70-26,392 YEOH, Peng Nam, 1941- A PHARMACOLOGICAL EVALUATION OF SEIZURES INDUCED BY ELECTRICAL STIMULATION OF THE HIPPOCAMPUS. The Ohio State University, Ph.D., 1970 Pharmacology University Microfilms, A XEROX Company, Ann Arbor, Michigan THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED A PHARMACOLOGICAL EVALUATION OF SEIZURES INDUCED BY ELECTRICAL STIMULATION OF THE HIPPOCAMPUS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Peng Nam Yeoh, B. Pharm., M.Sc. I.,.s The Ohio State University 1970 Approved by ft*#*Adviser wA V College of Pharmacy ACKNOWLEDGMENTS I wish to thank Dr. Harold H. Wolf for his advice, help and encouragement. The critical appraisal of the manuscript by Drs. Allan M. Burkman, Michael C. Gerald, Popat N. Patil and John W. Nelson and their helpful suggestions are appreciated. I am also grateful to the Fulbright-Hays program and the College of Pharmacy of The Ohio State University for making this work possible. VITA December 16, 1941 . Born - Mentakab, Pahang, Malaysia 1964............... B. Pharm., University of Singapore, Singapore 1966 ............... M. Sc., The Ohio State University, Columbus Ohio 196 7 ............... Teaching Assistant, Division of Pharmacology, College of Pharmacy, The Ohio State Univer­ sity, Columbus, Ohio 1967-1970 .......... Research Associate, Division of Pharmacology, College of Pharmacy, The Ohio State Univer­ sity, Columbus, Ohio PUBLICATIONS Yeoh, P.N. and Wolf, H.H. : Effects of some adrenergic agents on low frequency electroshock seizures. J. Pharm. Sci. 57: 340, 1968. Yeoh, P.N. and Wolf, H.H. : The effect of some adrenergic agents on electrically induced hippocampal seizures. In : Abstracts symposia and contributed papers presented to the A.Ph.A Academy of Pharma­ ceutical Sciences at 116th. Annual Meeting of the American Pharma­ ceutical Association at Montreal. 105, 1969. Yeoh, P.N. and Wolf, H.H. : A pharmacological evaluation of seizures induced by electrical stimulation of the hippocampus. In Press. iii CONTENTS Page ACKNOWLEDGMENTS ..................................... ii VITA ............................................. Ill LIST OF TABLES...................................... vi LIST OF ILLUSTRATIONS............................... vii Chapter I. GENERAL INTRODUCTION............................ 1 A. Literature Review B. Basis for the Present Study C. Statement of the Problem II. GENERAL PROCEDURES.............................. 18 III. EFFECT OF PROTOTYPE ANTICONVULSANTS ON SEIZURES INDUCED BY ELECTRICAL STIMULATION OF THE HIPPOCAMPUS. 21 A. Introduction B. Methods C. Results and Discussion IV. THE IMPORTANCE OF ADRENERGIC TONE ON SEIZURES INDUCED BY ELECTRICAL STIMULATION OF THE HIPPOCAMPUS. 52 A. Introduction B. Methods C. Results D. Discussion V. GENERAL DISCUSSION.............................. 153 VI. SUMMARY AND C O N C L U S I O N S......................... 160 iv APPENDIX 162 A. Electrode Implantation B. Verification of Electrode Site and Tissue Damage C. Histochemical Assay for Norepinephrine D. Radiometric Assay for Norepinephrine BIBLIOGRAPHY 173 LIST OF TABLES Table Page 1. Neurotoxicity of drugs........................... 20 2. The Stability of Rat Resistance................... 35 3. Effects of Reserpine, c* -Methyl-p-Tyrosine and DL-threo-DOPS on Norepinephrine Levels in Dif­ ferent Brain Areas in the R a t ...................... 104 4. Effects of Reserpine, oL-Methyl-p-Tyrosine and DL-threo-DOPS on Norepinephrine Levels in Dif­ ferent Brain Areas in the R a t ...................... 105 vi LIST OF ILLUSTRATIONS Figure Page 1. Circuit diagram ......................... 31 2. Stability of seizure threshold over time..... 37 3. Effect of diphenylhydantoin on seizure t h r e s h o l d.............................. 44 4. Effect of trimethadione on seizure threshold. .. 47 5. The importance of adrenergic tone to seizure t h r e s h o l d.............................. 86 6. Effect of phentolamine on seizure threshold .... 108 7. Effect of phenoxybenzamine on seizure threshold . Ill 8. Effect of propranolol on seizure threshold. 114 9. Effect of pronethalol on seizure threshold. 117 10. Effect of sotalol (MJ1999) on seizure threshold . 119 11. Effect of INPEA on seizure threshold...... 121 12. Effect of phenoxybenzamine and phentolamine on seizure thresholds in animals with altered catecholamine levels..................... 145 13. A representative norepinephrine standard curve. 171 Plate I Minimal and maximal extent of the brain lesions projected on frontal sections (A-D) and saggital section (E) at the stated distances from frontal and saggital zeros, respectively ................. 41 vi i PI ate Page II. Histochemical studies of drug effects on central norepinephrine neurons . .................. 91 III. Histochemical studies of drug effects on central norepinephrine neurons ..................... 93 IV. Histochemical studies of drug effects on central norepinephrine neurons. ..................... 95 V. Histochemical studies of drug effects on central norepinephrine neurons . .................. 97 VI. Histochemical studies of drug effects on central norepinephrine neurons ..................... 99 viii I. GENERAL INTRODUCTION A. Literature Review The history of the seizure state is as old as man. In the past, patients suffering from epileptic convulsions were thought to be pos­ sessed by devine or evil forces. Hippocrates was, perhaps, the first investigator to associate the disease with a disfunction of the brain. At present, we recognize that seizures are varied in type and can result from "occasional, sudden, excessive, rapid and local discharges of gray matter" (for reviews see Streeter, 1931; Penfield and Erickson, 1941; Temkin, 1945; Tower, 1960; Millichap, 1965). The classification of seizures clinically is based on the patient's abnormal electroencephalogram (EEG) and the presence and nature of subject-auras. Seizures are generally treated by selective drug therapy and/or surgical removal of the epileptogenic focus (foci). Over the last 3 decades, a large number of laboratory tests have been developed for routine screening of potential anticonvulsant agents. This development has contributed to the availability of drugs for the management of epileptic seizures. In the laboratory, electrically- induced seizures were employed by some investigators (Putnum and Merritt, 1937; Merritt and Putnum, 1938), whose work resulted in the in­ troduction of diphenylhydantoin for the treatment of grand mal epilepsy. Similar laboratory studies yielded trimethadione as an effective agent 1 2 against petit mal epilepsies (Everett and Richards, 1944; Goodman et al., 1946; Richards and Everett, 1946; Richards and Perl stein, 1946). Electrical stimuli may be applied via corneal electrodes to induce a minimal electroshock seizure, or a maximal hind limb "flexor-extensor" seizure (Goodman et al., 1949; Swinyard, 1949; Swinyard et al., 1952). Direct electrical stimulationsof specific cortical or subcortical regions have also been employed in producing experimental seizures in the laboratory (Millichap, 1965; Swinyard, 1969). Stimuli other than elec­ trical current have also been used. For example, chemicals like metrazol, methionine sulfoximine, thiosemicarbazide, strychnine and picrotoxin have been used and are generally administered systemically. Other compounds (e.g., penicillin, metals, alumina cream) can induce the formation of chronic epileptogenic foci when placed directly into brain substance. Sensory stimulation has also been employed to induce audiogenic seizures in mice and rats (Plotnikoff and Green, 1957; Plotnikoff, 1958; Fink and Swinyard, 1959) and light-induced seizures in rabbits (Barnes, 1954) and in baboons (Killam, 1967; 1969; Killam et al., 1967). Still other techniques involve manipulations of body metabolism, e.g., imbalance of water and electrolytes in vivo, induced by administration of vasopressin or glucose CSwinyard, 1949; Swinyard et al., 1952), by carbon-dioxide withdrawal (Woodbury et al., 1952) and hyperthermia (Millichap, 1958; Millichap et al., 1960; Millichap, 1965). Furthermore, seizures have also been produced by hypophysectomy, or administration of insulin (DeSalva, 1962; 1963; Kreindler, 1965). The majority of laboratory studies use intact animals. Mice and rats are the most common animals employed, but cats, hamsters, rabbits and baboons have also been studied (Swinyard et al., 1952; Barnes, 1954; Esplin, 1957; Killam, 1967). In addition, other biological systems have also been used. These include the response of single neurons in different brain areas, isolated nerves, nerve-muscle preparations, tissue slices, cortical slabs and the spinal cord (Swinyard, 1969). Seizures produced in the laboratory are generally classified into minimal and maximal seizures. A minimal seizure is characterized by a "stun" response (a period of immobility and "frozen" behavior), facial clonus (including eye twitching and rhythmic jaw movements) and continuous flexion and extension of the fore limbs. In addition, when seizures are induced by sound, there is present a phase of "wild running" (Millichap, 1965). A maximal seizure occurs when tonic flexion and extension of both the fore and hind limbs are exhibited. This can be followed by unconsciousness and death due to respiratory failure (Millichap, 1965). Both minimal and maximal seizures may be induced by the same type of stimulus by varying its strength, duration and the route of applica­