Characterization of lithium potentiation of pilocarpine-induced status epilepticus in rats

EXPERIMENTAL

NEUROLOGY

g&471-480

(1986)

Characterization of Lithium Potentiation of PilocarpineInduced Status Epilepticus in Rats RICHARD

S. JOPE, RICHARD

A. MORRISETT,

AND 0. CARTER

SNEAD’

Departments of Pharmacology and Pediatrics and Neuroscience Program, University of Alabama at Birmingham, Birmingham, Alabama 35294 Received August 19, 1985; revision received October

23, 1985

Subcutaneous administration of pilocarpine to rats that were pretreated with a small dose of lithium chloride results in the evolution of generalized convulsive status epilepticus. The production of status epilepticus is absolutely reproducible, has a very consistent time to onset (22 min), has a duration of several hours, and is extremely severe with a high mortality rate. Experimental results show that this animal model of status epilepticus: (i) requires activation of muscarinic receptors because the initiation of seizures is blocked by atropine; (ii) requires presynaptic cholinergic activity because it is attenuated by hemicholinium-3; (iii) recruits noncholinergic cells because when status epilepticus is established it is not altered by atropine administration; and (iv) is blocked by pretreatment with diazepam and ongoing seizures are terminated by administration of diazepam, similar to certain forms of status epilepticus in humans. The reproducibility, prolonged nature, and involvement of a clearly defined neurochemical systemas the triggering mechanism, i.e., cholinergic activation, makes this a potentially valuable animal model of generalized convulsive status epilepticus. 0 1986 Academic Press. Inc.

INTRODUCTION Although the cholinergic system has not traditionally been emphasized in studies of epilepsy (19), recently there has been a resurgence of interest in cholinergic mechanisms in animal models of epilepsy, both as a kindling agent and directly as a convulsant. Carbachol, a cholinergic agonist, is now widely used to kindle animals (5,24,25) and two groups of investigators have Abbreviation: HC-3-hemicholinum-3. ’ The authors thank Helen Stephens for excellent technical assistance and Beverly Callens for manuscript preparation. This work was supported by a grant from the Epilepsy Foundation of America and by National Institutes of Mental Health grant 38752. 471 0014-4886/86 $3.00 Copyright 0 1986 by Academic Press, Inc. All rigbu of reproduction in any form nw-wd.

472

JOPE, MORRISETT,

AND SNEAD

shown that there is transfer between chemical (cholinergic agonist) and electrical kindling ( 1,26). Limited studies of genetic models of epilepsy in animals also indicate altered cholinergic metabolism in some instances (10). Earlier studies indicating that cholinergic agonists are convulsants when administered in large doses ( 19) have been followed by a series of detailed studies by Turski and his collaborators who have shown that both peripheral and central administration of cholinergic agonists produce limbic seizures accompanied by widespread brain damage (2 l-23). Olney et al. ( 16) showed that intraamygdaloid injection of cholinergic agonists or cholinesterase inhibitors produce seizure-brain damage syndrome and suggested that choline& mechanisms may play an important role in human epilepsy. These studies indicate a need for further elucidation of the interactions between activation of the cholinergic system and seizures. Honchar et al. (9) recently made the intriguing observation that treatment of rats with doses of lithium roughly equivalent to those used therapeutically in patients with affective disorders prior to administration of subconvulsive doses of cholinergic agonists caused seizures and suggested that altered phosphoinositide metabolism may play a role in this effect. Lithium is the major drug used to treat mania and we reported elsewhere that lithium alters cholinergic metabolism (11, 12). In the present study we characterized the proconvulsant effects of lithium prior to pilocarpine administration. We used EEG recordings to establish that this combination of drugs results in a longlasting state of status epilepticus. The production of status epilepticus is absolutely reproducible, there is an extremely consistent latency to onset of seizures, and the massive electrical activity, which occurs in all brain regions examined, remains constant for several hours. Pharmacological experiments indicate that the seizures are dependent on muscarinic receptor activation, are modulated by presynaptic cholinergic activity, and are rapidly terminated by diazepam. This reliable model of status epilepticus should be useful in studies of the genesis of seizures and of the biochemical and pathologic responses to seizures of long duration. MATERIALS

AND

METHODS

Adult, male Sprague-Dawley rats (Charles Rivers) were stereotaxically implanted with cortical and depth electrodes in the dorsal hippocampus, caudate nucleus, and basolateral amygdala while under halothane anesthesia. All rats were rested for 5 to 7 days after surgery. All EEG recordings were made with the rats freely moving in heated plexiglass recording chambers and baseline EEG recordings were made for at least 30 min prior to drug administration. EEG and behavioral observations were used to identify effects of drugs by (i) noting the latency to the appearance of prominent spike activity and noting

STATUS

EPILEPTICUS

473

the corresponding behavioral responses, (ii) recording the behavioral and EEG progression from the paroxysmal spike activity to generalized spike trains, (iii) recording the latency to tonic clonic seizure activity and status epilepticus, and (iv) recording the mortality rate that follows drug treatments. Lithium chloride, pilocarpine hydrochloride, and atropine sulfate were from Sigma Chemical Company, and were administered in saline in a volume of approximately 0.1 ml. Diazepam (Valium injectable, Hoffman-LaRoche Inc.) was injected in approximately 0.1 ml and propylene glycol was used in control rats. Hemicholinium-3 (Aldrich Chemical Co.) was administered in saline in 1 111. RESULTS Generalized convulsive status epilepticus occurred in 100% of rats treated with pilocarpine (30 mg/kg, s.c.) 24 h after administration of lithium chloride (3 meq/kg, i.p.) (Table 1). The onset of status epilepticus followed a very characteristic, uncompromising pattern (Fig. 1). Signs of peripheral cholinergic stimulation were evident within 5 min of pilocarpine administration, including piloerection, chromodacryorrhea, salivation, and diuresis. During the next 15 to 20 min the rats displayed several behavioral automatisms, including wet-dog shakes, grooming, scratching, and chewing. Prominent single spike activity appeared 20 + 1 (X + SE) min after pilocarpine treatment and was always associated with staring behavior. Spike activity appeared concurrently in all regions tested, and rapidly progressed during the next 2 to 3 min, with rats displaying head bobbing movements, associated with spike trains, followed by rearing and forelimb clonus. The latency to tonic clonic seizure activity and forelimb clonus was 22 f 1 min from the time of pilocarpine administration. During a period of 1 to 2 min the generalized spike activity was separated by intermittent low voltage activity after which the spike trains became continuous. Generalized convulsive status epilepticus occurred in every animal treated with this combination of lithium and pilocarpine. When status epilepticus occurred it continued unabated for several hours and resulted in death within 24 h in 11 of 12 rats. (The single rat that survived this period had persistently abnormal EEG 24 h after the prolonged seizure, displaying spike and slow wave activity from all brain regions). Using a smaller dose of pilocarpine (15 mg/kg) following LiCl (3 meq/kg, i.p., 24 h) administration reduced the occurrence of status epilepticus to 50% of the rats tested and increased the spike latency. Neither lithium (3 meq/kg) nor pilocarpine (30 mg/kg) caused any abnormal EEG responses when administered alone. AS cholinergic agonists alone can cause seizures, we tested several larger doses of pilocarpine to estimate the potentiation caused by preadministration of lithium. Administration of larger doses of pilocarpine (in lithium-free rats)

474

JOPE, MORRISETT,

AND SNEAD

TABLE 1 Seizure Activity and Mortality in Treated Rats’ Treatment

Number spikingb

Li + Pi10 (30)

12112

20*

Li + Pi10 (15)

414

33k

Li

o/10

(20)

114 Of4 O/4

(40)

o/2

Pi10 (30) (50) (1W

O/3 113

Li + Pilo + AT (1) (5)

20

aw

313

(400)

($6

Spike 1atencyC

status epilepticusd

Seizure 1atencyC

1

12112

22 + 1

l/12

I

214

26,30

214

-

O/IO

-

IO/f0

16 -

114 O/4 O/4

20 -

314 414 414

516

79 23,33 32 + 5

313 313 213 113 116

018

-

818

33 9, 41 17* 3 14* 3

012 O/3 O/3 113 213

24-h SUIViVd’

212

O/8

-

Li + HC-3 + Pi10

W8

48 + 10 (P < 0.025)

318

Li + Dz(5) + Pilo

O/3

-

O/3

-

313

Li + Dz( IO) + Pilo

O/3

-

O/3

-

313

Li + HC-3

68 + 9 (P < 0.00 1)

618

’ Adult, male Sprague-Dawley rats were administered the following drugs followed by observation of behavior and EEG recordings (Doses are in parenthesis in mg./kg; values are X * SE). Li-LiCl, 3 meq/kg, i.p., 22-24 h prior to experiment. Pilo-pilocarpine hydrochloride, 30 mg/ kg (unless stated otherwise), s.c.,24 h after Li. AT-atropine sulfate, i.p., 15 min prior to pilocarpine, l-40 mg/kg. HC-3-hemicholinium-3,50 nmol, i.c.v., 22 h after Li. Dz-diazepam, 5 or 10 mg/ kg, i.p., I5 min prior to pilocarpine. ’ Ratio of rats exhibiting paroxysmal spike activity versus total rats tested. ’ Average time (+ SE) following pilocarpine administration to paroxysmal spike activity (min). d Ratio of rats with status epilepticus versus total rats tested. e Average time (it_ SE) following pilocarpine administration to display of forelimb clonus accompanied by tonic-clonic seizure activity (min). ‘Ratio of rats surviving 48 h after lithium and/or 24 h after pilocarpine administration.

resulted in paroxysmal spike activity in some rats and one rat displayed status epilepticus at 100 mg/kg pilocarpine (Table 1). However, even 400 mg/kg pilocarpine did not produce status epilepticus in all rats. These results are similar to those reported by Turski et al. (2 1,22). Therefore, preadministration

STATUS

475

EPILEPTICUS

Cd CtX DH A Baseline

24 h Post Lithium

I Pilocorpme

21 Min (Staring)

Cd ctx DH A 25 Min (Tonic Clonic Seizure)

23 Min (Head Bobbing)

4 h Fbt Pbcorpne

FIG. 1. Serial EEG recordings from a rat which received pilocarpine (30 mg/kg, s.c.) 24 h after lithium (3 meq/kg, i.p.). The initial change in EEG was generalized single spiking seen 2 1 min after pilocarpine administration and was associated with staring behavior, rapidly progressing to head bobbing, tonic-clonic seizures, and generalized seizure activity which lasted continuously for at least 4 h. Cd-caudate, Ctx-cortex, DH-dorsal hippocampus, A-amygdala.

of lithium potentiated the convulsant effect of pilocarpine more than 13-fold. Honchar et al. (9) reported that administration of atropine at a dose of 150 mg/kg prior to pilocarpine in lithium-treated rats blocked seizures. Because that is a very large dose of atropine, we examined the effects of several smaller doses of atropine to determine its efficacy in blocking the initiation of seizures. When atropine was administered 15 min prior to pilocarpine in lithiumtreated rats (Fig. 2, top row) even a dose as small as 1 mg/kg blocked seizure activity in 75% of the rats tested (Table 1). This clearly indicated that atropine effectively blocked the initiation of seizure activity and that stimulation of muscarinic receptors was required for the genesis of this seizure activity. Our working hypothesis is that cholinergic stimulation is responsible for the initiation of these seizures and that when status epilepticus is attained other cell types are recruited. This would be consistent with results of others indicating that there is a wide variety of triggering mechanisms resulting in status epilepticus. To test this hypothesis, rats (four in each group) were treated immediately after the initial forelimb clonus or 20 min following the first forelimb clonus with a relatively large dose (20 mg/kg) of atropine. Typical

476

JOPE, MORRISETT,

AND SNEAD 20-25 min

40 min

50 mln

FIG. 2. Effects of atropine (20 mg/kg) administration on seizures produced by treatment with pilocarpine (30 mg/kg, s.c.) 24 h after lithium (3 meq/kg, i.p.). The top row depicts results when atropine was administered prior to pilocarpine and there was total blockade of seizures. The middle row depicts results when atropine was administered at the time of the first forelimb clonus. The bottom row depicts results when atropine was injected 20 min after the first forelimb clonus at which point atropine had no effect. At-atropine, Pilo-pilocarpine, other abbreviations as in Fig. 1.

results from rats treated with atropine are shown in Fig. 2. In the four rats that received atropine 20 min after forelimb clonus (Fig. 2, bottom row), there was no discernible effect of atropine, i.e., the rats remained in status epilepticus until death. This is similar to results in humans in which anticholinergics are not effective anticonvulsants for status epilepticus. In the four rats that received atropine near the initial period of forelimb clonus (Fig. 2, middle row) the seizures were terminated and all animals survived the experiment. The requirement for presynaptic cholinergic activity for the production of status epilepticus in rats treated with lithium and pilocarpine was tested by administering hemicholinum-3 (HC-3) prior to pilocarpine. HC-3 is a specific, potent inhibitor of high-affinity choline transport, thereby inhibiting acetylcholine synthesis (7). Intraventricular injection of HC-3 reduced acetylcholine concentrations to 10 to 20% of normal for the entire period of 1 to 4 h after treatment (4). In our experiments, rats were treated with LiCl(3 meq/kg, i.p.) 22 h prior to HC-3 (50 nmol; intraventricular). Pilocarpine (30 mg/kg, s.c.) was injected 2 h after HC-3. Paroxysmal spike activity was observed in all rats treated in this manner but the HC-3 treatment significantly (P < 0.025) increased the latency period from 20 to 48 + 10 min. Five of the eight rats treated in this manner never displayed status epilepticus. In these five rats, the paroxysmal spike activity displayed a very interesting pattern. The spike activity was evident only in the caudate [the region with the most rapid rate of acetylcholine turnover (17)], and it occurred in bursts of 5 to 10 spikes repeating every few seconds. The initial spike was highest followed by several

STATUS

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EPILEPTICUS

dampened spikes. With time the number of spikes increased and the interval between spike trains decreased. Each of these five rats survived the treatment; thus HC-3 treatment blocked the development of status epilepticus and decreased mortality. The remaining three rats did display status epilepticus with a significantly (P < 0.001) increased latency to tonic-clonic seizure activity (from 22 to 68 f 9 min). These results clearly showed that presynaptic cholinergic activity modulated the development of status epilepticus. Because diazepam is considered a drug of choice for treatment of status epilepticus in human patients (3), we tested the effects of diazepam on status epilepticus produced by the lithium-pilocarpine treatment. Rats were treated with lithium (3 meq/kg, i.p.) 24 h prior to diazepam (5 mg/kg or 10 mg/kg, i.p.) followed 15 min later by administration of pilocarpine (30 mg/kg, s.c.). Pretreatment with either dose of diazepam totally blocked the development of paroxysmal spike activity and of status epilepticus in all rats tested and all rats survived the experiment. The results presented above demonstrated that pretreatment with diazepam blocked the initiation of seizure activity. Of greater importance for an animal model of the human condition of status epilepticus is the ability of clinically useful anticonvulsants to terminate ongoing seizure activity in the animal

LF-LP A

RF-RP

F

LF-LP B

C

RF-RP

LF-LP RF-W IFC

D

IDZ

LF-LP

RF-RP ts

tDz

-

tFC

FIG. 3. Effect of diazepam on seizures. Rats were implanted with cortical electrodes and I week later treated with lithium (3 meq/kg, i.p.) followed 24 h later by pilocarpine (30 mg/kg, s.c.). Abaseline EEG prior to administration of pi&at-pine. B, C, and D show results from three different rats. B-paroxysmal spike activity was evident prior to diazepam (10 mgjkg) administration which rapidly normalized the EEG. C-spike trains and forelimb clonus occurred during diazepam administration. After a very short period the EEG returned to normal. D-diazepam was administered after spike trains were well established. After diazepam administration, forelimb clonus occurred but was short lived and the EEG returned to normal. LF, left frontal; LP, left parietal; RF, right frontal; RP, right parietal; S, spikes; FC, forelimb clonus; Dz, diazepam.

478

JOPE, MORRISETT,

AND SNEAD

model. Therefore, we tested the ability of diazepam to terminate seizure activity in this model of status epilepticus. Three rats were treated with LiCl(3 meq/ kg, i.p.) followed 24 h later by pilocarpine (30 mg/kg; s.c.). Paroxysmal spike activity occurred in all three rats. During this stage or during the initial tonicclonic stage they were treated with diazepam (10 mg/kg, i.p.). Within 1 min of diazepam treatment (Fig. 3) the EEG returned to normal in each rat. In Fig. 3, A shows the baseline recording from one rat and B, C, and D show the response of each rat. In B, spike trains induced by lithium and pilocarpine treatment were evident prior to treatment with diazepam (10 mg/kg, i.p.). The EEG became normal within a few seconds of administration of the diazepam. The animal represented in C had shown spike trains and was in the process of developing generalized convulsive seizures as manifested by forelimb clonus. This animal was treated with diazepam (10 mg/kg, i.p.) during forelimb clonus which subsequently ceased and the EEG normalized. In the third animal D, diazepam was administered after spike trains were well under way. Forelimb clonus occurred during and immediately after the diazepam administration but was short lived. The animal subsequently stopped seizing and the EEG normalized. All diazepam-treated animals survived and had no recurrent seizures after diazepam treatment. In a recent abstract, Olney et al. ( 15) also reported that diazepam at 10 mg/kg arrested seizure activity caused by the combination of lithium and pilocarpine. These experiments further validate the lithium-pilocarpine-treated animal as a relevant model of status epilepticus. DISCUSSION A variety of pharmacologic treatments have been used as trigger mechanisms to induce status epilepticus in animals (27). In the present model, activation of the cholinergic system is clearly required for the initiation of seizures as it is blocked by small doses of atropine. The mechanism by which lithium potentiates the seizure threshold to muscarinic agonists remains to be identified. However, it may involve stimulation of acetylcholine synthesis and release (8, 11) (as it is attenuated by HC-3) which would act in concert with administered agonists to stimulate cholinergic receptors to reach seizure threshold. The reported effects of lithium on phosphoinositide metabolism may play a role (9, 18). Hydrolysis of phosphoinositides results in the production of inositol phosphates, one of which, myo-inositol- 1,4,5trisphosphate, is reputed to increase the intracellular concentration of calcium (13,20) which has been linked to seizure activity (6, 14). These mechanisms may contribute presynaptically to facilitate acetylcholine release and postsynaptically to amplify the signal produced by receptor activation. When status epilepticus is initiated, the seizures rapidly become unresponsive to atropine treatment indicating recruitment of systems in addition to choline& cells.

STATUS

479

EPILEPTICUS

Convulsive status epilepticus is clinically defined as continuous, prolonged electrical and clinical seizure activity in which the patient does not regain consciousness to a normal alert state between repeated tonic-clonic attacks. This disorder is a neurologic emergency associated with a mortality rate of 10 to 12% and an even greater morbidity (2). However, potential animal models of convulsive status epilepticus are limited in their relevance to the clinical condition. The main difficulty in developing such animal models is in producing prolonged, severe seizures because most acutely induced experimental seizures are self-limiting (27). The seizures produced by the combination of lithium and pilocarpine are absolutely reproducible, extremely consistent in time of onset, are certainly continuous and prolonged, and are associated with a large mortality rate. In addition they are blocked by the anticonvulsant, diazepam, and, more importantly, when the seizures are underway they are rapidly terminated by diazepam and the rats are protected from the mortality of the seizures. An additional advantage of this model is that a well defined neurochemical system (i.e., cholinergic) is clearly involved in at least the initiation of this status epilepticus which should allow dissection of specific biochemical mechanisms involved in these seizures. The animal model described here should be useful for the study of the pathogenesis and treatment of generalized convulsive status epilepticus. REFERENCES 1. CAIN, D. P. 1983. Bidirectional transfer of electrical and carbachol kindling. Brain Rex 260: 135-138. 2. DELGADO-ESCUETA, A. V., AND J. G. BAJOREK. 1982. Status Epilepticus: mechanisms of brain damage and rational management. Epilepsia 23 (suppl. 1): S29-S4 1. 3. DELGADO-ESCUETA, A. V., C. WASTERLAIN, D. M. TREIMAN, AND R. J. PORTER. 1983. Current concepts in neurology: management of status epilepticus. N. Engl. J. Med. 306: 1337-1340. 4.

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