconditioned response measurements. Lower record (high sensitivity) used for more accurate measurement of the conditioned response. Each tension peak during the first second of the CS presentation is the response to individual stimuli comprising the CS.
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alone were 284 + 104 S.E., 306 + 74 S.E., and 338 * 86 S.E. grams for conditioning, sensitization and habituation groups respectively, and these did not differ significantly from one another (Duncan’s New Multiple Range Test). Subsequent 5 trial mean responses to the CS are shown for each group as a percentage of the control level responses. In the conditioning group (paired CS-US presentations) the response to the CS increased rapidly in magnitude during the first 10 trials (e.g., see Fig. 2A), and this level was maintained throughout subsequent conditioning trials. Beginning with Trial Block 4 the variability in response of the conditioning group began to increase (Fig. 3). This was because the responses of 4 animals began to decline toward the control level, while that of the other 6 either remained near the plateau level or increased in magnitude. The sensitization group (unpaired CS and US presentations) ex-
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hibited no reflex facilitation in response to the CS, the reflex remaining near control level throughout the first 30 CS presentations. With regard to the responses of individual sensitization animals, 3 of these exhibited small flexion increases in response to the CS over the initial 30 trials. The responses of these 3 sensitization animals did not appear to differ from those of the two lowest responders in the conditioning group. Repeated presentations of the CS alone (habituation group) resulted in a rapid decline from control level during the first and second 5 trial blocks, the reflex declining more gradually during subsequent trials. The responses of every animal in this group declined from control levels during the initial thirty trials. Significant differences were found among the mean responses of the different experimental groups during conditioning, F(2,27) = 24.91, p0.6). Tests for correlations between mean CR (conditioned response) scores and UR magnitude and changes over conditioning and sensitization were not significant. There were no significant correlations between an animal's 30 trial mean CR and: ( i ) t h e magnitude of the UR, (ii) the ratio of the control magnitude to the UR magnitude or (iii) the change in UR magnitude between the first and sixth five trial blocks. Statistical analyses of the extinction data in Fig. 3
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MINUTES FIG. 5. Group data (n = 10) from post-tetanic potentiation experiments. Mean responses of tibialis anterior muscle to single shocks to the saphenous nerve (S.N.) before and at intervals following a 40/sec, 0.5 sec stimulus to superficial peroneal nerve (SPN stim.) plotted as percent of pre-SPN stimulus responses. Vertical lines represent standard deviations of the mean. i n d i c a t e d t h a t significant d i f f e r e n c e s existed a m o n g m e a n responses of the t h r e e groups, F ( 2 , 2 7 ) = 4.39, p < 0 . 0 2 5 . T h e m u l t i p l e range test s h o w e d t h a t t h e 30 trial m e a n r e s p o n s e s of t h e c o n d i t i o n i n g g r o u p were significantly greater t h a n t h o s e of t h e s e n s i t i z a t i o n and h a b i t u a t i o n groups. O t h e r c o m p a r i s o n s o f g r o u p m e a n d a t a and regression c o e f f i c i e n t s were n o t significant. Several animals were r u n o n m o r e t h a n o n e b e h a v i o r a l paradigm and t h e single trial data f r o m o n e of t h e s e are p r e s e n t e d in Fig. 4. This a n i m a l was initially tested as an h a b i t u a t i o n a n i m a l a n d t h e d a t a f r o m t h e first 65 CS alone (Fig. 4, o p e n circles) p r e s e n t a t i o n s were used along w i t h similar d a t a f r o m o t h e r cats t o f o r m t h e h a b i t u a t i o n g r o u p curve in Fig. 3. S u b s e q u e n t tests o n t h e s e n s i t i z a t i o n and c o n d i t i o n i n g paradigms were carried o u t in this a n i m a l t o c o m p a r e t h e results of d i f f e r e n t m o d e s o f s t i m u l u s present a t i o n o n flexor reflex e x c i t a b i l i t y in a single animal. T h e h a b i t u a t i o n d a t a in Fig. 4 b e a r a close r e s e m b l a n c e t o t h e h a b i t u a t i o n g r o u p d a t a p i c t u r e d in Fig. 3. T h e r e was a rapid decline in t h e r e s p o n s e to t h e CS b e t w e e n t h e s i x t h a n d t w e l f t h CS p r e s e n t a t i o n s and this data is similar t o t h e g r o u p d a t a in Fig. 3. S u b s e q u e n t decline in t h e r e s p o n s e was m o r e gradual.
F o l l o w i n g a 1 h r rest period t h e a n i m a l was tested o n t h e sensitization p a r a d i g m exactly as n o r m a l s e n s i t i z a t i o n animals had b e e n tested. However, t h e d a t a f r o m this and s u b s e q u e n t tests are n o t i n c l u d e d in Fig. 3. T h e r e s p o n s e to the initial CS o f this series was similar in m a g n i t u d e to t h a t observed in the first series o f CS p r e s e n t a t i o n s over 2 h r before. This was followed in t h e s u b s e q u e n t 4 CS presentat i o n s b y a decreased r e s p o n s e to levels near t h o s e r e a c h e d at the end of t h e h a b i t u a t i o n series. With t h e i n t r o d u c t i o n of a l t e r n a t e CS and US p r e s e n t a t i o n s (Fig. 4, b l a c k triangles) t h e r e was an increase in t h e r e s p o n s e to t h e CS to magnitudes n o t r e a c h e d since t h e initial 5 CS's were p r e s e n t e d . This was followed b y a decrease in t h e r e s p o n s e over t h e last h a l f o f the s e n s i t i z a t i o n series and t h e s u b s e q u e n t 30 CS alone p r e s e n t a t i o n s . N o t e t h e similarities o f these individual a n i m a l d a t a w i t h t h e curves o f t h e s e n s i t i z a t i o n g r o u p in Fig. 3. A n o t h e r 1 h r rest period was followed b y t h e same series of stimuli t h a t t h e a n i m a l s o f t h e c o n d i t i o n i n g g r o u p had received. T h e responses t o t h e initial CS alone trials again showed evidence of some s p o n t a n e o u s r e c o v e r y f r o m t h e previous CS alone r e s p o n s e levels o f the p r e c e d i n g series. Pairing of CS w i t h US (Fig. 4, solid circles) resulted in
SPINAL CONDITIONING evidence of flexor reflex facilitation beginning at trial four with a continuous increase through trial fourteen. The CS alone (extinction) trials which followed produced a rapid decrement in the response but the response did not decline to the levels reached in previous CS alone series. This last set of data corresponds closely to the conditioning group data of Fig. 3. The behavioral data show that a facilitation of the flexor reflex response to the CS takes place when the CS is paired closely in time with the US (conditioning). However, if the CS is presented alone (habituation) or alternated in a series with the US (sensitization) such reflex facilitation does not occur. One explanation of the difference between conditioning and sensitization animals in this experiment could be that the US produces a post-tetanic potentiation of the response to the CS which is minimal 30 seconds after delivery of the US, but which increases significantly after one minute. The post-tetanic potentiation experiments shown in Fig. 5 were designed to test this possibility. It was found that following presentation of the US (40/sec stimulation of superficial peroneal nerve for 0.5 sec) there was a potentiation of the response to single saphenous nerve stimuli. However, the time course of the post-tetanic potentiation decayed monotonically. Furthermore, the only point found to be significantly different from pre-US control levels was the response observed five seconds after the US presentations (t = 8.633, d f = 108, p
