Drug-induced audiogenic seizure and its suppression

Transcription

1 Drug-induced audiogenic seizure and its suppression Item Type text; Thesis-Reproduction (electronic) Authors Wong, Franklin Chiu-Leung Publisher The University of Arizona. Rights Copyright is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 21/04/ :27:23 Link to Item

2 DRUG-INDUCED AUDIOGENIC SEIZURE AND ITS SUPPRESSION by Franklin Chiu-Leung Wong, A Thesis Submitted to the Faculty of the COMMITTEE ON PHARMACOLOGY (GRADUATE) In Partial Fulfillment of the Requirements for the Degree of.master OF SCIENCE In the Graduate College THE UNIVERSITY OF ARIZONA

3 STATEMENT BY AUTHOR This thesis has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. APPROVAL BY THESIS DIRECTOR This thesis has been approved on the date shown below: ^ LINCOLN CHIN Date * Professor of Pharmacology and Toxicology

4 This thesis is dedicated to those who know I love them.

5 ACKNOWLEDGMENTS If induction of decent ideas and suppression of inappropriate notions are. the two basic modes of the approach to Truth, the author wishes to express his appreciation to his thesis committee who have taught him their correct sequence along his thesis research. Specially, the author wishes to thank Dr. Chin for generous provision of experimental materials and patient guidance along the course of the authorts graduate study. The author is indebted to Dr. Picchioni for his close concern along the research project, and to Dr. Laird for critical discussions. Dr. Consroe1s generous provision of raw data of his anticonvulsant studies helped greatly in the author s anticonvulsant studies and is appreciated very much. Finally, but not the least, the author wishes to thank his parents who provided him financial assistance from the other side of the earth so that his graduate studies could be completed.

6 TABLE OF CONTENTS Page LIST OF TABLES.... vii LIST OF ILLUSTRATIONS... ix ABSTRACT... x 1. INTRODUCTION... 1 Epilepsy and Experimental Epilepsy Electrically-induced Seizures Chemically-induced Seizures Seizures Induced by Metabolic Alteration... 3 Seizures Induced by Sensory Stimulation... 3 Audiogenic Seizure Susceptible Rats Drug-induced Audiogenic Seizure Susceptibility OBJECTIVES M E T H O D O L O G Y... ' General P r o c e d u r e A n i m a l s Audiogenic Seizure and Audiogenic Response Score (ARS) Neurotoxicity T e s t Statistical Analysis D r u g s Induction of Audiogenic Seizure Susceptibility by Drugs in Genetically Nonaudiogenic Rats Suppression of Pentylenetetrazol-induced Audiogenic Seizure RESULTS Induction of Audiogenic Convulsion Susceptibility Isoniazid (INH), Ro , and Combinations of INH and Ro * 18 Pentylenetetrazol (PTZ) by i.p. Injection or in Combination with Ro Pentylenetetrazol (PTZ) by s.c. Injection v

7 TABLE OF CONTENTS Continued Suppression of PTZ-induced Audiogenic Seizure by Anticonvulsants Clonazepam»... Valproic Acid... Phenytoin... DISCUSSION 00, SUMMARY AND CONCLUSION... o...., «Summary, ,....., Conclusion..... APPENDIX A: DRUG SOLUTIONS.... REFERENCES

8 LIST OF TABLES Table Page 1. Responses to Auditory Stimulus in Nonaudiogenic IHA Rats Pretreated with Isoniazid, i.p Responses to Auditory Stimulus in Nonaudiogenic IHA Rats Pretreated with Ro (5 mg/kg, i.p., 1 Hour Before Belling) and Isoniazid, i.p Responses to Auditory Stimulus in Nonaudiogenic Rats Pretreated with Ro , 5 mg/kg, i.p., 1 Hour Prior to Belling Responses to Auditory Stimulus in Nonaudiogenic HA Rats Pretreated with Isoniazid, i.p Responses to Auditory Stimulus in Nonaudiogenic HA Rats Pretreated with Ro (i.p., 1 Hour Prior to Belling) and Isoniazid, i.p Responses to Audiogenic Stimulus in Nonaudiogenic HA Rats Pretreated with Pentylenetetrazol (i.p., 5 Minutes Prior to Belling) Responses to Audiogenic Stimulus in Nonaudiogenic HA Rats Pretreated with Ro (2.5 mg/kg, 1 Hour Prior to Belling) and Pentylenetetrazol (i.p., 5 Minutes Prior to Belling) Response to Auditory Stimulus in Nonaudiogenic HA Rats Pretreated with-pentylenetetrazol (s.c.) Anticonvulsant Activities of Clonazepam on PTZ-Induced Audiogenic Seizure (AS) in HA Rats Neurotoxicity of the Anticonvulsants, as Determined by the Rolling Roller Method Protective Indexes (PI) and Potencies (ED50) of the Anticonvulsants in Audiogenic Seizures and PTZ S e i z u r e vii

9 viii LIST OF TABLES Continued Table 12o Page Anticonvulsant Activities of Valproic Acid on PTZ-induced Audiogenic Seizure (AS) in HA R a t s Anticonvulsant Activities of Phenytoin on PTZ-induced Audiogenic Seizure (AS) in HA Rats (

10 LIST OF ILLUSTRATIONS Figure Page 1. Audiogenic Response Score (ARS) as illustrated by Brown (1974)... 6 ix

11 ABSTRACT Audiogenic animals are potentially useful for investigating mechanisms of convulsive seizure and evaluating antiepileptic drugs. However9 audiogenic rats are not readily available to all investigators. The purposes of this investigation are to find a simple pharmacological procedure for inducing susceptibility to audiogenic seizure in commercially available rats and to evaluate the potential usefulness of such a procedure. Of the methods reported in the literature and those tested in this project, subcutaneous pentylenetetrazol, 70 mg/kg, injected 15 minutes prior to belling is the simplest procedure for consistently inducing susceptibility to audiogenic seizure. This induced audiogenic seizure is suppressed by the antiepileptic drugs, clonazepam, valproic acid, or phenytoin. The potency ranking of these anticonvulsants against the pentylenetetrazol-induced audiogenic seizure is identical to that based on genetically determined audiogenic seizure. It is thus concluded that these two types of audipgenic seizure may be similar. An additional feature of the induced audiogenic seizure is that both spontaneous pentylenetetrazol-seizure and audiogenic seizure can be evaluated in the same sample. x

12 CHAPTER 1 INTRODUCTION Epilepsy and Experimental Epilepsy Epilepsy is derived from the Greek term meaning "to fall upon or seize from without," This definition reflects the practice of ancient man to perceive spiritual or demonological possession as explanations for unusual phenomena (Scott 1973). It was Hippocrates who first suggested that epilepsy originated in the brain. Today epilepsy is defined as a group of paroxysmal recurring disturbances of the central nervous system which may be manifested as episodic impairment of consciousness5 abnormal motor phenomena (usually convulsion), psychic or sensory disorder, or disturbance of the autonomic nervous system. Such disturbances are usually associated with abnormal or excessive EEG discharge (Woodbury and Fingl 1975)". On the basis of origin, epilepsy is either idiopathic (cryptogenic or genetic) or symptomatic (organic or acquired). On the basis of clinical and electroencephalographic observations, two subdivisions are recognized: partial seizure and generalized seizure (Gastaut 1970). The exact etiology of epilepsy is not known. Since defective inhibition or excessive neuronal discharge within the brain appears common to all the epilepsies (Jasper 1972)9 it appears that many types of cerebral pathology may lead to epileptic seizure (Neidermeyer 1974). Individual susceptibility to seizure development and propagation vary greatly and

13 seems to be related to genetic, prenatal and postnatal factors (Metrakos and Metrakos 1960; Neidermeyer 1974) Experimental epilepsy in man is subject to restrictions, and basic studies for the evaluation of anticonvulsants and on mechanisms of epileptic seizure are usually carried out in animal models Four common animal models have been identified by Swinyard (1969). Electrically-induced Seizures Generalized electrical stimulation involves either supramaximal or threshold current for producing maximal or minimal seizure. The maximal electroshock seizure is characterized by tonic contraction of all skeletal muscles. It has been shown that anti-grand mal drugs block hind leg extension of maximal electroshock seizure (Swinyard 1949), During minimal seizure, animals display clonus of the limbs. Drugs that are effective against petit mal epilepsy elevate minimal electroshock seizure threshold (Toman 1964). Local electrical stimulation of discrete brain areas and the spinal cord can also produce seizures that are useful in pharmacological studies (Esplin and Curto 1960; Jobe, Rey et al. 1977). Chemically-induced Seizures A variety of chemicals which act at different sites and different levels in the central nervous system to produce convulsions have been studied. These chemicals include pentylenetetrazol (PTZ), strychnine, picrotoxin, methionine sulfoximine and hydrazides such as isoniazid and thiosemicarbazide. Central or systemic administration of convulsant

14 drugs can produce either minimal (clonic) seizure or maximal (tonic) seizure (Goodman et al. 1953). In addition, topical application to the brain of irritating agents such as penicillin or metal powders can also provoke convulsions in animals (Gutnick and Prince 1971; Goldberg et al. 1972)., Seizures Induced by Metabolic Alteration Experimental convulsions may also be induced by causing metabolic changes. Examples of such procedures include alteration of water and electrolyte balance with intraperitoneal glucose or vasopressin (Swinyard, Brown and Goodman 1952), carbon dioxide withdrawal (Woodbury et al. 1958), hyperthermia (Millichap 1958) and endocrine ablation or hormone administration (DeSalva 1962, 1963). Such technics have contributed to the understanding of seizure mechanisms, and some of them may be potential methods for the assay of anticonvulsant drugs. Seizures Induced by Sensory Stimulation Photic Stimulation. A species of baboon, papio papio, from Senegal has been found to display seizure activity in response to flickering light (Naquet 1973). This light-induced convulsion is similar to photic epilepsy in man. However, despite a relatively high incidence of response, there is great variability in sensitivity to photic stimulation and severity of seizure (Killam 1969).

15 4 Sound Stimulation. When subjected to sound stimulation, audiogenic seizure-prone rats and" mice display a distinct pattern of responses (Lindsley, Finger and Henry 1942). Such audiogenic seizure occurs consistently in susceptible rats and has been shown to be genetically linked (Bevan 1955; Krushinsky et al. 1970; Sterc 1963; Consroe, Picchioni and Chin 1979). Although audiogenic seizure in several mammalian species, ranging from mice, rats, cats, dogs, goats, and hamsters to man has been documented (Jobe 1970), audiogenic rats and audiogenic mice have been most extensively studied (Consroe, Picchioni and Chin 1979; Seyfried 1979). Audiogenic Seizure Susceptible Rats Sound-induced seizure in rats was first reported in 1924 (Donaldson 1924). It was not until 1941 that the primacy of auditory stimulus in producing the convulsive reaction was recognized (Morgan and Waldman 1941). Since then, the term "audiogenic seizure" has been used to describe such behavior. The electric bell is the most widely used sound source for provoking audiogenic seizure in mice and rats although jingling keys and high-frequency drivers have been employed (Dice 1935). The pattern of audiogenic seizure consists of several phases. In the case of the rat, when the bell is first turned on there is a startle response, which is followed by a period of latency and then wild running. The running phase may be followed by convulsion or a period of quiescence, considered to be a central inhibitory phenomenon (Sterc 1963), resumption of violent running, and then convulsion followed by

16 postictal depression. The convulsions range from minimal clonus to maximal tonic-clonic seizure, but the audiogenic seizure pattern is relatively consistent in individual animals. Audiogenic rats seldom die of audiogenic seizure (Finger 1947). A descriptive ordinal scale of seizure severity has been published (Jobe, Picchioni and Chin 1973). This scale consists of ten scores ranging from 0 to 9, depending on the severity of convulsion and the number of running phases (Figure 1). Much work has been done in attempts to shed some light on the pathogenesis of audiogenic seizures although much is still unknown. Eiectrophysiological studies of the brains of audiogenic rats have shown that subcortical areas are more important for the genesis of audiogenic seizure than cortical areas (Duplisse 1976; Krushinsky et al. 1970; Sterc 1963). Both excitatory and inhibitory neural networks are activated by sound stimulation. However, since inhibition is more labile in audiogenic rats, seizure discharges develop along subcortical.areas and spread to other parts of the brain (Sterc 1963). Since seizure discharges are subject to modulation by the inhibitory activities of various areas such as the hippocampus, caudate nuclei and cerebellum, the interplay of inhibitory activities determines the severity of the seizure (Krushinsky et al. 1970; Sterc 1963). Interestingly, when audiogenic rats are subjected to other seizure-inducing stimuli, such as electroshock or chemoshock, they exhibit lower thresholds to these stimuli than nonaudiogenic rats (Duplisse et al. 1973; Laird and Huxtable 1978; Krushinsky et al, 1970). These observations are in congruence with the hypothesis that there is a

17 6 ARS RESPONSE TO SOUND STIMULATION 0 = No response 1 * Running only; no convulsion 2 = Two running phases separated by a refractory phase; convulsive endpoint consists of generalized clonus involving forelimbs, hindlimbs, pinnae and/or vibrissae 3 = Same as 2 except only one running phase and no refractory phase 4 = Two running phases separated by a refractory phase; convulsive endpoint consists of " J '' J ' 1 * 1 " J '* ' * ' us of hindlimbs 5 * Same as 4 except only one running phase and no refractory phase. 6 Two running phases separated by a refractory phase; convulsive endpoint similar to 4 except hindlimbs are in partial tonic extension (i.e., tonic extension of thighs and legs with clonus of feet). 7 = Same as 6 except only one running phase and no refractory phase. 8 = Two running phases separated by a refractory phase; convulsive endpoint similar to 4 except hindlimbs are in complete tonic extension (i.e., animal 9 = Same as 8 except only one running phase and no refractory phase. Figure 1. Audiogenic Response Score (ARS) as illustrated by Bourn (1974).

18 general deficit of central inhibitory function in audiogenic rats (Krushinsky et al. 1970). In fact9 5-hydroxytryptamine (5HT) and norepinephrine (NE) levels are lower in the cortex and midbrain of audiogenic rats than in nonaudiogenic rats (Laird 1974). Furthermore, susceptibility to audiogenic seizure can be antagonized by centrally administered putative inhibitory neurotransmitters such as norepinephrine, 5-hydroxytryptamine or dopamine (Jobe 1970) However, central administration of amino acid neuromodulators such as taurine, GABA, glycine or aminoisobutyric acid also blocks audiogenic seizure. Yet, contents of taurine, GABA, glycine, serine, threonine, alanine, glutamate, glutamine, aspartate and phosphoethanolamfne in various brain regions of audiogenic rats are not different from those of nonaudiogenic rats (Huxtable and Laird 1978). Treatment of audiogenic rats with a catecholamine and 5HT depleting agent, such as reserpine or Ro , increased the severity of sound-induced convulsions (Picchioni et al. 1963; Duplisse 1976; Jobe, Dailey and Brown 1977). Yet, similar treatment which lowered electroshock and chemoshock thresholds in mice (Chen, Ensor and Bohner 1954, 1968) failed to induce susceptibility to audiogenic seizure in rats (Jobe et al. 1973; Duplisse 1976). On the other hand, injection of bicuculline into the inferior colliculi produced running and convulsions, an audiogenic seizure-like response, in audiogenic rats as well as nonaudiogenic rats. The main differences in response in the two types of rats is that the audiogenic rats require lower doses of bicuculline and their dose-response slope is steeper. Such seizure

19 can be antagonized with injections of y-aminobutyric acid (GABA) into the inferior colliculi (Duplisse 1976). These findings suggest that deficit in GABA transmission may be a determinant of susceptibility to audiogenic seizure in rats (Duplisse 1976; Consroe et al. 1979). Drug-induced Audiogenic Seizure Susceptibility. Attempts to precipitate seizure with auditory or visual stimuli in dogs pretreated with cortical administration of strychnine have been reported as early as 1929 by Clement! (Jasper 1972). Since only some of the dogs showed audiogenic seizure, the conclusion was that susceptibility must be due to some form of genetic predisposition (Jasper 1972). Intraperitoneal administration of subconvulsive doses of strychnine in rats also produced audiogenic seizure in 45-50% of the subjects (Arnold 1944; Snee, Ferrence and Crowley 1942). In a strain of rats which has 5% frequency of audiogenic seizure, physo- stigmine increased the frequency to 70% (Humphrey 1942a). However, because Mecholyl failed to sensitize rats to sound-induced convulsion, it was implied that the parasympathetic nervous system is not involved in the genesis of audiogenic seizure. Nicotine produced only a modest change, increasing the frequency of audiogenic seizure to 12% 'in the rats described by Humphrey (1942b). Intraperitoneal administration of a subconvulsive dose of pentylenetetrazol (PTZ) can render 30-60% of the rats susceptible to sound-induced seizure (Maier and Sack 1941; Maier, Sack and Glaser 1941; Snee et al. 1942; Karn, Lodowski and Pattin 1941). On the other hand, lesioning the cortex of nonaudiogenic rats and then lowering the inhibitory mechanism with subconvulsive

20 doses of pentylenetetrazol rendered all these rats susceptible to audiogenic seizure (Sterc 1963). Other central stimulants such as methionine sulfoximine and caffeine have been tested in rats, dogs and cats (Snee et al. 1942; Lodin 1959; Wada and Ikeda 1966) and induced audiogenic seizure susceptibility in up to 70% of the animals. Other procedures for inducing susceptibility to audiogenic seizure in rats include low magnesium diet, thyro-parathyroidectomy and low calcium diet, methods which are 40-50% effective (Greenberg, Boeler and Knoff 1942). In studying the genetic characteristics of offsprings of audiogenic rats, Jobe, Dailey and Brown (1977) reported that depletion of brain catecholamines and 5HT with Ro resulted in consistent conversion of Mrunners1' (ARS = 1) to maximal audiogenic convulsions (ARS = 9). Identical treatment of non-audiogenic offsprings (ARS = 0) of audiogenic rats resulted in audiogenic seizure in 60% of the subjects when they were exposed to belling (Jobe, Dailey and Brown 1977). On the other hand, Ro treatment of audiogenic rats that had been rendered refractory to sound stimulation failed to restore audiogenic seizure susceptibility (Duplisse 1976). However, when such rats were treated with subconvulsive doses of isoniazid, audiogenic seizure susceptibility was restored in half of the fats (Duplisse 1976). Since isoniazid is known to deplete GABA (Wood and Peesker 1972), it was suggested that deficiency in GABA transmission may be a determinant of audiogenic seizure susceptibility (Duplisse 1976; Consroe, Picchioni and Chin 1979).

21 Various methods have been employed to induce susceptibility to 10 audiogenic seizure. However, all of the procedures except for the method of combined lesion and PTZ, transformed only a portion of nonaudiogenic rats to audiogenic seizure susceptible rats. Therefore, it is desirable to find a simple procedure which is consistent for converting nonaudiogenic rats to audiogenic seizure susceptible animals.

22 CHAPTER 2 OBJECTIVES Audiogenic animals are potentially useful for investigating mechanisms of convulsive seizures (Duplisse 1976; Laird 1974; Jobe 1970; Humphrey and Vicarri 1960), as well as for evaluation of antiepileptic drugs (Consroe et al. 1979; Swinyard 1969). A major shortcoming of using audiogenic rats for such studies is that there are only a few colonies of these animals in the world, and animals from these colonies are not readily available to most investigators. Therefore, the purpose of this investigation is to find a pharmacological procedure that will consistently induce susceptibility to audiogenic convulsion in nonaudiogenic, rats. The potential usefulness of such drug-induced audiogenic seizure as a model for studying epilepsy will be evaluated by testing it against established antiepileptic drugs. 11

23 CHAPTER 3 METHODOLOGY General Procedure Animals Two nonaudiogenic strains of male rats were used. One strain consists of male Holtzman Albino Rats, inbred at The University of Arizona College of Pharmacy for 32 months (IHA rats). The second strain of rats consists of male HoItzman Albino Rats purchased directly from the same supplier. HoItzmann Company, Madison, Wisconsin (HA rats). Throughout this investigation, the animal colonies were housed at 25±2 C in a room illuminated with incandescent lights from 6 A.M. to 6 P.M. daily. The animals ( g) were maintained on dry pellets (Wayne Laboratory Blox, Allied Mills Inc., Chicago, Illinois) and water. At least one week was allowed for the rats to recover after any drug treatment, before they were used again. Audiogenic Seizure and Audiogenic Response Score (ARS) For audiogenic testing individual rats were put into a sound" chamber consisting of a galvanized metal cylinder, 16 inches in diameter and 20 inches high, and enclosed in a sound-insulated wooden cabinet A- sound level of approximately 115 db relative to 1 x 10 dyne/cm was 12

24 generated inside the chamber by means of two electric bells (Jobe 1970; 13 Jobe et ale 1973)- Sound stimulation was started shortly after placement of a rat in the chamber. The sound stimulation was continued for 120 seconds, or until a convulsion was observed," whichever was the shorter duration. The response to sound stimulation was scored by the method of Jobe et al. (1973), as illustrated and defined in Figure 1. All rats were screened once a day on 3 alternate days for response to sound stimulation. Only non-responders, i.e., rats that showed no running or convulsion (ARS = 0) during the three trials, were used in the induction and anticonvulsant studies. Neurotoxicity Test Neurotoxicity was determined by a modification of the method of Dunham and Miya (1957). Rats were trained to walk on an 8 cm diameter rotating rod revolving at 15 r.p.m. Rats were considered trained when they remained on the rotating rod without falling for 3 consecutive 2-minute trials. After the administration of each drug, rats were tested at the time of peak toxic effect of the drug. Neurotoxicity was defined as the inability of an animal to remain on the rotating rod at least one minute out of 3 consecutive trials. Statistical Analysis ^ The graphical method of probit analysis (Litchfiel 1 and Wilcoxon 1949) was used to determine the median effective dose (ED50) and median toxic dose (TD50) of each anticonvulsant. The protective index of each anticonvulsant was calculated as the ratio TD50/ED50.

25 14 Drugs Isoniazid, Rp , pentylenetetrazol, phenytoin, valproic acid and clonazepam were used for this investigation. Water, saline and/or Tween 81 (polysorbate) were used as solvents (Appendix A). Routes of administration are intraperitoneal, subcutaneous and/or oral, as specified in subsequent sections. Induction of Audiogenic Seizure Susceptibility by Drugs in Genetically Nonaudiogenic Rats 1. Isoniazid (INH) a. Optimal Time Groups of rats were injected with 150 mg/kg of INH, i.p., and then belled 7.5, 15, 30 or 60 minutes later, and ARS were recorded. b. Optimal Dose Groups of rats were injected with graded doses of INH, i.p., and then belled at the optimal time after injection, and the ARS were recorded. 2. Ro Groups of rats were injected with 5 mg/kg of Ro , i.p., then belled 1 hour later, and the ARS were recorded. 3. INH and Ro Groups of rats were injected with 2.5 or 5 mg/kg of Ro , i.p., 1 hour before belling. Then graded doses of INH, i.p., were

26 injected at various times prior to belling. The ARS were determined and recorded. 1 Pentylenetetrazol (i.p.) Groups of rats were injected with 15, 20, 30 or 40 mg/kg of pentylenetetrazol (PTZ). Five minutes later, they were belled for audiogenic seizure. Pentylenetetrazol (i.p.) and Ro Groups of rats were injected with 2.5 mg/kg of Ro , i.p., 1 hour prior to belling. Then 5 minutes prior to belling, they were injected with 15 or 20 mg/kg of PTZ, i.p. The ARS were determined and recorded. Pentylenetetrazol (s.c.) a. Optimal Time Groups of rats were injected with PTZ, 70 mg/kg, s.c., and then belled 5, 10, 15 or 25 minutes later. Prior to belling, the animals were observed for spontaneous PTZ convulsions. b. Optimal Dose Groups of rats were injected with graded doses of PTZ, s.c. At the optimal time they were belled for audiogenic seizure. Anticonvulsant Test Suppression of Pentylenetetrazol-induced Audiogenic Seizure Test subjects consisted of HA rats which were fasted for hours prior to testing. Time of peak anticonvulsant effect was

27 chosen according to previous findings of*consroe (Consroe and Wolkin 1977; personal.communication 1979)«The subjects were observed for spontaneous PTZ-seizures, sound-induced seizures, and death. The responses to sound stimulation were recorded as ARS. a. Clonazepam Groups of rats were given graded oral doses of clonazepam. Forty-five minutes later (15 minutes prior to belling) they were injected with 70 mg/kg of PTZ, s.c. b. Valproic Acid Groups of rats were given graded oral doses of valproic acid. Forty-five minutes later (15 minutes prior to belling) they were injected with 70 mg/kg of PTZ, s.c. c. Fhenytoin Groups of rats were given graded oral doses of phenytoin. Three hours and 45 minutes later (15 minutes prior to belling) they were injected with 70 mg/kg of PTZ, s.c. Toxicity Test Test subjects consisted of HA rats which were fasted for hours prior to testing. Time of peak toxicity was chosen according to previous findings of Consroe (Consroe and Wolkin 1977; personal communication 1979). a. Clonazepam Groups of rats were given graded oral doses of clonazepam. One hour later, they were tested for neurotoxicity on the rolling roller.

28 Valproic Acid Groups of rats were given graded oral doses of valproic acid. One hour later, they were tested for neurotoxicity on the rolling roller. Phenytoin One group of rats was given an oral dose of 1000 mg/kg of phenytoin. Four hours later, the animals were tested for neurotoxicity on the rolling roller.

29 CHAPTER 4 RESULTS Induction of Audiogenic Convulsion Susceptibility Isoniazid (INH), Ro , and Combinations of INH and Ro In the IHA rats, INH induced audiogenic seizure susceptibility in 33-67% of the animals during all four test periods (Table 1) The optimal time for inducing audiogenic seizure (AS) by INH occurred at 15 minutes; the highest mean audiogenic response and highest range of responses were observed at this time. The 150 and 200 mg/kg doses of INH appeared to be optimal for inducing AS susceptibility because these doses produced the highest mean ARS scores and highest range of responses. Isoniazid, in combination with 5 mg/kg of Ro , i.p., induced a higher incidence of audiogenic response (Table 2) than by INH alone. Isoniazid, 150 mg/kg, i.p., 15 minutes prior to belling, in combination with 5 mg/kg of Ro (1 hour prior to belling) rendered all 8 test rats (100%). susceptible to audiogenic seizure, and the ARS for each animal was 9 (maximal). However, 5 mg/kg of Ro by itself did not induce AS susceptibility in any of the rats (Table 3). In the HA rats, INH induced AS susceptibility in 0-50% of rats during the 3 test periods (Table 4). Most of the animals showed no response (ARS = 0) or only running (ARS = 1) when subjected 18

30 19 Table 1* Responses to Auditory Stimulus in Nonaudiogenic IHA Rats Pretreated with Isoniazid, i.p. Isoniazid Time of Injection Prior to Belling (minutes) Dose (mg/kg) Number of Animals Tested Number of Audiogenic Responses Mean Scorea (Range) (1-2) (1-3) (3-9) (3-9) (2-4) (1-2) a Mean audiogenic response score is based on responses of 1 or greater.

31 / 20 Table 2. Responses to Auditory Stimulus in Nonaudiogenic ISA Rats Pretreated with Ro (5 mg/kg, i.p., 1 Hour before Belling) and Isoniazid, i.p. Isoniazid Time of Injection Prior to Belling (minutes) Dose (mg/kg) Number of Animals Tested Number of Audiogenic Responses Mean Score3 (Range) (2-9) (3-7) a Mean audiogenic response score is based on responses of 1 or greater.

32 21 Table 3. Responses to Auditory Stimulus in Nonaudiogenic Rats Pretreated with Ro , 5 mg/kg, i.p., 1 Hour Prior to Belling. Rats Dose of Ro (mg/kg) Number of Animals Tested Number of Audiogenic Responses Mean Audiogenic Response Score IHA HA

33 Table 4. Responses to Auditory Stimulus in Nonaudiogenic HA Rats Pretreated with Isoniazid, i. Isoniazid Time of Injection Prior to Belling (minutes) Dose (mg/kg) Number of Animals Tested Number of Audiogenic Responses Mean Score3.(Range) Deaths Due to Audiogenic. Convulsion (1-2) (3-4) a Mean audiogenic response score is based on responses of 1 or greater.

34 23 to sound stimulation. audiogenic convulsions. Only 5 of 32 test animals exhibited minimal The addition of Ro pretreatment to various doses of INH did not greatly increase the: incidence of induction of AS susceptibility (Table 5), compared to INH alone (Table 4), but some of these combination pretreatments considerably enhanced the severity of the audiogenic response. A few animals died following convulsion at higher doses of INH in combination with Ro On the other hand, Ro by itself did not induce AS susceptibility in any of the HA animals (Table 3). Pentylenetetrazol (PTZ) by i.p. Injection or in Combination with Ro Usually subconvulsive doses of PTZ (20-40 mg/kg) induced a low incidence of AS susceptibility in HA rats (Table 6). In combination with 2.5 mg/kg of Ro , PTZ in doses of 15 and 20 mg/kg also induced AS susceptibility in some animals and caused death in rats that exhibited tonic seizure, ARS of 9 (Table 7). Pentylenetetrazol (PTZ) by s.c. Injection Pentylenetetrazol, in adequate doses, induced AS susceptibility 5, 10, 15 or 25 minutes following injection (Table 8). Some animals exhibited spontaneous minimal PTZ-seizures before belling, but no difference in the audiogenic seizure pattern was observed whether the AS test was performed before or after appearance of spontaneous PTZ- convulsion. In a dose of 70 mg/kg, s.c., PTZ caused spontaneous minimal convulsion in all animals 4-11 minutes (mean time of 7.5 minutes) following injection. All 4 rats that,were belled 10 minutes after

35 Table 5. Responses to Auditory Stimulus in Nonaudiogenic HA Rats Pretreated with Ro (i p<,9 1 Hour Prior to Belling) and Isoniazid, i,p. Isoniazid Time of Injection Prior to Belling (minutes) Dose (mg/kg) Dose of Ro (mg/kg) Number of Animals Tested Number of Audiogenic Responses Mean Score3 (Range) Deaths Due to Audiogenic, Convulsion (1-9) (1-9) ' a Mean audiogenic response score is based on responses of 1 or greater.

36 Table 6. Responses to Audiogenic Stimulus in Nonaudiogenic HA Rats Pretreated with Pentylenetetrazol (i.p,, 5 Minutes Prior to Belling). Pentylenetetrazol (mg/kg) Number of Animals Tested Number of Spontaneous PTZ-convulsions Number of Audiogenic Responses Mean ^ Score Deaths Due to Audiogenic Convulsion T B Observed prior to belling. ) Mean audiogenic response score is based on responses of 1 or greater. to Ln

37 26 Table 7» Responses to Audiogenic Stimulus in Nonaudiogenic HA Rats Pretreated with Ro (2.5 mg/kg,. 1 Hour Prior to Belling) and Pentylenetetrazol (i.p«, 5 Minutes Prior to Belling). PTZ Number of Number of Mean Deaths Due to (mg/kg) Animals Audiogenic Score3 Audiogenic Tested Responses Convulsion _ a Mean audiogenic response score is based on responses of 1 or greater.

38 Table 8. Response to Auditory Stimulus in Nonaudiogenic HA Rats Pretreated with Pentylenetetrazol (s.c.). Pentylenetetrazol Time of Injection Dose Prior to Belling (mg/kg) (minutes) Number of Animals Tested Spont. PTZ- Seizure Number of Audiogenic Responses Mean Score (Range) Deaths Due to Audiogenic Convulsion (5-7) (5-7) (5-7) (3-7) Observed prior to belling, occurring in 4 to 11 minutes after injection of 70 mg/kg of PTZ. k Mean audiogenic response score is based on responses of 1 or greater.

39 70 mg/kg of PTZ convulsed and died of the audiogenic seizure; whereas9 only 2 of the 8 animals that received the same dose of PTZ died when they were belled at 15 minutes. Suppression of PTZ-induced Audiogenic Seizure by Anticonvulsants Clonazepam Pentylenetetrazol-induced AS were blocked by clonazepam in a dose-related fashion (Table 9) and there were no deaths due to audiogenic convulsion. In addition, spontaneous PTZ seizure was suppressed at doses of clonazepam ranging from 0.1 to 0.4 mg/kg. The ED50 against induced AS was calculated to be 0.18 mg/kg with a 95% fiducial interval of mg/kg. In the toxicity test, ataxia and depression were the first signs of intoxication although some rats with such signs passed the roller test. The TD50 of clonazepam was calculated to be 6.8 mg/kg with a 95% fiducial interval of mg/kg (Table 10). The protective index is calculated to be 37.7 with a 95% fiducial interval of (Table 11). Valproic Acid Valproic acid in a dose range of mg/kg protected HA rats against PTZ-induced AS in a dose-related fashion (Table 12). Spontaneous PTZ seizure was also suppressed, and there were no deaths due to AS. The ED50 was calculated to be 308 mg/kg with a 95% fiducial interval of mg/kg. first sign of neurotoxicity. In the toxicity test, ataxia was usually the Relaxation and loss of muscle tone was observed.with administration of mg/kg of valproic acid. The

40 Table 9. Anticonvulsant Activities of Clonazepam on PTZ-induced Audiogenic Seizure (AS) in HA Rats. Clonazepam3, (mg/kg) Number of Rats Tested Spontaneous Seizure13 Audiogenic Seizure Deaths Number Protected Number Protected Mean Score0 (Range) Due to AS (3-5) (2-5) a Clonazepam was administered orally 1 hour prior to belling. k Spontaneous Seizure due to PTZ, 70 mg/kg, s.c,, observed up to time of belling (15 min.). u Mean audiogenic response score is based on responses of 1 or greater. K) VO

41 30 Table 10. Neurotoxicity of the Anticonvulsants, as Determined by the Rolling Roller Method. Drug TD50 (mg/kg) 95% Fiducial Interval Clonazepam Valproic Acid Phenytoin3 >1000 ) 3 TD50 cannot be determined mg/kg of phenytoin produced 11.1% toxicity.

42 Table 11 o Potencies (ED50) and Protective Indexes (PI) of the Anticonvulsants in Audiogenic Seizures and PTZ Seizure. scptz-induced Audiogenic Seizure3 Genetic ^ Audiogenic Seizure PTZ Seizure0 Drugs ED50 (mg/kg) (95% C.I.)d PI (95% C.I.)d ED50 (mg/kg) PI ED50 (mg/kg) PI Clonazepam 0.18 ( ) 37.4 ( ) Phenytoin 125 ( ) >8 42 >24 not active - Valproic Acid 308 ( ) 4.2 ( ) a Results from this investigation. k Data from Consroe and WoIkin (1977) and Consroe et al. (1979). C Data from Krall et al. (1978). Fiducial Interval.

43 Table 12» Anticonvulsant Activities of Valproic Acid on PTZ-induced Audiogenic Seizure in HA RatSo Valproic Acida (mg/kg) Number of Rats Tested b Spontaneous Seizure Audiogenic Seizure Deaths Due to Number Number Mean Score*- Audiogenic Protected Protected (Range) Convulsion (3-5) a Valproic acid was administered orally 1 hour prior to. belling, k Spontaneous Seizure due to PTZ, 70 mg/kg, s«c»9 observed up to time of belling (15 min.)». c Mean audiogenic response score is based on responses of 1 or greater»

44 TD50 was calculated to be 1270 mg/kg with a 95% fiducial interval of mg/kg (Table 10). The protective index was calculated to be 4»2 with a 95% fiducial interval of (Table 11). Phenytoin Phenytoin in a dose range of mg/kg protected the HA rats against PTZ^induced AS in a dose-related fashion (Table 13)- However, spontaneous PTZ seizure was not suppressed. The spontaneous PTZ seizures prior to belling showed enhanced severity, consisting of violent incoordinated convulsive movements of the limbs. Except for the group treated with the 400 mg/kg dose, phenytoin did not appear to protect the animals from death during or following AS. There were no deaths in the animals treated with 400 mg/kg of phenytoin. The ED50 of phenytoin against induced-as was calculated to be 125 mg/kg with a 95% fiducial interval of mg/kg (Table 12). When the rats were tested for neurotoxicity, very few signs were observed even at a dose of 1000 mg/kg. Only 2 of the 18 rats tested (11.1%) showed neuro-. toxicity at this dose. It was not practical to test larger doses of phenytoin for toxicity because the volume for oral administration of 1000 mg/kg of phenytoin was already about 3 ml for each rat. The TD50 was thus considered as greater than 1000 mg/kg, and no further doses were evaluated.

45 Table 13. Anticonvulsant Activities of Phenytoin on PTZ-induced Audiogenic Seizure (AS) in HA Rats. Phenytoin3 (mg/kg) Number of Rats Tested Spontaneous Seizure Number Protected Audiogenic Seizure Number Mean Score0 Protected (Range) Deaths Due to AS or to Subsequent Convulsions ,(3-5) (3-5) (3-5) 4 ' a Phenytoin was administered 4 hours prior to belling Spontaneous seizure due to PTZ, 70 mg/kg, s.c.» observed up to time of belling (15 min.). c Mean audiogenic response score is based on responses of 1 or greater. Uo 4^

46 CHAPTER 5 DISCUSSION There are inhibitory mechanisms in the central, nervous system which appear to provide feedback control of neural circuits to limit spread of discharges in the brain.' These inhibitory mechanisms may involve presynaptic or postsynaptic inhibition. The mechanism of most convulsant drugs has been linked to their ability to remove these inhibitory mechanisms (Straighan 1975). Isoniazid (INH) which'lowers brain GABA levels and can cause convulsions (Wood and Peesker 1972; Saad, ElMasry and Scott 1972), can render human subjects sensitive to photic and auditory stimuli, so that flickering light or repeated sound produces spikes of high potentials in the EEC and myoclonic movement of the extremities (Reilly et al. 1953). In the dose range tested, INH pretreatment induced a similar incidence of susceptibility to audiogenic seizure (AS) in IRA rats as in HA rats. However, the IHA rats that became AS susceptible exhibited more severe convulsions than the corresponding HA rats (compare Table 1 and Table 4). The catecholamine and 5HT depleting drug, Ro did not induce AS, but its addition to the INH pretreatment resulted in a higher incidence of induction of susceptibility to AS and an increase in severity of AS in IHA rats (compare Table 1 and Table 2). In comparison, the incidence of induction in HA animals was not noticeably increased although the animals that responded to sound 35

47 stimulation exhibited marked increase in severity of convulsions (compare Table 4 and Table 5). It is also noteworthy that peak induction occurs earlier in IHA rats than in HA rats. The reason for the apparent greater sensitivity of IHA rats to AS induction, compared to HA rats, is unknown. However, it is possible that deficit(s) of genetic origin in the IHA rats may have been amplified by the random in-breeding of a small colony. Indeed, it was observed during the screening procedure that 50% of the IHA rats exhibited sound-induced running, whereas only 10% of the HA rats did so. The i.p. pretreatment of HA rats with subconvulsant doses of PTZ (15-40 mg/kg) made 0-25% of the animals susceptible to audiogenic seizure and caused some animals to die during AS. The addition of Ro to the pretreatment marginally increased the rate of induction and also enhanced the severity of response (compare Table 6 and Table 7). The subcutaneous pretreatment of HA rats with PTZ, 70 mg/kg, induced AS susceptibility in 100% of the subjects (Table 8). However, it was noted that all of these subjects exhibited spontaneous PTZ convulsions a few minutes prior to sound stimulation (i.e. PTZ convulsion occurred 4-11 minutes after pretreatment). In addition, 2 of 8 animals died as the result of AS. Fifteen minutes appears to be the optimal pretreatment time; a longer interval results in a lower incidence of induction and a shorter interval results in a higher incidence of fatality. It is not surprising that 70 mg/kg of PTZ, s.c., resulted in some fatality because the LD50 for s.c. PTZ is 100 mg/kg (Gross and Featherstone 1946). Although the convulsant mechanism of

48 37 PTZ is uncertain, it has been suggested that it works through selective reduction of transmitter-induced chloride conduction, which results in a disinhibitory effect (Pellman and Wilson 1977). Initial apprehensions that spontaneous PTZ convulsion may cause postictal depression and interfere with induction of susceptibility to AS were dispelled by subsequent observations. Not only was there no apparent interference, but spontaneous PTZ convulsions and induction of susceptibility to audiogenic seizure by PTZ injection suggest that 2 convulsion tests may be possible in a single animal model. The potential advantages of such an animal model are obvious, and the only additional equipment needed is a sound chamber. It would be desirable to investigate this experimental procedure in nonaudiogenic mice, since this species of laboratory animal is most commonly employed for the subcutaneous PTZ seizure threshold test (Krall et al. 1978) The practicability of inducing 2 types of seizure in mice remains to be established, but its practicability in rats has been demonstrated. Of the methods available for inducing susceptibility to AS in rats, only two are consistently reliable, the method of Sterc (1963) which requires surgery plus i,p. pentylenetetrazol in sub convulsive doses, and the use of s.c. pentylenetetrazol in a 97% convulsant dose of 70 mg/kg (Swinyard 1972). Of the two procedures, s.c. PTZ is the simpler and was selected for further study. Since different seizure models can be evaluated on the basis of pattern of responses to clinically established antiepileptic drugs (Toman 1964), anticonvulsant studies were performed to evaluate the s.c. PTZ-induced AS model. The anticonvulsants selected for the present study, clonazepam, phenytoin

49 38 and valproic acid have markedly different potencies and protective indexes in the genetic AS model. These published data from the genetic model (Consroe et al. 1979; Consroe and Wolkin 1977) were used as a basis for comparison with the data derived from the new model. Clonazepam is one of the benzodiazepines which are known for their broad spectrum of antiepileptic activities. It was found to be the most potent of the three drugs tested for suppressing s.c. PTZ- induced AS. Clonazepam also protected against spontaneous PTZ convulsion as well as de&th due to induced audiogenic convulsions. These findings are parallel to the ability of clonazepam to elevate the threshold for electroshock seizure in rodents and to protect baboons against photomyclonic seizure (Reynolds 1978; Stone and Javid 1978). The mechanism of anticonvulsant action of clonazepam has been speculated to be an activation of GABA receptor and hence increased efficiency of GABA inhibition (Baraldi et al ). Valproic acid is effective in suppressing s.c. PTZ-induced AS at high doses (Table 12). It is the least potent of the three anticonvulsants against PTZ-induced AS. However«, like clonazepam, it also protects against spontaneous PTZ seizure and death due to induced AS. Its mechanism of anticonvulsant action has been suggested to be through an elevation of GABA level in the brain (Simler et al. 1973). Phenytoin is a classical drug for the treatment of grand mal epilepsy. It failed to protect against spontaneous PTZ convulsion, and it exaggerated and prolonged clonus, as expected (Woodbury and Fingl 1975). Surprisingly, it protected against PTZ-induced AS, although it failed to

50 39 protect against lethality, A few test subjects which were not protected against AS died during audiogenic convulsion. Of the remaining animals that died, death occurred during repeated convulsions subsequent to the audiogenic test. It is of interest to note that the largest dose of phenytoin (400 mg/kg) protected against death, despite repeated convulsions following the audiogenic test. The basic mechanism by which phenytoin exerts its anticonvulsant effect remains to be determined. However, there is considerable evidence that its ability to prevent post-tetanic potentiation and to stabilize neuronal membrane may result from effects on the movement of ions across cell membranes (Watson and Woodbury 1972; Fertziger and Ranck 1970; Woodbury 1972). The observation that phenytoin protects against PTZ-induced audiogenic seizure but does not protect against spontaneous minimal PTZ convulsion implies that the two types of experimental seizures may involve different neural mechanisms or pathways. It was not possible to rank accurately the protective indexes of the three anticonvulsants based on studies with PTZ-induced AS in rats because it was not possible to determine the median toxic dose (TD50) of phenytoin. However, ranking of the three anticonvulsants in terms of the median effective dose (ED50) against PTZ-induced AS revealed that clonazepam is the most effective, phenytoin is of intermediate effectiveness, and valproic acid is the least effective. This ranking agrees with the ranking based on studies involving genetically determined audiogenic rats (Consroe et al. 1979), and implies that PTZ- induced AS in rats bears a similarity to genetically determined AS in

51 rats. Therefore, the possibility exists that the PTZ-induced AS model may be useful for certain studies in.which the genetic AS model is employed. Ranking of the three anticonvulsants on the basis of their ability to protect against spontaneous PTZ seizure (Tables 9, 12, 13) reveals that clonazepam is most effective, valproic acid is of intermediate effectiveness, and phenytoin is ineffective. This ranking agrees with the s.c. PTZ test in mice (Krall et al. 1978), and serves to emphasize the possibility that a single animal model may be used for eliciting two different types of experimental convulsions.

52 CHAPTER 6 SUMMARY AND CONCLUSION Summary The results of this investigation may be summarized as follows: Subconvulsive doses of isoniazid (INH) or pentylenetetrazol (PTZ) inconsistently induce susceptibility to audiogenic seizure in genetically nonaudiogenic rats. Treatment of nonaudiogenic rats with Ro does not result in induction of susceptibility to audiogenic seizure. However, addition of Ro to INH or PTZ pretreatment of nonaudiogenic rats results in increased severity of induced audiogenic seizure. Pretreatment of nonaudiogenic rats with PTZ, 70 mg/kg, s.c., 15 minutes prior to belling consistently results in spontaneous minimal convulsion and also consistently induces susceptibility to audiogenic seizure. Clonazepam, phenytoin and valproic acid, in this order of relative effectiveness, suppress PTZ-induced audiogenic seizure. This ranked order agrees with published data on the ranking of the three anticonvulsants against genetic audiogenic seizure in rats. Clonazepam and valproic acid, in this order of relative effectiveness, suppress spontaneous minimal PTZ seizure in rats, but phenytoin is inactive in this regard. This ranked

53 42 order agrees with published data on the ranking of the three anticonvulsants against s.c. PTZ seizure in mice. Conclusion Subcutaneous injection of PTZ, 70 mg/kg, is a simple procedure for inducing susceptibility to audiogenic seizure in genetically nonaudiogenic rats. On the basis of studies with three antiepileptic drugs, this induced audiogenic seizure appears to be similar to genetically determined audiogenic seizure. Additionally, the PTZ- induced audiogenic seizure model may be useful for providing two convulsion tests with use of one population sample. Although this latter possibility appears promising, additional studies are needed to determine whether it will fulfill the hope of the author that this model will serve as another effective tool for contributing to the knowledge arid treatment of epilepsy.

54 APPENDIX A DRUG SOLUTIONS 1) Isoniazid : Sigma Chemical Co.. Dose: 125, 150, 200, or 250 mg/kg Route: Vehicle; Intraperitoneal Deionized water Concentration: 100 mg/ml 2) Ro (2-hydroxy-2-ethyl-3-isobutyl-9,10-dimethoxy- 1,2,3,4,6,7-hexahydrox-ll-bH-benzo(a)quinolizine): Hoffmann-LaRoche Inc. Dose: Route: Vehicle: 2.5 or 5 mg/kg Intraperitoneal Saline (0.9% NaCl in deionized water) Concentration: 2.5 mg/ml 3) Pentylenetetrazol: Knoll Pharmaceutical Co. Dose: Route: Vehicle: 15, 20, 40, 50, 60 or 70 mg/kg Intraperitoneal or subcutaneous Saline Concentration: 20 mg/ml 4) Tween 81 (polyoxyethene (5) Sorbitan Monooleate): Ruger Chemical Co. 43

55 44 5) Clonazepam: Hoffmann-LaRoche Inc. Dose : Route: 0.1, 0.2, 0.4, 2.0, 4.0 or 8.0 mg/kg Oral Vehicle:- 10% Tween 81 in saline Concentration: 0.1 mg/ml or 2 mg/ml 6) Valproic Acid: Saber Laboratories Inc. Dose: Route; Vehicle: 200, 300, 500, 600, 900, 1500 or 2000 mg/kg Oral 1% Tween 81 in saline Concentration: 100 mg/ml or 500 mg/ml 7) Phenytoin; Aldrich Chemical Co. Dose- Route; Vehicle; 25, 50, 100, 200, 400 or 1000 mg/kg Oral 1% Tween 81 in saline Concentration: 25 mg/ml or 100 mg/ml

56 REFERENCES Arnold9 M. B. Emotional factors in experimental neuroses, Exp, Psychol,^ 34: Baraldi, M., A. Guidotti, J. P. Schwartz and Z. Costa. Benzodiazepines and GABA receptor: Clonal cell studies. Science 205: Bevan? W. Sound-precipitate convulsion: 1947 to Psychol. Bull. 52: Bourn3 W. M. Modulation of Audiogenic Seizures by Cortical Norepinephrine in the Rat. (Doctoral Dissertation). Tucson, The University of Arizona, Chen, G., C.R. Ensor and B. Bohner. A facilitative action of reserpine on the central nervous system. Proc. Soc. Exp. Biol. N.Y. 86: , 1954., Studies on drug effects on electrically-induced extensor seizures and clinical implications. Arch. Int. Pharmacodyn. Ther. 172: , Consroe, P. Associate Professor of Pharmacology and Toxicology, The University of Arizona, Tucson, Consroe, P., A. Picchioni and L. Chin. Audiogenic seizure susceptible rats. Fed. Proc. ^8: , Consroe, P. and A. Wplkin. Cannabidiol - antiepileptic drug comparisons and interpretations in experimentally induced seizures in rats. J. Pharmacol. Exp. Ther. 2_0:26-32, DeSalva, S. Effects of centrally acting drugs in intact and hypophysectomized rats on electroshock threshold. Arch. Int. Pharmcodyn. 137: , 1962., EST effects of diphenyihydantoin in hypophysectomized rats. Arch. Int. Pharmacodyn. 142: , 1963 Dice, L. R. Inheritance of waltzing and of epilepsy in mice of the genus Pero myscus.. J. Mamol. _16:25-35, Donaldson, H. H. The Rat: Data and Reference Tables, 2nd ed., p. 134, Wistar Institute, Philadelphia,

57 46 Dunham9 N» W. and T. S. Miya. A note on a simple apparatus for detecting neurological deficit in rats and mice. J. Amer. Pharm. Assoc. 46:208^209, Duplisse, B. R, Mechanism of Susceptibility of Rats to Audiogenic Seizure (Doctoral Dissertation). Tucson. The University of Arizona, Duplisse, B. R., A. L. Picchioni, L. Chin and P. Consroe, Electroshock seizure threshold: differences between audiogenic and nonaudiogenic rats. J. Int. Res. Comm, 1:9, Esplin, D. W. and E. M. Curto. Physiological and pharmacological analysis of spinal cord convulsions. J. Pharmacol. Exp. Ther. 130:68-80, Fertziger, A. P. and J. B. Ranck. Potassium accumulation in interstitial space during epileptiform seizure. Expl. Neurol. 26: , Finger, F. W. Convulsive behavior in the rat. Psychol. Bull. 44: , Gastaut, H. Clinical and electroencephalographical classification of epileptic seizure. Epilepsia 11: , Goldberg, A. M., J. J. Pollock, E. R. Hartman and C. R. Craig. Alterations in cholinergic enzymes during the development of cobaltinduced epilepsy in the rat. Neuropharmacol. J_l: , Goodman, L. S., M. S. Grewak, W. C. Brown and A. Swinyard. Comparison of maximal seizure evoked by pentylenetetrazol (metrazol) and electroshock in mice and their modification by anticonvulsants. J. Pharmacol. Exp. Ther. 108: , Greenberg, D. M., M. D. D. Boeler and B. W. Knoff. Factors concerned in the development of tetany by the rat. Amer. J. Physiol. 137: , Gross, E. G. and R. M. Featherstone. Studies of tetrazole derivatives. 1. Some pharmacologic properties of aliphatic substitutes pentamethylene tetrazol derivatives. J. Pharmacol. Exp. Ther. 87: , Gutnick, M. J., D. A. Prince. Penicillinase and the convulsant penicillin. Neurology 2_1: , Humphrey, G. Experiments on the physiological mechanism of noiseinduced seizures in the albino rat. I. The action of parasympathetic drugs. J. Comp. Psychol. 3_3: , 1942a.

58 47 ' 5 Experiments on the physiological mechanism of noise-induced seizures in the albino rat. II. The site of action of the parasympathetic drugs. J. Comp. Psychol. 33: , 1942b. Humphrey, M. and E. Vicarri. A study of some physiological mechanisms underlying susceptibility to audiogenic seizure in mice. J. Neuropathol..Exp. Neurol. 19^: , Huxtable, R. J. and H. E. Laird. Are amino acid patterns necessarily abnormal in epileptic brain? Studies on the genetically seizure-susceptible rats. Neurosci. Lett. 10^: , Jasper, H. H. Application of experimental models to human epilepsy. In: Experimental Models of Epilepsy, ed. by D. P. Purpura, J. K. Penry, D. Tower, D. M. Woodbury and R. Walter. New York, Raven, 1972, pp Jobe, P. C. Relationship of Brain Amine Metabolism to Audiogenic Seizure in the Rat. (Doctoral Dissertation). Tucson, The University of Arizona, Jobe, P. C., J. W. Dailey and R. D. Brown. Effects of Ro (RO) in audiogenic seizure susceptibility and intensity in rats. Neurosci. Abst, 3_:141, Jobe, P. C., A. L. Picchioni and L. Chin. Role of brain norepinephrine in audiogenic seizure in the rat. J. Pharmacol. Exp. Ther. 184:1-10, Jobe, P. C., T. B. Rey, P. F. Geiger and W. M. Bourn. Effects of Ro on electrically induced spinal cord seizures and on spinal cord norepinephrine and 5-hydroxytryptamine levels. Res. Comm. Chem. Pathol. and Pharmacol. 18(4): , Kam, E. W., C. H. Lodowski and R. A. Pattin. The effect of metrazol on the susceptibility of rats to sound-induced seizure. J. Comp. Psychol. 32: , Killam, K. R. Experimental Epilepsy in Primates. Ann. N.Y. Acad. Sci. 162: , Krall, R. L., J. C. Penry, B. G. White, H. J. Kupferberg and E. A. Swinyard. Antiepileptic drug development. II. Anticonvulsant drug screening. Epilepsia 19: , Krushinsky, L. V., L. N. MoTodkina, D. A. Fless, L. P. Debrokjotova, A. P. Steshenko, A.Z. Semiokhina, A. Z. Zorina and L. B. Romanova. The functional state of the brain during sonic stimulation. In: Physiological Effects of Noises, ed. by B. L. Welch and A. S. Welch. New York, Plenum, p , 1970.

59 48 Laird, H. E. 5-Hydroxytryptamine as an Inhibitory Modulator of Audiogenic Seizure (Doctoral Dissertation). Tucson, The University of Arizona, Laird, H. E. and R. Huxtable. Taurine and audiogenic epilepsy. In: Taurine and Neurological Disorders, ed. by A. Barbeau and R. Huxtable. New York, Raven, p , 1978* Lindsley, D. B., F. W. Finger and C. E. Henry. Some physiological aspects of audiogenic seizures in rats. J. Neurophysiol, : , Litchfield, J. T. and F. Wilcoxon. A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96:99-133, Lodin, Z. The effect of some external stimuli on seizure produced by methionine sulfoximine. Physiol. Bohemoslov. 8^: , Maier, N. R. F. and J. Sack. Studies of abnormal behavior in the rat. VI. Patterns of convulsive reaction to metrazol. J. Comp. Psychol. 32: , Maier, N. R. F., J. Sack and N. M. Glaser. Studies of abnormal behavior in the rat. VIII. The influence of metrazol on seizure occuring during auditory stimulation. J. Comp. Psychol. 32: , Metrakos, J. D. and K. Metrakos. Genetics of convulsive disorders. I. Introduction, problem, methods and base lines. Neurology 10: , Millichap, J. G. Effects of drugs on experimental febrile seizures: development of a new specific therapy. Amer. J. Dis. Chil. 96: , Morgan, C. T. and H. Waldman. Conflict and audiogenic seizures. T. Comp. Psychol. 31:1-11, Naquet, R. Contribution of experimental epilepsy to understanding some particular forms in man. In: Epilepsy, its Phenomena in Man, ed. by M. A. B. Brazier, New York, Academic Press, p , Niedermeyer, E. Comparison of the Epilepsies. Illinois, Thomas, 1974* Pellman, T. C. and W. A. Wilson. Synaptic mechanism of pentylenetetrazol: selectivity for chloride conductance. Science 192: , Picchioni, A. L., L. Chin, D. Choisser and C. Brietner. 5-Hycroxytryptamine and Norepinephrine: Relationship to audiogenic seizure. Pharmacologist 5^:238, 1963.

60 49 Reilly, R. H», K, F. Killam, E. H. Jenney, W. Ho Marshall, T. Tansig, No So Apter and Co C. Pfeiffer.' Convulsant effects of isoniazido JAMA 152 (14): , Reynolds, E. H. How do anticonvulsants work? Brit. J. Hosp. Med. 19: , Saad, S. F., A. M. ElMasry and P. M. Scott. Influence of certain anticonvulsants on the concentration of GABA in the cerebral hemisphere of mice. Eur. J. Pharmacol. 17: , Scott, D. About Epilepsy. International Universities Press, New York, Seyfired, T. N. Audiogenic seizure in mice. Fred. Proc. 38(10): , Simler, S., L. Ciesielski, M. Maitre, H. Randrianarisoa and P. Mandel. Effects of Na-n-Dipropylacetate on audiogenic seizure and brain GABA level. Biochem. Pharmacol. 22_: 1701, Snee,T. J., C. F. Ferrence and M. E. Crowley. Drug facilitation of the audiogenic seizure. J. Physiol. JL3: , Stare, J. Experimental reflex epilepsy (audiogenic epilepsy). In: Reflex mechanisms in the genesis of epilepsy, ed. by Z. Servit. Amsterdam: Elsevier, p , Stone, W. E. and M. J. Javid. Benzodiazepine and phenobarbital as antagonists of dissimilar chemical convulsants. Epilepsia 19: , Straighan, D. W. Convulsant drugs and inhibitory mechanisms in the mammalian central nervous system. Arch, de Farmacol. y Toxicol. I: 7-36, Swinyard, E. A. Laboratory assay of clinically effective antiepileptic drugs. J. Amer. Pharm. Assoc. 3^8: , Swinyard, E. A. Laboratory Evaluation of antiepileptic drugs. Epilepsia.10: , Swinyard, E. A. Assay of antiepileptic drug activity in experimental animals: Standard Tests. In:Anticonvulsant Drugs. International Encyclopedia of Pharmacology and Therapeutics. Section 19(1), Pergamon Press, Oxford and New York, p , 1972.

61 Swinyard, E. A., W. C. Brown and L. S. Goodman. Comparative assays of antiepileptic drugs in mice and rats. J. Pharmacol. Exp. Ther. 106: , Toman, J. E. P. Animal technique for evaluating anticonvulsants. In: Animal and Clinical Technique in Drug Evaluation, ed. by J. H. Nodine and P. E. Seigler, p , Year Book Medical, Chicago, Wada, J. A. and H. Ikeda. The susceptibility to auditory stimuli of animals treated with methionine sulfoximine. Exp. Neurol. 15: , Watson, E. and E. M. Woodbury. Effects of diphenylhydantoin on active sodium transport in frog skin. J. Pharmacol. Exp. Ther. 180: , Wood, J. D. and S. J. Peesker. A correlation between changes in GABA metabolism and isoniotinic acid hydrazide-induced seizures. Brain Res. 45: , Woodbury, D. M. Applications to Drug Evaluation. In: Experimental Models of Epilepsy, ed. by D. P. Purpura, J. K. Penry, D. B. Tower, D. M. Woodbury and R. Walter. New York, Raven, p , Woodbury, D. M. and E. Fingl. Drugs effective in the therapy of the epilepsies. In: The Pharmacological Basis of Therapeutics, ed. by L. S. Goodman and A. Gilman, p , New York, 1975 Woodbury, D. M., L. T. Pollins, M. D. Gardner, W. L. Hirschi, J. R. Hogan, M. L. Rallison, G. S. Tanner and D. A. Brodie. Effects of carbon dioxide on brain excitability and electrolytes. Amer. J. Physiol. 192:79-90,

62