Journal of Comparative and Physiological Psychology 1975, Vol. 88, No. 1, 329-334

HIPPOCAMPAL ABLATION PROLONGS IMMOBILITY RESPONSE IN RABBITS (ORYCTOLAGUS CUNICULUS) MICHAEL L. WOODRUFF, DANIEL C. HATTON, AND MERLE E. MEYER University of Florida The present study was performed to test the hypothesis that damage to the hippocampus reduces tonic immobility in rabbits. Two measures were used: the number of successful inductions of tonic immobility in a test session and the duration of each successful immobilization. Both of these measures were found to increase in rabbits with hippocampal lesions rather than to decrease as expected. It is suggested that the hippocampus may act to suppress any ongoing dominant or prepotent response, whether the response involves movement or cessation of movement.

Many research reports that have dealt are about to be shocked (Green, Beatty, & with hippocampo-behavioral relationships Schwartzbaum, 1967; Thomas, Hostetter, & during the last decade have employed some Barker, 1968). The tendency of rats and type of learning paradigm. It is apparent cats to freeze when anticipating shock has from the reviews of Douglas (1967), Kimble been termed the "defensive immobility (1968), Jarrard (1973), and Altman, Brun- reaction" (Blanchard, Blanchard, & Fial, ner, and Bayer (1973) that hippocampally 1970). Hippocampal lesions presumably reablated infrahuman animals do not exhibit duce this tendency. The hippocampus, then, unequivocal signs of memory impairment. is generally viewed as acting to inhibit or Isaacson (1972) has even suggested that the brake ongoing motoric responses. memory deficits in humans subsequent to Rabbits react to a certain group of enbilateral hippocampal ablation (Penfield & vironmental stimuli with an inhibition of Milner, 1958) may not be due to the destruc- movement that may last as long as 20 min. tion of the hippocampus. It may be that the During this period of tonic immobility, the preoperative epileptic state of the patients rabbit exhibits a reduction in reactivity to described in these communications is a external stimulation, loss of the righting necessary condition for the observed post- reflex, and a depression of flexor and extensor operative memory impairment and not the polysynaptic reflexes (Klemm, 1971). Imactual destruction of the hippocampus. mobility can be induced by repetitive stimuThese observations led Altman et al. to lation, pressure on body parts, inversion, question whether approaches other than and restraint from movement. A combinalearning tasks might not be more useful in tion of the last 3 manipulations is particuthe study of hippocampal functioning. larly effective in inducing the state in rabHowever, Altman et al. (1973), Douglas bits. If the hippocampus is part of a system (1967), and Kimble (1968) have all been that acts to brake motor responses, as has able to derive theories of hippocampal func- been suggested by Altman et al. (1973), then tioning that do seem to account for much of removal of this structure in the rabbit should the available data. These theories all propose serve to reduce the effectiveness of mathe hippocampus as a substrate of behavioral nipulations that produce tonic immobility. "inhibition" or "braking." For example, Furthermore, tonic immobility in rabbits animals with hippocampal lesions have diffi- appears similar in some respects to the deculty in passive avoidance (Isaacson & fensive immobility reaction in rats. If this Wicklegren, 1962; Papsdorf & Woodruff, is the case, hippocampal ablation could act 1970). Usually this task requires that the to eliminate or shorten the immobility reanimal suppress a previously learned motoric sponse in rabbits. Finally, it has been sugresponse. It is also known that animals with gested that limbic structures, particularly hippocampal lesions do not freeze as much the hippocampus, are intimately involved as normals in response to a signal that they in the production of this state (Davis, 1963; 329

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readily without loss of animals due to overdose if the corneal reflex and reaction to pinching of the toe were used as indices of depth of anesthesia. A piece of cord was tied about the upper rib cage to facilitate breathing by reflex excitation of the reMETHOD spiratory centers (Lumb, 1963). The scalp was shaved, and under clean surgical Subjects conditions, a midline incision was made in the to expose the dorsolateral skull. A burr and The subjects were 20 Dutch Belt rabbits scalp rongeurs were used to remove a portion of the (Oryctolagus cuniculus) of both sexes. They were skull, thereby exposing the dura and neocortex approximately 160 days old at the time of preoper- overlying the hippocampus. The dura was reative testing, They were housed in groups of 2 or moved with scissors and forceps. For Group C, 3 and maintained on ad-lib food and water the neocortex was aspirated bilaterally to exthroughout the experiment. A 12:12 light/dark pose the dorsal hippocampus. For Group H, cycle was in effect for their colony room. Testing aspiration was continued until the hippocampus was done during the first 6 hr. of the light period. was transected at the dorsal convexity, and as much of the upper ventral and anterior hippocamPreoperative Behavioral Testing pus was removed as possible. Hemostasis was achieved, and the wound closed with 9-mm. ClayOne day preoperatively, each rabbit was sub- Adams wound clips. One hundred thousand units jected to an immobilization test. The rabbit was oi Bicillin were given, and tetracycline was placed transported from its home cage to the test room in the rabbits' drinking water for 4 days to combat in a large box. The test room was illuminated by a possible postoperative infections. 150-w. surgical lamp that was focused on a Vshaped trough in which the rabbits were immo- Postoperative Behavioral Testing bilized. The trough was constructed after the method of Ratner (1958) from 6.35-mm.-thick unThirteen days of recovery time were allowed. painted plywood. It was 47.63 cm. long; the sides The same IR test procedure was followed as had were 23.60 cm. high and joined at a right angle. been used in the preoperative test. The vertex of the angle was 9.50 cm. from the floor. The surgical lamp was approximately 2 m. above Histology the trough. Following the postoperative test the operated The rabbit was taken from the transport box and placed in an upright position in the trough. rabbits were sacrificed with an overdose of sodium Immobilization was induced 15 sec. later by rap- pentobarbital. They were then intracardially peridly inverting the rabbit and forcefully restraining fused with .9% saline followed by 10% formalin. it in the inverted position by pressing the thorax The brains were removed, embedded in celloidin, with one hand for 15 sec. The hand was slowly and sectioned at 30 ,um. Every tenth section was withdrawn after 15 sec., and a clock was started retained, slide mounted, and stained with thionin. to time the duration of the immobility response The sections were examined for extent of the le(IR). The clock was stopped when the rabbit sion. Lesions were reconstructed utilizing the atrighted itself. If the rabbit did not become im- las of Monnier and Gangloff (1961). mobile within 15 sec., it was allowed to right itRESULTS self and remain in the upright position in the trough 15 sec. Three inductions were given in this Histology way preoperatively for each rabbit. Reconstructions of the largest and smallOperative Procedure est lesions are plotted in Figure 1 for both The ablations were performed the day after the Groups C and H at the anterior, middle, preoperative test for IR. The subjects were divided into 3 groups: Group H-—bilateral hippocampal and posterior levels of the hippocampus. ablation (n = 7); Group C—bilateral neocortical The anterior and dorsal hippocampus was ablation (n = 7); Group N—unoperated controls almost completely removed bilaterally in (n = 6). Because the number of successful induc- all subjects in Group H. The ablation also tions of the IR and the duration of the successful immobilizations was found to vary greatly among included most of the lateral convexity of the rabbits, rabbits with long, medium, and short the hippocampus. Ventral hippocampus was immobilization times were placed in each group. spared in all subjects, though small lesions Surgical anesthesia was induced in each oper- were observed in the extreme caudal porated animal by means of an injection of sodium pentobarbital through the marginal ear vein. No tions of the structure in 4 rabbits. Extrastandard dose was used as previous experience had hippocampal damage was limited to overindicated that anesthesia could be induced more lying dorso-lateral neocortex and the subKlemm, 1971). The present study investigated the effect of ablation of the hippocampus on tonic immobility in rabbits.

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FIGDHE 1. Minimum (darkened area) and maximum (cross-hatched) lesion extents for subjects in Group C (upper row) and Group H (lower row).

cortical medullary layer. No lesions included any portion of the thalamus, but some cell loss and gliosis were observed in the lateral nucleus as a consequence of deafferentation of this area. More neocortex was removed in the subjects in Group C than in the subjects in Group H. The neocortical lesions extended more caudally and slightly more laterally in the subjects in Group C. Some cell loss and gliosis were observed in the lateral nucleus of thalamus in the subjects in Group C. The amount of thalamic reaction was the same for rabbits in Group C and Group H. Pre- and Postoperative Immobility A Friedman 2-way analysis of variance (Siegel, 1956) was conducted on the durations of the IRs on each trial for each rabbit both pre- and postoperatively to determine if a trials effect was present. No statistically significant differences were found. The 3 preoperative IR durations for each rabbit in each group were added together, as were the 3 postoperative scores, to give a composite duration for each subject. The mean and standard deviation of the composite scores for each group are presented in Table 1. The composite preoperative scores were compared with the composite postoperative durations by means of the Wilcoxon

matched-pairs signed-ranks test. Only the group with hippocampal lesions demonstrated a significant change in IR duration from the first series of IR" inductions to the second (T = 0, n = 7, p < .02). As can be seen in Table 1, the duration of the IRs produced in the rabbits in Group H increased as a result of the hippocampal lesion. A Kruskal-Wallis one-way analysis of variance was performed on the preoperative scores, and no group differences were found. The same procedure was performed on the postoperative scores, and a significant difference was found (H = 8.6, df = 2, p < .02). A subsequent Mann-Whitney U test indicated that the rabbits given hippocampal lesions had significantly longer postoperative IR durations than either control group (U = 5, p < .01, in both cases). The control groups did not statistically differ from one another. The number of failures to induce the IR both pre- and postoperatively was analyzed. Preoperatively, the rabbits in Group H failed to immobilize 8 times in 21 attempts. The rabbits in Group C did not immobilize 8 times in 21 attempts, and the rabbits in Group N gave no IR 7 times in 18 attempts. A chi-square for unrelated samples indicated no significant differences preoperatively for the number of failures to induce IR. Post-

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TABLE 1 MEAN AND STANDARD DEVIATION OF COMPOSITE DURATION (IN SEC.) OF THE IMMOBILITY RESPONSE Preoperative

M

Bilateral hippocampal Bilateral neocortioal Unoperated control

SD

Postoperative

M

SD

315.9 287.9 1,327.9 730.2 227.1 207.0 415.4 297.3 219.3 210.3 322.5 481.0

operatively, Group H immobilized 21 times in 21 attempts. Group C failed to immobilize 9 times in 21 attempts, and Group N failed to immobilize 10 times in 18 attempts. A chi-square for unrelated samples indicated a significant effect postoperatively (p < .02). Chi-squares for related samples were conducted for each group between the number of preoperative and postoperative failures to immobilize. Only Group H gave a significant value (p < .01). DISCUSSION The results of this experiment indicate that bilateral ablation of the hippocampus, or at least of the dorsal hippocampus, increases susceptibility of the rabbit to tonic immobility and that the duration of the immobility is increased. These data are difficult to interpret within the framework of current theories of hippocampo-behavioral functioning (Altaian et al., 1973; Douglas, 1967; Kimble, 1968). Kimble (1968, 1969) has suggested that the hippocampus provides the neural substrate for Pavlovian internal inhibition of response. Pavlov (1960) considered the IR (animal hypnosis) to be the result of internal inhibition. Taken together, these propositions suggest that, if activation of the hippocampus has any effect on IR, it should act to potentiate the response. Conversely, removal of hippocampus should result in attenuation of the response. Our results are in contrast to these predictions. Altaian et al. (1973) also assign to the hippocampus the role of controlling motor action such that "movement can be brought to a sudden halt if necessary [p. 581]." This

proposal is supported by the results of studies cited above in which rats with hippocampal lesions did not freeze when presented with a stimulus signaling shock. Freezing in this situation is a prepotent response for the normal rat (Green et al., 1967; Thomas et al., 1968). However, freezing is not the initial prepotent or ongoing response in these experiments. Rather, the rats were moving about, perhaps engaging in exploratory behavior. With the onset of the stimulus signaling shock, normal rats suppressed the ongoing exploratory behavior. Rats with hippocampal lesions did not. Both the initial exploratory movements and the signal of impending shock occurred in the same experimental setting, and the rats with hippocampal lesions failed to suppress the ongoing response. Therefore, they did not inhibit movement. In the present study, the rabbit was taken from a carrying box, placed in a different setting (i.e., a wood trough), and inverted 15 sec. later. It was not permitted any other response sequence in its new environment. The only response with a high probability of emission for this species in this situation is immobility. The IR does not compete with an ongoing response in this case and is the behavior that is potentiated. It may be, then, that the hippocampus does not function only to inhibit movement. It may act to suppress any dominant or prepotent response, whether the response is movement or cessation of movement. One function of the intact hippocampus in the rabbit may be as part of a system that attenuates the IR in order to provide the rabbit with the opportunity to initiate other behavior. Developmental evidence also supports the hypothesis that the hippocampus may act to suppress the IR. McGraw and Klemm (1969) and Svorad (1957) have found that neonatal rats up to 20 days of age readily exhibit the IR. After about 25 days, the response is almost impossible to obtain in rats. Though these authors have interpreted their data in terms of neocortical development, the time course is also appropriate for development of the fascia dentata (Altman, 1967). These data agree with the theory of Altman et al. (1973) in which they pro-

HIPPOCAMPAL ABLATION AND TONIC IMMOBILITY

pose that animals with hippocampal lesions are behaviorally somewhat like preweanling juveniles. The immobility response may be a very good unlearned behavior to use in investigating hippocampo-behavioral relationships on a physiological level. Some data are available that suggest a possible mechanism whereby activation of hippocampus could terminate the IR. Klemm (1969) has observed an increase in multiple unit activity in the reticular formation correlated with the IR in rabbits and has also observed that reticular stimulation will increase the duration of the response (Klemm, 1965). It is known that both poly- and monosynaptic connections exist between hippocampus and at least the rostral brain stem reticular formation in the rabbit (Sprague & Meyer, 1950). Furthermore, stimulation of the hippocampus has been reported to inhibit reticular activity (Adey, Segundo, & Livingston, 1957). It could be, then, that activation of hippocampus serves to inhibit reticular activity and thereby terminate the; IR. Several problems exist with this hypothesis. For example, Klemm (1965) found no effect on IR as a result of low intensity electrical stimulation of the hippocampus. MacLean (1957) has observed that the afterdischarge in cat hippocampus correlates with behavioral arrest, though this type of activity may result in a functional ablation of the structure. Finally, Grantyn, Margnelli, Mancia, & Grantyn (1973) have observed that hippocampal stimulation produces primarily excitation of mesencephalic and bulbopontine neurons. These studies serve to illustrate the problems associated with any attempt to specify precisely the neurophysiological and neuroanatomical substrates of the influences of the hippocampus on the IR at this time. However, this response could serve as a good model of a comparatively simple, unlearned behavior that is influenced by ablation of the hippocampus and could be used to study hippocampo-behavioral relationships on a neurophysiological level. REFERENCES Adey, W. R., Segundo, J. P., & Livingston, R. B. Cortifugal influences on intrinsic brainstem con-

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duction in cat and monkey. Journal oj Neurophysiology, 1957, 20,1-16. Altman, J. Postnatal growth and differentiation of the mammalian brain, with implications for a morphological theory of memory. In G. D. Quarton, T. Melnechuk, & F. O. Schmitt (Eds.), The neurosciences: A study program. New York: Rockefeller University Press, 1967. Altman, J., Brunner, R. L., & Bayer, S. A. The hippocampus and behavioral maturation. Behavioral Biology, 1973, 8, 557-596. Blanchard, R. J., Blanchard, D. C., & Fial, R. A. Hippocampal lesions in rats and their effect on activity, avoidance, and aggression. Journal oj Comparative and Physiological Psychology, 1970, 71,92-102. Davis, W. M. Neurophysiological basis and pharmacological modification of inhibitory emotional behavior in the rabbit. Archives Internationales de Pharmacodynamie et de Therapie, 1963, 142, 349-360. Douglas, R. J. The hippocampus and behavior. Psychological Bulletin, 1967, 67, 414-442. Grantyn, R., Margnelli, M., Mancia, M., & Grantyn, A. Postsynaptic potentials in the mesencephalic and ponto-medullar reticular regions underlying descending limbic influences. Brain Research, 1973, 56,107-121. Green, R. H., Beatty, W. W., & Schwartzbaum, J. S. Comparative effects of septo-hippocampal and caudate lesions on avoidance behavior in rats. Journal of Comparative and Physiological Psychology, 1967, 64, 444-452. Isaacson, R. L. Hippocampal destruction in man and other animals. Neuropsychologia, 1972, 10, 47-64. Isaacson, R. L., & Wickelgren, W. 0. Hippocampal ablation and passive avoidance. Science, 1962, 138, 1104-1106. Jarrard, L. E. The hippocampus and motivation. Psychological Bulletin, 1973, 79, 1-12. Kimble, D. P. Hippocampus and internal inhibition. Psychological Bulletin, 1968, 70, 285295. Kimble, D. P. Possible inhibitory functions of the hippocampus. Neuropsychologia, 1969, 7, 235244. Klemm, W. R. Potentiation of "animal hypnosis" with low levels of electric current. Animal Behaviour, 1965, 13, 571-574. Klemm, W. R. Mechanisms of the immobility reflex ("animal hypnosis"): II. EEG and multiple unit correlates in the brain stem. Communications in Behavioral Biology, 1969, 3, 43-52. Klemm, W. R. Neurophysiologic studies of the immobility reflex ("animal hypnosis"). In S. Ehrenpreis & O. C. Solnitzky (Eds.), Neurosciences research. Vol. 4. New York: Academic Press, 1971. Lumb, W. V. Small animal anesthesia. Philadelphia: Lea & Febiger, 1963. MacLean, P. D. Chemical and electrical stimulation of hippocampus in unrestrained animals:

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II. Behavioral findings. AMA Archives of Neurology and Psychiatry, 1957, 78, 128-142. MoGraw, C. P., & Klemm, W. R. Mechanisms of the immobility reflex ("animal hypnosis"): III. Neocortical inhibition in rats. Communications in Behavioral Biology, 1969, 3, 53-59. Monnier, M., & Gangloff, M. Rabbit brain research. Vol. 1. Amsterdam: Elsevier, 1961. Papsdorf, J. P., & Woodruff, M. Effects of bilateral hippocampectomy on the rabbit's acquisition of shuttle-box and passive-avoidance responses. Journal of Comparative and Physiological Psychology, 1970, 73, 486-489. Pavlov, I. P. [Conditioned reflexes.] (G. V. Anrep, Ed. and trans.) New York: Dover, 1960. Penfield, W., & Milner, B. Memory deficit produced by bilateral lesions on the hippocampal zone. AMA Archives of Neurology and Psychiatry, 1958, 79, 475-497.

Ratner, S. C. Hypnotic reactions of rabbits. Psychological Reports, 1958, 4, 209-210. Siegel, S. Nonparametric statistics for the behavioral sciences. New York: McGraw-Hill, 1956. Sprague, J. M., & Meyer M. An experimental study of the fornix in the rabbit. Journal of Anatomy, 1950, 84,354-368. Svorad, D. "Animal hypnosis" (Totstell reflex) as experimental model for psychiatry. AMA Archives oj Neurology and Psychiatry, 1957, 77, 533-539. Thomas, G. J., Hostetter, G., & Barker, D. J. Behavior functions of limbic system. In E. Stellar & J. M. Sprague (Eds.), Progress in physiological psychology. Vol. 2, New York: Academic Press, 1968. (Received October 10,1973)

Hippocampal ablation prolongs immobility response in rabbits (Oryctolagus cuniculus).

The present study was performed to test the hypothesis that damage to the hippocampus reduces tonic immobility in rabbits. Two measures were used: the...
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