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Long-lasting habituation in spinal frogs
BRAIN RESEARCH
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LONG-LASTING HABITUATION IN SPINAL FROGS
PAUL B. FAREL Department of Psychology, University of California, Los Angeles, Calif. 90024 (U.S.A.)
(Accepted April 25th, 1971)
INTRODUCTION Habituation, a centrally mediated response decrement to repeated stimulation 10, has many features in common with more complex sorts of behavioral plasticity - dependence on experience, specificity to particular stimuli, and, occasionally, strong persistance over time - - yet it can be demonstrated in preparations with highly restricted nervous systems. For these reasons, as emphasized by Thompson and Spencer z2, the study of habituation in spinal animals has considerable value as a model system in which to investigate the basic neuronal substrates of behavioral plasticity. In most previous studies of spinal habituation2,S,O, 1z,18-24, complete recovery occurred within an hour. However, several investigators have reported response depressions to repeated stimulation that persisted over days which, because their time course is more on the order of learning in the intact animal, would appear to greatly enhance the value of habituation as a model phenomenon. Most of these reports~,7,17 of long-lasting habituation did not include explicit controls for the possibility that the decline in responsiveness interpreted as centrally mediated habituation was due to decreased afferent input resulting from damage caused by repeated stimulus application. Kozak et al. ~1, however, demonstrated that the decline in responsiveness over days shown by 3 chronic spinal kittens was indeed centrally mediated because the response depression generalized to stimuli exciting a completely different set of primary afferent fibers than that excited by the habituating stimulus. This demonstration of generalization or transfer of habituation to independent input channels means that changes in effective stimulus strength cannot account for the response depression seen over days. The present study shows that long-lasting habituation can be reliably seen in chronic spinal frogs. Frogs were chosen as subjects for 3 major reasons: (1) a fairly advanced form of behavioral plasticity might be more easily demonstrated in a lower vertebrate whose spinal cord still mediated functions perhaps encephalized in higher animals - the literature on spinal frogs indicates that they indeed have a large behavioral repertoire ~; (2) spinal frogs are more easily cared for than spinal mammals in that problems Brain Research, 33 (1971) 405-417
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of feeding, excretion, and control of body temperature are greatly reduced; (3) the neuronal circuitry of the frog spinal cord is such that certain hypotheses, very difficult to test in mammals, may be subject to investigation in the frog3-5,14,16. METHODS
Subjects. Approximately 100 bullfrogs (Rana catesbiana) with body lengths of 65-100 mm were obtained from a local supplier, stored in a refrigerator overnight, and usually operated the next day. The frogs were not fed after spinalization although, in later subjects, 3 ml of 20% glucose-Ringer solution were injected every few days into the dorsal lymph sac. Spinal frogs were kept at 19°C. Operation. The animals were placed in a closed dish with ether-soaked cotton and left in a freezer for 15 min. Under a dissecting microscope, the vertebral column was exposed and the roof of the second and part of the first vertebra removed. All blood vessels on the dorsal surface of the cord, including the large dorsal vein, were cauterized before removing the inner meninges. The exposed cord was then cut through with fine scissors just rostral to the second pair of spinal roots. (The adult frog has 10 pairs of spinal roots; the second pair, easily identifiable by their large size, provides the main innervation to the forelimbs.) The severed ends of the cord separate slightly, allowing a clear view of the floor of the vertebral canal. Thus, it could be immediately determined whether the section was complete. Animals in which excessive bleeding prevented clear visualization were discarded. Finally, Oxycel (ParkeDavis) was placed over the severed cord, and the skin was sewn closed with nylon suture. This operation is a traumatic one; about 55 70 of the animals died within 24 h or, more often, developed areflexia and flaccid paralysis caudal to the section, despite the apparent normality of more rostral reflexes. During the course of the experiment, several animals developed signs correlated with poor condition (pupil constriction, reflex attenuation, and skin lightening) and were discarded. The data presented are from animals surviving 2-11 days (,X ---- 6.2 days) following completion of the experiment. Apparatus. Stimulation and recording were through bipolar stainless steel 00 insect pins insulated to within 1 mm of their tips. Recorded muscle potentials were run in parallel through a Tektronix 122 preamplifier to a Tektronix 502 oscilloscope and through a Grass 5P3B preamplifier, which full-wave rectified and integrated the EMG, to a Grass 5C polygraph. One msec monophasic pulses of from 1 to I0 mA were delivered by ELS constant current stimulators through bipolar electrodes 1 mm apart. Procedure. The spinal frogs were allowed to recover for 2-7 days following the operation and then placed in a pan filled with hard wax into which pins were inserted to brace and to immobilize the animals with their hindlimbs in a semi-extended position (Fig. 1). Animals were kept in these pans, partially immersed in water, at all times. Five pairs of electrodes were inserted into each animal, 2 pairs of recording electrodes and 3 pairs of stimulating electrodes. One pair of recording electrodes was
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placed deep in the back o f each thigh. Of the 3 pairs o f stimulating electrodes, 2 were placed in the foot to be habituated, and the third pair was inserted into the contralateral foot. The distance between the electrodes labeled 'habituation' and 'transfer' was 25-30 mm. The E M G was recorded from the set o f electrodes ipsilateral to the stimulating electrodes being used. Subjects were left in this situation for the duration of the experiment. Animals were considered to be substantially habituated when a criterion of 10 consecutive responses in which the peak o f the integrated E M G was less than 100 #V had been met. On the first day of habituation training, animals were given about 200 more trials than they required to reach this criterion. Any given animal received the same number of trials each day, but over all the animals the number of trials given per day ranged from 400 to 700. In all cases, trials occurred 30 see apart (1/30 see). The current level used with a particular animal was chosen on the basis of trial stimulations given several hours before habituation training began. A stimulus level was chosen which elicited a peak integrated E M G of 450 -4- 25 #V, and this level was kept constant for a given animal for the duration o f the experiment. In addition, before and after habituation training, each animal was subjected to certain control procedures (described in Results) which involved stimulation through the transfer and control electrodes. Although these control procedures included habituation to the same criterion as in habituation training, two modifications were added to minimize the number of stimulations an animal received in order to lessen the chance of the control stimulations themselves having an effect on responsiveness: (1) stimulation was halted as soon as an animal reached criterion, and (2) the stimulus level chosen
HABITUATION
CONTROL
Fig. 1. Placement of electrodes in habituation experiments. The habituation and transfer electrodes are 25-30 mm apart. Control electrodes were either homologous to the transfer electrodes or midway between the homologous points for habituation and transfer. Brain Research, 33 (1971) 405-417
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200./JV I 20 msec Fig. 2. Changes in the EMG recorded from electrodes deep in the back of an animal's thigh during an habituation training session. A, Trial 10. B, Trial 496, Traces have been retouched to restore losses during reproduction.
was one which elicited a peak integrated E M G of 300 ffV in trial stimulations given several hours before the control procedures themselves were begun. Once a current level was chosen for a given site, that level was maintained for the duration of the experiment. RESULTS
Description of the response Hindleg position of a spinal frog is very similar to that of the intact animal - the upper part of the leg being drawn up next to the body. When free spinal animals relaxed their legs to the semi-extended position in which the experimental animals were restrained and were shocked through electrodes on the foot, they responded with a rapid leg flexion. The response of experimental animals when restraint was removed for a trial was also flexion. Fig. 2 shows the E M G recorded from electrodes deep in the back of a restrained animal's thigh in response to foot shock, presumably activating cutaneous and, perhaps, muscle afferents. The 14 msec latency demonstrates clearly its reflex nature.
Decline in responsiveness over days Subsequent to certain control procedures described in the next section, each of 18 spinal frogs received 400-700 constant current stimulations per day for 3-5 days (habituation training). The stimuli were delivered 1/30 sec through the habituation electrodes (Fig. 1). Current level and the number of trials a given animal received were both constant over days. tn Fig. 3, it can be seen that the peak amplitude of the integrated E M G decreased within each day. Between the end of one day's training and the beginning of training on the following day, the integrated E M G recovered completely; however, the fact that over successive days the response amplitudes declined progressively more rapidly and reached progressively lower levels indicates that a record of training is retained from one day to the next. This finding is brought out Brain Research, 33 (1971) 405-417
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Fig. 3. Habituation of the peak integrated E M G over days. An animal's score for a given block of 50 trials was determined by averaging the peak integrated E M G s on the first 10 trials of that block. All scores are presented as percents of the score on Day 1, Block 1. Note the recovery of the peak integrated E M G amplitude on the initial block of each day, but the more rapid decline to a lower level on successive days.
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Fig. 4. Decline in the mean number of trials to reach criterion over days of habituation training. Scores are expressed as percents of the number of trials required to reach criterion on Day 1. The standard error of the mean and the standard deviation are indicated above and below each point.
more clearly in Fig. 4, which shows that the number of trials required to reach a criterion of 10 consecutive responses in which the peak integrated EMG was less than 100 #V declines progressively over days. Every animal reached criterion faster on the last day of habituation training than on the first day (P < 0.01, two-tailed Wilcoxon signed-ranks matched-pairs test), and the mean scores fell by about two standard errors from day to day. Therefore, the fall is statistically very reliable. Brain Research, 33 (1971) 405-417
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Controls for stimulus change and deterioration of the preparation Demonstrating transfer of habituation to stimuli exciting different afferents than those responding to the habituation stimulus provides assurance that a response decrement was not solely the result of decreased stimulus effectiveness due to, for example, skin damage. The day before habituation training began, stimuli presented 1/30 sec were applied through the transfer electrodes (Fig. 1). The number of trials an animal required to reach criterion (10 consecutive responses less than 100 #V) was its pretraining transfer score. The next day, the 3-5 days of habituation training described above began. After completion of this training, stimuli were again applied to the transfer site to criterion (posttraining transfer score). Comparison of the preand posttraining transfer scores was used as a measure of transfer of habituation. If the declines in responsiveness seen at the transfer and habituation sites were due to worsening condition of the preparation, then other responses besides those affected by habituation training would be depressed as well. A test for general deterioration was provided by comparing responsiveness of the leg contralateral to the one receiving habituation training before and after that training. Several hours after the pretraining transfer test, stimuli were delivered through the control electrodes (Fig. 1) 70060O bJ N
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Fig. 5. Results of transfer and deterioration controls. For each animal, trials to criterion on each test were expressed as perce~t__a£~_ of the trials to criterion o n the relevant p r e t r a i n ~ : test. Histograms show the medians and semi-interquartite ranses of these scores. Note that all pretraining scores a r e 100 ~o by definition. Posttralning tests were halted if an animal's score had ~ gO0~ and criterion still had not been met.
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until the animal reached criterion (pretraining control score). Likewise, several hours after the posttraining transfer test, stimuli were again delivered through the control electrodes (posttraining control score) to criterion. The distributions of pretraining transfer and control scores were similar. The median pretraining transfer score was 41.5 while the median pretraining control score was 42.0. The variability about these medians was fairly large; however, this variability could in no way account for the results of this experiment, which were very uniform across animals despite the range of initial scores. Fig. 5 summarizes results of these control procedures. The most conspicuous feature of these results is the rise in excitability of the control leg (P < 0.01, twotailed Wilcoxon signed-ranks matched-pairs test comparing the pre- and posttraining control scores). Fourteen of 18 animals showed increased responsiveness of the control leg. An effect of transfer of habituation, then, would have to occur against a background of generally enhanced excitability. If transfer were occurring, it could be seen, not as an absolute decline of responsiveness at the transfer site, but, rather, as a lesser rise than seen at the control site. In fact, only 11 of 18 animals did show an absolute decline at the transfer site after habituation training (not significant). However, in contrast to responsiveness at the control site, responsiveness at the transfer site did fail to rise. In 17 of 18 animals, the normalized posttraining transfer score was less than the normalized posttraining control score (P < 0.01, two-tailed Wilcoxon signed-ranks matched-pairs test). Subject to later discussion, this relative depression can be interpreted as evidence of transfer of habituation, and the conclusion drawn that the response decline of the habituated limb does depend on central changes.
Further controls Stimulus spread. The interpretation of the transfer data is based on the assumption that the effective range of the habituation stimulus does not extend to the region excited by the transfer stimulus. In the above experiment, stimulation was through bipolar electrodes 1 mm apart, and the habituation and transfer electrode pairs were 25-30 mm apart. To determine the degree of effective stimulus spread, crushed-end recordings with Ag-AgCI electrodes were made from the cut distal portions of all the large nerves in the frog's leg. Habituation and transfer stimulating electrodes were placed as in previous experiments. The highest intensity stimulus used in habituation training was also used in these experiments. Fig. 6 shows the results of one such experiment. Successive incisions, extending deep into the muscle, were made in the frog's foot, starting near the habituation electrodes and working toward the transfer electrodes (see inset, Fig. 6). By this means, impulses initiated distal to an incision were prevented from traveling up the nerve to the point where recordings were being made, yet impulses initiated by current spread beyond the region of the incision would be relatively unaffected. The results for all 9 experiments performed indicate that spread of the stimulating current is not capable of exciting fibers 10 mm rostral to the stimulation site. Brain Research, 33 (1971) 405--417
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Fig.6. Recordings from distal stump ofcut sciatic nerve. Inset shows relative placements of incisions and electrodes. A, Response to stimulation at transfer site. B, Response to stimulation at habituation site. C, As in B, but after incision 2 mm rostral. D, As in C, but after incision t0 mm rostral. E, Response to stimulation at transfer site at end of experiment. The discrepancy between the compound action potentials elicited by stimulation at the transfer and habituation sites was seen in all experiments, presumably because the transfer site is much less richly innervated.
Afferent volley. In an attempt to determine directly if profound peripheral changes occurred over trials, 3 spinal frogs were prepared to allow monitoring of the afferent volley from the cut distal portion of the sciatic nerve. The foot was stimulated using the strongest stimulus used in habituation training, which, however, evoked a submaximal response. Seven hundred stimuli were presented at 1/30 sec. The recorded compound action potential tended to be somewhat variable, but the distribution of responses was the same in early and late trials. Fig. 7 shows responses recorded at the beginning and end of one experiment. The stimulus artifact has changed over the almost 6-h experiment; however, the two compound action potentials are almost perfectly superimposable. Movement of transfer electrodes. Stimuli delivered through the habituation electrodes often caused the foot to twitch, which caused a slight movement of the transfer electrodes but not of the control electrodes on the other foot. It is possible that stimulation caused by these movements resulted in the relative decline of responsiveness seen at the transfer site that was interpreted as centrally mediated transfer of habituation. To test this possibility, 4 spinal frogs were prepared as usual, but with only transfer and control stimulating electrodes. Animals were given pretraining training transfer and control tests as described above, and, on the following day, the transfer Brain Research, 33 (1971) 405-417
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I Fig. 7. A, Compound action potential evoked by 10 mA foot stimulation recorded from cut distal stump of sciatic nerve. B, As in A, but approximately 700 stimulations later. Note that, although the stimulus artifact has changed over the almost 6 h duration of the experiment, the traces of evoked activity are almost perfectly superimposable. electrodes were attached to the armature o f a large relay. When the armature moved, the protruding ends of the elctrodes were moved through an arc o f 5-8 mm. The maxim u m movement of the transfer electrodes seen during habituation was 4 ram. The frogs were given 4 days o f such trials, the number and spacing o f which mimicked the trials of animals given the most habituation training. Posttraining tests were given on the same day as the last electrode movement trials and were halted if 10 times the number of pretraining trials had been given and the criterion of habituation still had not been reached. I f movement of the transfer electrodes accounted for the relative depression seen at the transfer site, then this experiment should produce the same pattern of results. In fact, both transfer and control sites showed an increase in excitability with no consistent differences between them. The posttraining transfer scores, as percents of the pretraining transfer scores, were 346, 537, > 1000, and > 1000%. The 4 posttraining control scores, as percents of the pretraining control scores, were respectively 343,483, > 1000, and > 1000%. Effectorfatigue. To the extent that the same set of neuromuscular connections is activated reflexively by stimulation at the transfer and habituation sites, it is possible that the decreased responsiveness interpreted as centrally mediated could be due to changes of the neuromuscular junctions or of the muscles themselves. Direct stimulation o f m o t o r fibers was used to indicate whether neuromuscular changes could account for the transfer results. Six spinal animals were prepared as usual, except no stimulating electrodes and only one set of recording electrodes were used. Twenty-four hours after being placed in the wax-filled pans, the eighth spinal nerve, which innervates many of the muscles in the thigh region, was dissected free, cut close to its exit from the cord, and the cut distal portion placed on Ag-AgC1 stimulating electrodes. Submaximal stimuli were used to avoid masking a possible depression with a supramaximal response. The nerve was stimulated 1/30 sec and the integrated E M G recorded as in the habituation experiments.
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Fig. 8. Effects of stimulating motor nerve versus habituation training on the peak i n t u i t e d EMG. An animal's score for each block of trials was determined as in Fig. 4. Standard errors of the mean are indicated above and below each point.
Fig. 8 compares the results of these experiments to the results on the first day of habituation training. While animals in the habituation training condition show a large decline, particularly in the early trials, the responses to motor nerve stimulation tend to remain fairly constant. DISCUSSION
The purpose of this study was to show that the brain-isolated frog spinal cord can mediate habituation cumulative over days. Controls were included to insure that neither peripheral changes nor deterioration of the preparation contributed to the decline interpreted as centrally mediated habituation. The transfer of habituation to a skin area 25-30 mm from the site where habituation stimuli were applied makes the possibility that damage to receptors or primary afferent fibers accounted for the decline extremely remote. Lindblom 13 has reported that, in toads, single fibers innervate a leg region having a longitudinal extent o f about 5 mm. Because the habituation and the (generally weaker) transfer stimulus spread less than 10 ram, they do not stimulate common areas of skin, nor, presumably, common receptors or common afferent fibers. Stimulation at the habituation site could affect the peripheral excitability of entities stimulated by the transfer electrodes only if the receptive fields of cutaneous fibers overlapped the two stimulus zones and if stimulation in one part of the receptive field somehow reduced excitability of other parts (e.g., by damaging the afferent fiber generally). The extent of current spread was determined using a 10 mA stimulus, the most intense stimulus used in the habituation experiments. In fact, with only two animals were transfer and habituation stimuli both 10 mA. The mean transfer stimulus was Brain Research, 33 (1971) 405-417
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4.2 mA and the mean habituation stimulus was 5.3 mA - - both about half the current level used to make estimates o f stimulus spread. To be stimulated by both the mean transfer and the mean habituation stimuli, a primary afferent fiber would have to have a receptive field of about 15 mm. To the extent that receptive fields in the bullfrog are not more than 3 times larger than those in the toad, it seems unlikely that the same primary afferent fbers were excited by both transfer and habituation stimuli. Also, if the depression seen at the transfer site (relative to the control site) were due to damage caused by the habituation stimulus, then those animals in which the habituation stimulus current was high, presumably activating more afferents also fired by the transfer stimulus, should show a greater relative depression than those in which it was low. However, the ratio o f the posttraining transfer score to the posttraining control score was not different for the 6 animals in which the habituation stimulus was 10 mA and those 5 in which it was 2 mA or less. In addition, all 6 animals in the high current group had transfer stimuli of at least 5 mA while the transfer stimuli o f the low current group were all 4 mA or less, and the same number of trials was given to the members of each group. Finally, the failure to find profound changes in the compound action potential over the course of 700 stimulations further supports the conclusion that the reported response depressions are not the result of changes in afferent inflow. Changes in the effector system were shown not to operate within the course of a daily session. To the extent that response decrements over days reflect the same processes as do decrements within days, it can be assumed that changes efferent to the spinal cord do not play a role in habituation over days. Deterioration of an animal's condition would be expected to change his responsiveness nonspecifically. Because the leg not given habituation training showed an increase in responsiveness in the period between pre- and posttraining tests, the possibility that responsiveness at the habituation site declined because the animal's condition worsened can be rejected. Conversely, one could argue that, while habituation is not the result of deterioration, the increased responsiveness o f the control leg might be due to a decline in the animal's general condition. For example, McAfee 15 has shown that the excised frog spinal cord maintained in an artificial chamber goes through a brief phase of heightened polysynaptic responsiveness when subjocted to hypoxia. To test whether deterioration could account for the increased general responsiveness, the posttraining control scores o f 3 animals dying within 3 days of the end of the experiment were compared to the scores of 3 animals surviving at least 10 days. The normalized control scores of the short-lived animals were each less than any of the scores of the long-lived animals. The mean posttraining control score for the short-lived animals was 272 % of their mean pretraining score as compared to a mean of 601% for the longer-lived animals. This finding that the excitability o f the control leg showed a 440 % median increase between pre- and posttraining tests was unexpected. The question whether this increase is dependent upon habituation training or whether it represents a general change in the animals' excitability as a consequence o f spinal transection is the subject Brain Research, 33 (1971) 405-417
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of a later investigation. Support for the latter hypothesis is found in the results of the experiment run to control for electrode movement in which all 4 animals showed increased excitability of both legs, although essentially no habituation intervened between pre- and posttraining tests. The experiments here provide little information on what the neuronal mechanisms of long-lasting habituation might be. It should be pointed out, however, that, to the extent that different primary afferent fibers are excited by the transfer and habituation stimuli, the demonstration of transfer of habituation means that a change which affects only the activated primary afferent pathway, such as transmitter depletion, cannot account for habituation over days. Because so little information is available, none of the classic neurophysiological phenomena can be rejected as possible mediators of long-lasting habituation in this preparation, provided one postulates some way that the effect could persist over days. The time course of habituation described in these experiments is so vastly longer than that of phenomena studied in most electrophysiological experiments that any attempt to explain the one by means of the other must be primarily speculative. SUMMARY
It was shown that the brain-isolated frog (Rana catesbiana) spinal cord can mediate response depressions cumulative over 24-h periods. The peak integrated EMG recorded from the back of the thigh, evoked by a constant current foot shock (1-10 mA, 1 msec, presented 1/30 sec), declined more rapidly on successive days of habituation training and reached a lower asymptote. The following controls were performed to insure that this decline was not the result of peripheral receptor or effector changes or of deterioration of the preparation: (1) habituation transferred to a skin site not excited by the habituation stimulus; (2) the volley recorded directly from afferent fibers remained relatively constant; (3) no response decrement was found when the motor fibers were stimulated directly; and (4) the leg not subjected to habituation training showed increased responsiveness over time. ACKNOWLEDGEMENTS
Supported by NINDS Grant No. 1 R01 NB08108-01NP awarded to Dr. Franklin Krasne. The author was a U.S. Public Health Service predoctoral trainee. This research forms part of a dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Psychology. Considerable help in all phases of this study was provided by my dissertation adviser, Franklin Krasne. This help is gratefully acknowledged. The author is currently an N I M H postdoctoral fellow at the Department of Psychobiology, University of California, Irvine, Calif. 92664, U.S.A.
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REFERENCES 1 AFELT,Z., Variability of reflexes in chronic spinal frogs. In E. GUTMANNAND P. HNIK (Eds.), Central and Peripheral Mechanisms of Motor Functions, Czechoslovak Academy of Science, Prague, 1963, pp. 37-41. 2 BUCHWALD,J. S., HALAS,E. S., ANDSCHRAMM,S., Progressive changes in efferent unit responses to repeated cutaneous stimulation in spinal cats, J. Neurophysiol., 28 (1965) 200-215. 3 BROOKHART,J. M., AND FADIGA,E., Potential fields initiated during monosynaptic activation of frog motoneurons, J. Physiol. (Lond.), 150 (1960) 633-655. 4 BROOKHART,J. M., MACHNE,X., ANDFADIGA,E., Patterns of motor neuron discharge in the frog, Arch. ital. Biol., 97 (1959) 53-67. 5 FADIGA,E., AND BROOKHART,J. M., Monosynaptic activation of different portions of the motor neuron membrane, Amer. J. Physiol., 198 (1960) 693-703. 6 FRANZISKET,L., Characteristics of instinctive behavior and learning in reflex activity of the frog, Anita. Behav., 11 (1963) 318-324. 7 GRIFFIN,J. P., ANDPEARSON,J. A., Habituation of the flexor reflex in spinal rats, and in rats with frontal cortex lesions followed by spinal transection, Brain Research, 6 (1967) 777-780. 8 GROVES,P. M., DEMARcO, R., AND THOMPSON,R. F., Habituation and sensitization of spinal interneuron activity in acute spinal cat, Brain Research, 14 (1969) 521-525. 9 GROVES,P. M., LEE, D., AND THOMPSON,R. F., Effects of stimulus frequency and intensity on habituation and sensitization in acute spinal cat, Physiol. Behav., 4 (1969) 383-388. 10 HARRIS,J. O., Habituatory response decrement in the intact organism, Psychol. Bull., 40 (1943) 385-422. 11 KOZAK,W., MACFARLANE,W. V., AND WESTERMAN,R., Long-lasting reversible changes in the reflex responses of chronic spinal cats to touch, heat, and cold, Nature (Lond.), 193 (1962) 171173. 12 LEHNER, G. F., A study of the extinction of unconditioned reflexes, J. exp. Psychol., 29 (1941) 435-456. 13 LINDBLOM,U. F., Excitability and functional organization within a peripheral tactile unit, Acta physiol, scand., 44, Suppl. 153 (1958). 14 Liu, C. N., AND CHAMBER~,W. W., Experimental study of anatomical organization of frog's spinal cord, Anat. Rec., 127 (1957) 326. 15 McAFEE,D. A., A study of mechanisms by which hypoxia influences nervous activity, unpublished doctoral dissertation, Univ. of Oregon, 1969. 16 MACHNE,X., FADIGA,E., AND BROOKHART,J. M., Antidromic and synaptic activation of frog motor neurons, J. NeurophysioL, 22 (1959) 583-603. 17 NESMEIANOVA,T. N., The inhibitionoftbe motor reflex in spinal dogs under conditions of chronic experimentation, Sechenov physiol. J. U.S.S.R., 42 (1957) 281-288. 18 PROSSER,C. L., AND HUNTER, W. S., The extinction of startle responses and spinal reflexes in the white rat, Amer. J. Physiol., 117 (1936) 609-618. 19 SPENCER,W. A., THOMPSON,R. F., ANDNEILSON,D. R., Response decrement of the flexion reflex in the acute spinal cat and transient restoration by strong stimuli, J. Neurophysiol., 29 (1966) 221-329. 20 SPENCER,W. A., THOMPSON,R. F., ANDNEILSON,D. R., Alterations in responsiveness of ascending and reflex pathways activated by iterated cutaneous afferent volleys, J. NeurophysioL, 29 (1966) 240-252. 21 SPENCER,W. A., THOMPSON,R. F., AND NEILSON,D. R., Decrement of ventral root electrotonus and intracellularly recorded P.S.P.'s produced by iterated cutaneous afferent volleys, J. Neurophysiol., 29 (1966) 253-274. 22 THOMPSON,R. F., AND SPENCER,W. A., Habituation: A model phenomenon for the study of neuronal substrates of behavior, Psychol. Rev., 173 (1966) 16-43. 23 WICKELGREN,B. G., Habituation of spinal motoneurons, J. NeurophysioL, 30 (1967) 1404-1423. 24 WICKELGREN,B. G., Habituation of spinal interneurons, J. Neurophysiol., 30 (1967) 1424-1438.
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LONG-LASTING HABITUATION IN SPINAL FROGS
PAUL B. FAREL Department of Psychology, University of California, Los Angeles, Calif. 90024 (U.S.A.)
(Accepted April 25th, 1971)
INTRODUCTION Habituation, a centrally mediated response decrement to repeated stimulation 10, has many features in common with more complex sorts of behavioral plasticity - dependence on experience, specificity to particular stimuli, and, occasionally, strong persistance over time - - yet it can be demonstrated in preparations with highly restricted nervous systems. For these reasons, as emphasized by Thompson and Spencer z2, the study of habituation in spinal animals has considerable value as a model system in which to investigate the basic neuronal substrates of behavioral plasticity. In most previous studies of spinal habituation2,S,O, 1z,18-24, complete recovery occurred within an hour. However, several investigators have reported response depressions to repeated stimulation that persisted over days which, because their time course is more on the order of learning in the intact animal, would appear to greatly enhance the value of habituation as a model phenomenon. Most of these reports~,7,17 of long-lasting habituation did not include explicit controls for the possibility that the decline in responsiveness interpreted as centrally mediated habituation was due to decreased afferent input resulting from damage caused by repeated stimulus application. Kozak et al. ~1, however, demonstrated that the decline in responsiveness over days shown by 3 chronic spinal kittens was indeed centrally mediated because the response depression generalized to stimuli exciting a completely different set of primary afferent fibers than that excited by the habituating stimulus. This demonstration of generalization or transfer of habituation to independent input channels means that changes in effective stimulus strength cannot account for the response depression seen over days. The present study shows that long-lasting habituation can be reliably seen in chronic spinal frogs. Frogs were chosen as subjects for 3 major reasons: (1) a fairly advanced form of behavioral plasticity might be more easily demonstrated in a lower vertebrate whose spinal cord still mediated functions perhaps encephalized in higher animals - the literature on spinal frogs indicates that they indeed have a large behavioral repertoire ~; (2) spinal frogs are more easily cared for than spinal mammals in that problems Brain Research, 33 (1971) 405-417
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of feeding, excretion, and control of body temperature are greatly reduced; (3) the neuronal circuitry of the frog spinal cord is such that certain hypotheses, very difficult to test in mammals, may be subject to investigation in the frog3-5,14,16. METHODS
Subjects. Approximately 100 bullfrogs (Rana catesbiana) with body lengths of 65-100 mm were obtained from a local supplier, stored in a refrigerator overnight, and usually operated the next day. The frogs were not fed after spinalization although, in later subjects, 3 ml of 20% glucose-Ringer solution were injected every few days into the dorsal lymph sac. Spinal frogs were kept at 19°C. Operation. The animals were placed in a closed dish with ether-soaked cotton and left in a freezer for 15 min. Under a dissecting microscope, the vertebral column was exposed and the roof of the second and part of the first vertebra removed. All blood vessels on the dorsal surface of the cord, including the large dorsal vein, were cauterized before removing the inner meninges. The exposed cord was then cut through with fine scissors just rostral to the second pair of spinal roots. (The adult frog has 10 pairs of spinal roots; the second pair, easily identifiable by their large size, provides the main innervation to the forelimbs.) The severed ends of the cord separate slightly, allowing a clear view of the floor of the vertebral canal. Thus, it could be immediately determined whether the section was complete. Animals in which excessive bleeding prevented clear visualization were discarded. Finally, Oxycel (ParkeDavis) was placed over the severed cord, and the skin was sewn closed with nylon suture. This operation is a traumatic one; about 55 70 of the animals died within 24 h or, more often, developed areflexia and flaccid paralysis caudal to the section, despite the apparent normality of more rostral reflexes. During the course of the experiment, several animals developed signs correlated with poor condition (pupil constriction, reflex attenuation, and skin lightening) and were discarded. The data presented are from animals surviving 2-11 days (,X ---- 6.2 days) following completion of the experiment. Apparatus. Stimulation and recording were through bipolar stainless steel 00 insect pins insulated to within 1 mm of their tips. Recorded muscle potentials were run in parallel through a Tektronix 122 preamplifier to a Tektronix 502 oscilloscope and through a Grass 5P3B preamplifier, which full-wave rectified and integrated the EMG, to a Grass 5C polygraph. One msec monophasic pulses of from 1 to I0 mA were delivered by ELS constant current stimulators through bipolar electrodes 1 mm apart. Procedure. The spinal frogs were allowed to recover for 2-7 days following the operation and then placed in a pan filled with hard wax into which pins were inserted to brace and to immobilize the animals with their hindlimbs in a semi-extended position (Fig. 1). Animals were kept in these pans, partially immersed in water, at all times. Five pairs of electrodes were inserted into each animal, 2 pairs of recording electrodes and 3 pairs of stimulating electrodes. One pair of recording electrodes was
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placed deep in the back o f each thigh. Of the 3 pairs o f stimulating electrodes, 2 were placed in the foot to be habituated, and the third pair was inserted into the contralateral foot. The distance between the electrodes labeled 'habituation' and 'transfer' was 25-30 mm. The E M G was recorded from the set o f electrodes ipsilateral to the stimulating electrodes being used. Subjects were left in this situation for the duration of the experiment. Animals were considered to be substantially habituated when a criterion of 10 consecutive responses in which the peak o f the integrated E M G was less than 100 #V had been met. On the first day of habituation training, animals were given about 200 more trials than they required to reach this criterion. Any given animal received the same number of trials each day, but over all the animals the number of trials given per day ranged from 400 to 700. In all cases, trials occurred 30 see apart (1/30 see). The current level used with a particular animal was chosen on the basis of trial stimulations given several hours before habituation training began. A stimulus level was chosen which elicited a peak integrated E M G of 450 -4- 25 #V, and this level was kept constant for a given animal for the duration o f the experiment. In addition, before and after habituation training, each animal was subjected to certain control procedures (described in Results) which involved stimulation through the transfer and control electrodes. Although these control procedures included habituation to the same criterion as in habituation training, two modifications were added to minimize the number of stimulations an animal received in order to lessen the chance of the control stimulations themselves having an effect on responsiveness: (1) stimulation was halted as soon as an animal reached criterion, and (2) the stimulus level chosen
HABITUATION
CONTROL
Fig. 1. Placement of electrodes in habituation experiments. The habituation and transfer electrodes are 25-30 mm apart. Control electrodes were either homologous to the transfer electrodes or midway between the homologous points for habituation and transfer. Brain Research, 33 (1971) 405-417
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200./JV I 20 msec Fig. 2. Changes in the EMG recorded from electrodes deep in the back of an animal's thigh during an habituation training session. A, Trial 10. B, Trial 496, Traces have been retouched to restore losses during reproduction.
was one which elicited a peak integrated E M G of 300 ffV in trial stimulations given several hours before the control procedures themselves were begun. Once a current level was chosen for a given site, that level was maintained for the duration of the experiment. RESULTS
Description of the response Hindleg position of a spinal frog is very similar to that of the intact animal - the upper part of the leg being drawn up next to the body. When free spinal animals relaxed their legs to the semi-extended position in which the experimental animals were restrained and were shocked through electrodes on the foot, they responded with a rapid leg flexion. The response of experimental animals when restraint was removed for a trial was also flexion. Fig. 2 shows the E M G recorded from electrodes deep in the back of a restrained animal's thigh in response to foot shock, presumably activating cutaneous and, perhaps, muscle afferents. The 14 msec latency demonstrates clearly its reflex nature.
Decline in responsiveness over days Subsequent to certain control procedures described in the next section, each of 18 spinal frogs received 400-700 constant current stimulations per day for 3-5 days (habituation training). The stimuli were delivered 1/30 sec through the habituation electrodes (Fig. 1). Current level and the number of trials a given animal received were both constant over days. tn Fig. 3, it can be seen that the peak amplitude of the integrated E M G decreased within each day. Between the end of one day's training and the beginning of training on the following day, the integrated E M G recovered completely; however, the fact that over successive days the response amplitudes declined progressively more rapidly and reached progressively lower levels indicates that a record of training is retained from one day to the next. This finding is brought out Brain Research, 33 (1971) 405-417
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Fig. 3. Habituation of the peak integrated E M G over days. An animal's score for a given block of 50 trials was determined by averaging the peak integrated E M G s on the first 10 trials of that block. All scores are presented as percents of the score on Day 1, Block 1. Note the recovery of the peak integrated E M G amplitude on the initial block of each day, but the more rapid decline to a lower level on successive days.
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more clearly in Fig. 4, which shows that the number of trials required to reach a criterion of 10 consecutive responses in which the peak integrated EMG was less than 100 #V declines progressively over days. Every animal reached criterion faster on the last day of habituation training than on the first day (P < 0.01, two-tailed Wilcoxon signed-ranks matched-pairs test), and the mean scores fell by about two standard errors from day to day. Therefore, the fall is statistically very reliable. Brain Research, 33 (1971) 405-417
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Controls for stimulus change and deterioration of the preparation Demonstrating transfer of habituation to stimuli exciting different afferents than those responding to the habituation stimulus provides assurance that a response decrement was not solely the result of decreased stimulus effectiveness due to, for example, skin damage. The day before habituation training began, stimuli presented 1/30 sec were applied through the transfer electrodes (Fig. 1). The number of trials an animal required to reach criterion (10 consecutive responses less than 100 #V) was its pretraining transfer score. The next day, the 3-5 days of habituation training described above began. After completion of this training, stimuli were again applied to the transfer site to criterion (posttraining transfer score). Comparison of the preand posttraining transfer scores was used as a measure of transfer of habituation. If the declines in responsiveness seen at the transfer and habituation sites were due to worsening condition of the preparation, then other responses besides those affected by habituation training would be depressed as well. A test for general deterioration was provided by comparing responsiveness of the leg contralateral to the one receiving habituation training before and after that training. Several hours after the pretraining transfer test, stimuli were delivered through the control electrodes (Fig. 1) 70060O bJ N
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Fig. 5. Results of transfer and deterioration controls. For each animal, trials to criterion on each test were expressed as perce~t__a£~_ of the trials to criterion o n the relevant p r e t r a i n ~ : test. Histograms show the medians and semi-interquartite ranses of these scores. Note that all pretraining scores a r e 100 ~o by definition. Posttralning tests were halted if an animal's score had ~ gO0~ and criterion still had not been met.
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until the animal reached criterion (pretraining control score). Likewise, several hours after the posttraining transfer test, stimuli were again delivered through the control electrodes (posttraining control score) to criterion. The distributions of pretraining transfer and control scores were similar. The median pretraining transfer score was 41.5 while the median pretraining control score was 42.0. The variability about these medians was fairly large; however, this variability could in no way account for the results of this experiment, which were very uniform across animals despite the range of initial scores. Fig. 5 summarizes results of these control procedures. The most conspicuous feature of these results is the rise in excitability of the control leg (P < 0.01, twotailed Wilcoxon signed-ranks matched-pairs test comparing the pre- and posttraining control scores). Fourteen of 18 animals showed increased responsiveness of the control leg. An effect of transfer of habituation, then, would have to occur against a background of generally enhanced excitability. If transfer were occurring, it could be seen, not as an absolute decline of responsiveness at the transfer site, but, rather, as a lesser rise than seen at the control site. In fact, only 11 of 18 animals did show an absolute decline at the transfer site after habituation training (not significant). However, in contrast to responsiveness at the control site, responsiveness at the transfer site did fail to rise. In 17 of 18 animals, the normalized posttraining transfer score was less than the normalized posttraining control score (P < 0.01, two-tailed Wilcoxon signed-ranks matched-pairs test). Subject to later discussion, this relative depression can be interpreted as evidence of transfer of habituation, and the conclusion drawn that the response decline of the habituated limb does depend on central changes.
Further controls Stimulus spread. The interpretation of the transfer data is based on the assumption that the effective range of the habituation stimulus does not extend to the region excited by the transfer stimulus. In the above experiment, stimulation was through bipolar electrodes 1 mm apart, and the habituation and transfer electrode pairs were 25-30 mm apart. To determine the degree of effective stimulus spread, crushed-end recordings with Ag-AgCI electrodes were made from the cut distal portions of all the large nerves in the frog's leg. Habituation and transfer stimulating electrodes were placed as in previous experiments. The highest intensity stimulus used in habituation training was also used in these experiments. Fig. 6 shows the results of one such experiment. Successive incisions, extending deep into the muscle, were made in the frog's foot, starting near the habituation electrodes and working toward the transfer electrodes (see inset, Fig. 6). By this means, impulses initiated distal to an incision were prevented from traveling up the nerve to the point where recordings were being made, yet impulses initiated by current spread beyond the region of the incision would be relatively unaffected. The results for all 9 experiments performed indicate that spread of the stimulating current is not capable of exciting fibers 10 mm rostral to the stimulation site. Brain Research, 33 (1971) 405--417
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Fig.6. Recordings from distal stump ofcut sciatic nerve. Inset shows relative placements of incisions and electrodes. A, Response to stimulation at transfer site. B, Response to stimulation at habituation site. C, As in B, but after incision 2 mm rostral. D, As in C, but after incision t0 mm rostral. E, Response to stimulation at transfer site at end of experiment. The discrepancy between the compound action potentials elicited by stimulation at the transfer and habituation sites was seen in all experiments, presumably because the transfer site is much less richly innervated.
Afferent volley. In an attempt to determine directly if profound peripheral changes occurred over trials, 3 spinal frogs were prepared to allow monitoring of the afferent volley from the cut distal portion of the sciatic nerve. The foot was stimulated using the strongest stimulus used in habituation training, which, however, evoked a submaximal response. Seven hundred stimuli were presented at 1/30 sec. The recorded compound action potential tended to be somewhat variable, but the distribution of responses was the same in early and late trials. Fig. 7 shows responses recorded at the beginning and end of one experiment. The stimulus artifact has changed over the almost 6-h experiment; however, the two compound action potentials are almost perfectly superimposable. Movement of transfer electrodes. Stimuli delivered through the habituation electrodes often caused the foot to twitch, which caused a slight movement of the transfer electrodes but not of the control electrodes on the other foot. It is possible that stimulation caused by these movements resulted in the relative decline of responsiveness seen at the transfer site that was interpreted as centrally mediated transfer of habituation. To test this possibility, 4 spinal frogs were prepared as usual, but with only transfer and control stimulating electrodes. Animals were given pretraining training transfer and control tests as described above, and, on the following day, the transfer Brain Research, 33 (1971) 405-417
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I Fig. 7. A, Compound action potential evoked by 10 mA foot stimulation recorded from cut distal stump of sciatic nerve. B, As in A, but approximately 700 stimulations later. Note that, although the stimulus artifact has changed over the almost 6 h duration of the experiment, the traces of evoked activity are almost perfectly superimposable. electrodes were attached to the armature o f a large relay. When the armature moved, the protruding ends of the elctrodes were moved through an arc o f 5-8 mm. The maxim u m movement of the transfer electrodes seen during habituation was 4 ram. The frogs were given 4 days o f such trials, the number and spacing o f which mimicked the trials of animals given the most habituation training. Posttraining tests were given on the same day as the last electrode movement trials and were halted if 10 times the number of pretraining trials had been given and the criterion of habituation still had not been reached. I f movement of the transfer electrodes accounted for the relative depression seen at the transfer site, then this experiment should produce the same pattern of results. In fact, both transfer and control sites showed an increase in excitability with no consistent differences between them. The posttraining transfer scores, as percents of the pretraining transfer scores, were 346, 537, > 1000, and > 1000%. The 4 posttraining control scores, as percents of the pretraining control scores, were respectively 343,483, > 1000, and > 1000%. Effectorfatigue. To the extent that the same set of neuromuscular connections is activated reflexively by stimulation at the transfer and habituation sites, it is possible that the decreased responsiveness interpreted as centrally mediated could be due to changes of the neuromuscular junctions or of the muscles themselves. Direct stimulation o f m o t o r fibers was used to indicate whether neuromuscular changes could account for the transfer results. Six spinal animals were prepared as usual, except no stimulating electrodes and only one set of recording electrodes were used. Twenty-four hours after being placed in the wax-filled pans, the eighth spinal nerve, which innervates many of the muscles in the thigh region, was dissected free, cut close to its exit from the cord, and the cut distal portion placed on Ag-AgC1 stimulating electrodes. Submaximal stimuli were used to avoid masking a possible depression with a supramaximal response. The nerve was stimulated 1/30 sec and the integrated E M G recorded as in the habituation experiments.
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Fig. 8. Effects of stimulating motor nerve versus habituation training on the peak i n t u i t e d EMG. An animal's score for each block of trials was determined as in Fig. 4. Standard errors of the mean are indicated above and below each point.
Fig. 8 compares the results of these experiments to the results on the first day of habituation training. While animals in the habituation training condition show a large decline, particularly in the early trials, the responses to motor nerve stimulation tend to remain fairly constant. DISCUSSION
The purpose of this study was to show that the brain-isolated frog spinal cord can mediate habituation cumulative over days. Controls were included to insure that neither peripheral changes nor deterioration of the preparation contributed to the decline interpreted as centrally mediated habituation. The transfer of habituation to a skin area 25-30 mm from the site where habituation stimuli were applied makes the possibility that damage to receptors or primary afferent fibers accounted for the decline extremely remote. Lindblom 13 has reported that, in toads, single fibers innervate a leg region having a longitudinal extent o f about 5 mm. Because the habituation and the (generally weaker) transfer stimulus spread less than 10 ram, they do not stimulate common areas of skin, nor, presumably, common receptors or common afferent fibers. Stimulation at the habituation site could affect the peripheral excitability of entities stimulated by the transfer electrodes only if the receptive fields of cutaneous fibers overlapped the two stimulus zones and if stimulation in one part of the receptive field somehow reduced excitability of other parts (e.g., by damaging the afferent fiber generally). The extent of current spread was determined using a 10 mA stimulus, the most intense stimulus used in the habituation experiments. In fact, with only two animals were transfer and habituation stimuli both 10 mA. The mean transfer stimulus was Brain Research, 33 (1971) 405-417
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4.2 mA and the mean habituation stimulus was 5.3 mA - - both about half the current level used to make estimates o f stimulus spread. To be stimulated by both the mean transfer and the mean habituation stimuli, a primary afferent fiber would have to have a receptive field of about 15 mm. To the extent that receptive fields in the bullfrog are not more than 3 times larger than those in the toad, it seems unlikely that the same primary afferent fbers were excited by both transfer and habituation stimuli. Also, if the depression seen at the transfer site (relative to the control site) were due to damage caused by the habituation stimulus, then those animals in which the habituation stimulus current was high, presumably activating more afferents also fired by the transfer stimulus, should show a greater relative depression than those in which it was low. However, the ratio o f the posttraining transfer score to the posttraining control score was not different for the 6 animals in which the habituation stimulus was 10 mA and those 5 in which it was 2 mA or less. In addition, all 6 animals in the high current group had transfer stimuli of at least 5 mA while the transfer stimuli o f the low current group were all 4 mA or less, and the same number of trials was given to the members of each group. Finally, the failure to find profound changes in the compound action potential over the course of 700 stimulations further supports the conclusion that the reported response depressions are not the result of changes in afferent inflow. Changes in the effector system were shown not to operate within the course of a daily session. To the extent that response decrements over days reflect the same processes as do decrements within days, it can be assumed that changes efferent to the spinal cord do not play a role in habituation over days. Deterioration of an animal's condition would be expected to change his responsiveness nonspecifically. Because the leg not given habituation training showed an increase in responsiveness in the period between pre- and posttraining tests, the possibility that responsiveness at the habituation site declined because the animal's condition worsened can be rejected. Conversely, one could argue that, while habituation is not the result of deterioration, the increased responsiveness o f the control leg might be due to a decline in the animal's general condition. For example, McAfee 15 has shown that the excised frog spinal cord maintained in an artificial chamber goes through a brief phase of heightened polysynaptic responsiveness when subjocted to hypoxia. To test whether deterioration could account for the increased general responsiveness, the posttraining control scores o f 3 animals dying within 3 days of the end of the experiment were compared to the scores of 3 animals surviving at least 10 days. The normalized control scores of the short-lived animals were each less than any of the scores of the long-lived animals. The mean posttraining control score for the short-lived animals was 272 % of their mean pretraining score as compared to a mean of 601% for the longer-lived animals. This finding that the excitability o f the control leg showed a 440 % median increase between pre- and posttraining tests was unexpected. The question whether this increase is dependent upon habituation training or whether it represents a general change in the animals' excitability as a consequence o f spinal transection is the subject Brain Research, 33 (1971) 405-417
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of a later investigation. Support for the latter hypothesis is found in the results of the experiment run to control for electrode movement in which all 4 animals showed increased excitability of both legs, although essentially no habituation intervened between pre- and posttraining tests. The experiments here provide little information on what the neuronal mechanisms of long-lasting habituation might be. It should be pointed out, however, that, to the extent that different primary afferent fibers are excited by the transfer and habituation stimuli, the demonstration of transfer of habituation means that a change which affects only the activated primary afferent pathway, such as transmitter depletion, cannot account for habituation over days. Because so little information is available, none of the classic neurophysiological phenomena can be rejected as possible mediators of long-lasting habituation in this preparation, provided one postulates some way that the effect could persist over days. The time course of habituation described in these experiments is so vastly longer than that of phenomena studied in most electrophysiological experiments that any attempt to explain the one by means of the other must be primarily speculative. SUMMARY
It was shown that the brain-isolated frog (Rana catesbiana) spinal cord can mediate response depressions cumulative over 24-h periods. The peak integrated EMG recorded from the back of the thigh, evoked by a constant current foot shock (1-10 mA, 1 msec, presented 1/30 sec), declined more rapidly on successive days of habituation training and reached a lower asymptote. The following controls were performed to insure that this decline was not the result of peripheral receptor or effector changes or of deterioration of the preparation: (1) habituation transferred to a skin site not excited by the habituation stimulus; (2) the volley recorded directly from afferent fibers remained relatively constant; (3) no response decrement was found when the motor fibers were stimulated directly; and (4) the leg not subjected to habituation training showed increased responsiveness over time. ACKNOWLEDGEMENTS
Supported by NINDS Grant No. 1 R01 NB08108-01NP awarded to Dr. Franklin Krasne. The author was a U.S. Public Health Service predoctoral trainee. This research forms part of a dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Psychology. Considerable help in all phases of this study was provided by my dissertation adviser, Franklin Krasne. This help is gratefully acknowledged. The author is currently an N I M H postdoctoral fellow at the Department of Psychobiology, University of California, Irvine, Calif. 92664, U.S.A.
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REFERENCES 1 AFELT,Z., Variability of reflexes in chronic spinal frogs. In E. GUTMANNAND P. HNIK (Eds.), Central and Peripheral Mechanisms of Motor Functions, Czechoslovak Academy of Science, Prague, 1963, pp. 37-41. 2 BUCHWALD,J. S., HALAS,E. S., ANDSCHRAMM,S., Progressive changes in efferent unit responses to repeated cutaneous stimulation in spinal cats, J. Neurophysiol., 28 (1965) 200-215. 3 BROOKHART,J. M., AND FADIGA,E., Potential fields initiated during monosynaptic activation of frog motoneurons, J. Physiol. (Lond.), 150 (1960) 633-655. 4 BROOKHART,J. M., MACHNE,X., ANDFADIGA,E., Patterns of motor neuron discharge in the frog, Arch. ital. Biol., 97 (1959) 53-67. 5 FADIGA,E., AND BROOKHART,J. M., Monosynaptic activation of different portions of the motor neuron membrane, Amer. J. Physiol., 198 (1960) 693-703. 6 FRANZISKET,L., Characteristics of instinctive behavior and learning in reflex activity of the frog, Anita. Behav., 11 (1963) 318-324. 7 GRIFFIN,J. P., ANDPEARSON,J. A., Habituation of the flexor reflex in spinal rats, and in rats with frontal cortex lesions followed by spinal transection, Brain Research, 6 (1967) 777-780. 8 GROVES,P. M., DEMARcO, R., AND THOMPSON,R. F., Habituation and sensitization of spinal interneuron activity in acute spinal cat, Brain Research, 14 (1969) 521-525. 9 GROVES,P. M., LEE, D., AND THOMPSON,R. F., Effects of stimulus frequency and intensity on habituation and sensitization in acute spinal cat, Physiol. Behav., 4 (1969) 383-388. 10 HARRIS,J. O., Habituatory response decrement in the intact organism, Psychol. Bull., 40 (1943) 385-422. 11 KOZAK,W., MACFARLANE,W. V., AND WESTERMAN,R., Long-lasting reversible changes in the reflex responses of chronic spinal cats to touch, heat, and cold, Nature (Lond.), 193 (1962) 171173. 12 LEHNER, G. F., A study of the extinction of unconditioned reflexes, J. exp. Psychol., 29 (1941) 435-456. 13 LINDBLOM,U. F., Excitability and functional organization within a peripheral tactile unit, Acta physiol, scand., 44, Suppl. 153 (1958). 14 Liu, C. N., AND CHAMBER~,W. W., Experimental study of anatomical organization of frog's spinal cord, Anat. Rec., 127 (1957) 326. 15 McAFEE,D. A., A study of mechanisms by which hypoxia influences nervous activity, unpublished doctoral dissertation, Univ. of Oregon, 1969. 16 MACHNE,X., FADIGA,E., AND BROOKHART,J. M., Antidromic and synaptic activation of frog motor neurons, J. NeurophysioL, 22 (1959) 583-603. 17 NESMEIANOVA,T. N., The inhibitionoftbe motor reflex in spinal dogs under conditions of chronic experimentation, Sechenov physiol. J. U.S.S.R., 42 (1957) 281-288. 18 PROSSER,C. L., AND HUNTER, W. S., The extinction of startle responses and spinal reflexes in the white rat, Amer. J. Physiol., 117 (1936) 609-618. 19 SPENCER,W. A., THOMPSON,R. F., ANDNEILSON,D. R., Response decrement of the flexion reflex in the acute spinal cat and transient restoration by strong stimuli, J. Neurophysiol., 29 (1966) 221-329. 20 SPENCER,W. A., THOMPSON,R. F., ANDNEILSON,D. R., Alterations in responsiveness of ascending and reflex pathways activated by iterated cutaneous afferent volleys, J. NeurophysioL, 29 (1966) 240-252. 21 SPENCER,W. A., THOMPSON,R. F., AND NEILSON,D. R., Decrement of ventral root electrotonus and intracellularly recorded P.S.P.'s produced by iterated cutaneous afferent volleys, J. Neurophysiol., 29 (1966) 253-274. 22 THOMPSON,R. F., AND SPENCER,W. A., Habituation: A model phenomenon for the study of neuronal substrates of behavior, Psychol. Rev., 173 (1966) 16-43. 23 WICKELGREN,B. G., Habituation of spinal motoneurons, J. NeurophysioL, 30 (1967) 1404-1423. 24 WICKELGREN,B. G., Habituation of spinal interneurons, J. Neurophysiol., 30 (1967) 1424-1438.
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