Brain Research &&in, Vol. 30, pp.597405, 1993 Printed in the USA. All rights reserved.
0361-9230193 $6.00 + .oO Copyright 0 1993 Pergamon Press Ltd.
Clozapin~ I~ibits Limbic System ~ndling: Implications for Antipsychotic Action SHELLY R. GRAHAM
AND LARRY KOKKINIDIS’
Department of Psychology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 0 WO, Canada Received 4 May 1992; Accepted 12 August 1992 S. AND L. KOKKINIDIS. Clozapine inhibits limbic system kindling: Implications for antipsychotic uction. BRAIN RES BULL 30(5/6) 597-605, 1993.-Clozapine and haloperidol were tested for their ability to influence the acquisition of kindled seizures following electrical stimulation of the amygdala and ventral hippocampus. While haloperidol pretreatment did not alter kindling genesis from either limbic region, preexposure to clozapine delayed the rate at which kindling evolved. Analysis of the number of seizure behaviors expressed during ep~lept~enesis revealed that clozapine produced a relative an~gonism of seizure development arresting kindling at the stage-3 level. The kindling inhibition was dependent upon the daily administration of clozapine during the kindling process and was not evident afier withdrawal from a chronic schedule of clozapine exposure. A subconvulsive dose of pilocarpine (80.0 me/kg) produced an overall enhancement of kindling rate, a finding consistent with the excitatory role of acetylcholine (ACh) in kindling. Lower doses of pilocarpine (20.0 and 40.0 mg/kg) that did not alter seizure advancement partially antagonized the clozapine-elicited inhibition of amygdaloid kindling. Pilocarpine, however, did not affect the clozapine-induced increase in the number of stage-3 behaviors exhibited during amygdaloid kindling, suggesting that other neur~hemi~l effects of clozapine, not related to its anticholiner~c properties, modulate the kindling suppression. Clozapine’s unique actions on limbic system sensitization were discussed in relation to its effectiveness as an antipsychotic agent. GRAHAM,
Clozapine Pilocarpine
Haloperidol Dopamine
Kindling Amygdala Acetylcholine
Hippocampus
HALOPERI~L is a widely prescribed neuroleptic drug used in the treatment of schizophrenia. In addition to its antipsychotic actions, this classic neuroleptic causes extrapyramidal motor side effects and can result in the development of tardive dyskinesia (5,lO). Clozapine, an atypical neuroleptic, has become an increasingly popular choice as a drug therapy for schizophrenia because it produces minimal motor disturbances (1 I ,23,33) and is more eff&ctive as an ~tipsychotic agent than the typical neuroleptics ( f&19). Evidence from electrophysiological studies suggests that the antipsychotic properties of classic and atypical neuroleptics involve their ability to influence dopamine (DA) functioning in the mesolimbic system. Acute administration of haioperidol and clozapine enhance DA neuronal firing rates in the A 10 cell body region of the ventral tegmental area (VTA), ultimately resulting in a depolarization inactivation of these neurons after repeated drug exposure (7,15,44). Because both classes of neuroleptics are able to silence firing patterns of VTA neurons, it would appear that other characteristics of clozapine treatment, not shared by the typical neuroleptics, are responsible for its efficacy in the treatment of schizophrenia (26). Kindling refers to the genesis of epileptiform events elicited by repeated low-intensity electrical stimulation of limbic structures (14). With continued intermittent stimulation, kindling acquisition entails the progressive development of electrophys-
iological and behavioral seizure activity through five defined stages, culminating in bilateral clonic convulsions (30). It has been proposed that schizophreniform symptoms associated with temporal lobe epilepsy (36,37,41) evolve via a kindling-like mechanism (6,39). One aim of this research was to evaluate and compare the effects of clozapine and haloperidol pretreatment on kindling of the amygdala and ventral hipp~ampus (Experiment I), and a second objective was to assess further some of the characteristics of clozapine’s actions on amygdaloid kindling (Experiments 2 and 3). Additional experiments were conducted to evaluate the role of clozapine’s anticholinergic properties (32) on the drug’s capacity to influence kindling development (Experiments 4 and 5). METHOD
Experiment 1: Effects ofClozapine and Haloperidol on Amygdaloid and Ventral Hippocumpal Kindling Fifty-six naive, male Wistar rats (approximately 250 g) were individually housed under standard laboratory conditions and permitted free access to food and water throu~out the duration of the experiment. Animals were maintained on a 12L: 12D cycle and behavioral testing was conducted during the light portion of the cycle. Subjects were anesthetized with sodium pentobarbital (60.0 mg/kg) and a bipolar nichrome stimulating electrode
’ To whom requests for reprints should be addressed.
597
598
GRAHAM 50
KOKKINIDIS
At the termination of the experiment, subjects were deeply anesthetized with sodium pentobarbital and perfused intracardially with saline followed by 10.0% formalin. Brain sections (40 pm) were stained with thionine and electrode placements were verified.
40 [i
Experiment 2: IZfects of‘ Withdruwui From Chronic Clozapine Treutment on Amygdaloid Kindling
AMYGDALA
@I HIPPOCAMPUS
Eighteen naive, male Wistar rats served as subjects in this experiment. All particulars concerning housing, surgical, histological, ADT, and kindling procedures were similar to those described in Experiment 1. Animals were implanted with a bipolar stimulating electrode in the central nucleus of the amygdala. Following a 7-day recovery period, one half the animals received two daily IP injections of clozapine (10.0 mg/kg) 12 h apart for I4 consecutive days, while the remaining half was treated with saline. Twenty-four hours after the last clozapine treatment, AD thresholds were determined for each animal, and these current intensities were used for the subsequent kindling phase of the experiment. No drug treatments were administered during the kindling procedure.
30
20
AND
4
10
Exprrimmt 3: E&cts of' Ckzapine on Established A mygduloid Seizures
0 SALINE
HALOPERIDOL
CLOZAPINE
DRUG TREATMENT FIG. I. The effects of preexposure to saline, haloperidol (1 .O mg/kg). and clozapine (10.0 mg/kg) on the mean number (+SEM) of afterdischarges (ADS) necessary to elicit a stage-5 kindled seizure after daily electrical stimulation of the amygdala and ventral hippocampus. Administration of clozapine 30 min prior to each kindling stimulation inhibited kindling acquisition from both limbic regions.
Eighteen naive. male Wistar rats were stereotaxically implanted in the central amygdaloid nucleus with a bipolar stimulating electrode. AD thresholds were determined for each subject and these current intensities were used to kindle animals to two consecutive stage-5 seizures. The kindling procedure was continued and 30 min prior to each daily amygdaloid stimulation
4o (MS-303/ 1, Plastics One, Roanoke, VA) was implanted in either the central nucleus of the amygdala (AP, -0.5 mm from bregma; L, +4.5 mm from the midline suture; and V, -8.5 mm from the skull surface) or the CA I region of the ventral hippocampus (AP, -3.2 mm from bregma; L, +5.2 mm from the midline suture; and V. -8.0 from the skull surface). Electrodes were implanted perpendicular to the horizontal plane and the incisor bar was adjusted for each animal such that the horizontal plane was level for the posterior and anterior portions of the skull. A jeweler’s screw anchored in the frontal pole of the skull served as an indifferent lead during electroencephalographic (EEG) recording. Following a 7-day postsurgery recovery period, the minima1 electrical current intensity necessary to elicit an afterdischarge (AD) from these limbic regions was determined 30 min after IP injection of either saline, haloperidol ( 1.O mg/kg), or clozapine (10.0 mg/kg). Subjects were placed in a clear Plexiglas box (20 X 10 X 20 cm) and electrical stimulation provided from a constant-current stimulator (monophasic square wave, 0. I-ms pulse duration, 100 Hz, 2-s pulse train) was presented starting at 5 FA [root mean square (rms)] and progressively increased in ~-PA increments each minute until an AD was observed. Each individual animal’s AD threshold (ADT) current was used during the kindling procedure, which involved a single stimulation administered daily until a stage-5 generalized rearing and falling seizure developed. The kindling procedure was initiated 24 h after the ADT trial and 30 min prior to each daily kindling stimulation rats were treated with either haloperidol (I .O mg/kg), clozapine (10.0 mg/kg), or saline.
AMYGDALA
35
30
25 n
20
STAGE
1
STAGE
2
STAGE
3
‘I STAGE
4
15
10
SALINE
HALOPERIDOL
CLOZAPINE
DRUG TREATMENT FIG. 2. Mean number (*SEM) ofstage- I, -2, -3, and -4 seizure behaviors expressed during amygdaloid kindling after pretreatment with saline, haloperidol (1 .O mg/kg). and clozapine (10.0 mg/kg). Animals receiving clozapine spent more time at the stage-3 level of seizure acquisition.
CLOZAPINE,
HALOPERIDOL,
4o
’ HIPPOCAMPUS
n :
be summarized as follows: saline-saline, saline-pilocarpine (20.0 mg/kg), saline-pilocarpine (40.0 mg/kg), clozapine-saline, clozapine-pilocarpine (20.0 mg/kg), and clozapine-pilocarpine (40.0 mg/kg). Thirty minutes after the second injection, ADTs were determined for each animal. The ADT current was used for the kindling procedure, which began 24 h after the ADT test. Kindling stimulation was administered once daily 30 min after the second drug treatment until a stage-5 rearing and falling generalized seizure was observed.
STAGE
1
H STAGE
2
1-1 STAGE
3
II
4
STAGE
SALINE
RESULTS
Experiment I: Effects of Clozapine and Haloperidol on Amygdaloid and Hippocampal Kindling
HALOPERIDOL
CLOZAPINE
DRUG TREATMENT FIG.
3. Effects of saline, haloperidol (1 .O mg/kg), and clozapine ( 10.0
mg/kg) preexposure on the mean number (SEM) of stage-l, -2, -3, and -4 seizure behaviors expressed during kindling of the ventral hippocampus. Clozapine pretreatment increased the number of stage-3 behaviors exhibited during kindling.
one half the subjects received an IP injection of clozapine (10.0
mg/kg) and the remaining drug pretreatment/kindling stage-5 seizure developed.
599
AND KINDLING
half was administered saline. The schedule proceeded until a third
Experiment 4: Effects of Pilocarpine on Amygdaloid Kindling
Forty naive, male Wistar rats were implanted with a bipolar nichrome stimulating electrode in the central nucleus of the amygdala. Seven days postoperatively, ADTs were determined after IP injection of either saline or one of three doses of pilocarpine nitrate salt (20.0,40.0, and 80.0 mg./kg). Each individual animals’s ADT current was used during the kindling procedure, which was initiated 24 h after the ADT test. Animals were pretreated with either saline, 20.0, 40.0, or 80.0 mg/kg pilocarpine and 30 min after drug injection the amygdala was stimulated. This procedure was continued daily until a stage-5 seizure was observed. Experiment 5: Clozapine-pilocarpine of the Amygdala
Interaction and Kindling
Sixty naive, male Wistar rats were implanted with a bipolar stimulating electrode into the central nucleus of the amygdala. One week later, half the animals were administered an IP injection of saline and the remaining animals were treated with clozapine (10.0 mg/kg). Immediately after drug administration, animals in each drug group were subdivided and received a second injection of either saline, 20.0, or 40.0 mg/kg pilocarpine nitrate salt. The doses for pilocarpine were determined from the results of Experiment 3. The six drug treatment conditions can
A one-way analysis of variance (ANOVA) of the number of ADS that elicited a class-5 motor seizure was conducted separately for the amygdaloid and hippocampal kindling data (Fig. 1). In both cases, a significant drug treatment effect was evident for the rate at which stage-5 kindled seizures evolved, F(2, 24) = 25.26 and 22.96, p < 0.000 1 (for kindling of the amygdala and hippocampus, respectively). Newman-Keuls ((u = 0.05) multiple comparisons of the drug treatment means revealed that clozapine significantly increased the number of ADS required for the expression of a stage-5 seizure following repeated stimulation of these limbic sites. As can be seen in Fig. 1, haloperidol pretreatment did not modify kindling acquisition from either limbic region. Racine (30) described five levels of seizure development during kindling, characterized by rhythmic facial movements (stage l), headbobbing (stage 2), bilateral forelimb clonus (stage 3), bilateral forelimb clonus and rearing (stage 4), and convulsions with the loss of postural control (stage 5). ANOVAs of the number of class-l, -2, -3, and -4 behaviors expressed during amygdaloid (Fig. 2) and ventral hippocampal (Fig. 3) kindling yielded a significant drug treatment effect for stage-3 behaviors, F(2,24) = 22.49, p < 0.0001 (amygdala); F(2, 20) = 34.19, p < 0.0001 (hippocampus). Posthoc comparisons showed that animals subjected to clozapine preexposure exhibited more stage-3 seizure behaviors relative to haloperidol and control subjects from both stimulation sites, indicating a relative antagonism of kindling development (8). The effects of drug treatments on AD thresholds, AD durations, latency to clonus, and clonus durations are shown in Table 1. Neither clozapine nor haloperidol pretreatment influenced these seizure-related variables after kindling of the amygdala. However, a significant drug treatment effect was observed for hippocampal kindling with respect to the overall AD, F(2, 24) = 4.01,~ < 0.05, and clonus, fl2,24) = 7.96,~ <: 0.01, durations. Subsequent posthoc comparisons found these variables to be significantly enhanced following clozapine treatment. Experiment 2: Eflects of Withdrawal From Chronic Clozapine on Amygdaloid Kindling
Analysis of the AD current thresholds found no significant effects as a result of long-term exposure to clozapine (65.6 + 5.8 and 58.9 f 9.9 for the chronic saline and clozapine groups, respectively). As well, drug treatment effects were not evident with respect to kindling rate. The mean (+SEM) number of ADs necessary to elicit a stage-5 generalized seizure after withdrawal from chronic clozapine treatment was 12.4 f 2.1 as compared to 11.9 + 2.3 for the saline-treated group. Clozapine withdrawal also did not influence the latency to clonus, AD, and clonus duration data.
600
GRAHAM
AND KOKKINIDIS
TABLE 1 EFFECTS OF SALINE (SAL). HALOPERIDOL (HAL), AND CLOZAPINE (CLOZ) PRETREATMENT ON THE MEAN (+SEM) AD THRESHOLDS, AD DURATIONS, CLONUS LATENCIES, AND CLONUS DURATIONS DURING KINDLING OF THE AMYGDALA
AND
VENTRAL
HIPPOCAMPUS
Latency to Cionus (seconds)
Clonus Durations
(-+6.8) 72.9 (k6.3)
3.9 (k2.5) 1.4 (iO.6) 3.3 (+ 1.O)
39.9 (k3.8) 28.4 (i3.1) 35.9 (52.4)
44.2 (k7.5) 59.3 (112.3) 83.9* (29.7)
2.6 (50.8) .31 .._ (il.7) 2.8 (lrO.5)
22.6 (?2.8) 24.7 ii2.4) 34.8* (+l,5f
ADT
AD Durations
n
(PA)
(seconds)
SAL
9
66.1 (*I 1.0)
61.7 (28.2)
HAL
9
62.2
59. I
9
(16. I) 57.8 (?I 1.5)
78.9 (+10.1) 93.3 (*I 1.5) 87.8 (i 10.5)
(seconds)
Amygdala
CLOZ
Hippocampus SAL
9
HAL
9
CLOZ
9
* p < 0.05.
Group differences were not evident with respect to the rate of kindling to two consecutive stage-5 generalized seizures prior to the initiation of drug treatment. The mean (+_SEM) number of ADS for the two groups were 23.6 2 3.8 and 2 1.4 _t 2.0. Figure 4 shows the mean @EM) number of ADS to a third stage-5 seizure following clozapine and saline pretreatment. Animals preexposed to clozapine required significantly more ADS to exhibit a third generalized motor seizure, t( 16) = 4.5, p < 0.05. Per-stage analysis of the number of seizure behaviors expressed during continued amygdaloid stimulation indicated that clozapine-treated animals regressed in terms of seizure stage development and exhibited significantly more stage-3. f( 16) = 4.0, p < 0.05, and stage-4, t( 16) = 3.0, p < 0.05, seizure behaviors than control animals (see Fig. 5). No significant effects were evident with respect to the latency to clonus, AD, and clonus duration data as a function of clozapine treatment,
One-way ANOVA showed that animals exposed to pilocarpine required fewer ADS to reach a stage-5 seizure, F(3. 36) = 3.18, p < 0.05. Newman-Keuls multiple comparisons (a = 0.05) indicated that only the 80.0-mg/kg dose of pilocarpine accelerated kindling rate (see Fig. 6). ANOVA of the number of seizure stage behaviors expressed during the kindling process found no significant changes following pilocarpine pretreatment, indicating that pilocarpine (80.0 mg/kg) administration resulted in an overall acceleration of kindling evolution. The seizure-related variables as a function of pilocarpine treatment are shown in Table 2. A significant drug treatment effect was observed for AD durations, F(3, 36) = 4.3 1, p < 0.05. Multiple comparisons of the drug dose means revealed that 80.0 mg/kg pilocarpine decreased AD durations relative to control values.
A 2 X 3 (clozapine X pilocarpine) ANOVA of the stage-5 AD data yielded a significant clozapine X pilocarpine interaction, 92, 53) = 4.70, p < 0.05. Newman-Keuls multiple comparisons ((u = 0.05) revealed that clozapine administration significantly increased the number of ADS required to induce the first stage5 seizure. While pilocarpine pretreatment (20.0 and 40.0 mg/ kg) did not influence kindling evolution in saline-treated subjects, both doses of pilocarpine partially antagonized the suppressing effects of clozapine. The antagonism was not complete, however, and kindling still progressed at a slower rate after pilocarpine administration to clozapine pretreated animals relative to control subjects (see Fig. 7). Consistent with the results of Experiment I, analysis of the number of stage-3 behaviors expressed during kindling found that animals subjected to clozapine treatment exhibited significantly more stage-3 behaviors, F( 1, 53) = 141.04, p < 0.001 (main effect for clozapine) irrespective of pilocarpine treatment (see Fig. 8). ANOVA of the stage-4 data yielded a significant clozapine X pilocarpine interaction. F(2, 53) = 6.42. p < 0.01. As can be seen in Fig. 9, animals preexposed to clozapine exhibited a greater number of stage-4 seizure behaviors relative to control subjects. While a trend for clozapine to increase stage4 behaviors was observed in Experiment 1, this effect did not reach statistical significance. Coadministration of 20.0 mg/kg piloca~ine partially reduced the clozapine effect, and the incidence of stage-4 seizure activity following injection with the 40.0mg/kg dose of pilocarpine in clozapine-pretreated animals was similar to that seen in the control group. These results suggest that pilocarpine affects the clozapine-induced inhibition of kindling rate primarily by reducing the amount of time animals spend in stage-4 kindling. The seizure-related data are depicted in Table 3. Pilocarpine was observed to shorten AD durations, F(2. 52) = 2.5 1, p < 0.05. In the previous experiment involving the dose-response effects of pilocarpine, this result was evident after administration of
CLOZAPINE.
HALOPERIDOL,
601
AND KINDLING
SALINE
CLOZAPINE
DRUG TREATMENT FIG. 4. Mean number of afterdischarges (ADS) (KSEM) necessary to elicit a third stage-5 seizure from animals kindled to two consecutive stage-5 seizures following daily pretreatment with saline and clozapine (10.0 mg/kg). Clozapine administration significantly inhibited the development of a third stage-5 seizure.
iform region, whereas stage-4 and -5 convulsions involve the recruitment of brain stem mechanisms necessary for the expression of generalized seizures (8). Because, for the most part, kindling is arrested at the stage-3 level, it would appear that clozapine affects the mechanisms responsible for the advancement of kindling-associated behaviors related to the spread of epileptiform events from the forebrain to brainstem regions. In addition to delaying kindling evolution, clozapine was found to significantly increase the number of ADS required for a third rearing and falling seizure in animals already kindled to two consecutive stage-5 convulsions and provoked a regression in seizure stage development. These results suggest that although the neural circuitry responsible for generalized seizures has been established clozapine has the capability to retard the appearance of a subsequent seizure, indicating a possible anticonvulsant action. However, the results concerning the effects of clozapine on seizure-associated variables are not in agreement with this conclusion. Many clinically effective anticonvulsant drugs affect the rate of kindling as well as altering behavioral and EEG activity characteristic of seizure maintenance. For instance, administration of phenobarbital and valproic acid inhibit kindling evolution and decrease alterdischarge durations (2 1,42). Also, clonic motor seizures elicited by amygdaloid stimulation are suppressed by these anticonvulsant agents (2). While AD and clonus durations related to amygdaloid kindling were not influenced by clozapine treatment, they were increased during hippocampal kindling, an observation consistent with the proconvulsant properties of clozapine in a clinical setting (33). The finding that clozapine delays amygdaloid kindling without decreasing AD activity or affecting AD thresholds and aug-
20 18 j 16
80.0 mg/kg pilocarpine with a tendency to decreased AD durations after injection of the lower doses (see Table 1). It is noteworthy that the decrease in AD durations seen after administration of the 20.0- and 40.0-mg/kg doses of pilocarpine was not accompanied by a corresponding increase in kindling rate (see the Discussion section). DISCUSSION
The major finding of this study was that in contrast to haloperidol, which did not influence the rate at which amygdaloid and ventral hippocampal kindling evolved, clozapine pretreatment significantly delayed the development of limbic system kindling. To determine whether the clozapine-induced inhibition of kindling is dependent upon the daily administration of the drug prior to each kindling stimulation or is the outcome of neurochemical changes associated with long-term drug treatment, rats were exposed to the kindling procedure after withdrawal from chronic clozapine administration. The results of Experiment 2 showed that kindling of the amygdala after cessation of a chronic schedule of clozapine progressed normally, suggesting that the acute effects of daily clozapine pretreatment are necessary to retard kindling genesis. From our data, it seems that clozapine produces a relative antagonism of kindling development at the transitional stage-3 level of development. Class-3 seizures entail stimulation siteindependent forebrain circuitry primarily in the amygdala-pyr-
STAGE
3
1:; STAGE
4
14 12
!
10
8
64
SALINE
CLOZAPINE
DRUG TREATMENT FIG. 5. Effects of saline and clozapine (10.0 mg/kg) administration on the mean number (fSEM) of stage-3 and -4 seizure behaviors expressed during amygdaloid kindling when drug treatment commenced aRer the development of two stage-5 kindled convulsions. A regression to stage3 and -4 seizures was observed in clozapine-pretreated animals.
602
GRAHAM
AND
o
KOKKINIDIS
SALINE CLOZAPINE
SALINE
PILO
20
PILO
40
PILO
SO
SALINE
PILO
20
PILO
40
DRUG TREATMENT
DRUG TREATMENT FIG. 6. Effects of saline and pilocarpine (PILO 20.0.40.0, and 80.0 mg/ kg) on the mean (HEM) number of afterdischarges to a stage-5 seizure during kindling of the amygdala. The 80.0-mg/kg dose of pilocarpine increased kindling rate.
FIG. 7. Effects of saline, clozapine (10.0 mg/kg), and pilocarpine (PILO 20.0 and 40.0 mg/kg) on the mean (?SEM) number of afterdischarges to a stage-5 seizure during amygdaloid kindling. Both doses of pilocarpine partially antagonized the clozapine-induced inhibition of kindling acquisition.
hippocampal AD durations is not entirely surprising. It is known that the progression of kindled seizures rely upon the appearance of an AD, but ADS are not required for stimulation-induced threshold changes (29) and the development of motor seizures are not the result of kindling-induced increases in AD durations (30). Thus, many manipulations can alter kindling genesis independently of changes in these seizure-affiliated variables. For example, microinjection of the GABA agonist muscimol into the amygdala suppresses the expression of kindled seizures without modifying AD durations (4). Further, electrical
stimulation of the locus coeruleus (LC) elicits a pronounced inhibitory effect on amygdaloid kindling, yet the increases in AD durations typically seen as a function ofthe kindling process were unaffected by LC stimulation ( 17). Electrophysiological experiments have shown a selective enhancing effect for clozapine on single-unit firing rates in the amygdala that is accentuated after repeated drug treatment (3). This finding, coupled with the observed inability of haloperidol to affect amygdaloid kindling, suggests that the distinctive properties of clozapine on limbic system functioning involve mech-
menting
TABLE
2
EFFECTS OF PILOCARPINE (PIL) ADMINISTRATION (20.0, 40.0, AND 80.0 @kg) AND SALINE (SAL) ON AD THRESHOLDS, AD DURATIONS, CLONUS LATENCIES. AND CLONUS DURATIONS DURING AMYGDALOID KINDLING
Group
n
SAL
10
PIL 20 PIL 40
IO IO
AD7 (@A)
10
Clonus Durattons (seconds)
66.0
62.7
I.7
25.7
(k6.5)
(k0.5)
(42.0)
49.5
52.3
5.4
25.9
(k4.6)
(k7.0)
(12.4)
(42.7)
64.0 52.5 (k5.0)
* p < 0.05.
Latency to Clonus (seconds)
(t7.2)
(+I 1.4) PIL 80
AD Durations (seconds)
42.8
1.9
24.3
(k5.5)
(k0.4)
(t2.7)
34.5* (k3.7)
3.2
24.6
(21.8)
(k2.6)
CLOZAPINE,
HALOPERIDOL,
25 q
T
SALINE CLOZAPINE
20
51 0
IfSALINE
2 PILO 20
PILO 40
DRUG TREATMENT FIG. 8. Mean (+SEM) number of stage-3 seizure behaviors expressed during kindling of the amygdala as a function of saline, clozapine (10.0 mg/kg), and pilocarpine (PILO 20.0 and 40.0 mg/kg) administration. Clozapine pretreatment increased the number of stage-3 behaviors expressed during amygdaloid kindling, an effect that was not modified by coadministration of pilocarpine.
of its actions on DA neural dynamics. Moreover, the haloperidol results further indicate that DA is not a necessary requisite for kindling genesis in the rat (20). While earlier research found pretreatment with low doses of haloperidol to increase kindling rate (I 3) this effect is more congruent with haloperidol’s influence on norepinephrine synapses than DA activity (28). It is known that ACh has an excitatory role in kindling acquisition (9) and amygdaloid kindling is delayed by systemic administration of the antimuscarinic scopolamine (20,43). In agreement with these reports, we found a subconvulsive dose of the muscarinic agonist pilocarpine to increase the rate at which amygdaloid kindling develops. Low doses of pilocarpine that did not influence epileptogenesis significantly modified the inhibitory consequences of clozapine on kindling acquisition. However, the pilocarpine effect was incomplete and animals continued to kindle at a slower rate following clozapine preexposure. Moreover, coadministration of pilocarpine did not alter the relative antagonism produced by clozapine administration, suggesting that other neurochemical consequences of clozapine, not related to its anticholinergic actions, are responsible for the kindling inhibition. In addition to clozapine’s DA and ACh receptor antagonist properties, this atypical neuroleptic affects a variety of neurotransmitters (25,26,32). A potentially important effect of clozapine with respect to kindling acquisition involves changes in excitatory amino acid (EAA) functioning. While classic and atypical neuroleptics have been shown to decrease glutamate release in the amygdala (34,35), clozapine was found to be subanisms that are independent
603
AND KINDLING
stantially more effective than haloperidol in displacing [3H]MK80 1 binding to glutamate receptors in the striatum (22). Given the importance of EAA activity in kindling genesis (9), and the capability of the noncompetitive NMDA antagonist MK-80 1 to retard kindling development (27), the prospect that clozapine’s antiglutamate effects might be involved in arresting the kindling process needs to be explored. GABA might also play a role in clozapine’s ability to suppress limbic system kindling. Using in vivo microdialysis, Drew et al. ( 12) found acute administration of clozapine to increase GABA release in the ventral striatum. While this effect was specific to the fundus striati and clozapine’s influence on limbic system GABA remains to be determined, our results indicate that the acute actions of daily clozapine administration are necessary for its antikindling properties. The significance of reduced GABA activity in kindling genesis is well documented (4,27), and the possibility that enhanced GABA neurotransmission is involved in the clozapine-induced inhibition of kindling also requires investigation. Considerable interest has focused on limbic system EEG abnormalities underlying the development of schizophrenia (1,38,40). Heath (I 6) for example, found spiking in several limbit and related regions, including the septal nuclei, nucleus accumbens, amygdala, hippocampus, and olfactory bulb, in individuals with psychosis. It is known that kindling results in a restructuring of temporal lobe circuitry (24,3 l), and such a reorganization might be a mechanism by which the cognitive repercussions of limbic system dysfunction evolve. Given the compelling clinical evidence that clozapine is effective in the treatment of neuroleptic-resistant schizophrenia (18,19,33), the
15 -
12 -
q q
SALINE CLOZAPINE
9-
6-
T
SALINE
PILO 20
PILO 40
DRUG TREATMENT FIG. 9. Mean (BEM) number of stage-4 behaviors expressed during amygdaloid kindling after saline, clozapine (10.0 mg/kg), and pilocarpine (PILO 20.0 and 40.0 mg/kg) pretreatment. Clozapine increased the number of stage-4 behaviors during amygdaloid kindling, and this effect was partially inhibited by coadministration of 20.0 mg/kg pilocarpine and antagonized after injection of 40.0 mg/kg pilocarpine.
604
GRAHAM TABLE
AND
KOKKINIDIS
3
EFFECTS
OF SALINE (SAL), CLOZAPINE (CLOZ), AND PILOCARPINE (PIL) (20.0 AND 40.0 n&g) ON AD THRESHOLDS, AD DURATIONS, LATENCY TO CLONUS, AND CLONUS DURATIONS DURING KINDLING OF THE AMYGDALA
Group
SAL-SAL PIL-SAL 20 PIL-SAL 40 CLOZ-SAL CLOZ-PIL 20 CLOZ-PIL 40
n 10
9 10 10 10 10
ADT (PA)
AD Durations (seconds)
76.7 (k7.0) 46.5* (k3.6) 44.6* (k4.7) 64.1 (24.9) 52.1 (k4.9) 57.9 (k4.6)
45.5 (6.1)
48.5 (k5.4) 42.0 (k13.3) 51.0 (+-16.1) 48.3 (k16.1) 41.5 (k13.1)
Latency to Clonus (seconds)
2.1 (il.])
2.1 (kO.7) 2.4 (k3.5) 2.9 (kO.7) 1.6 (kO.4) 3.5 (?0.5)
Clonus Durations (seconds)
26. I
(t2.9) 28.0 (k2.3) 25.5 (t2.6) 34.6 (iO.9) 32.8 (k3.4) 28.0 (?2. I)
* p < 0.05.
results of this study raise the possibility that the efficacy of clozapine as an antipsychotic agent may lie in its unique ability to inhibit mechanisms involved limbic system sensitization.
in the behavioral
expression
of
ACKNOWLEDGEMENTS
This research was supported by Natural Sciences and Engineering Research Council of Canada Grant A70420 to L.K. Appreciation is extended to Sandoz for their gift of clozapine.
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