seizures produced by pilocarpine in mice: a behavioral, electroencephalographic and morphological...

Brain Research, 321 (1984) 237-253 237 Elsevier

BRE 10373

Seizures Produced by Pilocarpine in Mice: A Behavioral, Electroencephalographic and Morphological Analysis

WALDEMAR A. TURSKI 1,* , ESPER A. CAVALHEIRO 2, ZUNER A. BORTOLOTFO 2, LUIZ M. MELLO 2, MICHAEL SCHWARZ 1,** and LECHOSI_AW TURSKI 1

1Department of Pharmacology, Institute of Clinical Pathology, Medical School, Jaczewskiego 8, PL 20-090 Lublin (Poland) and 2Department of Neurology and Neurosurgery, Laboratory of Experimental Neurology, Escola Paulista de Medicina,

R. Napoleao de Barros 827, BR 04023 Sao Paulo, SP (Brazil)

(Accepted March 12th, 1984)

Key words: pilocarpine - - seizures - - brain damage - - temporal lobe epilepsy - - scopolamine - - diazepam - - mouse

Increasing doses of pilocarpine, 100-400 mg/kg, were given intraperitoneally to mice and the resulting behavioral, electroencephalographic and neuropathological alterations were studied. No behavioral phenomena were observed in mice treated with the lowest dose of pilocarpine. Occasional tremor and myoclonus of hindlimbs were found in animals which received pilocarpine in a dose of 200 mg/kg. At doses of 300, 325 and 350 mg/kg, pilocarpine produced a sequence of behavioral alterations including staring spells, limbic gustatory automatisms and motor limbic seizures that developed over 15-30 min and built up progressively into a limbic status epilepticus lasting for several hours. The highest dose of pilocarpine, 400 mg/kg, was generally lethal to mice.

Pilocarpine produced both interictal and ictal epileptiform activity in the electroencephalogram (EEG). The earliest EEG alterations appeared in the hippocampus and then spread to cortical areas. EEG seizures started 10-15 min after injection of large doses of pilocarpine, 300-350 mg/kg. Ictal periods lasted for 1-2 min, recurred every 5-10 min and were followed by periods of depression of the EEG activity. By 30-45 min paroxysmal activity resulted in a status epilepticus.

Examination of frontal forebrain sections with light microscopy revealed a widespread damage to several brain regions including the hippocampus, amygdala, thalamus, olfactory cortex, neocortex and substantia nigra. Scopolamine, 10 mg/kg, and diazepam, 10 mg/kg, prevented the development of convulsive activity and brain damage produced by pilocarpine.

The results emphasize that excessive and sustained stimulation of cholinergic receptors can lead to seizures and seizure-related brain damage in mice. It is proposed that systemic pilocarpine in mice provides a useful animal model for studying mechanisms of and therapeutic approaches to temporal lobe epilepsy.

INTRODUCTION

D e b a t e on the i n v o l v e m e n t of ace ty lchol ine ( A C h )

and cent ra l chol inerg ic m e c h a n i s m s in neuro log ica l

and psychiatr ic diseases in h u m a n s was d o m i n a t e d

for some years by a lack o f consensus on the specific

con t r ibu t ion of chol inerg ic m e c h a n i s m s to these dis-

orders16, 31. O n the o t h e r hand , da ta have b e e n pre-

sen ted d e m o n s t r a t i n g that chol inerg ic mechan i sms

are invo lved in A l z h e i m e r ' s s8 and H u n t i n g t o n ' s dis-

ease 31, and may have i m p o r t a n t ef fects on m e m o r y 4s.

F u r t h e r m o r e , chol inerg ic m e c h a n i s m s have s o m e in-

f luence on m o o d 44 and s e e m to be e n g a g e d in cer ta in

forms of h u m a n epilepsy31, 47 as well as in expe r imen-

tally induced seizures16,17,28,37,55,56. The possible rela-

t ionship o f A C h to ep i lepsy was ra ised when it was re-

alized (i) that A C h or its analogues, acetylcholinester-

ase inhibi tors and A C h precursors , when adminis-

t e red into the bra in of e x p e r i m e n t a l animals resu l ted

in p r o f o u n d seizure activityl,17,2s; (ii) that a t rop ine

de layed the p rogress ion of e lectr ical ly k ind led sei-

zures in rats40; and (iii) that the concen t r a t i on of A C h

in the h u m a n C S F was e l eva t ed af ter se izures 47.

M c C a n n et al. 29 were the first to p ropose that e leva-

ted bra in A C h concen t r a t i on e n h a n c e d the ra te of

kindl ing in rats. T h e p roposa l of A C h i n v o l v e m e n t in

* Present address: Wander Research Institute, Sandoz Research Unit, Monbijoustr. 116, P.O. Box 2747, CH-3001 Berne, Switzer- land.

Correspondence: L. Turski (present address) Department of Biochemical Pharmacology, Max-Planck-lnstitute for Experimental Medicine, Hermann-Rein-Str. 3, D-3400 G6ttingen, F.R.G.

0006-8993/84/$03.00 (~ 1984 Elsevier Science Publishers B .V.

Author's personal copy

238

the initiation and spread of seizures has been put foreward on the basis of kindling experiments in rats demonstrating that stimulation of muscarinic cholinergic receptors within the amygdala was an efficient means to induce a kindled state characterized by both evoked and spontaneous seizures s6. New interest in the relation of cholinergic mechanisms to epilepsy was generated by the finding that intraamygda- 10id37,55, intrahippocampa152.55 or systemic 51,55 injec-

tions of large doses of muscarinic cholinergic agonists in rats produce EEG and behavioral occurrence of limbic seizures accompanied by a widespread damage to forebrain structures resembling that frequently observed in autopsied brains of human epileptics 12. These findings have been confirmed and extended by the direct demonstration of the potential importance of the cholinergic pathways in the development of an epileptic cell loss within the rat brain 32,59. McGeer et al. 32 and Wong et al. 59 demonstrated that application

of folic acid into the substantia innominata damaged GABAergic neurons in several areas of the forebrain distal to the injection site, the majority of the latter being cholinoceptive. Consequently, it has been proposed that both systemic and intraamygdaloid application of cholinomimetics in rats may provide valuable animal models for studying mechanisms of temporal lobe epilepsy ~9,37,51,55.

Basic research on the mechanisms and the treatment of epilepsy has been performed in the classical mouse models of convulsions, including pentylenete- trazol-, bicuculline-, picrotoxin- and electroshock seizure models43, 47 as well as in genetic models of re- flex epilepsy 43. Convulsions in animal models produced by means of chemical and electrical methods could be divided into myoclonic and generalized clonic-tonic, and it seems possible to differentiate between antiepileptic drugs preferentially active against either type 43. A valuable and useful mouse

model for studying mechanisms of and therapeutic approaches to temporal lobe epilepsy has not been developed so far. In fact, the results of neuroanatom- ical studies have established that systemic administration of kainic acid in mice may result in convulsions and damage to forebrain structures39; however, the practical efficacy of this preparation appeared hampered by relatively high doses of the drug re- quired and by a narrow margin of safety 39. The convulsive properties of intracerebral kainic acid in mice

have not been studied in detail, and no animal model of temporal lobe epilepsy has been created in this

species, although a rough description of limbic seizures and hippocampal pathology following intra- cerebroventricular kainic acid in mice has already appeared13,14,21.

The main objective of the present study on mice was to characterize pharmacologically and electroen- cephalographically the effects of systemically applied pilocarpine, a potent muscarinic cholinergic agonist, in order to determine whether a disturbed function of the central cholinergic mechanisms may cause either epileptiform activity in the limbic structures, behavioral manifestations of limbic seizures or seizure-related brain damage. Furthermore, it will be shown that diazepam, a potent anticonvulsant 18, was capa- ble to affect convulsive properties of systemically applied pilocarpine in mice.

MATERIALS AND METHODS

Adult male, albino Swiss mice 25-30 g body weight were used. For at least one week prior to the experiments, animals were housed in groups of 5-10 on a standard light-dark cycle with ad libitum access to food (mice chow pellets) and tap water. Mice were assigned to experimental groups by means of a randomized method and singly allocated to new cages not less than 30 min prior to experiments. To determine the subsequent time of behavioral and EEG testing for different treatment groups, a randomized schedule was employed. All behavioral assessments took place between 08.30 and 18.00 h. Experimental groups consisted of 8-16 animals.

Behavior

Behavioral assessments were carried out in com- partments (40 x 25 x 17 cm) built of plexiglass. Prior

to administration of the drug solutions each animal was habituated for 30-45 min. After the habituation mice were removed, injected s.c. or i.p. with the ap- propriate drug and rapidly returned to the experimental cage. A total of 144 mice was studied with different doses of pilocarpine. Eighty-one mice were used to determine the dose-response relationship and time-course of behavioral alterations after pilocarpine. Thirty-six animals were used to determine the effect of scopolamine or diazepam on convulsive


action of pilocarpine. Twenty-seven mice were used for EEG recordings. Twelve additional mice served as controls.

Surgery and electrophysiological procedures Under sodium pentobarbital (Nembutal; Ceva,

Neuilly-sur-Seine, France) anesthesia (50 mg/kg, i.p.), bipolar twisted wire electrodes (tip diameter 100 ktm, interelectrode distance 500/~m) were placed stereotaxically in the dorsal hippocampus (AP 2.5; L 2.0; V 3.536) and fixed to the skull with dental acrylic. Surface recordings were made from jeweller screws placed bilaterally over the sensory motor cortex. An additional screw placed over the frontal sinus served as a reference (indifferent) electrode. Signals under investigation were amplified by a Beckman model RM polygraph (time constant 0.03 s, high cut- off filter 15). EEG recordings began 5-7 days after surgery. Recordings and visual observations of the animal's behavior were carried out in a plexiglass compartment (30 x 30 x 45 cm). Before EEG recording session animals were singly placed in the recording compartment, connected to the recording plug, and allowed for at least 30 min for habituation to the experimental setup. Following the period of habituation, baseline EEG recordings were made for 15-30 min. EEG recordings were made continuously and behaviour noted for periods ranging from 6 to 8 h following pilocarpine or saline injection. Addi- tional recordings were made between 10 and 12 h and 24 and 27 h following the injection. The correct location of the implanted deep electrodes was histo- logically controlled in cresyl violet-stained serial sec-

tions.

Drugs Pilocarpine hydrochloride was obtained from Sig-

ma (St. Louis, MO, U.S.A.), freshly dissolved in saline and administered i.p. in doses of 100,200, 300, 325,350 and 400 mg/kg. Methyl scopolamine nitrate (Sigma), 1 mg/kg, was injected s.c. 30 min prior to all dosages of pilocarpine in order to minimize peripher- al cholinergic effects 51. Scopolamine hydrochloride (Sigma), 10 mg/kg, was dissolved in saline and administered s.c. Diazepam (Polfa, Poznafi, Poland) was suspended in a 3% solution of Tween 81 (Loba Chemie, Wien, Austria) and given i,p. in the dose of 10 mg/kg. Scopolamine and diazepam were adminis-

239

tered either 30 min prior to, or 1 h after, the injection of pilocarpine, 325 mg/kg.

Histology For examination by light microscopy, brains were

histological processed 48-72 h after administration of pilocarpine. Mice were deeply anesthetized with an overdose of sodium pentobarbital and perfused through the heart with saline followed by 10% forma- lin solution. The brains were removed, stored in for- malin, embedded in paraffin and coronally sectioned at 10ktm. Every 10th section was preserved and stained with cresyl violet. Extensive histological changes after application of pilocarpine were identified by the presence of widespread degeneration of neurons and an extensive disruption of the surrounding neuropil. A moderate neuronal degeneration was identified by the presence of a majority of shrunken and dark staining cells, while surrounding neuropil appeared relatively unaffected. In areas less severely damaged, cellular degeneration was largely restricted, neurons were shrunken and pyknotic compared with control tissue, and the cell nuclei often displaced to the periphery. Consequently, three types of histological changes were noted, a mild form with a partial neuronal loss and gliosis, severe form where entire nuclear groups or areas underwent necrosis, and moderate form where intensity of changes was limit- ed and less severe. Mild, moderate and severe changes could occur in the same animal.

RESULTS

Behavior Injection of 100 mg/kg (n = 8) of pilocarpine did

not result in any abnormal behavioral phenomena with the exception of mild tremor displayed by 2 out of 8 mice. With the higher dose, 200 mg/kg (n = 14), all animals tested were motionless (14/14), displayed a body tremor (6/14) which started 10 min after treatment or earlier, and occasionally showed a myoclonus of the hindlimbs (3/14). This activity persisted up to 45-60 min after pilocarpine, 200 mg/kg. With the two higher doses (300 and 325 mg/kg) pilocarpine produced a sequence of behavioral alterations including an initial akinesia (16/16 for both doses), a tremor of the whole body (9/16 and 12/16), ataxic lurching (7/16 and 14/16) and incomplete limbic gus-


240

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Fig. 1. Electroencephalographic recordings illustrating the sequence of alterations produced by intraperitoneal injection of pilocarpine in mice. A: pre-drug control recordings. B: electroencephalographic correlates 2-5 min after injection of pilocarpine in the dose of 325 mg/kg demonstrating low-voltage fast activity in cortical recordings and a significant hippocampal theta rhythm. C and D: 10-15 min after injection of pilocarpine. High-voltage fast activity (C) and spikes superpose (D) over the hippocampal theta rhythm. Isolated low-voltage spikes are initially registered in hippocampal leads (D), while cortical recordings display no or minor changes. E: high-voltage spiking similar to that shown in panel D spreads to cortical leads 15-30 min after pilocarpine.

tatory automatisms (12/16 and 16/16) with salivation

(6/16 and 8/16). This type of preconvulsive behavior persisted for up to 5-10 min after injection and built up progressively into motor limbic seizures with up-

per extremity clonus, rearing, falling and intense salivation. During the period of motor limbic seizures

mice exhibited additionally stereotyped behaviors like repeated head twitches, grooming and rearing,

and occasionally jumping fits. Motor limbic seizures

commenced after 10-18 min (mean 17.89 + 7.64 min, n = 16, for 300 mg/kg; and mean 11.67 _ 3.26 min, n = 16, for 325 mg/kg), recurred every 5-10 min and reached a maximal occurrence of 6 -8 per 30 min. The period of limbic convulsive behavior lasted for up to 30-45 min after injection and rapidly developed into a status epilepticus (mean 27.63 + 12.7, n = 12, for 325 mg/kg) which sometimes led to the death of the animal. With the dose of 325 mg/kg myoclonic seizures could rarely be observed; how-

ever, clonic-tonic seizures with death of the animal were observed in 4 out of 16 mice. With the dose of 350 mg/kg of pilocarpine the development of convul-

sive activity was rapid (mean 6.2 + 2.3 min, n -- 12) and the lethal toxicity in mice increased over 50% (7/12). With this dose, a short-lasting period of motor

limbic seizures rapidly developed into the status epi-

lepticus with generalized clonic-tonic convulsions which ended in death of the animals. At the dose of

400 mg/kg (n = 15) pilocarpine was generally lethal to mice (14/15). Pretreatment of mice with scopolamine, 10 mg/kg (n = 10) or diazepam, 10 mg/kg (n -- 10) prevented the development of convulsive alterations produced by pilocarpine, 325 mg/kg. Animals which were sedated and stuporous after injection of diazepam, began to walk and explore following the injection of pilocarpine, but no typical behavioral alterations produced by pilocarpine in drug-naive mice could be observed. Diazepam, 10 mg/kg (n = 8) re-


241

duced the convulsive activity when administered 1 h after pilocarpine, 325 mg/kg, while scopolamine, 10 mg/kg (n = 8) failed to disrupt pilocarpine-induced seizure activity and did not lessen the lethal toxicity of the drug (2/8).

Electroencephalography The nature of EEG alterations occurring after i.p.

application of pilocarpine in mice is illustrated in Figs. 1-3. Alterations produced by i.p. injections of pilocarpine in mice were dose- and time-dependent. Pilocarpine caused both interictal and ictal epileptiform activity in the EEG. Typical EEG changes consisted of (i) a fast activity of low or high voltage, (ii) slow or fast high voltage spiking and (iii) fully developed ictai activity. The sequence of behavioral alterations evoked by pilocarpine correlated well with the EEG changes produced by the drug. Immediately (2-5 min) after injection of pilocarpine in doses of

100-300 mg/kg, the normal background activity was replaced with low-voltage fast activity in the cortex (Fig. 1B), while significant theta rhythm (6-7 Hz) appeared in the hippocampus (Fig. 1B). By 5-10 min, high-voltage fast activity superposed over the hippocampal theta rhythm and isolated low-voltage spikes were registered initially in hippocampal leads, while cortical recordings displayed no or only minor changes (Fig. 1C, D). At this time a striking feature was that activity changes originated in the hippocampus and then rapidly spread to the cortex (Fig. 1D, E). In animals receiving low (100 mg/kg) and intermediate (200 mg/kg) doses of pilocarpine, low- or high-voltage fast activity and spiking, which appeared at variable frequencies and were initially restricted to the hippocampus, prevailed for at least 1-2 h after injection. Fully developed electrographic seizures did not appear with these doses. At the time of spiking activity with low and intermediate doses of

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Fig. 2, Electrographic correlates of behavioral seizures produced by pilocarpine in mice. A: continuous recording demonstrating electrographic seizure registered in hippocampal leads 25 min after intraperitoneal injection of 325 mg~g of pilocarpine. The fast activity develops into high-voltage spiking resulting rapidly in an electrical seizure registered in the hippocampal lead (A), while cortical recording displays minor changes restricted exclusively to the appearance of the fast activity and isolated high-voltage spikes. During the time in which an electrical seizure occurs in the hippocampus, the animal remained frozen in a motionless staring spell. B: electrographic seizure observed 33 min after injection of pilocarpine. The waxing and waning of electrical seizure activity is highly synchronized in both hippocampal and cortical leads. The fast activity and isolated high-voltage spikes registered in both recordings preceded the development of a seizure. C: electrographic recordings illustrating the development of a seizure 40 min after injection of pilocarpine. The fast activity develops into prominent high-voltage spiking, which built up progressively into a seizure activity. Note a rapid evolu- tion as well as prolonged waning of the seizure activity in the hippocampal recording. D: electrographic recordings to illustrate clonic- tonic seizures observed 2-4 h after injection of 350 mg/kg pilocarpine.


242

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Fig. 3. Electrographic recordings illustrating the sequence of alterations observed at longer delays after intraperitoneal injection of pilocarpine in mice. A: electrographie correlates of interictal activity observed 2-4 h after injection of 325 mg/kg pilocarpine. Note fast activity and highly synchronized high-voltage spiking that occurs in all recordings. B: electrographic recordings to illustrate the electrical alterations observed 2-6 h after injection of 325 mg/kg pilocarpine during a status epilepticus. C: electrographic correlates 8 h after injection of pilocarpine. High-voltage fast activity and prominent spiking still occurs in hippocampal lead, while cortical recording displays less pronounced changes. D: 24 h post-injection the EEG recordings are indistinguishable from pre-injection control activity, although isolated spikes are infrequently registered in hippocampal leads.

pilocarpine, animals remained motionless and often displayed mild limbic automatisms. By 24 h after pilocarpine, 100 and 200 mg/kg, EEG recordings were indistinguishable from the pre-drug background activity. Doses of 300 mg/kg of pilocarpine or greater, reproducibly elicited a sequence of E E G alterations and concomitant seizure-like behaviors described with low and intermediate doses; however, the laten- cy period decreased and clonic or clonic-tonic motor convulsions (Fig. 2D) frequently appeared on increasing the dose. With higher doses of pilocarpine, 300, 325 and 350 mg/kg, typically, fully developed EEG seizures occurred, which started 10-15 min after injection. A high-voltage fast activity and prominent high-voltage spiking usually preceded the development of a seizure (Fig. 2A-C) . Characteristically, these activity changes originated in the hippocampus

and built up progressively into an electrographic seizure registered in the hippocampal recordings (Fig. 2A), while cortical recordings displayed minor changes restricted to the appearance of the fast activity and isolated spikes (Fig. 2A). Later, the electrographic seizures became highly synchronized in the hippocampus and cortex (Fig. 2B, C); however, faster buildup, as well as a prolonged waning of the seizure activity in the hippocampal recordings, was reproducibly registered (Fig. 2C). The ictal periods lasted 1-2 min, recurred every 5-10 min and were followed by periods of depression of the electrographic activity. By 30-45 min paroxysmal activity, which progressively grew in duration and complexi- ty, resulted in a status epilepticus (Fig. 3B). This pattern of EEG changes lasted for 4 -6 h and was followed by a progressive normalization of the EEG ac-


tivity. By 8 h after the injection of pilocarpine,

300-350 mg/kg, high-voltage fast activity and promi-

nent spiking was often seen in hippocampal recordings (Fig. 3C), while cortical recordings displayed

less pronounced changes. By 24 h after injection of

higher doses of pilocarpine, E E G recordings were indistinguishable from pre-drug background activity,

although isolated high-voltage spikes were occasionally registered in the hippocampal leads (Fig. 3D).

The development of electrographic seizure activity

usually preceded the buildup of clinical seizures.

243

First electrographic seizures were usually restricted

to the hippocampus (Fig. 2A) and freezing of ani-

mals in a motionless staring spell coincided with this type of activity. Motor limbic seizures appeared

when electrical seizure activity became synchronized in the cortex and hippocampus (Fig. 2B, C). Head

twitches occurred infrequently after electrical seizures, and never preceded them. When seizure activ-

ity built up progressively and had became well developed (Fig. 2C), the motor manifestation of l imbic

convulsions became more severe. With doses greater than 350 mg/kg seizures were accompanied by in-

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Fig. 4. Electrographic recordings illustrating the effect of scopolamine on the convulsant activity of pilocarpine in mice. Scopolamine in the dose of 10 mg/kg was injected subcutaneously 30 min prior to injection of pilocarpine (325 mg/kg, i.p.). A: pre-drug control recording. B: electrographic correlates 5-10 min after injection of scopolamine. A normal background activity is replaced with the high- voltage slow activity. No theta activity could be registered in hippocampal leads. C: 20 min after injection of pilocarpine in scopolamine-pretreated mouse. Pilocarpine progressively decreases the voltage of the slow activity produced by scopolamine (B) resulting in the appearance of low-voltage activity in both leads (C), which is slower than that registered during control recordings (A) but faster than that observed after scopolamine alone (B). D and E: electrographic correlates 1-4 h after intraperitoneal injection of pilocarpine in scopolamine-pretreated mice. By 1 h after injection of pilocarpine, high-voltage slow activity alternates with and then superposes over low-voltage fast activity induced by the injection of the drug and prevails in hippocampal recordings up to 4 h post-injection (E). F: by 4-8 h after injection of pilocarpine the voltage of the slow activity gradually decreases (E) and is progressively replaced with faster activity (F). By 24 h after injection of pilocarpine in scopolamine-pretreated mice all recordings are indistinguishable from pre- drug background activity (A).


244

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Fig. 5. Electrographic recordings illustrating the effect of diazepam on the convulsant activity of pilocarpine in mice. Diazepam in the dose of 10 mg/kg was injected intraperitoneally 30 min prior to systemic injection of 325 mg/kg of pilocarpine. A: pre-drug control recording. B: electrographic correlates 5-10 min after injection of diazepam. A normal background activity is replaced with the low-voltage fast activity which prevails in both leads. No theta activity could be registered in hippocampal recordings. C-E: electrographic correlates 15 min to 1 h after injection of pilocarpine in diazepam-pretreated mice. Injection of pilocarpine results in a short-lasting reappearance of the theta activity in the hippocampal recordings (15 min after application of pilocarpine) (C). Low-voltage fast activity rapidly superposes over or alternates with the hippocampal theta activity within 30-60 min after pilocarpine and then predominates in hippocampal leads (D). F: by 8 h after injection of pilocarpine in diazepam-pretreated mice hippocampal and cortical recordings are indistinguishable from pre-drug background activity (A).

tense salivation, prominent cyanosis, conspicuous respiratory disturbances and death. Therefore electrophysiological analysis of effects produced by these doses of pilocarpine was not justified.

Pretreatment of mice with scopolamine, 10 mg/kg s.c., completely prevented the occurrence of convulsive activity in the E E G produced by 325 mg/kg of pilocarpine (Fig. 4). After injection of 10 mg/kg of scopolamine the background activity was replaced with the high-voltage slow activity, while the hippocampal theta rhythm was instantaneously blocked (Fig. 4B). Injection of pilocarpine, 325 mg/kg, led to a decrease of the voltage of the slow activity resulting in the appearance of low-voltage activity in both hippocampal

and cortical recordings (Fig. 4C). Characteristically, this type of low-voltage activity was slower than that registered during the pre-drug recordings (Fig. 4B). By 1 h after injection of pilocarpine low-voltage fast activity produced by the drug alternated with high- voltage slow activity and finally superseded the latter (Fig. 4C). This pattern of activity prevailed in hippocampal recordings up to 4 h post-injection (Fig. 4D). The voltage of the slow activity gradually decreased by 4-8 h after 325 mg/kg pilocarpine and faster activity progressively replaced it (Fig. 4E). By 24 h after injection of pilocarpine in scopolamine-pretreated mice the E E G in all recordings returned to the pre-drug pattern (Fig. 4F). After the injection of


diazepam, 10 mg/kg, the background activity (Fig. 5A) was replaced with a low-voltage fast activity which prevailed in both recordings (Fig. 5B). Fif- teen minutes after application of pilocarpine, 325 mg/kg, a short-lasting reappearance (up to 30-45 min) of the theta activity in the hippocampal recordings occurred (Fig. 5C). Low-voltage fast activity alternated with the former pattern of EEG and then rapidly replaced it (Fig. 5D, E). Eight hours after application of pilocarpine in diazepam pretreated mice, no consistent qualitative differences between electrographic activity registered from both hippocampus and cortex of these and drug-naive animals were found (Fig. 5F).

Neuropathology Examination of frontal forebrain sections with

light microscopy revealed widespread brain damage in mice treated systemically with pilocarpine. The development of neuropathological alterations produced by pilocarpine in mice was related to the dose of the drug. Intraperitoneal application of the low dose of pilocarpine, 100 mg/kg, which elicited slight behavioral and EEG alterations did not result in de- tectable pathological changes within the brain. With intermediate dose of pilocarpine, 200 mg/kg, mild neuropathological alterations, largely confined to piriform cortex and olfactory nucleus, could be en- countered. When the three highest doses of pilocarpine, 300, 325 and 350 mg/kg, which reproducibly elicited consistent and long-lasting behavioral and electrographic changes including motor limbic seizures and limbic status epilepticus, were used, extensive neuropathological alterations were found within the olfactory cortex, thalamus, amygdaloid complex, hippocampal formation, neocortex and substantia nigra. The anatomical terminology used in the present paper conforms to that used by Kovac and Denk 23 and Montemurro and Dukelow 36. With pilocarpine, 325 mg/kg, extensive degeneration of neurons and complete disruption of the surrounding neuropil were found within the olfactory system, i.e. piriform cortex, anterior olfactory nucleus and periamygda- loid cortex. The piriform cortex appeared severely swollen and edematous. The majority of cells within the piriform cortex were dark and shrunken (Fig. 6F) and the surrounding neuropil appeared frequently fragmented. Severely shrunken and darkened neu-

245

rons could be observed in the anterior olfactory nu-

cleus and the entorhinal cortex (Fig. 6G), as well. The olfactory tubercle was less sensitive to pilocarpine and neuronal degeneration has not been found in this area. The amygdaloid nuclear complex appeared to be particularly sensitive to pilocarpine (Fig. 6I). Degeneration and disapparance of virtual- ly all neurons with prominent edema, fragmentation and disruption of the neuropil were observed within this area (Fig. 6I). Within the hippocampal formation prominent neuronal degeneration was observed in the regio superior of the dorsal hippocampus (Fig. 6C). Destruction of almost all pyramidal cells was identified in the regio inferior (Fig. 6B) as well as in the area dentata. Within the area dentata de- generated neurons were usually observed in the hilus and at the granule cell-hilar border. Ventral hippocampus displayed extensive damage as well, including degeneration of pyramidal neurons within areas in continuity with the olfactory cortices. The ventral part of the subiculum and lateral septal nucleus were also consistently affected (Table I). Within the thalamus, most consistently affected were anterior, lateral, dorsomedial, reuniens, rhomboideus, paraventricular and parathenial nuclei; however, the extent of damage in the thalamus varied among mice treated with 325 mg/kg of pilocarpine (Table I). Exten- sive neuronal degeneration with almost complete disruption of the neuropil usually occurred in the lateral (Fig. 6D) and dorsomedial thalamic nuclei. Ventral, ventromedial and posterior thalamic nuclei were less severely damaged. The lateral geniculate nucleus was extensively damaged as well (Table I). Within the neocortex, severe damage was found in the outer molecular layers, but also deep layers could be affected. The areas most consistently affected included areas dorsal to the rhinal sulcus, cingulate cortex (Fig. 6H), portions of the perirhinal areas and soma- tomotor cortices. Shrunken and dark staining cells were found within the substantia nigra pars ventralis and dorsalis (Table I). Neuropathological alterations were never observed in the dorsal part of the striatum, while the ventral part was consistently affected. Little neuronal degeneration was encoun- tered within the hypothalamus. The typical pattern and distribution of the brain damage produced by pilocarpine, 325 mg/kg, is illustrated in Figs. 6 and 7. The animals pretreated with either scopolamine,


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10 mg/kg, or diazepam, 10 mg/kg, 30 min before the

application of 325 mg/kg pilocarpine, displayed no

epileptiform activity, and neuropathological alterations were not detected in their brains.

DISCUSSION

Three major conclusions can be drawn from the

experiments presented here: (i) systemically applied pilocarpine in mice produced profound convulsive

activity preferentially confined to limbic structures;

(ii) a major component of this activity is muscarinic; and (iii) the seizures induced by the drug may result

in a widespread damage to forebrain structures re-

sembling that frequently observed in brains of human epileptics 12.

Electroencephalographic observations of the epileptic response to pilocarpine in mice provide evi-

dence for the involvement of the hippocampus in the

buildup of epileptiform activity following the drug

administration and emphasize that hippocampus has a particularly low threshold for pilocarpine-induced

convulsions. This finding is related to the hypothesis

that there exists a powerful positive-feedback mech-

anism within discrete regions of the hippocampus and septum, and the assumption that its activation allows

the development of long-term changes in excitability within the hippocampal subfields 2,8,9,24. Further-

more, anatomical studies emphasize the high degree

of interconnections between hippocampus, entorhinal cortex, amygdala and other parts of the limbic

forebrain, which may facilitate the rapid spread of

seizures originating in one part of this system to all otherslt,57.

From the results of neuropathological analysis of brains from mice treated with pilocarpine it is apparent that pyramidal cells of the hippocampal forma-

tion, as well as of the piriform cortex and the amygdala, are particularly sensitive to pilocarpine neuro-

toxicity. The topography of the hippocampal damage in mice resembles that described after systemic application of either pilocarpine19,51, 55, arecoline 19 or

kainic acidT, 26,45 in rats, and that reported in mice

treated intracerebroventricularly with the latter drug t334. This finding may have interesting implica-

tions for the elucidation of the causative mechanism of pathological aspects of seizures produced by pilocarpine in mice. Firstly, the conformity of hippocam-

pal pathology in mice to that reported in other models of temporal lobe epilepsy 5,7,26,35,37,45,49,50,51,55 es-

t ab l i shed in different animal species (rat, cat, mon-

key) implicates similar, and very likely seizure-related, mechanisms of neuronal damage. Indeed,

from the studies with pilocarpine in rats, it is appar-

ent that an ischemic factor does not seem to be of ma-

jor importance for the hippocampal damage observed in these experiments. Specifically in the rat,

the regio superior of the dorsal hippocampus, a region which typically undergoes extensive neuronal degeneration following experimental ischemia, is

considerably less sensitive to pilocarpine neurotoxicity 51.

Secondly, the topography of brain damage follow-

ing pilocarpine in mice resembled that observed after

Fig. 6. Neuropathological alterations in the mouse brain produced by systemic administration of pilocarpine. Cresyl violet stain. Sur- vival time: 48-72 h. A: photomicrograph of the regio inferior of the dorsal hippocampus of a drug-naive control mouse. B: representa- tive section illustrating the destruction of the regio inferior of the hippocampus of the mouse treated i.p. with 300 mg/kg of pilocarpine. Note the nearly total disruption of the cytoarchitecture of the pyramidal layer with extensive cellular degeneration and glial infil- tration. Shrunken and dark staining cells are visible throughout the pyramidal layer. Some changed cells contain narrowings which lead to fragmentation and disintegration. Dark staining and severely shrunken cells are interspersed among normal appearing pyramidal neurons. C: high-power photomicrograph demonstrating the type of tissue reaction observed within the regio superior of the dorsal hippocampus of the mouse treated i.p. with 325 mg/kg of pilocarpine. Severely shrunken and darkened pyknotic pyramidal neurons are prominent in the pyramidal layer. D: photomicrograph illustrating the destruction of the lateral thalamic nucleus after i.p. injection of 300 mg/kg of pilocarpine. Note the nearly total disruption of the cytoarchitecture of the lateral thalamus. Neurons stain darkly and appear shrunken. E: photomicrograph of a portion of the piriform cortex of a drug-naive control mouse. F: photomicrograph illustrating the destruction of the posterior piriform cortex of the mouse injected i.p. with 325 mg/kg of pilocarpine. Shrunken and darkened neuronal somata are widely interspersed among normal appearing cells and are present throughout all layers of the piriform cortex. G: entorhinal cortex in the mouse injected systemically with 300 mg/kg of pilocarpine. The cells become dark and severely shrunken. H: cingulate cortex in the mouse injected with 325 mg/kg of pilocarpine. Shrunken and darkened pyknotic neurons are present in the outer layers of the cingulate cortex. Dark staining cells are present within the deep layers as well. I: photomicrograph of a portion of the amygdaloid complex of the mouse injected with 325 mg/kg of pilocarpine. Note the nearly total destruction of the neuronal population within the amygdala accompanied by edema and disruption of the neuropil. A-I: × 196. (Publisher's reduction factor: 0.75.)


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249

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A1.5 cpf Fig. 7. Schematic reconstruction of the distribution of neuropathological alterations in the mouse brain after intraperitoneal administration of 325 mg/kg pilocarpine relying on atlases of Kovac and Denk e3 and Montemurro and Dukelow 36. The lesions were estimated in cresyl violet-stained serial sections of the entire brain. Survival time: 48-72 h. Dark areas represent nearly total destruction of neurons accompanied by edema and disruption of the neuropil. Hatched areas represent moderate cellular destruction with partial neuronal loss apparent as numerous shrunken and dark staining neurons. Dotted areas represent mild cellular destruction with more isolated degenerating neurons which were found in several scattered locations. Abbreviations for anatomical structures: am, amygdaloid nuclear complex; ce, entorhinal cortex; cpf, piriform cortex; gm, medial geniculate nucleus; s, subiculum; sn, substantia nigra; tan, anterior thalamic nucleus; tl, lateral thalamic nucleus; tmd, dorsomedial thalamic nucleus; tpo, posterior thalamic nucleus; tpt, pare nte- nial thalamic nucleus; tpv, paraventricular thalamic nucleus; trh, rhomboideus thalamic nucleus; tv, ventral thalamic nucleus.

either intraamygdaloid37, 55 or intrahippocampa152,55

injections of cholinomimetics in rats which induced behavioral and electrographic occurrence of limbic seizures and no symptoms of respiratory distress. The destruction of the regio superior of the dorsal hippocampus is rather characteristic for the hippocampal pathology found after these treatment37,55.

Lemercier et al. 2s very recently formulated a tentative hypothesis that both seizure-related and ischemic factors may contribute to the neuropathological effects of acute and chronic poisoning of rats with soman, an organophosphorous compound with irre- versible anticholinesterase activity. These authors favored a predominant role for an ischemic factor, related to prolonged nicotinic cholinergic stimulation, as a tentative cause of the brain damage produced by soman 25. In contrast, McDonough et al. 30, on the basis of the metabolic activation of forebrain

structures in rats submitted to sustained seizures produced by soman, suggest that the brain damage induced by the organophosphate is linked to the pres-

ence of seizure activity per se. Whatever the underlying mechanism of brain damage produced by soman intoxication may be, the EEG monitoring and behavioral observation of mice and rats 5a after pilocarpine

treatment suggests that a major link between prolonged seizure activity within the areas undergoing extensive neuropathological changes and distribution of the brain damage exists.

The topography of neuropathological alterations detected in brains of mice treated with pilocarpine resembled the pattern of metabolic activation of forebrain structures following prolonged hippocampal seizures 57. Experimental evidence provided by Wat- son et al. 57 indicates that the generation of the dorsal hippocampal seizures with the prolonged electrical


250

stimulation of the granule cells of the dentate gyrus in the rat results in the metabolic activation bilaterally within the amygdala, piriform and entorhinal cortex, .claustrum, olfactory tubercle, cingulate and prefron- tal cortex, mediodorsal, reuniens and ventral thalamic nuclei as well as within the substantia nigra and substantia innominata. The topography of metabolic activation resulting from the prolonged dorsal hippocampal seizures may be understood in terms of a progressive development of secondary epileptic loci, the ventral subiculum being regarded to serve as a crit- ical region in the propagation of afterdischarges from, the hippocampus to the amygdala, lateral septum and entorhinal cortex 22. A consideration of the com- mitment of multiple (secondary) seizure loci in the propagation of afterdischarges within the limbic cir- cuitry suggests a relevant role for gradual recruit- ment of sensitive regions as a function of the duration of seizures and its severity in the development of electrographic and pathological aspects of temporal lobe epilepsy 11. The importance of anatomical pathways in the propagation of abnormal electrical activity is demonstrated not only by the similarities in the topographical distribution of a brain damage linked to different mechanisms which interact in the initiation of limbic seizure activity 5~7,10,11,26,35,37,38,45,46,

50.51,54,55, but in the limitation of the metabolic activa-

tion to the first synapse, when the stimulation of the specific area did not elicit seizures57, as well. This ob-

servation does not rule out the role of cholinergic pathways in the pathogenesis of the epileptic brain damage, but it indicates that sustained stimulation of muscarinic cholinergic sensitive regions elsewhere within the limbic system, e.g. within the projection fields of the septum, nucleus of diagonal band and substantia innominatalS, may result in the recruit- ment of multiple epileptic loci, the situation being re- flected in a progressive buildup of behavioral and electrographic seizures finally resulting in a status epilepticus. This hypothesis indicates that early stages of the development of limbic seizure activity produced by pilocarpine in mice are relatively specific and dependent on the stimulation of cholinoceptive parts of the limbic system, but it does not necessarily mean that this is the only mechanism responsible for the final consequences of cholinergic stimulation, particularly those observed during advanced stages of the convulsive activity. Such a hypothesis predicts

the role for other excitatory mechanisms in the pathogenesis of limbic seizures produced by pilocarpine, regardless of where those mechanisms function. In fact, it is well known that ACh mediates long- term increases in excitability within the hippocampus and neocortex: 4.8.9.24 and may account for increases

in the effectiveness of other excitatory inputs to these structuresS,4~. It seems likely that this mechanism may be responsible for either lowering the seizure threshold or facilitating triggering seizure discharges, and could develop a higher susceptibility to recurrent seizuresS.

The pivotal role for cholinergic pathways in deli- miting the extent and topography of the epileptic brain damage is demonstrated by both the localiza- tion of destructions within the projection fields of the cholinergic forebrain system, prevention of seizure activity and brain damage with scopolamine, and by the finding that chemical stimulation of cholinergic structures of the basal forebrain produced sustained epileptiform activity along their efferent pathways 32 and epileptic brain damage which parallels the densi- ty of cholinergic innervation from these nuclei32.59. Evidence from electrophysiological experiments rein-

forces the assumption that ACh may be involved in certain neurodegenerative processes within the brain. Muscarinic cholinergic excitation within the mammalian CNS occurs as a result of a reduced voltage-dependent and Ca-dependent K + conductance, and is mediated by voltage-dependent Ca 2÷ and Na + conductances 2,3. Such an action favors inward move- ments of Ca2+ and Na +, an effect which is primarily responsible for a prolonged membrane depolarization and might be relevant for the epileptic cell death 33,34. In fact, prolonged seizure activity shifted the balance between inward and outward Ca 2+ cur- rents, resulting in an excess accumulation of Ca 2+

within the ce1127,42; the nature of calcium-dependent

cell death is based on the failure of the cell to extrude or sequester Ca 2+ in the mitochondria 33,34.

Thus, two hypotheses may be offered for the neurotoxicity of pilocarpine and other muscarinic cholinergic agonists: either that the drug directly destroys neurons by the changes in membrane conductance resulting in a tonic depolarization or by inducing intense epileptic discharges. The results of our experiments are consistent with both of these hypotheses, but do not conclusively prove any specific mechanism


for pilocarpine-induced brain damage. Although the distribution of the brain damage in mice reflects well the areas within the projection fields of the basal forebrain cholinergic system 15, it does not allow us to specify the cholinergic mechanisms as a factor indica- tive of and responsible for the pathogenesis of the neuronal degeneration.

The data on the abortive effect of scopolamine against pilocarpine-induced seizures reveal a neurotransmitter specificity in the generation of the epileptic response to pilocarpine. However, if the cholinergic overstimulation was a sufficient condition for the neurotoxicity of pilocarpine, then the muscarinic cholinergic antagonist scopolamine should be able to prevent the development of pilocarpine-induced seizure activity as well as to stop seizures after they have emerged and progressed into the status epilepticus. From the observations in rats, it is evident that scopolamine prevented the buildup of seizures, but failed to disrupt pilocarpine-induced status epilepticus 53. In mice, scopolamine given 1 h after injection of 325 mg/kg of pilocarpine, when the status epilepticus has been well developed, failed to disrupt the seizures and reduced neither the severity of them nor the lethal toxicity of pilocarpine. Whatever the basic mechanism of the brain damage produced by pilocarpine may be, these experiments show that cholinergic circuits are very important for the generation of sustained seizure activity, and emphasize the relevance of seizure activity in pathological sequelae of pilocarpine action.

From the results of the intraamygdaloid injections of cholinomimetics37. 55, kainic acid 5, folates 38, GABA antagonists 50 and morphine 54 in rats, which revealed that initiation of abnormal electrographic activity within the limbic circuits linked to divergent receptor mechanisms resulted in very similar brain pathology, it seems evident (i) that epileptic brain damage in experimental animals involves the same mechanism regarding the development, and (ii) that one neurotransmitter system cannot be considered pivotal in the genesis of the seizures and their pathological sequelae.

Diazepam specifically prevented the seizure activity and brain pathology reproducibly observed in animal models of limbic seizures 5-7,52. Diazepam prevented the development of a sequence of seizure-related behavioral and EEG alterations, and wide-

251

spread damage to forebrain structures produced by pilocarpine in mice, also. The preferential effect of

diazepam on the limbic type of seizure activity produced by pilocarpine fits well with the potency of the drug in inhibiting the limbic seizure activity and brain damage produced by both intraamygdaloid and systemic injections of kainic acid in rats 5,6. The mechanism of action of benzodiazepines within the CNS is currently interpreted in terms of an enhancement of the GABA-mediated synaptic inhibition TM. Autora- diographic studies have shown that hippocampus, amygdala and cerebral cortex are particularly rich in GABA and benzodiazepine receptors 18. Further- more, there is evidence indicating that the anticonvulsant activity of benzodiazepines is specifically confined to the limbic system and dependent on the intactness of both the hippocampus 13,14 and the amygdala 20. Accordingly, one would expect that diazepam potentiates the effects of endogenous GABA in affected brain areas which counteracts the neurotoxic activity of pilocarpine. The finding of Mc- Geer et al. 32 and Wong et al. 59 on the loss in both GAD activity and [3H]quinuclidynylbenzilate binding within the amygdala, piriform and frontal cortex following the chemical stimulation of the substantia innominata with folic acid makes this hypothesis likely.

In summary, the pharmacological demonstration of a potent convulsant and brain damaging action of pilocarpine in mice supports and extends the propo- sal of usefulness of this treatment as a model for iden- tifying drugs preferentially active in the treatment of temporal lobe epilepsy. The relevance of this model is stressed by a broad margin of safety between seizures and mortality. Further evaluation of this hypothesis will depend on the results of studies specifically designed to find out whether drugs such as carbamazepine, phenytoin, valproate and clonazepam affect the development of behavioral, electroencephalographic and neuropathological alterations produced by pilocarpine in mice.

ACKNOWLEDGEMENTS

The authors wish to express their gratitude to Dr. John W. Olney for critically reading the manuscript. This work was supported by grants from FAPESP and CNPq of Brazil to E.A.C., Z.A.B. and L.M.M.


252

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seizures produced by pilocarpine in mice: a behavioral, electroencephalographic and morphological...

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