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Withdrawal-produced increase in susceptibility to kindled seizures following a single injection of alcohol… Mucha, Ronald Francis 1975

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WITHDRAWAL-PRODUCED INCREASE IN SUSCEPTIBILITY TO KINDLED SEIZURES FOLLOWING A SINGLE INJECTION OF ALCOHOL IN RATS by. RONALD FRANCIS MUCHA B.A.j University of B r i t i s h Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF ARTS i n the Department of Psychology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1975. In presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permission. Department of Psychology  The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 D a t e ,9th September. 1975 - i -ABSTRACT: It has been well-established i n both humans and laboratory animals that the withdrawal of alcohol after a period of chronic exposure can have convulsive effects. In the present studies, however, such convulsive effects were detected i n naive rats following the metabolism of a single ethanol i n j e c t i o n . These effects were assessed by measuring the duration of kindled motor seizures (MSs) and electrographic after discharges (ADs) e l i c i t e d by low-intensity amygdaloid stimulation at intervals before, during, and after the exposure. Kindled MSs are those which can be r e l i a b l y e l i c i t e d only after an organism has been stimulated p e r i o d i c a l l y with an i n i t i a l l y i n e f f e c t i v e low-intensity current. Thus, before each experiment the subjects were kindled with the periodic amygdaloid stimulation u n t i l MSs and ADs of stereotypical duration and pattern were r e l i a b l y e l i c i t e d . The consistency of the kindled seizures provided a stable base-l i n e against which to assess the convulsive effects of the withdrawal of ethanol. In Experiment 1 kindled seizures were e l i c i t e d at fixed intervals before and after a single intraperitoneal i n j e c t i o n of ethanol. Changes i n the AD and MS duration revealed a potent anticonvulsive effect i n the 3 hr immediately following the i n j e c t i o n , followed about 12 hr l a t e r by a convulsive effect l a s t i n g about 5 hr. In Experiment 2 th i s effect was replicated and d i r e c t l y related to changes i n the l e v e l of blood ethanol. The anticonvulsive effect was related to the presence of the ethanol while the convulsive effects seemed to be triggered by i t s metabolism. In Experiments 3 ,and 4 a different experimental design was used to study the - i i -effects of single intraperitoneal and intragastric injections, respectively. Since in these experiments only one seizure was e l i c i t e d in each subject after the alcohol injection, the possibility that the increase in seizure duration was an artifact of the repeated testing procedure rather than a bona fide withdrawal-produced effect was ruled out. Thus, the results confirmed previous demonstrations of convulsive effects following the metabolism of single injections of ethanol.- This established the generality of the phenomenon to other species, modes of ethanol administration, and to different experimental designs; and the u t i l i t y of the kindling paradigm for studying the time course of anti-convulsive and convulsive effects. The changes underlying the manifes-tation of severe convulsive effects obvious in humans after withdrawal from chronic ethanol exposure appears to develop with an individual's f i r s t exposure. i i i TABLE: OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES v i ACKNOWLEDGMENTS v i i INTRODUCTION 1' PURPOSE 4 LITERATURE REVIEW 6 Alcohol-Withdrawal-Produced Convulsive Effects 6 1. Spontaneous Withdrawal Effects 6 2. Increases i n S u s c e p t i b i l i t y to Experimentally-Elicitedd Convulsions 10 3. Duration of Alcohol Exposure and Convulsive Effects of Withdrawal 13 Kindling Effect 15 1. General Description 15 2. Assessment of Seizure S u s c e p t i b i l i t y with Kindled Seizures 17 Recapitulation 19 EXPERIMENT 1' 21.' EXPERIMENT 2 \ - 3 0 EXPERIMENT 3 36 EXPERIMENT 4 41' GENERAL DISCUSSION 45 Convulsive Effects of a Single, Brief Exposure to Ethanol 45 Kindled Seizures and the Assessment of Seizure S u s c e p t i b i l i t y 47. Other Effects of Brief Ethanol Exposure 49 iv Hangover .'51' Relation of Pathological Effects of Alcohol Exposure to Withdrawal Seizures 52 Concept of Physical Dependence 58 CONCLUSION 60 REFERENCES 61 APPENDICES A. Analysis of Variance Tables for Experiment 1 B. Analysis of Variance Tables for Experiment 2 C. Analysis of Variance Tables for Experiment 3 D. Analysis of Variance Tables for Experiment 4 E R R O R S A W b U i s r e . k 72 73 76 78 80 -v-LIST OF TABLES Page Table 1. Methods of administering ethanol for producing withdrawal convulsions in various species-. 8 - v i -- v i i -ACKNOWLEDGMENTS The author wishes to express his gratitude to Dr. J. Pinel for his support, advice and assistance throughout a l l phases of this research. The author i s also deeply indebted to R. Skelton for assisting with the experimentation, R. Perkins for his help wi'tH the blood assays, M. MacDoriald for her centrifuge, D. Wilkie and A. G. Phil l i p s for comments and criticisms of the manuscript, and to Brenda Carriere for her expert typing assistance. A thanks is also extended to Derek Vanderkooy and Donna Steele for assisting with some of the figures. A very special thanks goes to Kathleen Mucha for her patience and moral encouragement and for her editing and typing assistance. INTRODUCTION Ethanol i n moderate to high doses has well-known depressant effects on various levels of nervous system functioning (cf. Himwich & C a l l i s o n , 1972; I s r a e l , 1970; Kalant, 1970). Ethanol i n h i b i t s the u t i l i z a t i o n of glucose i n the brain (Roach, 1970), i n h i b i t s action.potentials (Armstrong & Binstock, 1964), decreases i n both frequency and amplitude spontaneous electroencephalographic (EEG) a c t i v i t y (Horsey & Akert, 1953), and reduces the amplitude of evoked potentials i n various parts of the mammalian brain (Himwich & C a l l i s o n , 1972) . Ethanol has also been shown to have strong anticonvulsant properties (Allan & Swinyard, 1949; McQuarrie & F i n g l , 1958). In apparent contradiction to these depressant effects i s the well-established r e l a t i o n between the use of ethanol and the occurrence of seizures. One of the e a r l i e s t available reports of such a r e l a t i o n was by a ship's surgeon (Trotter, 1804) who noted that some e p i l e p t i c f i t s and f a c i a l tremors were sometimes associated with consumption of alcoholic beverages. A si m i l a r report of the r e l a t i o n between chronic alcohol consumption and seizures was published by Hubbard i n 18811 Since these early reports, alcohol-related convulsions have been repeatedly described i n both the c l i n i c a l and experimental l i t e r a t u r e (cf. Bowman & J e l l i n e k , 1942; Echeverria, 1881J Essig, 1972; I s b e l l , Fraser, Wikler, B e l l e v i l l e , & Eisenman, 1955; Vic t o r , 1968). The most obvious interpretation of these alcohol-related convulsions was that the presence of the alcohol triggered the convulsions; . However, lack of experimental evidence showing that alcohol was a convulsant (cf. Berry, 1952) led investigators to consider the p o s s i b i l i t y . t h a t the -2-production of seizures was mediated by intermediate pathological effects of the alcohol. Pellagra (Langworth, 1931), vitamin Bg deficiency (Lerner, De C a r l i , & Davidson, 1958), s h i f t s i n blood plasma volume (Nicholson & Taylor, 1940), magnesium deficiency (Klingman, Suter, Green, & Robinson, 1955), hepatic diseases (Froment, Masson, & Brun, 1939), cerebral edema (Cobb, 1932), and cerebral atrophy (Lafron, Pages, Pussouant, Labunge, Mi n v i e l l e , & Cadilhac, 1956) have a l l at one time been considered as factors i n the production of alcohol-related convulsions. Some authors believed that alcohol precipitates convulsions i n only those already susceptible (Lennox, 1941) while others attributed the r e l a t i o n between alcohol consump-tion and convulsions to the presumed tendency of e p i l e p t i c s to consume large amounts of alcohol (Echeverria, 1881). More recent c l i n i c a l and experimental research has discounted these interpretations of alcohol-related convulsions by providing clear evidence that i t i s the sudden reduction i n alcohol consumption following sustained intoxication which e l i c i t s the convulsions ( I s b e l l et a l . , 1955; Kalinowsky, 1942; Victor & Adams, 1953; Victor & Brausch, 1967). Kalinowsky (1942), for example, i n an early report emphasized the s i m i l a r i t y between alcohol-related convulsions and those e l i c i t e d when chronic exposure to barbiturates and sedatives i s stopped. Among the more widely quoted studies providing evidence that alcohol withdrawal i s responsible for triggering alcohol-related convulsions are the reports of Victor and his colleagues (Victor & Adams, 1953; Victor & Brausch, 1967) based on the care f u l l y documented observations of alcoholics requiring hospital attention for seizures. Their major finding was that convulsions t y p i c a l l y occurred between 6 and 48 hr after the cessation of drinking rather than during -3-periods of intoxication. Only a small percentage of patients had any evidence of spontaneous and recurrent•convulsions or EEG epileptiform discharge, or a history of severe head trauma, thus the p o s s i b i l i t i e s of latent epilepsy producing the seizures and of epilepsy giving r i s e to alcohol use were discounted. Victor and his coworkers found withdrawal seizures to be e l i c i t e d only i f the cessation of drinking followed a long period of r e l a t i v e l y continuous alcohol consumption. Five days was the shortest period of exposure which they observed to culminate i n withdrawal seizures. The results of Victor and coworkers' c l i n i c a l studies are similar to those of an experiment by I s b e l l et a l . (1955). The subjects were healthy former morphine addicts who were maintained on nutritious diets and ethanol for periods ranging from 48 to 87 days. The subjects v o l u n t a r i l y consumed 388-489 ml of alcohol d a i l y maintaining average blood ethanol levels between 200 and 300 mg per 100 ml of blood over the drinking period." When alcohol administration was discontinued, a l l s i x subjects completing the study became tremulous,.hyper-reflexic, sweated profusely, l o s t weight, and experienced insomnia; two had convulsions, f i v e developed hallucinations, and three became disoriented. Dysrhythmias i n the form of spike-and-wave discharges were present i n the three subjected to EEG analysis (Wikler, Pescor, Fraser, & I s b e l l , 1956). The four patients that withdrew from the study within 34 days displayed only minor withdrawal-produced Symptoms; -4-PURPOSE Thus, i t i s now well-established that alcohol-related convulsions are triggered by the withdrawal rather than the administration of alcohol. C l i n i c a l investigations and experiments on both human and animal subjects have consistently found that withdrawal-produced convulsions are not elicited u n t i l there have been many weeks of exposure (Deneau, Yanagita, & Seevers, 1969; Essig & Lam, 1968; Isbell et a l . , 1955). There i s some evidence, however, which suggests that some withdrawal-produced convulsive effects may be observed following an organism's f i r s t brief exposure to ethanol. Goldstein (1972) and McQuarrie and Fingl (1958) found a small but detectable increase in the susceptibility to experimentally-elicited convulsions in naive mice a few hours following administration of a single dose of ethanol. The study of this indication of early withdrawal-produced convulsive effects may eventually provide valuable insights into the etiology of alcohol-related convulsions and of various disorders related to alcohol consumption. However, with the exception of the two afore-mentioned studies there is very l i t t l e research on convulsive effects after single doses of ethanol. Thus, the purpose of the present experiments was to study these convulsive effects produced by exposing a naive organism to a single injection of ethanol. In the present series of experiments the antifrconvulsant and convulsant consequences of a rat's f i r s t exposure to ethanol were assessed by measuring the duration of kindled seizures at various intervals before and after the administration of the ethanol. Kindled seizures are those el i c i t e d in animals previously kindled with periodic low-intensity bipolar stimulation -5-of the brain (Goddard, Mclntyre, & Leech, 1969). In the.present studies, rats were kindled with periodic amygdaloid stimulation administered at an intensity which was i n i t i a l l y too low to e l i c i t motor seizures (MSs) . Eventually, however, each stimulation e l i c i t e d a f u l l clonic MS and i t was the changes in the MS and the after discharge (AD) durations which were used to measure the effects of a single alcohol exposure. The development of this sensitive technique of measuring the time course of convulsive and anticonvulsive effects was a secondary purpose of the present investigation. -6-LITERATURE REVIEW Thus, i n view of the two purposes, t h i s series of experiments i s relevant to two separate experimental phenomena; alcohol-withdrawal-produced convulsive effects and kindling. There are three aspects of alcohol-withdrawal-produced convulsive effects that are p a r t i c u l a r l y pertinent. These are 1) studies of convulsions occurring spontaneously following withdrawal, 2) studies of increased s u s c e p t i b i l i t y to experi-mentally-elicited seizures after alcohol withdrawal, and 3) studies of the effects of the duration of the alcohol exposure on the convulsive effects of withdrawal. The kindling l i t e r a t u r e relevant to the present paper deals s p e c i f i c a l l y with the use of kindled seizures to assess changes i n seizure s u s c e p t i b i l i t y . Alcohol-Withdrawal-Produced Convulsive Effects With a few notable exceptions ( I s b e l l et a l . , 1955; Mendelson, Stein, & McGuire, 1966) investigations into the physiological basis of alcohol withdrawal seizures have t y p i c a l l y involved laboratory animals. The experimental control necessary to attack problems related to withdrawal convulsions i s not usually possible i n studies involving human subjects. 1. Spontaneous Withdrawal Effects Withdrawal-produced convulsions have been d i f f i c u l t to e l i c i t i n experimental animals because they w i l l not readily consume high levels of alcohol (Myers & Veale, 1972). Thus, the main problem has been to administer s u f f i c i e n t quantities of ethanol to maintain long periods of ex-posure. A number of methods of administering ethanol so that withdrawal -7-convulsions are eventually produced have been reported. These methods and their effects are summarized in Table 1. Until 1968 there was very l i t t l e direct evidence that alcohol withdrawal convulsions could be produced in animals. Indirect evidence consisted of observations of convulsions produced by barbiturate withdrawal in rats (Essig, 196%), dogs (Fraser & Isbell, 1954). and cats (Essig & Flanary, 1959). Essig and Lam (1968) provided the f i r s t published demonstration of alcohol withdrawal convulsions in animals. Ethanol was administered via an intragastric cannula several times a day to dogs maintained on a vitamin-enriched diet; ; the dogs were water deprived to encourage additional oral intake of ethanol. After 54 days of exposure the alcohol was withheld and five of eight subjects experienced convulsions between the 11th and 48th hr of abstinence. The intragastric route of administering alcohol has been the most widely used method of inducing withdrawal convulsions. However, with the exception of one study on cats by Guerrero-Figueroa, Rye, Gallant, & Bishop (1970) in which permanent gastric fistulae were used, gavage has been the method of choice for administering the ethanol. The primary advantage of gavage is that i t does not involve the surgical implantation of the cannulae; a tube is introduced into the stomach of the subject via the esophagus, the alcohol injected, and the tube removed. This technique has been effective in producing withdrawal effectsiincdogs(Ellis & Pick, 1972), monkeys ( E l l i s & Pick, 1970, 1971), and rats (Hunt, 1973; Majchrowicz, 1973; Mucha, Pinel, & Van o|.t, 1975; Wallgren, Kosunen, & Ahtee, 1973). Another method of producing alcohol withdrawal convulsions in laboratory animals that, like the intragastric techniques, permits s t r i c t -8-Table 1. Methods of administering ethanol for producing withdrawal convulsions in various species. / -8a-I n v e s t i g a t o r ^ * Spectra Method of Ethanol Administration Durstles of Kx 7 4 sure t p i i e p t i a ty*pto«a * Branchey, RAuscher 4 rat s t h a n o l - r l c h d i e t In 4.3/nL/rat/day 21 day* Eleaen (1971) w e i g h t - r e s t r i c t e d anlaale Cannon, Baker, Bereuto, rat 4 Atklnaoa (19:4) Deneau, Yanaglta, g Seevcra (1969) E l l l a h Flc*. (1972) dog Esalg 4 U a (1968, dot 1969) Intravenous s e l f -e d a l n l s t r s t l o n E l l l a 4 Pick (1970, nonkey nasogastric gavage 1972) B-12g/k«/dey S.6gAg/day *-*8/Wd«y 2 to 4 day* •bout S atoattta 10 to IB days 3.8-6\3g/kg7day 2 to 4 veefca I n t r a g a s t r i c Infusion 4-4.Snl/k« of 401 34 day* at 4 hr I n t e r v a l * c l o n i c convulsion* Increased M I * * c e p t l b l l l t y t * audiogenic s e i z u r e * c m v u l i t o m , c l o n i c - t o n i c convulsions c l o n i c - t o o f c convulalone convulsions convulsion* F a l k , Season, 4 Winger (1972) Freund (1969) ra t schedule-Induced p o l y i 13.1g/kg/day 3 wrath* d l p s l a e t h a n o l - r l e h d i e t l a .51-.53nl/day 4 days w e i g h t - r e s t r i c t e d animals Increased sus-c e p t i b i l i t y t o audiogenic oclaures generalized'tooic-cIonic convulsions Freund (1971) ethanol-rleh d i e t In .40ml/day 4 day* v e l g h t - r e s t r l c t e d animal* tonlc-clonle cos- . vulslons and n w ' -nlng f i t s Freund (1971) Cold«tein (1972) C o l d s t e l a 4 Pal (1971) • t h a n o j - r l c h d i e t l o mica .*0-.37»l/d*y 1 week with 4 C housing Inhalation of ethanol vapour with p y r a i o l * I n h a l a t i o n of ethanol vapour v l t h pyxazolc vapour coocentra- 1-9 days t i o n of l i n g / 1 1 t r a vapour coocentra- 4 days t l o o of 1 2 c g / l l t r e t o n l c - c l o n l e coo-vulslona and rus-nlng f i t s Increased suscep-t i b i l i t y to handling convulsions spontaneous c o o w l -slooa. Increased s u s c e p t i b i l i t y to ha ud l i n g coavulsloo Guerrero-Flgueroa, est Rye, Gallant, 4 Bishop (1970) Hunter, Boast, Walker, Tat 4 Zornetzer (1974) I n t r a g a s t r i c Infusion 18-31*l/day of 2-) month* 40X U-Ug/kg/day 7 days echanol-rlch d i e t i n 13.5g/kg/dey 13 days . w e i g h t - r e s t r i c t e d animals to n l c - c l o n l c con-vulsions c l o n i c - t o n i c con-vul s i o n s , lower pentylenel-tetraxol threshold convulsions, ETC e p l l e p t l f o r * a c t i v i t y I s b e l l , Fraser, Wilder,, nan, f o r w r o r s l Ingestion B e l l e v i l l e , 4 flsenaa morphine 3S8-4S9al/dsy 48-87 days convulalona, EEC epileptiform a c t i v i t y Meber 4 De C a r l l (1973) et h e n o l - r l c h d i e t 14-16g/kg/day 2-4 vaeka Increased suscep-t i b i l i t y to audio-genic seizures Kajchrovlcs (1973) KcQuarrl* 4 F l n g l (1938) gavage gavage 12-15gAf/dsy 3-3 days 5.4g/Vg/dsy 14 days convulalona decreased threshold Io electroconvulsive) seizures Kucha, F l n e l , 4 Van Oot (1975) B.V10.4g/kg/day 7-30 daya convulalona. I n -creased suscep-t i b i l i t y to audio-genic seizures, EEC i p l l e p t l f o r a a c t i v i t y O r t i z , G r i f f i t h s , 4 m t n i M t L i t t l e t o n (1973) l a t c l l f f e (1972) Roach, t*han, Ccffasn, rat Pennington, 4 bevls (W73) Walker 4 Zoraats«r (1974) Wallgren, rosutum, 4 rat Ant,. (1973) • i n h a l a t i o n of ethanol vapour vapour concentre- 10 daya t i o n of 8-25mg/ l i t r e Increasing concentrations (no data) ' of ethanol aa only a v a i l a b l e f l u i d I nhalation of ethanol vapour etha n o l - r l c h d i e t In v e l g h t - r a a t r l c t e d eiiLsale vapour concentre- 7 days t i o n of n-)0ag/ l i t r e 35.3ogA|/day 4 days 6-12g/kg/day 18-71 daya lncreaaed suscep-t i b i l i t y to handling convulsions Increased suscepti-b i l i t y to audiogenic seizures Increased s v s c e p t l * b l l l t y to hand 1 leg convol alone convulsions, EEC e p l U p r l f o r a a c t i v i t y -9-experimental control of ethanol administration involves housing animals i n an atmosphere of ethanol vapour^ Alcohol was f i r s t administered to experimental animals v i a t h i s route.by Goldstein and Pal (1971) who administered the vapour to mice injected with pyrazole, an i n h i b i t o r of alcohol dehydrogenase a c t i v i t y , which insured higher, more stable levels of blood ethanol. Withdrawal-produced convulsions were observed i n some mice after as l i t t l e as three days of exposure. Restricting consumption of weight-reduced animals to ethanol-rich diets i s another technique commonly employed i n the study of withdrawal-produced convulsions. Freund (1969, 1971) was the f i r s t to use t h i s i n the study of withdrawal convulsions. Mice were reduced to 65% or their normal weight and were then given diets r e s t r i c t e d to Metrecal and alcohol mixed so that 35% of the calories were supplied by the ethanol. Convulsions were/produced when the alcohol i n the diet, was replaced with sucrose after four days of exposure. Walker and Zornetzer (1974) produced convulsions after exposing mice r e s t r i c t e d to 85 to 90% of their normal weight to the ethanol diet for six days. In addition, EEG examination during withdrawal revealed wide-spread epileptiform a c t i v i t y i n forebrain structures. This technique has also been used to study withdrawal-produced convulsions i n rats. Branchey, Rauscher, and Kissen (1971) found that clonic convulsions were produced i n rats deprived to 66% of their normal weights when the alcohol-Metrecal solution was withdrawn after 21 days of exposure. Hunter, Boast, Walker, and Zornetzer (1973) found 15 days s u f f i c i e n t to produce convulsions and EEG epileptiform a c t i v i t y i n rats reduced to 75% of their o r i g i n a l weight. -10-Considerable c r i t i c i s m has been l e v e l l e d at the use of weight-reduced animals to study withdrawal convulsions ( L i t t l e t o n , G r i f f i t h s , & O r t i z , 1974; Mello, 1973; Ogata, Ogato, Mendelson, & Mello, 1972; Wallgren et a l . , 1973). Ogata et a l . , (1972) concluded that severe n u t r i t i o n a l d e f i c i t s contribute s i g n i f i c a n t l y to withdrawal effects. Of two groups of mice consuming the same quantity of ethanol, only animals i n the nutritionally-deprived group developed withdrawal symptoms. The fact that alcohol metabolism i n fasted animals i s decreased by as much as 50% (Owens & Marshall, 1955) may have been the basis for t h i s effect. Thus, techniques employing ethanol-liquid diets but not requiring weight reduction have been developed for the study of alcohol withdrawal seizures. Freund (1971) reported that convulsive symptoms could be seen i n normal-weight rats i f they were fed diets with 18% of the calories constituted by ethanol and housed at about 4°C for one week. S i m i l a r l y , Pieper, Skeen, McClure, and Bourne (1972) observed tonic-clonic withdrawal convulsions i n young, healthy chimpanzees after isix to 10 months of exposure to a l i q u i d diet i n which 45% of calories were comprised of ethanol. Intravenous self-administration by lever pressing was f i r s t shown to be useful for studying withdrawal-produced effects i n a morphine experiment by Weeks and C o l l i n s (1968). A year l a t e r Deneau et a l . 2 (1969) provided the f i r s t published report of animals self-administering ethanol. Monkeys chronically s e l f - i n j e c t e d up to 8.6 g/kg per day and developed convulsions when the ethanol administration was stopped. 2. Increases i n S u s c e p t i b i l i t y to Experimentally-Elicited Convulsions . The detection of withdrawal-produced convulsive effects by the presence or absence of spontaneous withdrawal convulsions has two major - 1 1 -l i m i t a t i o n s . F i r s t , there i s evidence of convulsive effects i n subjects not displaying spontaneous withdrawal seizures. Wikler et a l . , (1956), for example, found that alcohol withdrawal triggered epileptiform electro-graphic abnormalities i n subjects not displaying overt convulsions. Motor convulsions are generally all-or-none events and do not provide the investigator with a method of detecting mild;.-c6nv'ulsive-effects after alcohol withdrawal. Second, i n order to detect the presence of withdrawal convulsions, subjects must be observed continuously during the withdrawal period. Victor and Adams (1953) indicated that those patients experiencing withdrawal convulsions rarely have more than one or two during their entire abstinence period. An alternative means of identifying the convulsive effects of alcohol withdrawal i s to assess increases i n the s u s c e p t i b i l i t y to seizures triggered by convulsive agents during the withdrawal period. The rationale for t h i s method i s based on the well-established observation that e p i l e p t i c effects are additive (cf. Ajmone-Marson & Ralston, 1957). Thus, an e p i l e p t i c agent administered at doses which do not themselves e l i c i t convulsions w i l l i n t e n s i f y those produced by another convulsive agent. This additive feature has been widely used to detect e p i l e p t i c developments i n patients not displaying spontaneous convulsions or electrographic seizure a c t i v i t y . For example, a small dose of pentylenetetrazol, i n e f f e c t i v e i n healthy subjects, w i l l frequently trigger seizures i n suspected e p i l e p t i c s (cf. Ajmone-Marson & Ralston, 1957). .Theseris' i n d i r e c t r e l a t i o n between the presence of alcohol-withdrawal-produced spontaneous seizures and changes i n s u s c e p t i b i l i t y to convulsive agents. This r e l a t i o n was c l e a r l y evident i n a study by Victor and -12-Brausch (1967) i n which s e n s i t i v i t y to photic stimulation was assessed i n patients undergoing alcohol withdrawal. The photic stimulation consisted of flashes of l i g h t delivered at frequencies from 4 to 24 flashes per sec. This photic stimulation rarely provoked e p i l e p t i c responses i n normal individuals or i n diagnosed ep i l e p t i c s but patients undergoing withdrawal of alcohol developed convulsive responses i n about 50% of the cases. Furthermore, the periods i n which s u s c e p t i b i l i t y to l i g h t - e l i c i t e d effects was the greatest were the same periods i n which spontaneous convulsions were most prevalent. S i m i l a r l y , Goldstein (1972) reported a high positive correlation between spontaneous and handling-elicited convulsions i n mice following the cessation of exposure to ethanol vapour. R a t c l i f f e (1972) observed withdrawal-produced increases i n s u s c e p t i b i l i t y to audiogenic seizures i n 40 to 80% of the rats r e s t r i c t e d to ethanol-rich diets for f i v e to seven weeks, respectively. , Only three of 300 s i m i l a r l y exposed rats were observed to have spontaneous convulsions. Thus, i t seems that increases i n the s u s c e p t i b i l i t y to convulsive agents after alcohol with-drawal provide a more r e l i a b l e and sensitive measure of convulsive effects of alcohol withdrawal than the increases i n the incidence of spontaneous seizures. Methods employed to produce alcohol withdrawal convulsions i n experimental animals have increased the s u s c e p t i b i l i t y to a variety of convulsive agents (see Table 1). In r a t s , withdrawal following chronic alcohol exposure by gavage increases s u s c e p t i b i l i t y to audiogenic seizures (Cannon, Baker, Berman, & Atkinson, 1974; Mucha et a l . , 1975), s u s c e p t i b i l i t y to pentylenetetrazol-produced convulsions (Hunt, 1973; McQuarrie & F i n g l , 1958) and s u s c e p t i b i l i t y to convulsions e l i c i t e d by handling (Cannon et a l . , 1974). The withdrawal following presentation of -13-ethanol-liquld diets to weight-reduced rats (Hunter et a l . , 1974) and mice (Freund & Walker, 1971) and non-weight-reduced rats (Lieber & De C a r l i , 1973) has increased the s u s c e p t i b i l i t y to audiogenic seizures. Chronic alcohol consumption produced by schedule-induced polydipsia (cf. Lester, 1961J -'(Holman & Myers, 1968; Ogata et a l . , 1972) has been reported to result i n a withdrawal-produced increase i n the s u s c e p t i b i l i t y to audiogenic seizures (Falk, Samson, & Winger, 1972). In many of these experiments the increased s u s c e p t i b i l i t y to various convulsive agents occurred i n the absence of spontaneous convulsive effects. 3. Duration of Alcohol Exposure and Convulsive Effects of Withdrawal I s b e l l et a l . (1955) c l e a r l y showed that the severity of withdrawal reactions i s an increasing function of the amount of previous ethanol exposure. Subjects that were exposed to high levels of alcohol for 48 to 87 days experienced convulsions and epileptiform EEG a c t i v i t y , while subjects that were exposed for fewer than 34 days did not. This r e l a t i o n has also been demonstrated i n a variety of infra-human species. Mucha<- et a l . ,'(1975) , for example, administered ethanol to rats by gavage for 7, 15, or 30 days and found the incidence of spontaneous convulsive symptoms to be an increasing, negatively accelerated function of the duration of ethanol exposure. Using similar methods, Majchrowicz (1973) found a high incidence of withdrawal seizures after three to f i v e days but not after one or two days of exposure. The same r e l a t i o n has been observed when the convulsive effects of the alcohol withdrawal are gauged i n terms of the s u s c e p t i b i l i t y to e l i c i t e d seizures rather than i n terms of the incidence of spontaneous -14-seizures. Lieber and De C a r l i (1973) found that 10 to 14 but not 5 to 7 days of forced exposure to a diet i n which 36% of the calories were comprised of ethanol increased s u s c e p t i b i l i t y to audiogenic seizures. S i m i l a r l y , Goldstein (1972) found that the incidence of handling-elicited " withdrawal convulsions increased with the duration of intoxication produced by ethanol vapour. Two series of experiments demonstrating increased seizure s u s c e p t i b i l i t y following bri e f exposures to ethanol are p a r t i c u l a r l y relevant to the present studies. The f i r s t experiment was carried out by McQuarrie and F i n g l (1958). They found that following the administration of 4.0 g/kg of alcohol to mice v i a gavage there was a b r i e f period of increased s u s c e p t i b i l i t y to pentylene-tetrazol-induced seizures. Eight! andb.±2aHreaf ter:ethanol"administration threshold was decreased, while tests at 24 hr f a i l e d to reveal any difference between the thresholds of ethanol and control animals. McQuarrie and Fingl (1958) claim to have observed a similar pattern of change i n electro-shock thresholds i n a similar experiment, but did not publish the r e s u l t s . The second incidence of withdrawal-produced convulsive effects after a single i n j e c t i o n of ethanol was reported by Goldstein (1972). Mice injected with 5.0 g/kg of ethanol i.p. and 1.0 mmole/kg of pyrazole displayed mild increases i n s u s c e p t i b i l i t y to handling-elicited convulsions 10 to 11 hr after the i n j e c t i o n . The peak effect occurred at about 7 hr when no pyrazole was injected. A lower dose of alcohol, 2.0 g/kg, admini-stered without pyrazole increased the s u s c e p t i b i l i t y to handling convulsions 3 hr after the i n j e c t i o n . The only systematic determination of the blood alcohol levels was made on the animals receiving pyrazole. Ethanol elimination curves indicated that the peak withdrawal scores occurred while -15-some ethanol was s t i l l present i n the blood. Similar studies have not been.performed with human subjects. Human subjects employed i n alcohol experiments t y p i c a l l y have a long history of drinking. However, research has indicated that the duration of ethanol exposure required to produce withdrawal convulsions i n these subjects i s considerably less than once thought. Victor (1968) and Berry (1952) have reported that only one evening of heavy drinking i s enough to increase the severity and incidence of seizures i n some e p i l e p t i c s . S i m i l a r l y , Nagy, Zsadanyi, Nagy, and Zsigmond (1973) found that following the metabolism of only one or two small quantities of an alcoholic beverage there was pho t i c a l l y - or hyperventilation-induced e p i l e p t i c a c t i v i t y i n the EEG of some c l i n i c a l l y healthy subjects. In those few patients who had spike a c t i v i t y p r ior to the alcohol there was an amplification of the spike a c t i v i t y during the post-alcohol phase. Reports of withdrawal symptoms produced after very short drug exposures are much more common i n the opiate l i t e r a t u r e . Martin and Eades (1961) detected antagonist-precipitated withdrawal effects i n naive, spinal dogs after an 8rhr infusion of morphine. S i m i l a r l y , a single i n j e c t i o n of levorphenol i n mice (Cheney &r.Go'l'dsteintr.l971) and a single i n j e c t i o n of morphine i n rats (Smits, 1975) both result i n antagonist-precipitated withdrawal effects. ;The' Kindling:. Effect,! 1. General Description. The kindling effect refers to the discovery by Goddard (1967) that low-int e n s i t y , f o c a l , e l e c t r i c a l stimulation of the amygdala which i n i t i a l l y -16-produces no e p i l e p t i c response but does so with periodic presentation of the stimulation. Electrographic analysis has indicated that the periodic low-intensity .stimulation f i r s t lowers the afterdischarge (AD) threshold u n t i l ADs are r e l i a b l y e l i c i t e d at the t i p of the stimulating electrode (Racine, 1972a). With continued periodic e l i c i t a t i o n of ADs the degree to which they generalize to other parts of the brain increases u n t i l mild motor automatisms are e l i c t e d . I f even more stimulations are administered, the degree to which ADs generalize continues to increase u n t i l f u l l motor seizures (MSs) are e l i c i t e d by each stimulation (Racine, 1972a,b).^ The generality and permanence of the kindling phenomenon have been well established. Kindling has been demonstrated i n rats (Goddard et a l . , 1969), mice (Leech, 1972), cats (Morrell, 1973), rabbits (Tanaka, 1972), primates (Wada & Sato, 1973) and frogs (Morrell, Tsuru, Hoeppner, & Morgan, 1975). The permanence of the change i n neural function induced by periodic brain stimulation was f i r s t demonstrated by Goddard et a l . (1969) who stimu-lated rats after a 12-week stimulation-free period and found a savings of about 90% i n the number of stimulations required to e l i c i t a f u l l MS. Kindling has been shown to occur i n response to stimulation of the olfactory and limbic areas of the brain (Goddard et a l . , 1969) , of extra-pyramidal motor areas closely related to the limbic system (Goddard et a l . , 1969), and of some c o r t i c a l areas (Racine, 1975). Since the amygdala has proved to be the most easily kindled structure, most studies of kindling have employed amygdaloid stimulation. However, regardless of the 1 Due to the generally accepted terminology, overt and EEG components of a kindled seizure w i l l be represented as MS and AD respectively. Previously, convulsions and EEG epileptiform a c t i v i t y represented.these respective types of e p i l e p t i c a c t i v i t y . Convulsive effects and seizures referred'to both types of e p i l e p t i c a c t i v i t y ; the l a t t e r term also referred-'to increased seizure s u s c e p t i b i l i t y . -17-s i t e , stimulation must be presented p e r i o d i c a l l y i n order for kindling to take place. Racine, Burnham, Gardner, and Levitan (1973) and Goddard et a l . (1969) indicated that there i s a general inverse relationship between the number of stimulations required for kindling and the duration of the i n t e r v a l between stimulations. Goddard et a l . found intervals of 24 hr or greater to be optimal and intervals of less than 20 min to be completely i n e f f e c t i v e . Moreover, Morrell (1973) indicated that continuous stimulation not only did not produce kindling but retarded kindling s i g n i f i c a n t l y when distributed stimulation was subsequently administered. Racine (1972a) has provided convincing evidence that the e l i c i t a t i o n of ADs at the s i t e of stimulation i s a necessary prerequisite for kindling of MSs. Periodic stimulation maintained at a l e v e l below the AD threshold did not lead to the development of behavioural convulsions, nor did i t reduce the number of supra-threshold stimulations l a t e r required for kindling. Moreover, the main electrophysiological correlate of the kindling process appears to be the degree to which ADs generalize from the s i t e of stimulation to other neural structures (Racine, Gardner, & Burnham, 1972). 2. Assessment of Seizure S u s c e p t i b i l i t y with Kindled Seizures Kindling has been employed as a model of learning (Goddard et a l . , 1969; Mclntyre & Goddard, 1973),aasaa$methddc6fppc6dueing a.functional lesion (Mclntyre & Molino, 1972), as a model of epileptogenesis (Wada, Sato, & Corcoran, 1974; P i n e l , Mucha, & P h i l l i p s , 1975), as an amnesic agent (Mclntyre, 1970), and as a method for studying state-dependent learning (Mclntyre & Reichert, 1971). However, one of i t s most promising -18-applicatlons has been i n the assessment of the convulsive and anticonvulsive effects of ce n t r a l l y active agents. The effects of agents on the duration of ADs and MSs e l i c i t e d from various s i t e s i n the brain have been assessed (Babington & Wedeking, 1973). The features of the kindling phenomenon which make i t a valuable addition to the methods available for gauging the convulsive effects of centrally active agents have been discussed several times previously (Babington & Wedeking, 1973; P i n e l , P h i l l i p s , & MacNeill, 1973; Racine, Livingstone, & Joaquin, 1975). The locus, frequency, i n t e n s i t y , and timing of the test stimulation are a l l under s t r i c t experimental control. Moreover, i n the l a t e r stages of kindling the AD and MS duration of each animal i s remarkably consistent from t r i a l to t r i a l . P i n e l , P h i l l i p s , and MacNeill (1973) reported that following the development of f u l l MSs, subsequent stages of seizure development are characterized by a gradual increase i n the uniformity of seizures produced by successive stimulations. Eventually seizures were characterized by very low day-to-day v a r i a t i o n i n AD duration and MS duration and pattern. Thus, with these highly predictable seizures even agents with only subtle effects may be assessed r e l i a b l y . Kindled seizures have been employed to assess the anticonvulsant effects of antecedent footshock (Pinel et a l . , 1973), various components of canabis sativa (Fried & Mclntyre, 1973; Corcoran, McCaughran, & Wada, 1974), diazepam (Babington & Wedeking, 1973; Racine et a l . , 1975; Wise & Chinerman, 1974), phenobarbital (Babington & Wedeking, 1973; Wise & Chinerman, 1974), and a number of tra n q u i l i z e r s (Babington & Wedeking, 1973). Not only are kindled seizures useful for assessing -anticonvulsant effects but they also may be employed to detect convulsant effects. - 1 9 -Babington and Wedeking (1973), for example, noted that d-amphetamine prolongs the duration of kindled seizures. S i m i l a r l y , Racine et a l . (1975) found that diphenylhydantoin and procaine enhanced the duration and propagation^ of cthe AD. Seizures kindled from different s i t e s can be affected i n different ways by certain cen t r a l l y active agents. Babington and Wedeking (1973) assessed the effects of drugs on seizures kindled by stimulation of the neocortex and those from the amygdala. Since some drugs did not affect both types of kindled seizures i n the same way, they were able to suggest mechanisms or s i t e s of action. Racine et a l . (1975) also noted the d i f f e r e n t i a l effects of various drugs on c o r t i c a l and subcortically e l i c i t e d kindled seizures. Thus, on the basis of existing l i t e r a t u r e i t appears that the kindling paradigm could prove to be an extremely useful tool for assessing the changes i n seizure s u s c e p t i b i l i t y following b r i e f exposures to ethanol. Recapitulation Withdrawal-produced convulsive effects have been demonstrated with many techniques i n a variety of laboratory animals; However, increases i n s u s c e p t i b i l i t y to experimentally-induced convulsions seem to provide a more sensitive measure of such withdrawal effects than do increases i n the incidence of spontaneous withdrawal seizures. The use of such measures has indicated that the convulsive effects may be evident after an organism's f i r s t exposure to ethanol. The kindling paradigm has proven to be an extremely useful tool for assessing changes i n seizure s u s c e p t i b i l i t y . Thus, i n the present -20-experiments, an attempt was made to study the convulsive effects of a single dose of ethanol using the kindling method. -21-EXPERIMENT 1' One purpose was to determine anticonvulsive^ and convulsive N effects of a single i n j e c t i o n of alcohol. A second purpose was to demonstrate the use of kindled convulsions i n the assessment of these changes i n seizure s u s c e p t i b i l i t y . By noting the differences i n the kindled seizures e l i c i t e d by stimulation at various times before and after an alcohol i n j e c t i o n , the time course of the alcohol-produced changes i n seizure s u s c e p t i b i l i t y could be followed i n indiv i d u a l animals. METHODS Subjects: The subjects were 15, 250-310 g male, black-hooded rats purchased from the Canadian Breeding Laboratories (La P r a i r i e , Quebec). Throughout the experiment the subjects were housed singly i n st a i n l e s s - s t e e l cages under a 12-hr-light - 12-hr-dark cycle, with ad libitum access to food (Purina Lab Chow) and water. The rats weighed an average of 415:':±.'.ll g (± standard error of the mean, s.e.) on the test day. Electrodes and Surgical Treatment: A bipolar electrode was aimed at the median nucleus of the right amygdala of each subject. The electrodes were contructed of two 0.25 mm nichrome wires twisted together and insulated with Insul-X. The electrode t i p s were separated by 0.5 mm and scraped to expose a 0.5 mm length of stimulating surface. The surgery was conducted i n accordance with standard stereotaxic technique under combined sodium pentobarbital (30 mg/kg) and chlora l hydrate (125 mg/kg) anesthesia. The electrodes were implanted with a -22-Krieg stereotaxic instrument at coordinates of 1.5 mm posterior to Bregma, 1.5 mm l a t e r a l to the midline, and 8.8 mm below the dura. Following surgery each animal was routinely injected with 0.2' cc of pentylenetetrazol and 0.2 cc of p e n n i c i l l i n . The same dose of p e n n i c i l l i n was administered on each of two subsequent recovery days. Histology: At the termination of the experiment a l l animals were asphyxiated with carbon dioxide and thei r brains were removed and stored i n a buffered formalin solution for at least 1 week, at which time they were embedded i n para f f i n and sectioned at 20 microns. The sections were stained according to a modification of the Kluver-Bararra technique i n order to v e r i f y the electrode placements. Injection solutions: The ethanol was prepared by mixing 95% ethanol with 0.9% saline to produce a 20% by volume ethanol solution. These solutions were always prepared 24 hr prior to the injections and were stored at 4°C u n t i l t h e i r use. Stimulation and Recording: Tests were conducted i n a 25 x 25 x 40 cm clear Plexiglas chamber f i t t e d with a grid f l o o r and housed i n a grounded Faraday cage. Electrographic a c t i v i t y was monitored by a Grass 78B EEG polygraph through low noise, shielded recording leads. During stimulation the re-cording leads also served to deliver current across the electrode t i p s . The 1.0-sec, 400-uA (RMS) current, passed through a switching c i r c u i t which isolated the polygraph amplifiers during stimulation to reduce the post-stimulation interference i n the recording channel. Before each test session, each animal was allowed to acclimatize to -23-the leads and test chamber. The EEG a c t i v i t y was then recorded for 45 sec prior to current delivery and for 60 sec after the l a s t spike of the AD. MSs e l i c i t e d by the stimulation were c l a s s i f i e d according to the 5 stages of epileptogenesis described by Racine (1972b): 1) f a c i a l movements only; 2) f a c i a l movements and head-nodding; 3) f a c i a l movements, headnodding, and forelimb clonus; 4) f a c i a l movements, head-nodding, forelimb clonus, and rearing; and 5) f a c i a l movements, head-nodding, forelimb clonus, rearing, and loss of equilibrium. Kindling Procedure: Recording commenced 21 days after surgery. On the f i r s t day EEG a c t i v i t y was recorded for 10 min to check the connectors and recording electrodes and to habituate the subjects to the apparatus. Kindling began the following day. Three stimulations were given each day, 5 days each week for three weeks. The stimulations were given no less than 0.5 hr and no more than 18 hr apart. The animals were then switched to a stimulation regiment which involved one stimulation per day for 17 days. At that p o i n t , s a i l animals were consistently exhibiting class 5 MSs except for 5 animals consistently displaying class 4 MSs. The MS and AD duration of seizures e l i c i t e d i n each animal by the l a s t four stimulations of the series never differed by more than 25% on consecutive days. Experimental Procedure: Testing began on Day 18 with each rat receiving 18 stimulations, one every 1.5 hr. Following three baseline test stimulations the animals were assigned to two groups matched as much as possible for AD and MS duration and body weight. The injections of saline and ethanol were given 30 min prior to the fourth stimulation. The ethanol group (n = 8) received an i.p. i n j e c t i o n of 20% ethanol solution containing -24-2.0 g/kg of absolute alcohol, whereas the control group (n = 7) received an i.p. i n j e c t i o n of a similar quantity of saline. The quantity consisted of 12.6 ml/kg of f l u i d . The dose of 2.0 g/kg was chosen since t h i s l e v e l produced mild ataxia but no anesthesia when given to p i l o t animals. RESULTS AND DISCUSSION The results are summarized i n Fig. 1. The durations of AD and MSs e l i c i t e d by stimulations administered immediately after the alcohol i n j e c t i o n were reduced appreciably, but following this suppression there was a transient but marked increase i n the AD duration. The results of the ove r a l l analysis of variance and the tests of simple main effects (Kirk, 1968) are presented i n Appendix A. These j u s t i f i e d the use of multiple comparison tests. Tukey "A" tests carried out on the means of the ethanol group indicated that the mean AD duration 0.5 hr following the alcohol i n j e c t i o n was s i g n i f i c a n t l y shorter, and the mean AD duration at 15.5 hr was s i g n i f i c a n t l y longer, than the ADs e l i c i t e d by the three baseline stimulations i n the same animals (Tukey's test, HSD = 36.2 p<.05). Moreover, duration of the ADs e l i c i t e d 0.5 hr after the ethanol was s i g n i f i c a n t l y less than for a l l other test stimulations administered following the alcohol; while the duration of the ADs e l i c i t e d 15.5 hr after the inj e c t i o n was s i g n i f i c a n t l y greater than a l l other ADs e l i c i t e d i n the ethanol subjects except the ones at 12.5, 14.0, 17.0 and 21:5 hr (Tukey's test, HSD =36.2, p<.05). The pattern of significance was also i l l u s t r a t e d by the results of between-group comparisons; the ethanol subjects had s i g n i f i c a n t l y shorter ADs 0.5 and 2.0 hr after the i n j e c t i o n and s i g n i f i c a n t l y longerr ADs at 15.5 hr (Tukey's te s t , HSD ='36.2, p<.05). There was no -25-Mean (± s.e.) AD (top panel) and mean MS (Bottom panel) duration of kindled seizures e l i c i t e d in the rats of the two experimental groups at the 18 injection-test intervals in Experiment 1. M E A N M S D U R A T I O N i n sec . M E A N A D D U R A T I O N i n s e c e 00 ho cn ro o to ro CO CO ro CO cn o o ro * . CO CO cn ro en co o •A o co ro ro CO cn cn o • • i -26-s i g n i f i c a n t v a r i a t i o n i n the mean AD durations over the 18 stimulations i n the control conditions. In individual experimental animals the increase i n duration of the AD appeared at points between 11.0 and 20.0 hr after the alcohol i n f e c t i o n . These increases, however, were not consistently present over this period; they occurred interspersed with ADs of regular duration with the primary concentration of the long ADs occurring between 12.0 and 15.5 hr. Similar multiple comparison tests were carried out on the mean MS durations. Test stimulations 0.5, 2.0, and 3.5 hr after the ethanol i n j e c t i o n evoked shorter MSs than did any of the three pre-injection base-l i n e stimulations ( a l l Tukey's te s t , HSD = 8.56, p< .05). Comparisons of the means of animals receiving alcohol and those receiving saline were also s i g n i f i c a n t at 0.5, 2.0 and 3.5 hrs(Tukey's t e s t , HSD = 9.15, p< .05). At no point was the MS of the group receiving ethanol s i g n i f i c a n t l y longer than for the corresponding control points, and there were no mean MS durations following the ethanol i n j e c t i o n that were s i g n i f i c a n t l y longer than the mean MSs of the three baseline test stimulations. However, at 6.5 hr almost every experimental animal had a longer MS than those of the control animals. The duration of the MSs e l i c i t e d i n the saline controls also varied over the 18 stimulations. Multiple comparisons indicated that the f i r s t mean MS was s i g n i f i c a n t l y longer than the mean MS at 3.5, 5.0, 6.5, 8.0, 9.5, 11.0, and 12.5 hr ( a l l Tukey's te s t , HSD = 9.15, p< .05). The MS class and latency to MS onset were also monitored. In the experimental group the three baseline test stimulations e l i c i t e d class 4 and 5 MSs with MS latencies being less than 1 sec for a l l but one. The MS was blocked i n a l l but three experimental animals at the 0.5-hr i n t e r v a l . -27-These three animals experienced a seizure pattern of class 3 or less with a longer seizure latency. At the 2.0-and 3.5-hr intervals a l l animals exhibited MSs but they were of a lower class and had longer latencies. The MS class and MS latency measures proved to be of l i t t l e value i n detecting any increases i n seizure s u s c e p t i b i l i t y possibly because of " c e i l i n g and basement' effects, respectively. H i s t o l o g i c a l examination revealed that most electrodes had, i n fa c t , been implanted i n the medial amygdala (see Fig. 2). In one subject the t i p was located i n the pyriform area; however, th i s animal's behaviour could not be distinguished from the others. Attempts to correlate the placements with the behaviour of indi v i d u a l rats were unsuccessful. The r e l a t i v e s t a b i l i t y of seizures i n the control animals over the 18 stimulations reveals the potential of the kindling paradigm for tracing the course of the changes i n seizure s u s c e p t i b i l i t y produced by the alcohol. The ADs of most indi v i d u a l animals were very similar to one another during the regiment of 1.5-hr-interval stimulations. This was also the case for the MSs and although the mean duration of the MSs changed s i g n i f i c a n t l y over the course of the 18 stimulations, the change was gradual and consistent i n a l l animals. Against the stable control baseline the time course of changes i n seizure s u s c e p t i b i l i t y are easy to detect. Although kindled seizures have been previously used to assess convulsive and anticonvulsive effects, they have never been used, as i n th i s experiment, to follow the time course of these effects i n individual subjects. Although distributed stimulations are necessary for ki n d l i n g , once kindled, rats w i l l respond consistently to stimulation administered at r e l a t i v e l y short i n t e r v a l s . This i s not the case, however, for rats repeatedly stimulated with other convulsive agents. -28-Fig. 2 Electrode placements of subjects i n the four experiments. Each symbol represents one rat i n the experiments given at the ri g h t . Atlas diagrams are from Pellegririo and Cushman„(1967:). -28 a--29-For example, experiments i n our laboratory revealed that convulsions e l i c i t e d i n rats by electroconvulsive stimulation administered every hr undergo drastic decreases i n the severity and duration, and result i n heavy subject loss due to death and convulsion-produced i n j u r i e s (Van Oot & P i n e l , unpublished observations). Thus, i n comparison to other methods, kindled seizures provide a valuable method of following the time course of the effects i n ind i v i d u a l animals. The time course of the changes i n seizure s u s c e p t i b i l i t y suggests that the anticonvulsive effects were present during the period of high blood ethanol levels and the convulsive effects during a period immediately following alcohol metabolism. Owens and Marshall (1955) found that 2.0 g/kg administered to 300 to 400 g rats i s quickly absorbed into the blood and i s metabolized i n approximately 9 hr. Thus, the present results suggest that ethanol i s a potent anticonvulsive agent. Since the con-vulsive effects seem to have occurred following the metabolism of the alcohol they appear to be true withdrawal-produced effects. -30-EXPERIMENT 2 Comparisons with the results of Owens and Marshall (1955) suggest that the anticonvulsive and convulsive effects observed i n Experiment 1 were related to the presentation and withdrawal of alcohol respectively. The purpose of Experiment 2 was to replicate the results of Experiment 1 and to relate the anticonvulsive and convulsive effects to changes i n blood ethanol l e v e l s . The methods were similar to those of Experiment 1 except for the following innovations. F i r s t , only those kindled subjects that consistently displayed ADs and MSs of stereotyped duration and pattern were tested. Second, a higher dose of alcohol was administered i n an attempt to produce greater changes i n s u s c e p t i b i l i t y . Third, the test stimulations were administered at intervals of 3.0 hr rather than 1.5 hr i n an attempt to eliminate s h i f t s i n the baseline. Fourth, each animal was tested under both the experimental and control conditions so that a par t i c u l a r rat could act as i t s own control. METHODS Subjects: The subjects were 18 male rats similar to those used i n Experiment 1: They were maintained, implanted, and kindled as i n Experiment 1'. In the present experiment, however, animals were not i n the experiment proper unless they reached the c r i t e r i o n of seizure s t a b i l i t y employed by P i n e l , P h i l l i p s , and MacNeil (1973). The subjects were required to exhibit 10 consecutive class 5 MSs with no more than 20% -31-v a r i a t i o n of the MS and AD durations e l i c i t e d on consecutive days. Six of the 12 subjects that reached t h i s c r i t e r i o n did so after 18 d a i l y stimulations; the other s i x after 25 days. Ethanol Assays: The blood ethanol levels were determined according to:.a procedure similar to that of Baker, Alenty, and Zach (1969) and Roach and Creaven (1968). Blood was withdrawn from the t a i l vein 5 min after seizure e l i c i t a t i o n . Two 30 u.1 samples of blood were taken, each drawn into calibrated 75 _ul c a p i l l a r y tubes. Fifteen u.1 of 5% zinc sulfate and 15 u.1 of 1 normal sodium hydroxide, both cooled to 4°C, were immediately added to the blood. The tubes were sealed and stored at 4°C for at least 24 hr but no more than 48 hr before assaying. P r i o r to assaying the blood sample the c a p i l l a r y tubes were centrifuged at 10,000 x gravity for 10 min. A 1.0 u.1 quantity of clear supernatant was then withdrawn with a Hamilton syringe from the topp of the tube and injected into a Perkins-Elmer 900 programmable gas^liquid chromatograph. The helium c a r r i e r gas passed through a column packed with carbowax and maintained at a temperature of 100°C. The c a r r i e r gas then passed over an ionizing hydrogen flame where the presence of ethanol was detected and recorded on a chart recorder. The amount of ethanol was calculated by.making comparisons to i d e n t i c a l external standards of known ethanol concentration. Each of the two blood samples was analyzed twice. Experimental procedure: Testing commenced the day after the c r i t e r i o n of s t a b i l i t y was met. This occurred after the 18th d a i l y stimulation for six animals and after the 25th for the other s i x . Over the 33-hr testing period each animal was stimulated 12 times, once every 3.0 hr. Thirty min ^32-before the f i f t h stimulation s i x rats received an i.p. i n j e c t i o n of 2.5 g/kg of alcohol and six received an equivalent volume of saline (15.7 ml of f l u i d per kg). After t h i s testing period the animals were returned to the d a i l y stimulation regimen for 12 days before being tested as before over another 33-hr session. In this second session animals that had received saline i n the f i r s t test received ethanol, and vice versa. During both test sessions two samples of blood were taken from every animal f i v e min following the cessation of the response evoked by each of the 5th through to the 11th test stimulations. RESULTS AND DISCUSSION The major results of Experiment 2 are i l l u s t r a t e d by Fig. 3. The pattern of anticonvulsive and convulsive effects was comparable to that found i n Experiment 1. The results of the blood ethanol analyses confirmed the r e l a t i o n of the anticonvulsive and convulsive effects with the presence and absence of high blood ethanol l e v e l s , respectively. A summary of the ove r a l l analysis of variance of these results i s presented i n Appendix 'Bi. The alcohol-produced biphasic effects on AD duration similar to those observed i n Experiment 1; A p r i o r i multiple comparison tests indicated that the mean AD duration e l i c i t e d at 0.5 and 3.5 hr was s i g n i f i c a n t l y shorter, and the mean MS duration e l i c i t e d at 15.5 hr was s i g n i f i c a n t l y longer than the mean AD of the fourth baseline stimulation (Dunn's test, c r i t i c a l difference •= 11.5, p<.05). Comparisons between the ethanol and saline conditions at each i n t e r v a l also revealed the biphasic effects of alcohol. There was a s i g n i f i c a n t difference between the means at 0.5, -33-Fig. 3 Mean (± s.e.) concentration of blood ethanol (top panel), AD duration (middle panel), and MS duration (bottom panel) eli c i t e d by the test stimulations at the 12 injection-test intervals in Experiment 2. -34-3.5, and 15.5 hr (Dunn's test, c r i t i c a l difference =13.59, p< .05) while at 9..5, 12.5? and 18.5 hr there were no significant differences between the conditions. There were no systematic changes in the mean AD duration in the control conditions-. These AD data thus replicate the results and conclusions of Experiment 1. The changes in the MS duration were also similar to those observed in Experiment 1. A p r i o r i comparisons of means in the ethanol condition indicated that the mean durations of the MSs elicite d 0v5 and 3.5 hr after the ethanol injection were significantly less than the mean duration of the MSs evoked by the fourth baseline test stimulation (Dunn's test, c r i t i c a l difference = 5.07, p<.05). Comparisons between the means of the ethanol and saline conditions at each interval revealed that the MS durations of the ethanol group were significantly shorter at 0.5 and 3.5 hr and sig-nificantly longer at 9.5 and 12.5 hr after the ethanol injection (Dunn's test, c r i t i c a l difference = 4.99, p<.05). In addition, the,gradual changes in the MS duration observed* in the control condition of Experiment 1 were also observed in the present experiment. The durations of MSs elicited by the f i r s t stimulation in the saline condition were significantly different than the MS duration at 0.5, 3.5, 6.5, 9.5,and 21.5 hr (Dunn's test, c r i t i c a l difference = 5.07, p< .05). Thus, despite the procedural differences the MS data too were comparable to those of Experiment 1. The.results of the s t a t i s t i c a l analyses differed in one respect but this was i:ho.t.ja-Sef^lect"i^nv:of:'.'dif f erences-'in the^ data;.> In both experiments the MSs eli c i t e d after the anticonvulsive effect were longer than those elicited in the saline condition; however, this difference was not found to be significant in Experiment 1 without the use of a p r i o r i s t a t i s t i c a l tests. -35-The results of the blood ethanol assays confirmed that the Increases in AD and MS durations were true withdrawal-produced effects:. Significant increases in AD duration were observed at 15.5 hr and MSs were significantly longer at 9.5-and 12.5-hr intervals even though there was l i t t l e or no ethanol in the blood over this- period. Differences in the blood ethanol levels observed between animals at particular intervals were not systemati-cally related to d i f f erences in MS or AD duration. The electrode placements in the animals of Experiment 2 were similar to those of Experiment 1 (see Fig. 2). It is apparent that a l l electrodes were in the medial amygdala. No systematic relation between electrode placement and behavior of an individual animal could be found. -36-EXPERIMENT 3 The results of Experiment 1 and 2 suggest that the metabolism of an organism's f i r s t dose of ethanol can have convulsive effects. However, there i s another possible interpretation of the convulsive effects. They may simply be an a r t i f a c t of the testing procedure rather than of alcohol withdrawal per se. I t i s well-established that kindled seizures can have suppressive effects on subsequent kindled seizures (Mucha & P i n e l , 1975; P i n e l , P h i l l i p s , & Deol, 1974; Racine, 1972b). Thus, the longer seizures observed during the withdrawal period may have simply been a resu l t of ethanol's anticonvulsive effects on e a r l i e r seizures during the intoxication period: alcohol may have reduced the suppressive effects of e a r l i e r upon la t e r seizures. The purpose of Experiment 3 was to determine i f the long ADs and MSs observed during the withdrawal period were the direct result of the alcohol's anticonvulsant effects or the ind i r e c t result of the repeated testing procedure. Each subject was stimulated at only one i n t e r v a l following each i n j e c t i o n of alcohol or saline. METHODS Subjects: The subjects were the 10 rats that completed Experiment 2, and the 6 rats that f a i l e d to meet the s t a b i l i t y c r i t e r i o n by the 25th d a i l y stimulation. These 6 rats received d a i l y stimulation while the other 10 were being tested i n Experiment 2. By the end of th i s period a l l 6 rats had reached the c r i t e r i o n of s t a b i l i t y consisting of 10 consecutive class 5 MSs with less than 20% v a r i a t i o n i n MS and AD duration on consecutive days. A l l 16 animals were not stimulated i n the 4 weeks between Experiments 2 and 3. At the beginning of Experiment'3 they weighed between 400 and 560 g. Experimental Procedures: During the course of the experiment each rat was stimulated at the same time each day for 48 consecutive days. The f i r s t 14 days served as a baseline period during which 12 animals exhibited a high degree of s t a b i l i t y consisting of at least 5 consecutive class 5 MSs with no more than 20% difference i n the durations of either MSs or ADs e l i c i t e d on . consecutive days. At the end of t h i s 14-day period the 12 animals were divided into two equal groups matched according to AD duration. The remaining four rats were not included i n the present experiment. On days 15, 17, 19, and 21 one group of s i x animals received an i.p. i n j e c t i o n of 2.5 g/kg of ethanol and one group received an equivalent volume of saline (15.7 ml of f l u i d per kg). These injections were administered either 0.5, 7.0, 14.0, or 2110 hr before each d a i l y stimulation. A second series of injections was administered on days 36, 38, 40, and 42; however, during this second series the animals that had previously received the ethanol injections received the saline and vice versa. In a l l other respects the tests were the same. The order of the four i n j e c t i o n intervals was deter-mined according to a 4 x 4 l a t i n square. RESULTS AND DISCUSSION The results are presented i n Fig. 4. Even though a different experimental design was employed i n the present experiment, the results were similar to those of the i n i t i a l experiments; anticonvulsive and convulsive -38-F±g. 4 Mean (± s.e.) AD (top panel) and mean (± s.e.) MS (bottom panel) duration elici t e d at the four injection-test intervals in Experiment 3. -38a-INJECTION-TEST li'JTERVAL in hr -39-effects were observed following a single i n j e c t i o n of alcohol. A summary of the ove r a l l analysis of variance of the MS and AD durations i s presented i n Appendix C. The change;,- i n the mean AD duration was similar to that observed i n Experiments 1'and 2: a p r i o r i multiple comparisons between means of the experimental condition and of the control indicated that the ethanol i n j e c t i o n reduced s i g n i f i c a n t l y the duration of ADs e l i c i t e d 0.5 hr after the alcohol and s i g n i f i c a n t l y increased the duration of those e l i c i t e d at 14.0 hr (Dunn's te s t , c r i t i c a l difference = 13.05, p<.05) . Comparisons between the mean MS durations of the various ethanol and saline conditions indicated that at 0.5 and 7.0 hr the mean MS duration of the subjects i n the ethanol condition was s i g n i f i c a n t l y less than that of the subjects i n the saline condition (Dunn's te s t , c r i t i c a l difference = 6.77, p<\05). The mean difference between the data of subjects i n the ethanol and i n the saline conditions tended towards significance at 14.0 hr. At th i s time i n t e r v a l 9 of the 12 animals exhibited a longer MS following the ethanol i n j e c t i o n than following the saline i n j e c t i o n . The location of the electrode t i p s of the subjects are presented i n Fig. 2. A l l electrodes were i n the medial amygdala and, as i n previous experiments, no systematic r e l a t i o n was found between location and changes i n seizure s u s c e p t i b i l i t y of ind i v i d u a l animals. The results of th i s experiment, therefore, provide strong evidence that the withdrawal from a f i r s t exposure to ethanol can have convulsant effects. This convulsant effect does not appear to be due to a post-suppression "rebound" effect of the e a r l i e r anticonvulsive effects of alcohol. Alcohol resulted i n an increase i n mean seizure duration even when only a single -40-post-injection stimulation was used to assess the effect. EXPERIMENT 4 The purpose of Experiment 4 was to confirm the results of Experiment 3 and establish t h e i r generality by administering the ethanol i n t r a g a s t r i c a l l y rather than intraperitoneally as i n the f i r s t three experiments. Because of i t s rapid absorption into the blood, intraperitoneally administered ethanol may produce q u a l i t a t i v e l y different results than those following routes of administration with lower rates of absorption (cf. Kalant, 1961). METHOD Subjects: The subjects (N=19) and surgical and kindling procedures were similar to those employed i n Experiment 1. By the 25th d a i l y stimulation the three rats that had not reached the c r i t e r i o n of 10 consecutive days with less than a 25% difference on consecutive days i n either MS '^ o'f.SAB.sduracion'iVe'-Eex'excluded. Experimental Procedure: The experiment commenced on the day following the 25th d a i l y stimulation and lasted for seven days. Each day each animal received one stimulation at the same time of day. On days 1, 3, 5,and 7 each rat was exposed to one of four experimental conditions, 2.5 g/kg of ethanol administered by gavage at either 11.0, 14.0j;_'pr 17.0 hr before the stimulation or an equivalent volume of saline (15.7 ml/kg) administered 14.0 hr before the stimulation. The order i n which these four treatments were presented was determined by randomly assigning 4 rats to each row of a 4 x 4 l a t i n square. -42-RESTILTS AND DISCUSSION The results are summarized in Fig. 5. Only long ethanol-test intervals were looked at in the present experiment and changes in seizure susceptibility consistent with those of previous experiments were found. Thus, intragastrically administered as well as intraperitoneally administered ethanol results in increases in susceptibility to kindled seizures several hr after the ethanol administration. A summary of the overall analysis of variance for AD and MS durations i s presented in Appendix D. A p r i o r i comparisons indicated that the mean AD duration e l i c i t e d at 11.0 hr following the ethanol intubation was significantly longer than the mean AD duration following a similar saline intubation (Dunn's test, c r i t i c a l difference = 10.38, p<.05). The AD duration at 14.0 and 17.0 hr following the intubation was not s t a t i s t i c a l l y different than the mean duration e l i c i t e d following the saline. Multiple comparisons'of the mean MS durations of the experimental conditions to that of the control condition failed to reveal any s t a t i s t i c a l differences (Dunn's test, c r i t i c a l difference = 3.73, p< .05). In previous experiments the MS duration Increases were.-no;**:-;as.-*-rellabl-e-;as. those of the AD but they have nevertheless been present. Since only three relatively similar intubation-test intervals were used the lack of an appreciable change in the MS duration of the present experiment may have been due to a failure to employ appropriate test intervals. The electrode tips of a l l the rats i n the present experiment were located in the medial amygdala (see Fig. 2). No systematic relation between -43-Fig. 5 Mean AD (top panel) and mean MS (bottom panel) duration e l i c i t e d by the test stimulation before and after an i n t r a -gastric i n j e c t i o n of ethanol or saline. The v e r t i c a l l i n e at each point i s the s.e. M E A N M S D U R A T I O N i n s e c M E A N A D D U R A T I O N i n s e c t o t o t o t o t o CO • J>» ' *» ' o» —I - J — J OO OS co t o t o t o tn o ->j co to <=> —x r o t o ert co —» -J o co c n 1 • « : r — — s , « i i i i i J -44-behaviour and electrode placement was found. There are two major findings^ of the present experiment. F i r s t , the results demonstrate convulsive.effects several hr after an intragastrlcally administered dose of ethanol. This indicates that the increased seizure susceptibility found in the previous experiments was not specific to i n t r a -peritoneally administered ethanol. There are a number of differences between these two methods of injecting alcohol. For example, ethanol administered intraperitoneally is absorbed very rapidly and produces relatively high blood ethanol levels, while .intragstrically given ethanol is absorbed slower and reaches a much lower blood level (cf. Wallgren, 1970). Thus, the convulsive effect noted in the present experiments i s not a result of either the rapid rate of ethanol absorption or the higher blood ethanol levels that are specific to the intraperitoneal route of ethanol administration. The second major finding of the present experiment was that convulsive effects after a brief ethanol exposure were demonstrated with a design using only a single test stimulation after each intubation of ethanol. This replicates the results of Experiment 3 and confirms the conclusion that the increased susceptibility to kindled seizures found in the f i r s t two experiments was not an artifact of the repeated testing procedure employed. -45-GENERAL DISCUSSION The primary purpose of the present investigation was to study the convulsive effects produced i n naive organisms following a single b r i e f exposure to ethanol. An additional purpose was to describe a method of using kindled seizures to measure the time course of changes i n seizure s u s c e p t i b i l i t y . Hence, the discussion w i l l begin with a consideration of these two aspects of the present investigation. This section w i l l continue with a discussion of the following topics: other effects of b r i e f alcohol exposure ^ l\;hangover;, the r e l a t i o n of pathological effects of alcohol exposure to withdrawal seizures, and the concept of physical dependence. Convulsive Effects of a Single, Brief Exposure to Ethanol The major finding of the present experiments was a s i g n i f i c a n t increase i n the duration of kindled seizures e l i c i t e d several hr after an organism's f i r s t i n j e c t i o n of ethanol. Thus, these results confirm the previous demonstrations of increases i n seizure s u s c e p t i b i l i t y following a single dose of ethanol (McQuarrie & F i n g l , 1958; Goldstein, 1972). The study of these convulsive effects extended those of these previous investigations i n three different ways. F i r s t , the generality of these effects was i l l u s t r a t e d . The demonstration of increased seizure s u s c e p t i b i l i t y following a single b r i e f exposure to ethanol i s not peculiar to either the laboratory mouse or to convulsions e l i c i t e d by handling or pentylenetetrazol (McQuarrie & F i n g l , 1958; Goldstein, 1972). Second, an increase i n seizure s u s c e p t i b i l i t y following a single i n j e c t i o n of ethanol was demonstrated using two e n t i r e l y -46-different experimental designs. In addition to assessing seizure sus^ cep t i b i l i t y at multiple fixed intervals following the injection, the method used by both McQuarrie and Fingl 0-9.58) and Goldstein Q.9-72), a design with only a single test after each alcohol injection was used. Therefore,, changes in seizure susceptibility several hr after the alcohol injection are not simply artifacts of the multiple-assessment procedure. Third, the present experiment provided the f i r s t clear demonstration that the convulsive effects following brief exposure are triggered by the metabolism of the ethanol rather than the presence. At low concentrations ethanol may have direct convulsive effects that w i l l decrease the amount of current required to e l i c i t an AD with focal stimulation of the cortex and w i l l prolong the AD once i t is elicit e d (Kalant, 1970). In Experiment 2 increased seizure susceptibility was clearly present after a l l the ethanol had been metabolized, thus precluding the convulsive effects of ethanol as the basis of these increases. Goldstein (1972) had previously related blood ethanol levels to increases in seizure susceptibility following single brief exposures; however, in her studies increases in susceptibility to handling convulsions peaked while some ethanol was s t i l l present in the blood. Thus, the results of the present experiments provide clear support for the generality of withdrawal-produced convulsive effects following brief exposures of ethanol to naive organisms. The implication of these findings is that the process responsible for the severe withdrawal-produced convulsive effects following chronic alcohol exposure in humans begins to develop with the f i r s t exposure of ethanol. Consistent with this view are the reports of epileptic effects in nonalcoholic epileptics (Victor, 1968; -47-Lennox, 1941) and in young healthy persons (Nagy, et a l . , 1973) after brief ethanol exposures. Although i t is strongly suggested that the convulsive effects after the anticonvulsive effects of the alcohol are withdrawal-related, the present experiments have not precluded the possibility that part of the convulsive effect may be produced indirectly. For example ethanol exposure produces an increase in blood pH (Wolfe & Victor, 1971) which in the epilepsy literature has been demonstrated to increase the susceptibility to audiogenic seizures (Goldman, Lisak, Matz, SmDavidson, 1970). Thus, the increases in seizure susceptibility may be due to a mild state of alkalosis produced by the brief ethanol exposure. Kindled Seizures arid the Assessment of Seizure Susceptibility The present investigation demonstrated a number of features of kindled seizures which make them extremely useful for assessing changes in seizure susceptibility. For example, since low-intensity stimulation w i l l eventually e l i c i t seizures which vary l i t t l e from day-to-day agents having only subtle effects on seizure susceptibility can be assessed without having to use large numbers of subjects. Stability was apparent not only when stimulation occurred every 24 hr, but also when i t occurred at intervals as short as 1.5 hr. Another feature of kindled seizures illustrated by the present studies was their potential for repeated testing. After an animal has been kindled, seizures can be reliably eli c i t e d even after long rest periods (cf. Goddard et a l . , -48-1969), and they rarely cause f a t a l i t i e s or injuries. In the present studies subjects were repeatedly tested in every experiment and the same animals were used in Experiments 2 and 3. An- additional feature is their sensitivity to both increases and decreases in seizure sus-cep t i b i l i t y . This was clearly evidenced by the detection of both anticonvulsive and convulsive effects of alcohol exposure. Thus, the present data, confirm and extendla number of previous demonstrations that kindled seizures could be used to assess seizure susceptibility (cf. Babington & Wedeking, 1973; Pinel et a l . , 1973; Racine et a l . , 1975). The major contribution of the present study was the finding that kindling may be used to accurately follow the time course of a convulsive or anticonvulsive agent in a single animal. Typically, most studies have measured the seizure susceptibility at only a single interval after each administration of a particular agent. This has lik e l y resulted from the early report that interstimulation intervals of 24 hr were the most effective for kindling (Goddard, et a l . , 1969). Thus, few experimenters have studied the effects of kindled seizures e l i c i t e d by stimulation separated by short intervals; the type of stimulation necessary for the study of time courses of changes in seizure susceptibility lasting only a few hr. The present data also i l l u s t r a t e the potential of kindling for understanding the mechanisms underlying changes in seizure susceptibility. For example, the lack of a correlation between the increases in mean AD duration and in mean MS duration indicates that alcohol withdrawal does not have nonspecific convulsive effects; otherwise, the AD and MS durations should have been similarly affected. The data also suggest that the convulsive effect -49-of the ethanol acts i n i t i a l l y on the area of the brain responsible for motor function. The f i r s t successful use of kindled seizures to s i m i l a r l y study mechanisms of drug actions was by Babington and Wedeking (1969, 1973). By studying d i f f e r e n t i a l effects of centrally active agents on amygdaloid, septal and c o r t i c a l kindling they concluded that the action of some drugs i s primarily i n one area of the brain and not i n others. The conclusions based on these d i f f e r e n t i a l influences on kindling were highly consistent with other data specifying s i t e s of the brain where the ind i v i d u a l drugs act. Thus, the present data suggest that the convulsive effects following the metabolism of ethanol may be subjected to a similar type of analysis of mechanisms using the -kindling ^m~e£frd*d. Other Effects of Brief Ethanol Exposure There are a number of other effects of long-term alcohol con-sumption that may be demonstrated following very short exposures. Tolerance, for example, has been generally assumed to be due to long-term abuse of alcohol. I t i s w e l l established that a dose of ethanol just s u f f i c i e n t to impair nondrinkers w i l l have l i t t l e effect on an alcoholic (Kalant et a l , 1970). Recent research, however, has indicated that even a single dose of ethanol can produce tolerance (LeBlanc, Kalant, & Gibbins, 1975). The effects of a single i n j e c t i o n of alcohol were assessed i n rats at various times after intraperitoneal administra-t i o n . Disruption of behaviour soon after the in j e c t i o n was markedly less than the disruptive effect at the same blood alcohol l e v e l a short period of time l a t e r . -50-Pronounced changes i n REM sleep, c o r t i c a l potentials evoked by somatosensory s t i m u l i , and alcohol-induced nystagmus due to b r i e f exposures of ethanol has been reported. Knowles, Laverty, and Kuechler (1968), for example, found that 3.5 oz of alcohol at bedtime was s u f f i c i e n t to i n h i b i t REM sleep during the f i r s t half of the night and to enhance i t during the second half i n a normal healthy subject. Six oz produced a complete suppression of REM a c t i v i t y and during the nights following such complete REM suppression the incidence of REM was increased. Beglieter, Poyecz, and Yerre-Grubstein (1974) found that 10 hr after consuming 3.2 g/kg/day for four days there was a s i g n i f i c a n t increase i n the recovery cycle of somatosensory evoked responses. Aschan, Bergstedt, Goldberg, and L a u r e l l (1956) reported that p o s i t i o n a l nystagmus can be induced i n humans after the metabolism of even small amounts of alcohol. The effects of a single i n j e c t i o n are not r e s t r i c t e d to the central nervous system where the aforementioned phenomena l i k e l y occur. For example, one very large dose of ethanol produces an accumulation of f a t t y acids i n the l i v e r which becomes more pronounced after prolonged ethanol intake, (cf. Lieber, 197j0,) . Ugarte and Valenzuela (1971) concluded that small doses of ethanol administered intravenously stimulate pancreatic secretion i n a manner analagous to the way more dramatic increases are produced by chronic alcohol exposures. - 5 1 -Hangover . Khan, Jensen, and Drough (1973) included vomiting, loss of appetite, heartburn, lassitude, thirst, tremor of hands and limbs, palpitation, weakness of joints, respiratory d i f f i c u l t i e s , sleeplessness, dizziness, headache, fatigue, sweating, disturbance of balance, pallor, nystagmus, general malaise, mood disturbance with anxiety and depression as possible symptoms of hangover-. Although these are the most widely experienced aftereffects of drinking, l i t t l e research has been done on them. One major problem has been the failure to come up with an appropriate operational definitionoofhh'angc-ver• Thus everything from a headache (Wolff, 1963) to delirium tremens (Karpman, 1957) hag' been included as a symptom of hangover. Another problem has been the lack of suitable methods of inducing hangover.-.. Paradoxically only 10 to 20 percent of the subjects in experiments on hangovers' actually report them (Chapman, 1970). As a result of these problems there is a general lack of studies attempting to identify factors which influence the incidence and severity of hangover (cf. Wolff, 1963). Several indirect lines of evidence suggest that at least some hangover effects may be a product of withdrawal-produced increases in seizure susceptibility. For example, the hangover is typically observed as an aftereffect of drinking (Karvinen, Miettinen, & Ahlman, 1962) and as such has a very similar time course to the increases in seizure susceptibility seen upon the withdrawal of alcohol. In addition the results of the present experiment show that increases in seizure susceptibility, like the hangover, may be produced following the f i r s t -1 -52-brief exposure to ethanol. Moreover, additional alcohol is one of the best known remedies for both hangover and the r e l i e f of the severe withdrawal effects after long-term ethanol consumption (Wallgren & Barry, 1970). An additional piece of evidence suggesting that hangover is a mild withdrawal effect is the similarity of symptoms of hangover to those following termination of long-term alcohol consumption (Wallgren & Barry, 1970). In fact, there i s l i t t l e , i f any, difference between subjective reports of a mild withdrawal syndrome and a severe hangover (cf. Victor & Wolfe, 1973). Thus, there is evidence to suggest that at least some of the symptoms of hangover may be related to changes in seizure susceptibility. Despite i t s obvious importance, this relation has gone unnoticed. Many theories of the hangover have been put forward (Chapman, 1970) however with only one exception (Wallgren & Barry, 1970) none have considered hangovers to be related to changes in seizure susceptibility. Failure to recognize the hangover as an early manifestation of withdrawal effects typically produced following long-term ethanol ex-posure is one of the main reasons that haa'goWf'sfeBa^e=a'6«'fS&ted; l i t t l e experimental interest. This is unfortunate since the hangover may play an important role in the development of more severe withdrawal effects and may provide a valuable means to follow the genesis of these withdrawal effects. Relation of Pathological Effects of Alcohol Exposure to Withdrawal Seizures There is considerable indirect evidence that alcohol consumption -53-in humans is related to increases in seizure susceptibility. In this section the possibility that some of the effects of alcohol consumption may be a direct consequence of these convulsive effects is considered. Three hypotheses w i l l be discussed: 1) that the serious withdrawal effects observed in.chronic alcholics are at least partially the result of the repeated experience of the convulsive effects of short ethanol exposures; 2) that electrographic epileptic activity related to the withdrawal of alcohol may be a factor in alcohol-related blackouts; and 3) that electrographic epileptic activity may be responsible for intellectual deficits related to chronic alcohol consumption. The f i r s t hypothesis concerns the possibility that repeated experience of the convulsive effects of brief ethanol exposures may play an important role in determining the severity of the alcohol withdrawal syndrome in cchronic alcoholics. It was pointed out earlier that there is a gradual increase in stimulus-induced epileptic activity when localized brain stimulation is repeatedly administered to the amygdala of various species. This was termed the kindling effect. The kindling effect has typically been associated with the progressive development of seizures in response to periodic, local brain stimulation, however, recent developments in this literature strongly suggest that the changes associated with brain stimulation may simply be specific manifestations of a phenomenon general to a l l convulsants. Kindling-like effects have been recorded after repeated administration of a variety of convulsive agents. For example, Mason and Cooper (1972) administered "subconvulsive" doses of pentylenetetrazol to rats at 3-day intervals. After a few -54-injections minor myoclonic responses developed and eventually grand mal seizures were eli c i t e d in some animals. Leech (1972) observed that mice which are i n i t i a l l y resistant to audiogenic seizures eventually become responsive to the stimulation i f they are exposed to a short burst of audio stimulation each day. At f i r s t the sound elicit e d l i t t l e more than a startle response in most mice of "resistant" strains but after several days of exposure, f i t s of running and jumping were e l i c i t e d . With a few additional stimulations grand mal convulsions were typically produced. Prichard, Gallagher, and Glaser (1969) found that i f seizures were elicit e d with flurothyl ether at a rate of not more than once a day there was a progressive reduction of seizure threshold in rats, mice, and guinea pigs. With each presentation of flurothyl, there was a decline in the latency to the f i r s t myoclonic jerk and to sustained convulsion, and an increase in the severity of the MS pattern. Vosu and Wise (1975) disco"yered^that;;icarbachol\. injected daily into the amygdala, hippocampus, or caudate of rats at "subconvulsive" doses eventually e l i c i t e d MSs. Finally, Ramer and Pinel (1974) found a gradual intensification of the seizures e l i c i t e d by periodic electroconvulsive shock. In each of these situations, a specific level of the convulsive agent comes to e l i c i t more severe seizures following the periodic presentation of the convulsant. Thus, because of the apparent generality of the kindling phenomenon, i t appears possible that the epileptic consequences of alcohol exposure may also be intensified with repeated presentation and withdrawal. There is l i t t l e research providing direct support for this suggestion, however there are several observations consistent with i t . -55-F i r s t are the observations by Walker and Zornetzer (1974) that withdrawal after a second ethanol exposure i n rodents produces withdrawal effects more severe than those observed after the f i r s t . Second, Mendelson et al.(1966) and I s b e l l et al.(1955) found that alcoholics experiencing withdrawal effects following the cessation of drinking, are more l i k e l y to develop withdrawal effects following subsequent periods of drinking. A t h i r d relevant observation comes from the barbiturate l i t e r a t u r e . Wulff (1960) found that patients who had previously exhibited e p i l e p t i c symptoms during barbiturate withdrawal had twice the incidence of withdrawal convulsions than those who had not previously experienced them. Although these data are consistent with the contention that alcohol with-drawal convulsive effects i n t e n s i f y they are by no means conclusive. In a l l of these studies the number of withdrawal episodes and the duration of drug exposure are confounded. Thus, the aforementioned evidence i s consistent with a number of equally plausible explanations. Nevertheless, the hypothesis deserves serious considerations because of the implications i t has for the treatment and understanding the effects of alcohol consumption', a sudden temporary cessation of drinking would not only result i n increases i n seizure s u s c e p t i b i l i t y , but would also increase the severity of subsequent withdrawal symptoms. Since the hangover may be an early manifestation of convulsive effects of alcohol withdrawal, i t i s an extremely important phenomenon with regard to the present discussion. The s u s c e p t i b i l i t y and frequency of hangovers may play an important role i n the gensis of more severe forms of alcohol-withdrawal-produced convulsive effects. The second hypothesis suggests a r e l a t i o n between the electrographic e p i l e p t i c a c t i v i t y associated with alcohol withdrawal and alcohol amnesia -56-or blackouts. Alcohol blackouts are usually defined as memory loss without loss of consciousness for events that occur during drinking (Lisman, 1974). It i s w e l l established that epileptiform electrographic discharges with no motor correlates can have amnesic effects i n both humans (Browne, Penry, Porter, & Dreifuss, 1,974) and i n animals (Woodruff, 1974). Thus, i t i s not unreasonable to believe that some of the memory problems of alcoholics are related to subconvulsive e p i l e p t i c a c t i v i t y triggered by alcohol withdrawal (Goodwin, Crane, & Guze, 1969a; Ryback, 1970). There i s some empirical evidence suggesting that blackout s are related to withdrawal-produced electrographic abnormalities. For example, Goodwin et a l . (1969) found that the severity of blackouts i n hospitalized alcoholics was p o s i t i v e l y related to the extent and duration of alcohol abuse and to a history of head trauma, two factors which have been shown to be related to the incidence of withdrawal-produced epileptiform abnormalities (cf. V i c t o r , 1968). In addition, i t i s now well-known that both blackouts and seizure-related amnesia t y p i c a l l y occur as losses of blocks of time (Goodwin et a l . 1969a,b; Ryback, 1970). The t h i r d hypothesis i s that learning and i n t e l l e c t u a l d e f i c i t s that sometimes occur for several years following the withdrawal from long-term alcohol exposure (Farmer, 1973; Kleinknecht & Goldstein, 1972; Page & Linden, 1974) may also be a result of e p i l e p t i c a c t i v i t y present i n the brain after the withdrawal of alcohol. This p o s s i b i l i t y has generally been overlooked since the gross behavioural manifestations of the underlying epileptiform discharges do not usually persist for more than a few days after withdrawal (cf. V i c t o r , 1968). Thus; the withdrawal effects.have been viewed as short-term events which cannot be responsible for the long-term cognitive -57-d e f i c i t s frequently observed following chronic alcohol exposure. However, i t i s now strongly suggested that withdrawal-produced epileptiform abnor-malities can persist for several months after alcohol withdrawal. Bennett (1960), for example, found abnormal spike and wave a c t i v i t y i n many patients' EEG for up to several months after the alcohol withdrawal. Epileptiform EEG a c t i v i t y has also been found i n rats for up to several months following the withdrawal from long-term alcohol exposure (Mucha & P i n e l , unpublished observations). There are two additional l i n e s of evidence that support t h i s hypothesis. F i r s t , the d e f i c i t s i n chronic alcoholics are similar to those seen i n some patients with epileptiform a c t i v i t y induced by other agents (cf. Woodruff, 1974). A second l i n e of evidence was provided by Bennett (1960) who noted that i n many alcoholics with EEG abnormalities the recovery of the behavioural d e f i c i t s paralleled recovery of the EEG. Relevant for a l l three hypotheses i s the study of subcortical EEG epileptiform a c t i v i t y during the withdrawal of alcohol. There i s almost no research on t h i s i n humans; however, recent animal research indicates that subcortical, rather than c o r t i c a l structures, play the primary role i n the genesis of e p i l e p t i c a c t i v i t y during the withdrawal period. This finding i s important since the hypotheses concern phenomena associated with subcortical structures. For example, the amygdala i s the most responsive structure for kindling seizures (Goddard et al,•1969). S i m i l a r l y , hippocampal and amygdaloid ADs with no MSs result i n profound learning and retention d e f i c i t s i n cats (Kesner & Doty, 1968) . Thus, the study of alcohol-related e p i l e p t i c effects has implications for the understanding of three additional areas of alcohol related -58-pathology. The evidence for the involvement of seizures i n these phenomena remains without direct support primarily because of the d i f f i c u l t y i n conducting the appropriate experiments on human patients. However, the formulation of these hypotheses was based on c l e a r l y established findings i n the epilepsy l i t e r a t u r e . Concept of Physical Dependence An indi v i d u a l i s said to be physically dependent on a drug when i t s withdrawal triggers an i l l n e s s called a withdrawal syndrome, whose exact form depends to some extent on the nature of the drug. For example, the withdrawal of barbiturates and alcohol produces a syndrome with convulsions as one of the most prominent symptoms (Essig, 1972). Withdrawal of morphine, however, rarely results i n convulsions (Seever & Deneau, 1963). Thus, the term "physical dependence" can be used to describe the unknown characteristics which d i f f e r e n t i a t e a healthy subject from one i n which a withdrawal syndrome can be ea s i l y triggered, and as such i t serves a useful function. Unfortunately, the concept of physical dependence has been of l i t t l e s c i e n t i f i c use because there i s no way of measuring i t other than i n terms of the withdrawal syndrome (Seevers & Deneau, 1963) . Some studies have noted that tolerance develops at approximately the same time as physical dependence (Isbeill, e t _ a l . , 1955). However, the l e v e l of tolerance i s not a good measure of physical dependence as defined by the severity of the withdrawal syndrome. The duration of alcohol exposure required to produce asymptotic withdrawal effects i s considerably greater than that required to produce maximum tolerance (Kalant et a l . , 1970). Thus, -59-because physical dependence cannot be defined without withdrawing ethanol, i t s many.studies in the literature are in fact studies of the alcohol withdrawal syndrome. Although the term "physical dependence" i s useful in some contexts, i t s use has had two unfortunate effects. F i r s t , individuals have used the term as i f physical dependence had an existence separate from the withdrawal symptoms that define i t , and, worse, they have used the term to explain the presence of the withdrawal symptoms. Secondly, the fact that the processes underlying the development of susceptibility to withdrawal effects are generally referred to by a single term has caused some investigators to lose sight of the fact that there is probably more than a single factor involved. Victor (1968), for example, described the appearance of two distinct groups of symptoms occurring at two different times after withdrawal from longterm intoxication. The group characterized by tremors, hallucinations and convulsions occurred between 6 and 48 hr after the withdrawal of alcohol. The other group of symptoms comprised of delirium and autonomic overactivity almost invariably came after the f i r s t group. Thus, although theiterm 'physical dependence' is useful when used appropriately, i t s overall effect has been to complicate the study of the production of various withdrawal reactions. Nevertheless, the present results suggest that physical dependence, as defined by withdrawal-produced convulsive effects, begins to develop with an organism's f i r s t exposure to ethanol. -60-CONGLUSION In the present experiment i t was clearly established that'an organism's f i r s t exposure to ethanol could have convulsive withdrawal effects. 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Electroencephalography and C l i n i c a l  Neurophysiology, 1960, Supplement 14. -72-APPENDICES -73-Appendix A Analysis of Variance Tables for Experiment 1 Analysis of Variance Table for Experiment 1 AD Duration Source SS df MS F ' Between Subjects Drug Subj/groups 4015.5 56184.1' 1 13 4015.5 432118 .90 Within Subjects Time Time x Drug Time x Subj/groups 65341.0 39490.8 92877.7 17 17 221 3843.6 2323.0 420.3 9.14* 5.50* * J><. 05 Analysis of Variance Table for Experiment 1 MS Duration Source SS df MS F Between Subjects Drug Subj/groups 678.1' 2352.7 1' 13 678.1' 180.9 3.74 Within Subjects Time Time x Drug Time x Subj/groups 4189.3 5152.3 5183.0 17 17 221 246.4 303.0 23,5 10.50* 12.90* •P<.05 -74-Analysis of Variance Table for Simple Effects of Experiment 1 AD Duration Source SS df MS F 168.3 1 168.3 .26 146.7 1 146.7 .23 531.2 1 531.2 .83 11640.6 1 11640.6 18.27 6862.9 1 6862.9 10.70 1884.3 1 1884.3 - 2.95 1845.2 1 1845.2 2.89 936.6 1 936.6 1.47 2099.4 1 2099.4 3.29 27.6 1 27.6 .043 21.2 1 21.2 .03 976.0 1 976.0 1.53 1788.5 1 1788.5 2.80 9812.5 1 9812.5 15.40* 891.5 1 891.5 1.39 1666.0 1 1666.0 2.61 .318.3 1 318.3 .49 .1901.3 1 1901.3 2.98 Between Subjects Drug at T T 2 4 T4 T5 T T 8 T 9 T 1 0 T 1 1 ™12 T T 1 3 T 1 4 T 1 5 T 1 6 T 1 7 18 Within c e l l 149061.7 234 637.0 Within subjects Time at D D2 Time x Drug Time x Subj/groups 106487.0 5114.0 39490.8 92877.6 17 17 17 '221 6263.0 300.0 2323.0 7.420.0 14.90** .71 5.50 *p< .05/18 **p< .05/2 -75-Analysis of Variance Table f o r Simple E f f e c t s of Experiment 1 MS Duration Source Between Subjects Drug at T 10 v11 p 1 2 ™13 r 1 4 115 16 17 18 Within c e l l Within subjects Time at Dn °2 Time x Drug Time x Subj/groups SS df MS F 112.2 1 112.2 3.40 63.8 1 63.8 1.98 107>.0 1 107.0 3.32 3226.3 1 3226.3 100.10* 1241.1 1 1241.1 38.54* 711.9 1 711.9 22.10* 28.3 1 28.3 .88 224.5 1 224.5 6.90 110.0 1 110.0 3.40 23.5 1 23.5 .73 3.5 1 3.5 .10 4.5 1 4.5 .13 16.8 1 16.8 .53 19.1 1 19.1 .59 6.5 1 6.5 .20 4.5 1 4.5 .13 19.4 1 19.4 .60 22.0 1 :'22.0 .68 7575.7 234 32.2 8269.0 17 486.4 20.70** 1522.2 17 89.5 3.60** 5152.3 17 303.0 5183.0 221 23.5 *p< .05/18 **p< .05/2 -76-Appendix B • Analysis of Variance Tables for Experiment 2 Analysis of Variance Table for Experiment 2 AD Duration Source SS df MS F;. Between Subjects Order 168.7 1 168.7 . 11 Subj/groups 11980.2 8 1497.5 Within Subjects Drug 11172.2 1 1 ' 11172.2 18.05 Drug x Order ;726.7 r 726.7 1.17 Drug x Subj/groups C4951.2 8 618.9 Time 34247.1 11' 3113.4 21:17 Time x Order 771:6 11' 70.1 .04 Time x Subj/groups 12938.0 88 147.0 Drug x Time 44609.9 11 4055.4 25.73 Drug x Time x Order 1884.4 11' 171.3 1:08 Drug x Time x Subj/groups 13866.8 88 157.6 -77-Analysis of Variance Table for Experiment 2 MS Duration Source SS df MS F Between Subjects Order .1:4 Subj/groups 1640.7 Within Subjects Drug 946.6 Drug x Order 176.0 Drug x Subj/groups 230.2 Time 13521:7 Time x Order 396.3 Time x Subj/groups 3006.6 Drug x Time 8069.0 Drug x Time x Order 208.5 Drug x Time x Subj/groups 2234.5 1' 8 1' 11 8 11' 11' 88 111 11' 88 1.4 205.1 946.6 176.0 28.8 1229.2 36.0 34.2 733.6 19.0 25.4 .01 32.89 6.11' 35.97 1.05 28.89 0.74 -78-'Appendix C Analysis of Variance vTaMes f or'-_ Experiment 3 Analysis of Variance Table for Experiment 3 AD Duration Source SS df MS F Between Subjects Order 1 1092.5 1 1092.5 ' 1.19 Order 2 709.4 3 236.5 0.25 Order 1 x Order 2 2623.3 3 874.4 0.95 Subj/groups 3644.4 4 911.1 Drug 1121.3 1 1121.3 13.39 Drug x Order 1 431.9 1 431.9 5.15 Drug x Order 2 298.3 3 99.4 1.18 Drug x Order 1 x Order 2 3208.2 3 .1069.4 12.77 Drug x Subj/groups 334.9 4 83.7 Time 16918.3 3 5639.4 72.95 Time x Order 1 104.6 3 34.8 0.45 Time x Order 2 471.3 9 52.3 0.67 Time x Order 1 x Order 2 2943.9 9 104.8 1.35 Time x Subj/groups 927.6 12 77.3 Drug x Time 18924.3 3 6308.1 46.80 Drug x Time x Order 1 427.5 3 142.5 1.05 Drug x Time x Order 2 1172.5 9 130.2 0.96 Drug x Time x Order 1 x Order 2 2502.0 9 278.0 2.06 Drug x Time x Subj/groups 1617.1 12 134.7 -79-Analysis of Variance Table for Experiment 3 MS Duration Source SS df MS F Between Subjects Order : 1 33.7 1 33.7 0.29 Order : 2 . 343.1 3 114.3 0.99 Order : 1 x Order 2 327.9 3 109.3 0.94 Subj/groups 461.3 4 115.3 Drug 2285.9 1 2285.9 254.32 Drug X Order 1 1.2 1 1.2 0.13 Drug X Order 2 134.9 3 44.9 5.00 Drug X Order 1 x Order 2 124.2 3 41.3 4.60 Drug X Sub j /g ;roups 35.9 4 8.9 Time 4685.2 3 1561.7 73.82 Time X Order 1 194.1 3 64.6 3.05 Time X Order 2 102.9 9 11.4 0.54 Time X Order 1 x Order 2 192.1 9 21.3 1.00 Time X Subj/groups 253.9 12 21.1 Drug X Time 6203.3 3 2067.7 50.97 Drug X Time x : Order 1 6.9 3 2.3 0.05 Drug X Time x : Order 2 233.9 9 25.9 0.64 Drug X Time x : Order 1 x Order 2 387.9 9 43.0 1.06 Drug X Time x : Subj/groups 486.7 12 40.5 -80-Appendix D Analysis of Variance Tables for Experiment 4 Analysis of Variance Table for Experiment 4 AD Duration Source SS df MS Order 6915.1 3 2305.0 2.07 Subj/groups 13327.6 ,12 1110.6 Condition 2015.2 3 671.7 . 3.84 Condition x Order 2021.9 9 224.6 1.28 Condition x Subj/groups 6281.7 36 174.4 Analysis of Variance Table for Experiment 4 MS Duration Source SS df MS Order Subj/groups Condition Condition x Order Condition x Subj/groups 435, 1656, 140.4 259.5 833.7 3 12 3 9 36 145.1 138.0 46.9 28.8 23.1 1.05 2.02 1.24 PUBLICATIONS (Cont'd) P i n e l , J . P. J . , & Mucha, R. F. Incubation effect i n the cockroach (Periplaneta Americana). Journal of Comparative and  Physiological Psychology, 1973, 132-138. P i n e l , J . P. J . , & Mucha, R. F. Role of footshock-produced a c t i v i t y and r e a c t i v i t y functions i n the production of incubation and kamin gradients. Behavioural Biology, 1974, 353-363. Mucha, R. F., P i n e l , J . P. J . , & Van Oot, P. H. Simple method for producing an alcohol withdrawal syndrome i n rat s . Pharmacology, Biochemistry and Behaviour, 1975, i n press. P i n e l , J . P. J . , Mucha, R. F., & P h i l l i p s , A. G. Spontaneous seizures generated i n rats by kindl i n g : A preliminary report. Physiological Psychology, 1974, 3_, 127-29. P i n e l , J . P. J . , Mucha, R. F., & Van Oot, P. H. The implications of the kindling phenomenon for the study and treatment of alcohol withdrawal seizures. Proceedings of the Symposium  on B i o l o g i c a l Correlates of Alcohol Consumption. Published by Alcohol Research Foundation of Alberta, Edmonton, 1975, i n press. P i n e l , J . P. J . , Van Oot, P. H., & Mucha, R. F. I n t e n s i f i c a t i o n of the alcohol withdrawal syndrome by repeated brain stimulation. Nature, 1975, 254, 510-11. P i n e l , J . P. J . , & Mucha, R. F. Suppression of voluntary ethanol consumption i n rats by electroconvulsive shock. Physiology and Behaviour, 1975, i n press. 

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