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Locomotor and convulsive responses to picrotoxin in amygdala-kindled rats
EXPERIMENTAL
NEUROLOGY
70, 167- 172 (1980)
RESEARCH Locomotor MICHAEL
NOTE
and Convulsive Responses to Picrotoxin in Amygdala-Kindled Rats W. KALICHMANAND
Department of Pharmacology,
W. MCINTYREBURNHAM'
University of Toronto, Toronto, Ontario M5S lA8, Canada
Received December 6, 1979; revision received April 6, 1980 Amygdala-kindled rats and handled controls were tested for differences in locomotion and convulsive activity produced by picrotoxin, a specific synaptic antagonist of the putative neurotransmitter GABA. Compared with controls, the gross activity of kindled rats decreased more rapidly with dose (0.5 to 2.0 mglkg, i.p.) and kindled rats were more likely to convulse with the higher doses (1.0 to 2.0 mg/kg). These results suggest a functional deficit in the GABA system of kindled rats as measured by locomotion and convulsive activity.
Repeated low-level electrical stimulation of cortical or subcortical sites in the brain results in a process called “kindling” (3, 6, 12). Although the subject initially shows little or no response to the stimulus, repeated stimulation leads to progressively stronger epileptiform responses. Eventually the stimulus, which initially induced only a short local afterdischarge, elicits a generalized seizure with a clonic convulsion. This increased convulsive sensitivity has been shown to persist for months following kindling and appears to reflect permanent changes in brain function (6, 14). Several different approaches have been used to study the neurochemical and physiological bases for the kindling-related brain changes (1, 2, 4, 5, 7, 8, 10, 11, 13-15). One approach which may be particularly valuable is to observe the behavioral responses of kindled subjects which have Abbreviation: GABA-y-aminobutyric acid. * Supported by grant MT 5611 from the Medical Research Council of Canada to Drs. Bumham, K. E. Livingston, and R. Racine. Mr. Kalichman was supported by a Garfield Weston Foundation grant to Dr. Livingston. 167 OO14-4886/8O/lOO167-06$02OO/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
168
KALICHMAN
AND BURNHAM
been treated with drugs which have relatively well-understood mechanisms. Such a paradigm would make it possible to consider kindlinginduced differences in function, for example, of a given neurotransmitter. The present study used this approach to consider the possibility that the y-aminobutyric acid (GABA) system may be functionally different in animals which have been kindled. Liebowitz er al. (7) reported an increase in K+-sensitive GABA release in kindled rats. Assuming that GABA-mediated inhibition is antagonistic to epilepsy (9), an increased release of GABA would be expected to antagonize the kindling process. A hyperfunctional GABA system is further supported by Corcoran (unpublished observation), who found that kindled rats were less sensitive to convulsions induced by 3-mercaptoproprionic acid, a competitive inhibitor of GABA synthesis. On the other hand, in preliminary experiments we found that kindled animals were more sensitive to convulsions induced by a specific synaptic GABA antagonist, picrotoxin. This result is exactly opposite to that which might have been predicted based on the experiments with GABA release and 3-mercaptoproprionic acid. The objective of the present study was to consider further the possibility of a functional change in the GABA system induced by kindling. This was accomplished by comparing amygdala-kindled and control rat locomotion and convulsions after picrotoxin pretreatment. Fourteen male Royal Victoria Hooded rats (Canadian Breeding Laboratories) were used. Subjects (200 to 250 g) were implanted with twisted stainless-steel bipolar electrodes (250 pm diameter) using conventional stereotaxic methods. The electrodes were aimed at the right amygdala using the following coordinates: posterior and lateral to bregma, 1.0 and 4.5 mm; and down from the dura, 8.0 mm. One week after surgery, the rats were randomly divided into two groups. One group received the kindling stimulation and the other group was handled identically, but was not stimulated. Stimulation and handling took place twice daily. The stimulus consisted of a l-s train of 60-Hz, 1-ms diphasic pulses with an intensity of 250 PA peak-to-peak. Kindling stimulation was terminated after the occurrence of six bilateral forelimb clonic convulsions [stage 4 or greater (12)]. The kindled group of seven subjects achieved this criterion in an average of 14 days. Picrotoxin testing was begun 2 months after the completion of kindling. By 2 months, any transient effects of the last kindled seizure should be minimal. All tests consisted of a lo-min period during which two rats, one kindled and one handled, were simultaneously monitored for both locomotor and convulsive activity. The test chamber was a sound-attenuating box which contained two observation fields (each field approximately 40 x 70 cm) separated by a wooden barrier. The effects of extraneous
PICROTOXIN
169
AND KINDLING
90
0
80
control ( n=7)
70
z 8
60
cn F:
50
5 i= z
40
Y 2
30
2 20
IO
0 0
0.5
PICROTOXIN
1.0
(mg/kg
, 2.0
i.p.1
FIG. 1. Effect of picrotoxin on locomotor activity in amygdala-kindled and control rats. Activity decreased with dose in both groups (P < 0.05, analysis of variance). Kindled group activity decreased more rapidly with dose (P < 0.05, t test). Data are average activity scores * SE.
noise were masked by white noise (Noise Generator 901B, GrasonStadler). The floor of each observation field consisted of a 3 x 6 pattern of rectangles underneath a Plexiglas base. The kindled and control rats were placed on opposite sides of the barrier for the test. After 2 min of accomodation, data were scored for each of the rats during alternate 30-set periods as long as 10 min. Three types of data were scored: (i) activity as the number of times both front paws of a subject crossed either a horizontal or vertical division of the 3 x 6 grid; (ii) myoclonic jerks as the total number during the specified period of observation; and (iii) tonic-clonic convulsions by incidence in the kindled and control groups. Injections of the vehicle (i.p., 0.9% saline) or picrotoxin (0.5, 1.0, or 2.0 mglkg) were administered 20 min before testing. Picrotoxin (Sigma) was dissolved in the vehicle by sonication 25 to 45 min (time
170
KALICHMAN
AND BURNHAM TABLE
Picrotoxin-Induced
1
Convulsions in Kindled and Control Rats Dose of picrotoxin (mg/kg, i.p.)”
Type of convulsions
Group of rats
0
0.5
1.0
2.0
Myoclonic jerks’
Control (N = 7) Kindled (N = 7)’
0 0
0 0
0 2.9 2 1.4
2.6 k 2.2 14.3 k 4.3
Tonic-cloni&
Control (N = 7) Kindled (N = 7)
o/7 017
o/7 o/7
o/7 o/7
l/7 517
n Drug administered 20 min before testing. * Data represent the mean number for the observation period ? SE. c P < 0.01 (Kruskal-Wallis test, H(1) = 6.86, test of hypothesis that the number of myoclonic jerks observed in the kindled rats differed from that of the controls). d Data represent the number of subjects having tonic-clonic seizures per total number of subjects.
depended on the concentration). All injections were made up in volumes of 1 ml/kg. The testing schedule consisted of 8 days of habituation to the test box (l- or 2-day intervals) and 4 drug test days (2-day intervals). The drug injections were randomized such that each subject received each dose once, but every dose was given on each drug test day. The results are presented in Fig. 1 (locomotor activity) and Table 1 (convulsive activity). The locomotor behavior of all subjects decreased (P < 0.05) and the kindled and control groups did not differ significantly at any dose. However the slopes for the activity data were steeper for the kindled subjects than the controls (P < 0.05). Convulsive activity occurred in both groups, but the kindled rats were more susceptible to the picrotoxin seizures as measured by myoclonic jerks (1 to 2 mg/kg) and tonic-clonic convulsions (2 mg/kg). Convulsions similar to those seen in kindling (forelimb clonus, rearing) were observed occasionally, but were not seen in all subjects. Such convulsions usually occurred late in the seizure and were seen in both kindled and control subjects. The present study demonstrates long-lasting changes in the response of kindled rats to picrotoxin. The results of Liebowitz et al. (7) and Corcoran (unpublished data) might predict a decrease in sensitivity to picrotoxin. However, kindling resulted in increased sensitivity to picrotoxin convulsions and a steeper dose-response curve for inhibition of locomotion by picrotoxin. An increased convulsive responsiveness in kindled animals was described previously for pentylenetetrazol (14), alcohol withdrawal (lo), and lidocaine (11). Those reports attributed the
PICROTOXIN
AND
KINDLING
171
increased convulsant sensitivity to activation of the kindled focus because the convulsions were similar to the kindled convulsion (10, 11, 14). The present study did not support this hypothesis because the picrotoxininduced convulsions did not mimic kindled convulsions preferentially in the kindled group. However, the enhanced sensitivity to picrotoxin convulsions is consistent with a hypothesis of long-lasting deficiency in the GABA system which could be related to the increased epileptogenicity of the kindled subjects. The tests of locomotor activity also provide evidence for a change in sensitivity to picrotoxin, but this effect may be secondary to seizure activity induced by picrotoxin. The present experiments indicated a change in sensitivity to picrotoxin after kindling. However, the altered sensitivity to picrotoxin does not distinguish between a primary change (kindling as a result of changes in the GABA system) or a secondary change (GABA changes induced by the process of kindling). In addition, it is possible that the GABA system could be intact, but the changes occurred at synaptic sites subsequent or parallel to the immediate site of GABA’s action. We are currently testing some of the possibilities by using other GABA-modulating drugs on kindled rats. REFERENCES 1. ARNOLD, P. S., R. J. RACINE, AND R. A. WISE. 1973. Effects of atropine, reserpine, 6-hydroxydopamine, and handling on seizure development in the rat. Exp. Neurol. 40: 457-470. 2.
3. 4. 5. 6.
7. 8. 9. 10.
BABINGTON, R. G., AND P. W. WEDEKING. 1973. The pharmacology of seizures induced by sensitization with low intensity brain stimulation. Pharmacol. Eiochem. Behav. 1: 461-467. BURNHAM, W. M. 1978. Cortical and limbic kindling: similarities and differences. Pages 507-519 in K. E. LIVINGSTON AND 0. HORNYKIEWICZ, Eds., The Continuing Evolution of the Limbic System Concept. Plenum, New York. ENGEL, J., AND N. S. SHARPLESS. 1977. Long-lasting depletion of dopamine in the rat amygdala induced by kindling stimulation. Brain Res. 136: 381-836. FARIO, I. B., AND D. H. R. BLACKWOOD. 1978. Reduction in tyrosine hydroxylase activity in the rat amygdala induced by kindling stimulation. Brain Res. 153: 423-426. GODDARD, G. V., D. C. MCINTYRE, AND C. K. LEECH. 1%9. A permanent change in brain function resulting from daily electrical stimulation. Exp. Neural. 25: 295-330. LIEBOWITZ, N. R., T. A. PEDLEY, AND R. W. P. CUTLER. 1978. Release of gammaaminobutyric acid from hippocampal slices of the rat following generalized seizures induced by daily stimulation of entorhinal cortex. Brain Res. 138: 369-373. MCNAMARA, J. 0. 1978. Muscarinic cholinergic receptors participate in the kindling model of epilepsy. Brain Res. 154: 415-420. MELDRUM, B. S. 1975. Epilepsy and gamma-aminobutyric acid-mediated inhibition. Int. Rev. Neurobiol. 17: l-36. PINEL, J. P. J., P. H. VAN OOT AND R. F. MUCHA. 1975. Intensification of the alcohol withdrawal syndrome by repeated brain stimulation. Nature (London) 254: 510-511.
172
KALICHMAN
AND
BURNHAM
POST, R. M., K. M. SQUILLACE, W. SASS, AND A. PERT. 1977. Drug sensitization and electrical kindling. Sot. Neurosci. Abstr. 3: 204. 12. RACINE, R. J. 1972. Modification of seizure activity by electrical stimulation: II. Motor seizure. Electroenceph. Clin. Neurophysiol. 32: 281-294. 13. REEDY, D. P., E. G. MCGEER, W. A. STAINES, AND M. E. CORCORAN. 1978. Amygdaloid kindling and central enzyme activity. Sot. Neurosci. Absfr. 3: 146. 14. WADA, J. A., M. SATO, AND M. E. CORCORAN. 1974. Persistent seizure susceptibility and recurrent spontaneous seizures in kindled cats. Epilepsia 15: 465-478. 15. WILKISON, D. M., AND L. M. HALPERN. 1979. Turnover kinetics of dopamine and norepinephrine in the forebrain after kindling in rats. Neuropharmacology 18: 219-222. 11.
NEUROLOGY
70, 167- 172 (1980)
RESEARCH Locomotor MICHAEL
NOTE
and Convulsive Responses to Picrotoxin in Amygdala-Kindled Rats W. KALICHMANAND
Department of Pharmacology,
W. MCINTYREBURNHAM'
University of Toronto, Toronto, Ontario M5S lA8, Canada
Received December 6, 1979; revision received April 6, 1980 Amygdala-kindled rats and handled controls were tested for differences in locomotion and convulsive activity produced by picrotoxin, a specific synaptic antagonist of the putative neurotransmitter GABA. Compared with controls, the gross activity of kindled rats decreased more rapidly with dose (0.5 to 2.0 mglkg, i.p.) and kindled rats were more likely to convulse with the higher doses (1.0 to 2.0 mg/kg). These results suggest a functional deficit in the GABA system of kindled rats as measured by locomotion and convulsive activity.
Repeated low-level electrical stimulation of cortical or subcortical sites in the brain results in a process called “kindling” (3, 6, 12). Although the subject initially shows little or no response to the stimulus, repeated stimulation leads to progressively stronger epileptiform responses. Eventually the stimulus, which initially induced only a short local afterdischarge, elicits a generalized seizure with a clonic convulsion. This increased convulsive sensitivity has been shown to persist for months following kindling and appears to reflect permanent changes in brain function (6, 14). Several different approaches have been used to study the neurochemical and physiological bases for the kindling-related brain changes (1, 2, 4, 5, 7, 8, 10, 11, 13-15). One approach which may be particularly valuable is to observe the behavioral responses of kindled subjects which have Abbreviation: GABA-y-aminobutyric acid. * Supported by grant MT 5611 from the Medical Research Council of Canada to Drs. Bumham, K. E. Livingston, and R. Racine. Mr. Kalichman was supported by a Garfield Weston Foundation grant to Dr. Livingston. 167 OO14-4886/8O/lOO167-06$02OO/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
168
KALICHMAN
AND BURNHAM
been treated with drugs which have relatively well-understood mechanisms. Such a paradigm would make it possible to consider kindlinginduced differences in function, for example, of a given neurotransmitter. The present study used this approach to consider the possibility that the y-aminobutyric acid (GABA) system may be functionally different in animals which have been kindled. Liebowitz er al. (7) reported an increase in K+-sensitive GABA release in kindled rats. Assuming that GABA-mediated inhibition is antagonistic to epilepsy (9), an increased release of GABA would be expected to antagonize the kindling process. A hyperfunctional GABA system is further supported by Corcoran (unpublished observation), who found that kindled rats were less sensitive to convulsions induced by 3-mercaptoproprionic acid, a competitive inhibitor of GABA synthesis. On the other hand, in preliminary experiments we found that kindled animals were more sensitive to convulsions induced by a specific synaptic GABA antagonist, picrotoxin. This result is exactly opposite to that which might have been predicted based on the experiments with GABA release and 3-mercaptoproprionic acid. The objective of the present study was to consider further the possibility of a functional change in the GABA system induced by kindling. This was accomplished by comparing amygdala-kindled and control rat locomotion and convulsions after picrotoxin pretreatment. Fourteen male Royal Victoria Hooded rats (Canadian Breeding Laboratories) were used. Subjects (200 to 250 g) were implanted with twisted stainless-steel bipolar electrodes (250 pm diameter) using conventional stereotaxic methods. The electrodes were aimed at the right amygdala using the following coordinates: posterior and lateral to bregma, 1.0 and 4.5 mm; and down from the dura, 8.0 mm. One week after surgery, the rats were randomly divided into two groups. One group received the kindling stimulation and the other group was handled identically, but was not stimulated. Stimulation and handling took place twice daily. The stimulus consisted of a l-s train of 60-Hz, 1-ms diphasic pulses with an intensity of 250 PA peak-to-peak. Kindling stimulation was terminated after the occurrence of six bilateral forelimb clonic convulsions [stage 4 or greater (12)]. The kindled group of seven subjects achieved this criterion in an average of 14 days. Picrotoxin testing was begun 2 months after the completion of kindling. By 2 months, any transient effects of the last kindled seizure should be minimal. All tests consisted of a lo-min period during which two rats, one kindled and one handled, were simultaneously monitored for both locomotor and convulsive activity. The test chamber was a sound-attenuating box which contained two observation fields (each field approximately 40 x 70 cm) separated by a wooden barrier. The effects of extraneous
PICROTOXIN
169
AND KINDLING
90
0
80
control ( n=7)
70
z 8
60
cn F:
50
5 i= z
40
Y 2
30
2 20
IO
0 0
0.5
PICROTOXIN
1.0
(mg/kg
, 2.0
i.p.1
FIG. 1. Effect of picrotoxin on locomotor activity in amygdala-kindled and control rats. Activity decreased with dose in both groups (P < 0.05, analysis of variance). Kindled group activity decreased more rapidly with dose (P < 0.05, t test). Data are average activity scores * SE.
noise were masked by white noise (Noise Generator 901B, GrasonStadler). The floor of each observation field consisted of a 3 x 6 pattern of rectangles underneath a Plexiglas base. The kindled and control rats were placed on opposite sides of the barrier for the test. After 2 min of accomodation, data were scored for each of the rats during alternate 30-set periods as long as 10 min. Three types of data were scored: (i) activity as the number of times both front paws of a subject crossed either a horizontal or vertical division of the 3 x 6 grid; (ii) myoclonic jerks as the total number during the specified period of observation; and (iii) tonic-clonic convulsions by incidence in the kindled and control groups. Injections of the vehicle (i.p., 0.9% saline) or picrotoxin (0.5, 1.0, or 2.0 mglkg) were administered 20 min before testing. Picrotoxin (Sigma) was dissolved in the vehicle by sonication 25 to 45 min (time
170
KALICHMAN
AND BURNHAM TABLE
Picrotoxin-Induced
1
Convulsions in Kindled and Control Rats Dose of picrotoxin (mg/kg, i.p.)”
Type of convulsions
Group of rats
0
0.5
1.0
2.0
Myoclonic jerks’
Control (N = 7) Kindled (N = 7)’
0 0
0 0
0 2.9 2 1.4
2.6 k 2.2 14.3 k 4.3
Tonic-cloni&
Control (N = 7) Kindled (N = 7)
o/7 017
o/7 o/7
o/7 o/7
l/7 517
n Drug administered 20 min before testing. * Data represent the mean number for the observation period ? SE. c P < 0.01 (Kruskal-Wallis test, H(1) = 6.86, test of hypothesis that the number of myoclonic jerks observed in the kindled rats differed from that of the controls). d Data represent the number of subjects having tonic-clonic seizures per total number of subjects.
depended on the concentration). All injections were made up in volumes of 1 ml/kg. The testing schedule consisted of 8 days of habituation to the test box (l- or 2-day intervals) and 4 drug test days (2-day intervals). The drug injections were randomized such that each subject received each dose once, but every dose was given on each drug test day. The results are presented in Fig. 1 (locomotor activity) and Table 1 (convulsive activity). The locomotor behavior of all subjects decreased (P < 0.05) and the kindled and control groups did not differ significantly at any dose. However the slopes for the activity data were steeper for the kindled subjects than the controls (P < 0.05). Convulsive activity occurred in both groups, but the kindled rats were more susceptible to the picrotoxin seizures as measured by myoclonic jerks (1 to 2 mg/kg) and tonic-clonic convulsions (2 mg/kg). Convulsions similar to those seen in kindling (forelimb clonus, rearing) were observed occasionally, but were not seen in all subjects. Such convulsions usually occurred late in the seizure and were seen in both kindled and control subjects. The present study demonstrates long-lasting changes in the response of kindled rats to picrotoxin. The results of Liebowitz et al. (7) and Corcoran (unpublished data) might predict a decrease in sensitivity to picrotoxin. However, kindling resulted in increased sensitivity to picrotoxin convulsions and a steeper dose-response curve for inhibition of locomotion by picrotoxin. An increased convulsive responsiveness in kindled animals was described previously for pentylenetetrazol (14), alcohol withdrawal (lo), and lidocaine (11). Those reports attributed the
PICROTOXIN
AND
KINDLING
171
increased convulsant sensitivity to activation of the kindled focus because the convulsions were similar to the kindled convulsion (10, 11, 14). The present study did not support this hypothesis because the picrotoxininduced convulsions did not mimic kindled convulsions preferentially in the kindled group. However, the enhanced sensitivity to picrotoxin convulsions is consistent with a hypothesis of long-lasting deficiency in the GABA system which could be related to the increased epileptogenicity of the kindled subjects. The tests of locomotor activity also provide evidence for a change in sensitivity to picrotoxin, but this effect may be secondary to seizure activity induced by picrotoxin. The present experiments indicated a change in sensitivity to picrotoxin after kindling. However, the altered sensitivity to picrotoxin does not distinguish between a primary change (kindling as a result of changes in the GABA system) or a secondary change (GABA changes induced by the process of kindling). In addition, it is possible that the GABA system could be intact, but the changes occurred at synaptic sites subsequent or parallel to the immediate site of GABA’s action. We are currently testing some of the possibilities by using other GABA-modulating drugs on kindled rats. REFERENCES 1. ARNOLD, P. S., R. J. RACINE, AND R. A. WISE. 1973. Effects of atropine, reserpine, 6-hydroxydopamine, and handling on seizure development in the rat. Exp. Neurol. 40: 457-470. 2.
3. 4. 5. 6.
7. 8. 9. 10.
BABINGTON, R. G., AND P. W. WEDEKING. 1973. The pharmacology of seizures induced by sensitization with low intensity brain stimulation. Pharmacol. Eiochem. Behav. 1: 461-467. BURNHAM, W. M. 1978. Cortical and limbic kindling: similarities and differences. Pages 507-519 in K. E. LIVINGSTON AND 0. HORNYKIEWICZ, Eds., The Continuing Evolution of the Limbic System Concept. Plenum, New York. ENGEL, J., AND N. S. SHARPLESS. 1977. Long-lasting depletion of dopamine in the rat amygdala induced by kindling stimulation. Brain Res. 136: 381-836. FARIO, I. B., AND D. H. R. BLACKWOOD. 1978. Reduction in tyrosine hydroxylase activity in the rat amygdala induced by kindling stimulation. Brain Res. 153: 423-426. GODDARD, G. V., D. C. MCINTYRE, AND C. K. LEECH. 1%9. A permanent change in brain function resulting from daily electrical stimulation. Exp. Neural. 25: 295-330. LIEBOWITZ, N. R., T. A. PEDLEY, AND R. W. P. CUTLER. 1978. Release of gammaaminobutyric acid from hippocampal slices of the rat following generalized seizures induced by daily stimulation of entorhinal cortex. Brain Res. 138: 369-373. MCNAMARA, J. 0. 1978. Muscarinic cholinergic receptors participate in the kindling model of epilepsy. Brain Res. 154: 415-420. MELDRUM, B. S. 1975. Epilepsy and gamma-aminobutyric acid-mediated inhibition. Int. Rev. Neurobiol. 17: l-36. PINEL, J. P. J., P. H. VAN OOT AND R. F. MUCHA. 1975. Intensification of the alcohol withdrawal syndrome by repeated brain stimulation. Nature (London) 254: 510-511.
172
KALICHMAN
AND
BURNHAM
POST, R. M., K. M. SQUILLACE, W. SASS, AND A. PERT. 1977. Drug sensitization and electrical kindling. Sot. Neurosci. Abstr. 3: 204. 12. RACINE, R. J. 1972. Modification of seizure activity by electrical stimulation: II. Motor seizure. Electroenceph. Clin. Neurophysiol. 32: 281-294. 13. REEDY, D. P., E. G. MCGEER, W. A. STAINES, AND M. E. CORCORAN. 1978. Amygdaloid kindling and central enzyme activity. Sot. Neurosci. Absfr. 3: 146. 14. WADA, J. A., M. SATO, AND M. E. CORCORAN. 1974. Persistent seizure susceptibility and recurrent spontaneous seizures in kindled cats. Epilepsia 15: 465-478. 15. WILKISON, D. M., AND L. M. HALPERN. 1979. Turnover kinetics of dopamine and norepinephrine in the forebrain after kindling in rats. Neuropharmacology 18: 219-222. 11.