BEHAVIORAL BIOLOGY, 13,139-143 (1975), Abstract No. 4186
BRIEF REPORT C o r t i c a l I n v o l v e m e n t in T o n i c I m m o b i l i t y ( " A n i m a l Hypnosis"): Effect of Spreading Cortical Depression
ERIC J. TESCHKE, JACK D. MASER, 1 and GORDON G. GALLUP, JR.
Department of Psychology, Tulane University, New Orleans, Louisiana 70118 Either 25% KC1 solution or .9% NaC1 was applied to the surface of the brain of male Sprague-Dawley rats. Subjects were restrained on their dorsal surface and duration of the tonic immobility response ("animal hypnosis") observed for 20 consecutive trials. Animals that received KC1 showed significantly longer durations of tonic immobility than did saline and intact control animals. A cyclic pattern of high to low durations was also found. The data support Klemm's (1971) neural model of tonic immobility which suggests that the neocortex inhibits a brainstem immobility control center.
In a wide range of species from at least five phyletic classifications, fear, in combination with manual restraint, is known to produce a prolonged but reversible state of immobility (Gallup, 1974). Although the precise neural mechanism of tonic immobility (TI) is not understood, studies have revealed the influence of several central loci. McBride and Klemm (1969) found TI persisted in frogs with transectional cuts as far posterior as the origin o f the eighth cranial nerve, which suggests a direct, descending motor inhibitory center at the level of the medulla. Maser, Klara, and Gallup (1973) found that chickens sustaining lesions in the anterior two thirds of the archistriatum showed an 8-fold increase in the duration of TI when compared with control birds and chicks sustaining septal damage. The avian archistriatum is considered by some to be "conjunctive" cortex (I~llgn, 1962; Karten, 1969). A more direct demonstration of cortical involvement was provided by McGraw and Klemm (1969). These investigators demonstrated an inhibitory influence of the neocortex by selecting young male rats for insusceptibility and subjecting them to partial or total decortication. The immobility response could not be induced in intact rats, whereas in order of increasing magnitude TI was induced in rats sustaining skull retraction, hemidecortication, and complete decortication. 1Reprints may be obtained from Dr. Jack D. Maser, Department of Psychology, Tulane University, New Orleans, LA 70118. 139 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
140
TESCHKE,MASERANDGALLUP
The present study extends the McGrawand Klemm(1969) investigation and replicates earlier work in which KCl was applied directly to the surface of the brain (Bure~ and Bure~ov~, 1956). This technique produces a functional ablation by inducing a series of waves of neuronal depolarization which travel over the entire cortex and block neural activity (Bure} and Bure}ov~, 1972). It is important to note that this so-called spreading cortical depression (SCD) is stopped medially in the cingulate cortex and laterally in the pyriform lobe and entorhinal cortex. Some penetration may occur into the caudate nucleus via the amygdala, but this is not believed to significantlycontribute to the effect (Bure~ and Bure}ov~, 1972). Furthermore, propagation of SCD to subcortical areas is prevented by a layer of white matter, thus preserving the integrity of thalamus, hippocampus, and septal region. Application of KCI to the surface of the brain thus provides a technique by which a relatively specific and reversible cortical ablation may be effected without direct infringement upon subcortical tissue. Twenty-four male rats of the Sprague-Dawley strain served as subjects. 2 The animals were approximately" 208 days of age at the beginning of the present study. All animals were housed in a constant-temperature environment of 72 ° F and had continuous access to Purina rat chow and water. All surgery was done under ether anesthesia. After incision and separation of the periostium, a circular hole (av diam = 5.0 mm) between lambda and bregma was inscribed in the skull using a dental burr. A sharp needle was used to raise the dura mater and to create bilateral incisions parallel to the midsaggital sinus. A cotton pledget, saturated in 25% KC1 solution was then applied to the exposed cortex of 10 subjects. Ether anesthesia was removed just prior to application of the drug and the woun d was sutured over the cotton plug. After surgery the animal was returned to its cage which remained in the operating room for 30 min. Three animals in this group died during the operation. The sham operation closely paralleled that of the experimental animals with the exception that a 0.9% solution of NaC1 was substituted for the KC1 solution. This group consisted of eight animals, and two fatalities occurred during the operation. Four intact control animals were simply transported to the testing room. The testing room was sound attenuated, and light was indirectly provided by a 7.5-W bulb. The animal was immobilized on a table, and a stopwatch was used to record the duration of immobility. As an independent measure of the extent of cortical depression animals were subjected to tests suggested by Bure~ and Buregov~i (1972). Adequacy of the placing response was judged by grasping the subject's tail and lowering 2All subjects had been used previously in a study of partial reinforcement. Care was taken to randomly assign subjects to groups.
CORTICAL INVOLVEMENTIN TONIC IMMOBILITY
141
him to the surface of the table. The animal was also placed along a ledge with both extremities allowed to hang over the edge. A normal rat in these circumstances will extend its paws to meet the surface ill the first test, and withdraw its limbs from the hanging position in the second. All observations occurred 30 min after the application of the drug treatment. The animal was then transported to the testing room where 30 rain was allowed to elapse prior to immobility testing. Immobility was induced using the method described in Klemm (1971). The animal was gently grasped in the cervical area with the right hand and in the lumbar region with the left hand and quickly inverted to his dorsal surface. Care was taken to avoid occlusion of neck vessels and trachea. Restraint was gradually reduced, unless the animal struggled whereupon mild pressure was reinstated until struggling ceased. Manual restraint was maintained until immobility was evident, or until 45 sec had elapsed. A stopwatch was activated when TI was obtained and deactivated when the animal exhibited a righting response. This procedure was followed for 20 consecutive trials, the intertrial interval approximating 5-10 sec. Paper covering the surface of the table was changed after each animal had been completely tested. Upon completion of the study, several naive rats were subjected to similar operations with the addition of recording electrodes applied to the surface of the brain. Approximately 1 hr after the trephine hole was drilled recording of brain electrical activity under the influence of physiological saline or KC1 was made between two surface electrodes, or one of these and a third electrode on the rat's ear. Although animals were fully conscious at the time recordings were made, there was clear EEG suppression under the influence of KC1. For 60 min electrical activity was observed. This suggests that the procedures did, in fact, produce spreading cortical depression. Mean durations of TI for each group are seen in Fig. 1. The 20-trial, mean durations were 19.41, 3.74, and 1.43 sec for the KC1, saline, and intact control groups, respectively. A square-root transformation was found to be the most appropriate for minimizing skew in the data (Dunlap and Duffy, 1974), and analysis of variance of the transformed values revealed a significant difference between groups ( F ( 2 , 1 4 ) = 8 . 4 4 , P < 0 . 0 0 4 ) . Duncan's multiplerange test for unequal cell frequencies indicated that the KC1 group was significantly different from both control groups (P < 0.05), but the differences between controls was not reliable. A groups-by-trials interaction was also obtained (F(38, 266)=2.72, P < 0 . 0 0 1 ) , and it was evident that under the influence of KC1 repeated trials resulted in a cycling or "oscillation" of immobility duration. McBride and Klemm have observed immobility oscillations in intact frogs subjected to repeated inductions with 5- and 30-sec intertrial intervals, but cyclical behavior has also been previously noted in rats under SCD
142
TESCHKE, MASERANDGALLUP
~
20
ILl
m..~.18 Z 016 l-n::::) a ~2 >-
I.-io J
o 6
4
z
I.LI2
CONTROL
NaCI
KCI
Fig. 1. Twenty-trial mean durations of tonic immobility in seconds for intact control (N = 4), 0.9% NaC1 (iV = 8), and 25% KC1 (N = 10) groups. (Freedman, 1969). Rather than an intrinsic component of TI, oscillations may only indicate periodic reversion of the functional ablation. Two other analyses further illustrate the nature of the KC1 effect. An analysis of variance was performed on the square-root transformation o f the three longest durations exhibited by each subject (F(2,48) = 10.19, P < 0 . 0 0 1 ) , indicating that the KC1 group had the longest individual durations within the 20 test trials. The untransformed means were 105.76, 14.63, and 8.20 sec. Also o f interest were the frequency o f zero-sec durations in each group. The frequency of 10 or more zero scores was significantly greater in the control groups than in the experimental group (X2 (2) = 11.64, P < 0.005). The various measures indicate that the cortex is involved in the control of tonic immobility, and similar research by Bureg and Bure~owi (1956) was replicated. These investigators found over 400 sec average duration for 10 consecutive inductions. The difference in magnitude between the two studies may be due to strain differences. The present data are of the same order of magnitude as that reported by McGraw and Klemm (1969) for decorticated Wistar rats. The SCD group was more susceptible in the sense that they had fewer zero scores and showed longer durations of tonic immobility. Subjects in the SCD group were ataxic and exhibited little in the way of placing responses, while subjects in the control group did not evidence ataxia and placed
CORTICAL INVOLVEMENT IN TONIC IMMOBILITY
143 t
themselves appropriately. The fact that all animals receiving functional ablation by KC1 were, on some trials, able to right themselves immediately, suggests that their longer durations of TI and greater susceptibility to TI were not merely a consequence of debilitating motor impairment. The data support Klemm's (1971) model of TI which views the neocortex as suppressing a brainstem control center underlying the immobility response.
REFERENCES Buret, J., and Bure~ov~, O. (1956). The influencing of reflex acoustic epilepsy and reflex inhibition ("animal hypnosis") by spreading EEG depression. Physiol. Bohemoslov. 5, 395-400. Buret, J., and Bure'~owl, O. (1972). Inducing cortical spreading depression. In R. D. Myers (Ed.), "Methods in Psychobiology" pp. 319-343. New York: Academic Press. Dunlap, W. P., and Duffy, J. A. (1974). A computer program for determining optimal data transformations minimizing skew. Behav. Res. Meth. lnstrum. 6, 46-48. Freedman, N. L. (1969). Recurrent behavioral recovery during spreading depression, or. Comp. Physiol. Psychol. 68, 210-214. Gallup, G. G., Jr. (1974). Animal hypnosis: Factual status of a fictional concept. Psychol. Bull. In press. Kalldn, B. (1962). Embryogenesis of brain nuclei in the chick telencephalon. Ergebn. Anat. Entwicklungsgesch. 36, 62-82. Karten, J. H. (1969). The organization of the avian telencephalon and some speculation on the phylogeny of the amniote telencephalon. Ann. N. Y. Acact. Sci. 167, 164-179. Klemm, W. R. (1971). Neurophysiologic studies of the immobility reflex ("animal hypnosis"). NeuroscL Res. 4, 165-212. Maser, J. D.,'Klara, J. W., and Gallup, G. G., Jr. (1973). Archistriatal lesions enhance tonic immobility in the chicken (Gallus gallus). Physiol. Behav. 11, 729-733. McBride, R. L., and Klemm, W. R. (1969). Mechanisms of the immobility reflex ("animal hypnosis"). I. Influences of repetition of induction, restriction of auditory-visual input, and destruction of brain areas. Commun. Behav. Biol. 3, 33-41. McGraw, C. P., and Klemm, W. R. (1969). Mechanisms of the immobility reflex ("animal hypnosis"). III. Neocortical inhibition in rats. Commun. Behav. Biol. 3, 53-69.
BRIEF REPORT C o r t i c a l I n v o l v e m e n t in T o n i c I m m o b i l i t y ( " A n i m a l Hypnosis"): Effect of Spreading Cortical Depression
ERIC J. TESCHKE, JACK D. MASER, 1 and GORDON G. GALLUP, JR.
Department of Psychology, Tulane University, New Orleans, Louisiana 70118 Either 25% KC1 solution or .9% NaC1 was applied to the surface of the brain of male Sprague-Dawley rats. Subjects were restrained on their dorsal surface and duration of the tonic immobility response ("animal hypnosis") observed for 20 consecutive trials. Animals that received KC1 showed significantly longer durations of tonic immobility than did saline and intact control animals. A cyclic pattern of high to low durations was also found. The data support Klemm's (1971) neural model of tonic immobility which suggests that the neocortex inhibits a brainstem immobility control center.
In a wide range of species from at least five phyletic classifications, fear, in combination with manual restraint, is known to produce a prolonged but reversible state of immobility (Gallup, 1974). Although the precise neural mechanism of tonic immobility (TI) is not understood, studies have revealed the influence of several central loci. McBride and Klemm (1969) found TI persisted in frogs with transectional cuts as far posterior as the origin o f the eighth cranial nerve, which suggests a direct, descending motor inhibitory center at the level of the medulla. Maser, Klara, and Gallup (1973) found that chickens sustaining lesions in the anterior two thirds of the archistriatum showed an 8-fold increase in the duration of TI when compared with control birds and chicks sustaining septal damage. The avian archistriatum is considered by some to be "conjunctive" cortex (I~llgn, 1962; Karten, 1969). A more direct demonstration of cortical involvement was provided by McGraw and Klemm (1969). These investigators demonstrated an inhibitory influence of the neocortex by selecting young male rats for insusceptibility and subjecting them to partial or total decortication. The immobility response could not be induced in intact rats, whereas in order of increasing magnitude TI was induced in rats sustaining skull retraction, hemidecortication, and complete decortication. 1Reprints may be obtained from Dr. Jack D. Maser, Department of Psychology, Tulane University, New Orleans, LA 70118. 139 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
140
TESCHKE,MASERANDGALLUP
The present study extends the McGrawand Klemm(1969) investigation and replicates earlier work in which KCl was applied directly to the surface of the brain (Bure~ and Bure~ov~, 1956). This technique produces a functional ablation by inducing a series of waves of neuronal depolarization which travel over the entire cortex and block neural activity (Bure} and Bure}ov~, 1972). It is important to note that this so-called spreading cortical depression (SCD) is stopped medially in the cingulate cortex and laterally in the pyriform lobe and entorhinal cortex. Some penetration may occur into the caudate nucleus via the amygdala, but this is not believed to significantlycontribute to the effect (Bure~ and Bure}ov~, 1972). Furthermore, propagation of SCD to subcortical areas is prevented by a layer of white matter, thus preserving the integrity of thalamus, hippocampus, and septal region. Application of KCI to the surface of the brain thus provides a technique by which a relatively specific and reversible cortical ablation may be effected without direct infringement upon subcortical tissue. Twenty-four male rats of the Sprague-Dawley strain served as subjects. 2 The animals were approximately" 208 days of age at the beginning of the present study. All animals were housed in a constant-temperature environment of 72 ° F and had continuous access to Purina rat chow and water. All surgery was done under ether anesthesia. After incision and separation of the periostium, a circular hole (av diam = 5.0 mm) between lambda and bregma was inscribed in the skull using a dental burr. A sharp needle was used to raise the dura mater and to create bilateral incisions parallel to the midsaggital sinus. A cotton pledget, saturated in 25% KC1 solution was then applied to the exposed cortex of 10 subjects. Ether anesthesia was removed just prior to application of the drug and the woun d was sutured over the cotton plug. After surgery the animal was returned to its cage which remained in the operating room for 30 min. Three animals in this group died during the operation. The sham operation closely paralleled that of the experimental animals with the exception that a 0.9% solution of NaC1 was substituted for the KC1 solution. This group consisted of eight animals, and two fatalities occurred during the operation. Four intact control animals were simply transported to the testing room. The testing room was sound attenuated, and light was indirectly provided by a 7.5-W bulb. The animal was immobilized on a table, and a stopwatch was used to record the duration of immobility. As an independent measure of the extent of cortical depression animals were subjected to tests suggested by Bure~ and Buregov~i (1972). Adequacy of the placing response was judged by grasping the subject's tail and lowering 2All subjects had been used previously in a study of partial reinforcement. Care was taken to randomly assign subjects to groups.
CORTICAL INVOLVEMENTIN TONIC IMMOBILITY
141
him to the surface of the table. The animal was also placed along a ledge with both extremities allowed to hang over the edge. A normal rat in these circumstances will extend its paws to meet the surface ill the first test, and withdraw its limbs from the hanging position in the second. All observations occurred 30 min after the application of the drug treatment. The animal was then transported to the testing room where 30 rain was allowed to elapse prior to immobility testing. Immobility was induced using the method described in Klemm (1971). The animal was gently grasped in the cervical area with the right hand and in the lumbar region with the left hand and quickly inverted to his dorsal surface. Care was taken to avoid occlusion of neck vessels and trachea. Restraint was gradually reduced, unless the animal struggled whereupon mild pressure was reinstated until struggling ceased. Manual restraint was maintained until immobility was evident, or until 45 sec had elapsed. A stopwatch was activated when TI was obtained and deactivated when the animal exhibited a righting response. This procedure was followed for 20 consecutive trials, the intertrial interval approximating 5-10 sec. Paper covering the surface of the table was changed after each animal had been completely tested. Upon completion of the study, several naive rats were subjected to similar operations with the addition of recording electrodes applied to the surface of the brain. Approximately 1 hr after the trephine hole was drilled recording of brain electrical activity under the influence of physiological saline or KC1 was made between two surface electrodes, or one of these and a third electrode on the rat's ear. Although animals were fully conscious at the time recordings were made, there was clear EEG suppression under the influence of KC1. For 60 min electrical activity was observed. This suggests that the procedures did, in fact, produce spreading cortical depression. Mean durations of TI for each group are seen in Fig. 1. The 20-trial, mean durations were 19.41, 3.74, and 1.43 sec for the KC1, saline, and intact control groups, respectively. A square-root transformation was found to be the most appropriate for minimizing skew in the data (Dunlap and Duffy, 1974), and analysis of variance of the transformed values revealed a significant difference between groups ( F ( 2 , 1 4 ) = 8 . 4 4 , P < 0 . 0 0 4 ) . Duncan's multiplerange test for unequal cell frequencies indicated that the KC1 group was significantly different from both control groups (P < 0.05), but the differences between controls was not reliable. A groups-by-trials interaction was also obtained (F(38, 266)=2.72, P < 0 . 0 0 1 ) , and it was evident that under the influence of KC1 repeated trials resulted in a cycling or "oscillation" of immobility duration. McBride and Klemm have observed immobility oscillations in intact frogs subjected to repeated inductions with 5- and 30-sec intertrial intervals, but cyclical behavior has also been previously noted in rats under SCD
142
TESCHKE, MASERANDGALLUP
~
20
ILl
m..~.18 Z 016 l-n::::) a ~2 >-
I.-io J
o 6
4
z
I.LI2
CONTROL
NaCI
KCI
Fig. 1. Twenty-trial mean durations of tonic immobility in seconds for intact control (N = 4), 0.9% NaC1 (iV = 8), and 25% KC1 (N = 10) groups. (Freedman, 1969). Rather than an intrinsic component of TI, oscillations may only indicate periodic reversion of the functional ablation. Two other analyses further illustrate the nature of the KC1 effect. An analysis of variance was performed on the square-root transformation o f the three longest durations exhibited by each subject (F(2,48) = 10.19, P < 0 . 0 0 1 ) , indicating that the KC1 group had the longest individual durations within the 20 test trials. The untransformed means were 105.76, 14.63, and 8.20 sec. Also o f interest were the frequency o f zero-sec durations in each group. The frequency of 10 or more zero scores was significantly greater in the control groups than in the experimental group (X2 (2) = 11.64, P < 0.005). The various measures indicate that the cortex is involved in the control of tonic immobility, and similar research by Bureg and Bure~owi (1956) was replicated. These investigators found over 400 sec average duration for 10 consecutive inductions. The difference in magnitude between the two studies may be due to strain differences. The present data are of the same order of magnitude as that reported by McGraw and Klemm (1969) for decorticated Wistar rats. The SCD group was more susceptible in the sense that they had fewer zero scores and showed longer durations of tonic immobility. Subjects in the SCD group were ataxic and exhibited little in the way of placing responses, while subjects in the control group did not evidence ataxia and placed
CORTICAL INVOLVEMENT IN TONIC IMMOBILITY
143 t
themselves appropriately. The fact that all animals receiving functional ablation by KC1 were, on some trials, able to right themselves immediately, suggests that their longer durations of TI and greater susceptibility to TI were not merely a consequence of debilitating motor impairment. The data support Klemm's (1971) model of TI which views the neocortex as suppressing a brainstem control center underlying the immobility response.
REFERENCES Buret, J., and Bure~ov~, O. (1956). The influencing of reflex acoustic epilepsy and reflex inhibition ("animal hypnosis") by spreading EEG depression. Physiol. Bohemoslov. 5, 395-400. Buret, J., and Bure'~owl, O. (1972). Inducing cortical spreading depression. In R. D. Myers (Ed.), "Methods in Psychobiology" pp. 319-343. New York: Academic Press. Dunlap, W. P., and Duffy, J. A. (1974). A computer program for determining optimal data transformations minimizing skew. Behav. Res. Meth. lnstrum. 6, 46-48. Freedman, N. L. (1969). Recurrent behavioral recovery during spreading depression, or. Comp. Physiol. Psychol. 68, 210-214. Gallup, G. G., Jr. (1974). Animal hypnosis: Factual status of a fictional concept. Psychol. Bull. In press. Kalldn, B. (1962). Embryogenesis of brain nuclei in the chick telencephalon. Ergebn. Anat. Entwicklungsgesch. 36, 62-82. Karten, J. H. (1969). The organization of the avian telencephalon and some speculation on the phylogeny of the amniote telencephalon. Ann. N. Y. Acact. Sci. 167, 164-179. Klemm, W. R. (1971). Neurophysiologic studies of the immobility reflex ("animal hypnosis"). NeuroscL Res. 4, 165-212. Maser, J. D.,'Klara, J. W., and Gallup, G. G., Jr. (1973). Archistriatal lesions enhance tonic immobility in the chicken (Gallus gallus). Physiol. Behav. 11, 729-733. McBride, R. L., and Klemm, W. R. (1969). Mechanisms of the immobility reflex ("animal hypnosis"). I. Influences of repetition of induction, restriction of auditory-visual input, and destruction of brain areas. Commun. Behav. Biol. 3, 33-41. McGraw, C. P., and Klemm, W. R. (1969). Mechanisms of the immobility reflex ("animal hypnosis"). III. Neocortical inhibition in rats. Commun. Behav. Biol. 3, 53-69.
