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Contingent and pharmacologic tolerance to the anticonvulsant effects of antiepileptic drugs Mana, Michael Joseph 1990

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No Title PageIn presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT The development of tolerance to anticonvulsant drug e f f e c t s has t r a d i t i o n a l l y been studied i n terms of pharmacological variables associated with the drug i t s e l f ; for example, the dose or the schedule of administration. This type of tolerance i s referred to as pharmacologic drug tolerance. In contrast, we have demonstrated that the development of tolerance to ethanol's anticonvulsant e f f e c t i s contingent upon the adminstration of convulsive stimulation during periods of ethanol exposure; we re f e r to t h i s as contingent drug tolerance. The purpose of the f i r s t two experiments i n the present thesis was to extend the phenomenon of contingent tolerance to the anticonvulsant e f f e c t s of three c l i n i c a l l y relevant a n t i e p i l e p t i c drugs: carbamazepine (CBZ), diazepam (DZP), and sodium valproate (VPA). In Experiment 1, kindled rats that received an i n j e c t i o n of CBZ (70 mg/kg, IP), DZP (2 mg/kg, IP), or VPA (250 mg/kg, IP) 1 hr before each of 10 b i d a i l y (one every 48 hr) convulsive stimulations displayed a s i g n i f i c a n t amount of tolerance to the drugs' anticonvulsant e f f e c t s on the tolerance t e s t t r i a l ; i n contrast, there was no evidence of tolerance i n the rats from the three vehicle control groups. In Experiment 2, the development of tolerance to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA, administered on a b i d a i l y basis, was shown to be contingent upon the administration of convulsive stimulation during the periods of drug exposure. Kindled rats i n the three drug-before-stimulation groups rapidly developed tolerance to the i i i anticonvulsant e f f e c t s of CBZ, DZP, and VPA; i n contrast, there was no evidence of tolerance i n the respective drug-after-stimulation groups, despite the fact that they had the same drug histo r y . The purpose of the f i n a l three experiments was to compare contingent and pharmacologic tolerance to the anticonvulsant e f f e c t s of DZP. Experiment 3 replicated e a r l i e r demonstrations of pharmacologic tolerance to DZP's anticonvulsant e f f e c t ; kindled r a t s that received chronic DZP (2 mg/kg, every 8 hr, for 10 days) developed tolerance to the drug's anticonvulsant e f f e c t even though they did not receive convulsive stimulation during the periods of drug exposure. In Experiment 4, the rate of d i s s i p a t i o n of pharmacologic and contingent tolerance to DZP's anticonvulsant e f f e c t was compared. Pharmacologic tolerance gradually dissipated over the 16-day retention i n t e r v a l ; i n contrast, there was no evidence of d i s s i p a t i o n of contingent tolerance a f t e r 16 days of drug withdrawal. These data suggest that d i f f e r e n t physiological changes are responsible for pharmacologic and contingent tolerance to DZP's anticonvulsant e f f e c t . This conclusion was supported by the r e s u l t s of Experiment 5, i n which a single i n j e c t i o n of the benzodiazepine receptor antagonist RO 15-1788 24 hr p r i o r to a tolerance-retention t e s t t r i a l s i g n i f i c a n t l y reduced the expression of pharmacologic tolerance, but not contingent tolerance, to DZP's anticonvulsant e f f e c t . The r e s u l t s of these f i v e experiments make two general i v points. F i r s t , concurrent convulsive stimulation can have an important e f f e c t on the development of tolerance to the anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs. And second, there are s i g n i f i c a n t differences i n the physiological changes responsible for the development and the d i s s i p a t i o n of contingent and pharmacologic tolerance to DZP's anticonvulsant e f f e c t . Because t r a d i t i o n a l theories do not address these differences, a new model of contingent and pharmacologic tolerance i s presented. V TABLE OF CONTENTS Abstract i i Table of Contents v L i s t of Figures v i i i Acknowledgements ix I. General Introduction 1 1. Drug Tolerance 3 D e f i n i t i o n 3 B i o l o g i c a l Mechanisms . . 5 Dispositional Tolerance. 5 Functional Tolerance 7 Pharmacologic Drug Tolerance 9 Shortcomings of the Pharmacologic View of Tolerance 11 2. Context-Specific Tolerance 13 3. Contingent Tolerance 17 Introduction 17 Early Studies 18 An Analogy 21 Generality of Contingent Tolerance 22 i . Psychostimulants 23 i i . Morphine 24 i i i . Barbiturates 25 i v . Delta-9-THC. . . 26 v. Benzodiazepines 26 v i . Ethanol 27 4. Contingent Tolerance to Ethanol's Anticonvulsant Ef f e c t 29 5. Pharmacologic Tolerance to the Anticonvulsant Ef f e c t of A n t i e p i l e p t i c Drugs 39 Tr a d i t i o n a l Seizure Paradigms 41 Tolerance: Carbamazepine 42 Tolerance: Diazepam 47 Tolerance: Sodium Valproate. 51 Summary: Tolerance to Anticonvulsant Drug Effects 57 6. General Rationale 57 v i I I . General Methods 60 The Kindling Paradigm 60 Subjects 62 Surgical Procedure 62 Kindling 63 Stimulation-Baseline 63 Drug-Baseline 63 Tolerance-Development T r i a l s 64 Tolerance-Test T r i a l 64 Histology 64 S t a t i s t i c a l Analyses 65 Figure Caption: Histology 66 I I I . Experiment 1 68 Methods 7 0 Results 7 2 Discussion 79 IV. Experiment 2 84 Methods 84 Results 87 Discussion 95 V. General Background for Experiments 3, 4, and 5 99 VI. Experiment 3 102 Methods 103 Results 104 Discussion 104 VII. Experiment 4 I l l Methods 112 Results 116 Discussion 122 VIII. Experiment 5 124 Methods 125 Results 129 Discussion 133 IX. General Discussion 136 1. The Role of Convulsive Stimulation In the Development of Contingent Tolerance to the Anticonvulsant Effects of CBZ, DZP, and VPA 13 6 v i i 2. Theories of Contingent Tolerance: The Importance of A c t i v i t y to Development of Drug Tolerance 14 0 i . The Reinforcement-Density Hypothesis 141 i i . The State-Dependency Hypothesis 145 i i i . The Homeostatic-Conditioning Hypothsis.... 147 i v . Summary 149 v. An Activity-Dependent Analysis of Contingent and Pharmacologic Tolerance to Anticonvulsant Drug Effects 150 a) The Neuromuscular Junction 152 b) Ocular Dominance Columns 154 c) Long-Term Potentiation 158 d) The Role of Neural A c t i v i t y i n the Development of Functional Drug Tolerance 160 e) Caveats and Fi n a l Comments 169 3. Contingent and Pharmacologic Drug Tolerance: Common or Independent Physiological Bases? 170 X. Implications 174 XI. References 178 v i i i LIST OF FIGURES Figure 1.... Contingent Tolerance to Ethanol's Anticonvulsant E f f e c t : (Pinel et a l . , 1983) 31 Figure 2.... Dissipation of Contingent Tolerance to Ethanol's Anticonvulsant Effect: (Mana & Pinel, 1987) 35 Figure 3....Representative H i s t o l o g i c a l V e r i f i c a t i o n of Electrode Placements 67 Figure 4.... Development of Tolerance to CBZ 1s Anticonvulsant E f f e c t 74 Figure 5.... Development of Tolerance to DZP's Anticonvulsant Ef f e c t 7 6 Figure 6....Development of Tolerance to VPA's Anticonvulsant Effect 78 Figure 7....Development of Contingent Tolerance to CBZ's Anticonvulsant Ef f e c t 89 Figure 8.... Development of Contingent Tolerance to DZP's Anticonvulsant Ef f e c t .. 91 Figure 9.... Development of Contingent Tolerance to VPA's Anticonvulsant Effect 9 3 Figure 10... Development of Pharmacologic Tolerance to DZP's Anticonvulsant Effect 106 Figure ll...Bimodal Dis t r i b u t i o n of Forelimb Clonus i n Rats from the Pharmacologic Tolerance group..... 110 Figure 12... Dissipation of Pharmacologic Tolerance to DZP's Anticonvulsant Effect 119 Figure 13... Dissipation of Contingent Tolerance to DZP's Anticonvulsant Ef f e c t 121 Figure 14...Effects of RO 15-1788 on the Retention of Contingent and Pharmacologic Tolerance to DZP's Anticonvulsant Effect 131 Figure 15...A Model of Activity-Dependent Drug Tolerance 165 i x ACKNOWLEDGEMENT S I thank D r . John P . J . P i n e l f o r h i s s u p p o r t , h i s c o n f i d e n c e , and h i s f r i e n d s h i p over the l a s t 6 y e a r s , and f o r h i s c o n t r i b u t i o n s i n word and thought t o much o f what I have done d u r i n g t h a t t i m e . I thank D r . Donald M. W i l k i e f o r h i s doorway and our c o n v e r s a t i o n s , and f o r s e t t i n g the p a c e . I a l s o thank D r . R i c h a r d C . T e e s , D r . C a t h e r i n e Rankin and D r . W i l l i a m J . J a c o b s f o r t h e i r s u p p o r t and i n s i g h t s ; C . Kwon Kim f o r h i s i n s p i r i n g a s s i s t a n c e i n a l l phases o f the f i r s t 2 exper iments and f o r h o u r s o f d i s c u s s i o n about t h i n g s c o n t i n g e n t ; B r i a n Moorehead and K e i t h Waldron f o r keep ing t h i n g s f u n c t i o n a l ; L u c i l l e Hoover f o r h e r a s s i s t a n c e d u r i n g the course o f the e x p e r i m e n t s ; and C . H . Jones f o r h i s c o n t r i b u t i o n t o Experiment 2. I thank D r . R o b e r t Douglas f o r thoughts about the p o s s i b l e bases o f a c t i v i t y -dependent change and f o r h i s c o n t r i b u t i o n s t o t h i s t h e s i s ; D r . D a r r e n Lehman, D r . C a t h e r i n e R a n k i n , D r . Ken B a i m b r i d g e , D r . R a l p h H a k s t i a n , and D r . Andrew J . Goudie f o r t h e i r c o n t r i b u t i o n s t o t h i s t h e s i s ; and the M e d i c a l Research C o u n c i l o f Canada f o r i t s s u p p o r t . And I thank F i n . 1 I. INTRODUCTION In the l a s t s i x years, Pinel and his associates have published a series of papers demonstrating that the development of tolerance to ethanol's anticonvulsant e f f e c t on kindled r a t s i s greatly influenced by the administration of convulsive stimulation during the periods of ethanol exposure (e.g., P i n e l , Colbourne, Sigalet & Renfrey, 1983; Pinel, Mana, & Renfrey, 1985; Pi n e l , Kim, Paul, & Mana, 1989). In each of these papers, tolerance to the anticonvulsant e f f e c t of ethanol developed only when kindled rats received convulsive stimulation during each period of ethanol exposure; kindled rats that were unstimulated, or stimulated p r i o r to each period of ethanol exposure, demonstrated l i t t l e tolerance on the test t r i a l even though these rat s received exactly the same ethanol exposure. We have referred to the tolerance that develops to ethanol's anticonvulsant e f f e c t only when convulsive stimulation i s administered during periods of ethanol exposure as contingent  tolerance (Pinel et a l . , 1985; Pinel & Mana, 1986; see also Carlton & Wolgin, 1971) because the development of tolerance i s contingent upon convulsive stimulation during periods of ethanol exposure rather than upon ethanol exposure per se. Although the development of tolerance to ethanol 1s anticonvulsant e f f e c t s i s c l e a r l y influenced by the administration of convulsive stimulation during the periods of ethanol exposure, to date there has been no systematic attempt to determine whether convulsive stimulation has a s i m i l a r e f f e c t on 2 the development of tolerance to the anticonvulsant e f f e c t s of c l i n i c a l l y relevant a n t i e p i l e p t i c drugs. This i s p a r t i c u l a r l y noteworthy i n l i g h t of the fact that there i s experimental evidence of tolerance to the anticonvulsant e f f e c t s of almost every a n t i e p i l e p t i c drug currently i n c l i n i c a l use (see Frey, 1987) . We have referred to t h i s type of tolerance, which develops i n the absence of concurrent convulsive stimulation, as pharmacologic drug tolerance (Mana, Kim, Pi n e l , & Jones, submitted; see also J 0 r g e n s e n , Fasmer, & Hole, 1986). The present experiments were conducted for two primary purposes: (1) to assess the role of convulsive stimulation i n the development of tolerance to the anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs, and (2) to compare contingent and pharmacologic tolerance to anticonvulsant drug e f f e c t s . The f i r s t two experiments were designed to assess the degree to which the development of tolerance to the anticonvulsant e f f e c t s of three c l i n i c a l l y relevant a n t i e p i l e p t i c drugs—carbamazepine, diazepam, and sodium v a l p r o a t e — i s contingent upon the concurrent administration of convulsive stimulation with drug exposure. The three remaining experiments compared contingent and pharmacologic tolerance to the anticonvulsant effects of diazepam. Accordingly, the f i r s t three sections of the Introduction focus on the following 3 topics: (1) the phenomenon of drug tolerance, with an emphasis upon the t r a d i t i o n a l concept of pharmacologic drug tolerance; (2) the contingent tolerance phenomenon, with an emphasis upon our research on contingent tolerance to ethanol's anticonvulsant e f f e c t s ; and (3) tolerance to the anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs, with an emphasis upon the three drugs—carbamazepine, diazepam, and sodium v a l p r o a t e — t h a t were the focus for the present experiments. The fourth and f i n a l section of the Introduction presents the general purpose for the f i v e experiments that compose t h i s thesis. 1. Drug Tolerance Because i t i s an interesting example of b i o l o g i c a l adaptation (see Cappell & LeBlanc, 1979) and because of i t s hypothetical r e l a t i o n to the phenomena of drug dependence, withdrawal, and abuse (Haefly, 1986; Kalant, 1985; Siegel & MacRae, 1984), tolerance i s one of the most widely studied drug-re l a t e d phenomena (see Goudie & Emmett-Oglesby, 1989a). Yet our understanding of tolerance remains at an elementary l e v e l . D e f i n i t i o n Drug tolerance i s usually defined as a decrease i n the e f f e c t of a given dose of a drug that occurs as the r e s u l t of previous exposure to the drug. In many instances, the development of tolerance to a drug e f f e c t r e s u l t s i n a s h i f t i n the dose-response curve for that e f f e c t to the r i g h t (so that the maximum drug e f f e c t can s t i l l be achieved i f the drug dose i s increased), but i n other cases the development of tolerance f l a t t e n s the dose-response curve (so that there i s a decrease i n the maximal drug e f f e c t , regardless of dose; e.g., Haigh, Gent, 4 Garrat, P u l l a r , & Feely, 1988; Le, Khanna, Kalant, & Grossi, 1986). Kalant (1989) has proposed that the type of change seen i n the dose-response curve following the development of tolerance to a drug's e f f e c t s provides an insight into the drug's s i t e of action. According to Kalant (1989), a p a r a l l e l rightward s h i f t i n the dose-response curve following the development of tolerance i s t y p i c a l of drugs with a nonspecific s i t e of action i n the nervous system (e.g., ethanol, general anaesthetics), whereas a rightward s h i f t and f l a t t e n i n g of the dose-response curve i s t y p i c a l of drugs that produce desensitization or down-regulation of a s t e r e o s p e c i f i c , receptor-mediated mechanism of action (e.g., opiates, benzodiazepines). Although tolerance develops to the ef f e c t s of many drugs, i t does not develop to the eff e c t s of a l l drugs. And i t does not necessarily develop to a l l of the effects of a p a r t i c u l a r drug; exposure to a p a r t i c u l a r drug may lead to the development of tolerance to some of i t s effects (often with a d i f f e r e n t time course for each e f f e c t ; e.g., Loscher & Honack, 1989; Rosenberg, Chiu, & T i e t z , 1986), while others may be unchanged or even increased i n magnitude (e.g., Woolverton, Kandel, & Schuster, 1978; see Le & Khanna, 1989). This picture i s further complicated by the fact that the development of tolerance to one of a drug's e f f e c t s may be obscured by the development of tolerance or s e n s i t i z a t i o n to another e f f e c t of the same drug. For example, Mucha, Kalant, and Kim (1987) found that tolerance develops to morphine's hypothermic e f f e c t (which i s apparent soon a f t e r administration of the drug) much more rapidl y than to i t s hyperthermic e f f e c t (which i s not apparent u n t i l some time a f t e r the drug i s administered). As a res u l t , subjects receiving repeated morphine injections appear to develop tolerance to the drug's hypothermic effects and s e n s i t i z a t i o n to i t s hyperthermic e f f e c t s (because the fast-developing tolerance that develops to morphine's hypothermic ef f e c t allows the hyperthermic e f f e c t to express i t s e l f e a r l i e r and to a greater degree), when i n fac t tolerance i s developing to both of these thermic e f f e c t s of morphine, but at a d i f f e r e n t rate. B i o l o g i c a l Mechanisms of Tolerance Development There are two general types of b i o l o g i c a l change to which tolerance to a p a r t i c u l a r drug e f f e c t can be attributed: d i s p o s i t i o n a l change or functional change. Although a given instance of tolerance i s often attributed to either a d i s p o s i t i o n a l or a functional change, both types of change can contribute to a given instance of drug tolerance (see J a f f e , 1980; Kalant et a l . , 1971; Wood & Laverty, 1979). The following two sections b r i e f l y review the various mechanisms assumed to be responsible for d i s p o s i t i o n a l and functional drug tolerance. D i s p o s i t i o n a l Mechanisms of Tolerance. Di s p o s i t i o n a l change re f e r s to any instance i n which previous exposure to a drug diminishes i t s e f f i c a c y by reducing i t s concentration at i t s s i t e of action (Kalant et a l . , 1971) or by decreasing the duration of time that the drug remains at i t s s i t e of action (Le & Khanna, 6 1989) . Di s p o s i t i o n a l tolerance has been attributed to one of four d i f f e r e n t mechanisms; to changes i n the 1) absorption, 2) d i s t r i b u t i o n , 3) breakdown, or 4) clearance of the drug after repeated administration. 1. Absorption. Before a drug can a f f e c t the central nervous system, i t must be absorbed from i t s s i t e of administration into the general c i r c u l a t i o n . As Le and Khanna (1989) point out, the importance of drug absorption to the development of tolerance i s dependent upon the route of drug administration; drug absorption i s r e l a t i v e l y unimportant when a drug i s administered by microinjection d i r e c t l y into the brain (because the drug avoids the general c i r c u l a t i o n ) or intravenously (because the drug i s administered d i r e c t l y into the general c i r c u l a t i o n ) , more important when a drug i s administered i n t r a p e r i t o n e a l l y (because the drug must be absorbed by the vascularization i n the peritoneal cavity to enter the general c i r c u l a t i o n ) , and very important when a drug i s administered o r a l l y (because the absorption of the drug into the general c i r c u l a t i o n i s influenced by g a s t r i c emptying and the vascularization of the gut and small i n t e s t i n e ) . 2. D i s t r i b u t i o n . Once a drug has entered the general c i r c u l a t i o n , i t may be distributed to a variety of f l u i d and tis s u e "compartments" before gaining access to i t s s i t e of action. For example, ethanol i s widely d i s t r i b u t e d throughout the en t i r e body water; the d i s t r i b u t i o n of other drugs may be r e s t r i c t e d by the degree to which they bind to plasma proteins 7 (e.g., albumin) or are absorbed by various body tissues (e.g., adipose t i s s u e or bone). Changes i n the d i s t r i b u t i o n of a drug may greatly a f f e c t the concentration at i t s s i t e of action, and thus i t s e f f e c t on the central nervous system. 3. Breakdown. The most well-known mechanism of d i s p o s i t i o n a l tolerance i s the increase i n drug breakdown that can occur when a drug i s repeatedly administered. This increase i n drug metabolism i s often the re s u l t of an induction of hepatic enzymes that are capable of metabolizing a wide var i e t y of drugs. As a r e s u l t , i n many instances the induction of hepatic enzymes by repeated exposure to one drug w i l l r e s u l t i n the development of tolerance to that drug's effects and cross-tolerance to the ef f e c t s of other drugs that are also susceptible to breakdown by these enzymes (see Kalant, 1989). 4. Clearance. The rate of a drug's elimination from the body w i l l obviously influence the duration and magnitude of a drug's e f f e c t s . Drugs and/or t h e i r metabolic byproducts are usually excreted i n urine by the kidney, though s i g n i f i c a n t drug elimination can also occur i n the feces v i a the l i v e r and through the lungs and sweat glands. Functional Mechanisms of Tolerance. Functional tolerance r e f e r s to a reduction i n the e f f i c a c y of a drug that i s a t t r i b u t a b l e to a decrease i n the s e n s i t i v i t y of the physiological systems affected by the drug rather than to a decrease i n the concentration of the drug i t s e l f (Jaffe, 1980; Kalant et a l . , 1971; Le & Khanna, 1989). That i s , functional tolerance i s an 8 adaptation to the e f f e c t s of a drug on the function of a p h y s i o l o g i c a l system rather than to a decrease i n i t s presence per se (see also Kalant et a l . , 1971; Jaffe, 1980; Kalant, 1989). I t i s important to keep i n mind that the changes underlying the development of functional drug tolerance do not necessarily have to occur at the drug's primary s i t e of action (e.g., tolerance may be mediated by "subsensitive" receptors on postsynaptic neurons that are exposed to an increase i n neurotransmitter release that i s produced by a drug's e f f e c t on presynaptic neurons); t h i s greatly complicates the study of the p h y s i o l o g i c a l bases of functional drug tolerance "as much of our e f f o r t , at the more basic l e v e l s , may be describing phenomena that are not obviously r e l a t e d to the phenomena of tolerance..." (Martin, 1984) . A v a r i e t y of physiological alterations have been proposed to underlie the development of functional drug tolerance. These include changes i n the s e n s i t i v i t y or number of neurotransmitter or drug receptors (e.g., Gallager & Gonsalves, 1988; Rosenberg et a l , 1986); changes i n the synthesis, release, or reuptake of various neurotransmitters (e.g., Loscher, 1986a; Melchior & Tabakoff, 1981), neuromodulators (e.g., V o l l i c e r & Ullman, 1985), or hormones (e.g., Wood, 1977; Tabakoff & Yanai, 1979); changes i n c e l l membrane composition (e.g., Goldstein, 1983); changes i n the a c t i v i t y of secondary messengers necessary for many neurotransmitters to have a postsynaptic e f f e c t (e.g., Siggins, 1979) ; or changes i n ion conductances across the neuronal 9 membrane (e.g., L i t t l e t o n & L i t t l e , 1989; Ross, Garrett, & Cardenas, 1979). In many instances, the development of functional drug tolerance to a single drug e f f e c t i s l i k e l y dependent upon a combination of physiological changes. Furthermore, functional tolerance to d i f f e r e n t e f f e c t s of a single drug have been attributed to a variety of d i s t i n c t and independent physiological changes (e.g., Rosenberg, Chiu, & Te i t z , 1986; Teitz & Rosenberg, 1988). Given the number and combination of physiological changes that may underlie a given instance of functional drug tolerance, i t i s not surp r i s i n g that few d e f i n i t i v e statements can be made about i t s p h y s i o l o g i c a l bases (see Kalant, 1989). Pharmacologic Drug Tolerance Conventional research on the phenomenon of drug tolerance has been greatly influenced by the assumption that the administration of a drug i s a s u f f i c i e n t impetus for the d i s p o s i t i o n a l and functional changes that underlie the development of tolerance to the drug's e f f e c t s . More s p e c i f i c a l l y , the assumption has been that drug exposure automatically produces the changes i n absorption, d i s t r i b u t i o n , metabolism, or clearance necessary for d i s p o s i t i o n a l tolerance; and that drug exposure inevitably produces the drug e f f e c t s that are necessary for the physiological adaptations underlying the development of functional tolerance. The implication of t h i s focus i s that drug tolerance i s a function of a vari e t y of pharmacologic factors and that factors such as the context i n which the drug i s administered or the ongoing p h y s i o l o g i c a l and behavioral a c t i v i t y of the drug recipient during periods of drug exposure are inconsequential. This pharmacologic view of drug tolerance can be summarized as follows: DISPOSITIONAL CHANGES DRUG EXPOSURE > AND/OR > DRUG TOLERANCE FUNCTIONAL CHANGES Because t h i s view of drug tolerance has guided much of the work i n the area, a considerable amount i s known about the e f f e c t of pharmacologic factors on the development or d i s s i p a t i o n of tolerance. For example, i t has been shown that the following factors f a c i l i t a t e the development of tolerance: 1) increasing the s i z e of the treatment dose (e.g., Le, Khanna, & Kalant, 1984) ; 2) increasing the t o t a l number of drug administrations (e.g., Le, Kalant, & Khanna, 1986); 3) using a shorter rather than longer interdose i n t e r v a l (e.g., Giknis & Damjanov, 1984); 4) using a drug with a long h a l f - l i f e (e.g., Okamato, 1984); 5) using subjects that have previously developed (and then lost) tolerance to the e f f e c t s of that drug (this i s referred to as tolerance carry-over; Kalant et a l . , 1971); 6) using subjects that have developed tolerance to the e f f e c t s of a drug with a s i m i l a r d i s p o s i t i o n a l p r o f i l e or mechanism of action (this i s referr e d to as cross-tolerance; e.g., Kalant et a l . , 1971); or 7) using subjects that are p a r t i c u l a r l y sensitive to the drug's e f f e c t (e.g., Khanna, Le, LeBlanc, & Shah, 1985). According to the pharmacologic view of drug tolerance, tolerance dissipates as a function of time since the cessation of drug treatment (e.g., Teitz & Rosenberg, 1988). I t has also been demonstrated that the d i s s i p a t i o n of tolerance to a drug's e f f e c t s can be f a c i l i t a t e d by the administration of pharmacologic agents that antagonize the effects of the drug (e.g., Gallager & Gonsalves, 1988). Shortcomings of the Pharmacologic View of Drucr Tolerance. The development and d i s s i p a t i o n of drug tolerance are obviously influenced by pharmacologic factors. However, i n the l a s t two decades i t has become increasingly apparent that the development and d i s s i p a t i o n of drug tolerance i s also influenced by a v a r i e t y of behavioral and physiological processes not d i r e c t l y r e l a t e d to a drug's a d m i n i s t r a t i o n — f a c t o r s such as the context of the drug experience, the behavioral tasks facing the subject during the periods of drug exposure, and the a c t i v i t y of the organism's nervous system during the periods of drug exposure (see also Balster, 1984; Goudie & Emmett-Oglesby, 1989). In general, these factors do not play a minor r o l e i n the phenomenon of drug tolerance. In many instances, the development or expression of the d i s p o s i t i o n a l and/or functional changes that underlie a p a r t i c u l a r instance of tolerance are completely dependent upon the physiological or psychological a c t i v i t y of the drug r e c i p i e n t during the periods of drug exposure or the context i n which the drug i s administered (see Le & Khanna, 1989; Demellweek & Goudie, 1983b; Siegel, 1989). The e f f e c t of these behavioral and physiological variables upon the development and d i s s i p a t i o n of drug tolerance has been demonstrated by two types of research. The f i r s t type of research has focused on the phenomenon of context-dependent or conditioned drug tolerance, i n which the expression of tolerance to a drug's e f f e c t i s greatly influenced by the presence or absence of environmental cues that have become associated with the drug's e f f e c t s (Baker & Tiffany, 1985; Eikelboom & Stewart, 1982; Paletta & Wagner, 1986; Siegel, 1975; 1977, 1989; Siegel & MacRae, 1984; Solomon, 1977; Wikler, 1948; 1973). The second type of research that has demonstrated the important e f f e c t that behavioral and physiological variables can have on the development of drug tolerance has focused upon the behavioral or p h y s i o l o g i c a l responses of the drug recipient during periods of drug exposure. I t has been repeatedly demonstrated that the development of tolerance to many drug ef f e c t s i s contingent upon the drug r e c i p i e n t engaging i n a p a r t i c u l a r a c t i v i t y during periods of drug exposure—such tolerance has been termed contingent tolerance (Carlton & Wolgin, 1971; see also Wolgin, 1989; Demellweek & Goudie, 1983b; Goudie & G r i f f i t h , 1985). The next section of the Introduction provides a b r i e f review of the phenomena of context-specific tolerance; t h i s w i l l be followed by a more extensive review of the contingent tolerance l i t e r a t u r e . 2. Context-Specific Drug Tolerance The manifestation of many types of drug tolerance has been shown to be dependent upon the context i n which the subjects have previously experienced the drug's e f f e c t s (see Baker & Tiffany, 1985; Goudie & Demellweek, 1986; Paletta & Wagner, 1986; Siegel, 1978; 1989; Wikler, 1973). In instances of context-dependent tolerance, subjects display considerable tolerance to the e f f e c t s of a drug i f they are tested i n the same context i n which i t s e f f e c t s had been previously experienced; i n contrast, the same subjects display l i t t l e or no tolerance to the drug's e f f e c t s i f the t e s t dose of the drug i s experienced i n a context with no drug-related history. Such context-specific tolerance has been demonstrated to the effects of: 1) amphetamine (e.g., Poulos & Hinson, 1984); 2) benzodiazepines (e.g., Greeley & Cappell, 1985); 3) caffeine (e.g., Rozin, Reff, Mark, & Sch e l l , 1984); 4) ethanol (e.g., Mansfield & Cunningham, 1980); 5) haloperidol (Poulos and Hinson, 1982); 6) morphine (e.g., Siegel, 1975, 1977); 7) pentobarbital (e.g., Hinson, Poulos, & Cappell, 1982; Siegel, 1988); and 8) scopolamine (e.g., Poulos, Wilkinson, & Cappell, 1981). According to Siegel (1975; 1989; Siegel & Macrae, 1984), the context s p e c i f i c i t y of t h i s tolerance i s the consequence of Pavlovian conditioning. It i s Siegel's view that the context i n which a subject repeatedly experiences the drug's e f f e c t s acts as a conditional stimulus (CS) that becomes associated with the unconditional e f f e c t s of the drug (the unconditional s t i m u l i or 14 UCS's). Siegel argues that as t h i s association i s strengthened, the context begins to e l i c i t a conditional compensatory response (CCR) that opposes the unconditional e f f e c t s of the drug and increases i n magnitude as the association between the context and the drug's e f f e c t s strengthens. Because the CCR i s expressed only when the drug i s administered i n the presence of drug-p r e d i c t i v e cues, the manifestation of tolerance i s context-s p e c i f i c . Siegel's Pavlovian explanation of context-specific tolerance i s supported by several l i n e s of evidence. F i r s t , the development of context-specific tolerance i s sen s i t i v e to a var i e t y of Pavlovian procedures. For example, the development of context-specific tolerance shows a CS preexposure e f f e c t ; that i s , i f subjects are repeatedly presented with the context that i s to become the CS p r i o r to the regimen of drug exposure, the development of tolerance when the context and the drug's e f f e c t s are subsequently paired i s much slower than i t would be i f the subjects had no p r i o r experience with the context (Siegel, 1977). S i m i l a r l y , instances of context-specific tolerance have been found to be sensi t i v e to extinction procedures (Siegel, 1975; Greeley, Le, Poulos, & Cappell, 1984), p a r t i a l reinforcement e f f e c t s (Siegel, 1978; Krank, Hinson, & Siegel, 1984), conditioned i n h i b i t i o n (Siegel, Hinson, & Krank, 1981), and overshadowing (Walter & Riccio, 1984). The second, and most di r e c t , l i n e of evidence supporting Siegel's Pavlovian theory of context-specific tolerance has been 15 provided by demonstrations of the conditional compensatory response, the hypothetical construct on which Siegel's theory i s based. The administration of a placebo to tolerant subjects i n the drug-predictive environment has frequently been reported to e l i c i t a response opposite to the i n i t i a l e f f e c t of the drug. For example, placebo injections i n the drug-predictive environment have been shown to e l i c i t hyperalgesia i n rats that have developed context-specific tolerance to the analgesic e f f e c t of morphine (Krank, Hinson, & Siegel, 1981), hypothermia i n rats tolerant to morphine's hyperthermic e f f e c t (Siegel, 1978), hyperthermia i n rats tolerant to chlordiazepoxide's hypothermic e f f e c t s (Greeley & Cappell, 1985), or hyperactivity i n rats tolerant to ethanol's hypoactive e f f e c t (Mansfield & Cunningham, 1980). Unfortunately, many attempts to demonstrate a conditional compensatory response have been unsuccessful, and Siegel's Pavlovian model of context-specific tolerance has been c r i t i c i z e d on t h i s basis by a number of researchers (e.g., Baker & Tiffany, 1985; Goudie & Demellweek, 1986; Goudie & G r i f f i t h s , 1985; Shapiro, Dudek, & R o s e l l i n i , 1983; Tiffany, Baker, Petrie, & Dahl, 1983). In what i s arguably the most well-developed a l t e r n a t i v e to Siegel's theory of context-specific tolerance, Baker and h i s colleagues (Baker & Tiffany, 1985; Kesner & Baker, 1981; Kesner & Cook, 1983) have argued that such tolerance can be a t t r i b u t e d to a conditioned habituation to the drug's e f f e c t s (see also Siegel, 1977; Solomon, 1977; Wagner, 1978; 1981, for 16 e a r l i e r versions of t h i s idea). According to t h i s theory of context-specific tolerance, repeated administration of a drug i n a p a r t i c u l a r environment leads to the development of an association between the contextual cues and the drug's e f f e c t s . As a r e s u l t of t h i s association, subsequent presentation of the contextual cues leads to the r e t r i e v a l from long-term memory of a representation of the drug's e f f e c t s . This " a s s o c i a t i v e l y generated priming r e s u l t [ s ] i n decreased neural processing of the drug stimulus. Such decreased processing of drug stimulus information r e s u l t s i n [sic] attenuated behavioral e f f e c t and constitutes tolerance." (Baker & Tiffany, 1985, p. 83). Paletta and Wagner (1986) have proposed a compromise between the positions taken by Baker and Tiffany(1985) and Siegel (e.g., Siegel & MacRae, 1984), i n which an environmental stimulus that has become a CS for a drug-effect US w i l l always produce a conditioned habituation to the US, but may also e l i c i t a conditional compensatory response as well. Paletta and Wagner (1986) argue that the c r i t i c a l factor i n the development of a conditional compensatory response i s the existence of a "compensatory" secondary UCR e l i c i t e d by the drug (e.g., a biphasic response; for example, hypothermia followed by hyperthermia). Context-dependent tolerance for a l l drugs involves a conditional habituation (as suggested-by Baker & Tiffa n y , 1985) ; when the CS overlaps the secondary, "compensatory" portion of the UCR a conditional compensatory response (as suggested by Siegel & MacRae, 1984) w i l l also be 17 e l i c i t e d . Regardless of the s p e c i f i c processes underlying context-s p e c i f i c tolerance, the wide recognition that the phenomenon received represented a major advance i n the study of drug tolerance. In studies of context-specific tolerance, various groups of subjects with i d e n t i c a l drug h i s t o r i e s display markedly d i f f e r e n t l e v e l s of tolerance depending on the contextual cues present during periods of drug exposure. This finding has, more than any other, been responsible for focusing the attention of researchers on the importance of behavioral processes i n the phenomenon of drug tolerance. 3. Contingent Drug Tolerance Introduction. Contingent drug tolerance i s a form of functional tolerance that develops p r e f e r e n t i a l l y to a drug's ef f e c t s on those a c t i v i t i e s that occur during periods of drug exposure. I t i s usually demonstrated i n terms of the difference i n tolerance development observed between the two groups of subjects i n what has been termed the before-and-after design (Kumar & Stolerman, 1977) . In t h i s design, the subjects i n one group (the drug-before group) receive the drug before engaging i n a p a r t i c u l a r a c t i v i t y (the c r i t e r i o n a c t i v i t y ) on each tolerance-development t r i a l so that the a c t i v i t y i s performed while the subject i s under the influence of the drug. The subjects i n the second group (the drug-after group) receive the drug aft e r engaging i n 18 the c r i t e r i o n a c t i v i t y . On the test t r i a l , a l l subjects receive the drug before the performance of the c r i t e r i o n a c t i v i t y so that the drug's e f f e c t s on i t can be assessed. Any evidence of greater tolerance i n the drug-before subjects i s attr i b u t e d to the r e l a t i o n between the c r i t e r i o n a c t i v i t y and the period of drug exposure because the subjects i n the two groups do not d i f f e r i n either t h e i r exposure to the drug or i n t h e i r opportunity to engage i n the c r i t e r i o n a c t i v i t y (though see Wolgin, 1989). The term behavioral tolerance has also been used to re f e r to instances of contingent tolerance (e.g., Chen, 1972; Dews, 1978; Hayes & Mayer, 197 8). However, the term behavioral tolerance i s also commonly used to describe any tolerance that develops to the ef f e c t s of a drug on behavior (e.g., Kumar & Stolerman, 1977), and when used i n t h i s fashion, i t has no implications whatsoever for the conditions underlying the development of tolerance. Therefore, the term contingent tolerance i s used throughout t h i s t h e s i s to avoid ambiguity. Early Studies of Contingent Tolerance. Newman and Card (1937) were perhaps the f i r s t to propose that the behavior of a subject during periods of drug exposure might influence the development of tolerance to the drug's e f f e c t s ; however, i t was not u n t i l the seminal reports of Schuster, Dockens, and Woods (1966), Chen (1968), and Carlton and Wolgin (1971) that the idea began to a t t r a c t s i g n i f i c a n t attention. Schuster et a l . (19 66) reported that the development of tolerance to amphetamine's effects on operant responding i n rats was dependent on the schedule of reinforcement that was used during the periods of drug exposure; rats developed tolerance to amphetamine's e f f e c t s on t h e i r bar-press behavior only when the drug's e f f e c t s decreased the rate of po s i t i v e reinforcement ( i . e . , decreased delivery of food) or increased the rate of negative reinforcement ( i . e . , avoidance of shock). Schuster and his colleagues (1966) concluded that "the common ph y s i o l o g i c a l mechanisms responsible for drug tolerance cannot be appealed to as an explanation (for the s p e c i f i c i t y of t h i s d i f f e r e n t i a l tolerance; p. 177)" and that "tolerance w i l l develop i n those aspects of the organism's behavioral repertoire where the action of the drug i s such that i t disrupts the organism's behavior i n meeting the environmental requirements for reinforcements." (p. 181). Unfortunately, Schuster et a l . (1966) f a i l e d to include a control group that received the same drug experience without the opportunity to respond during the periods of drug exposure; thus, i t was impossible for them to unequivocally conclude that the d i f f e r e n t i a l tolerance demonstrated by t h e i r subjects was a consequence of responding on a given schedule during periods of amphetamine exposure or rather than simple drug exposure. Chen (1968) avoided t h i s problem by introducing the before-and-after design to study the eff e c t that performance of a maze task while under the influence of ethanol had on the development of tolerance to ethanol's disruptive effects on the performance 20 of the task. He trained rats to perform a maze task and then assigned them to one of two groups. The rats i n one group received ethanol before running the maze on each tolerance-development t r i a l , whereas the rats i n the other group ran the maze before receiving ethanol. Chen found that only those subjects given the opportunity to practice the maze while under the influence of ethanol on the tolerance-development t r i a l s subsequently demonstrated tolerance to i t s disruptive e f f e c t s , i n spit e of the fact that the rats in the ethanol-after group had received the same number of ethanol injections and had the same amount of experience with the maze. Carlton and Wolgin (1971) used the before-and-after design to study the e f f e c t that eating during periods of drug exposure have on the development of tolerance to d-amphetamine1s anorexigenic e f f e c t . They found that rats given an i n j e c t i o n of amphetamine shortly before they were given access to a sweet milk solu t i o n developed tolerance to the drug's anorexigenic e f f e c t within an average of four treatment sessions. In contrast, rats that received the same dose of amphetamine afte r they had consumed the milk solution showed no sign of tolerance a f t e r eight treatment t r i a l s . Furthermore, the amphetamine-after group did not display an accelerated rate of tolerance development when they were subsequently switched to an amphetamine-before-milk regimen. Carlton and Wolgin (1971) coined the term contingent  tolerance to describe t h e i r observation that the development of tolerance to the anorexigenic e f f e c t of d-amphetamine i n rats i s contingent upon providing the subjects with an opportunity to eat during each period of drug exposure. An Analogy for Contingent Drug Tolerance. Demonstrations of contingent tolerance a l l support the idea that the performance of some c r i t e r i o n response during periods of drug exposure can influence the development of tolerance to the drug's e f f e c t on that response. The importance of drugged responding i n the development of contingent tolerance can best be understood by r e c a l l i n g that functional tolerance develops not to the systemic presence of a drug but to i t s effects (see Demellweek & Goudie, 1983b; J a f f e , 1980; Kalant, 1985; Kalant et a l . , 1971; Okamoto, Boisse, Rosenberg, & Rosen, 1978). In many instances, the expression of a drug's effects i s a normal consequence of drug exposure; accordingly, the development of tolerance i s assumed to be a function of drug exposure. In other instances, however, the expression of a drug's e f f e c t i s contingent upon, or i s f a c i l i t a t e d by, the a c t i v i t y of the drug r e c i p i e n t during the periods of drug exposure. In such cases, i t i s possible to show that the drug e f f e c t , rather than the exposure to the drug per se, i s the c r i t i c a l factor i n the development of tolerance. Poulos and his colleagues (Poulos & Hinson, 1984; Poulos et a l . , 1981) i l l u s t r a t e d the role of the c r i t e r i o n response i n the development of contingent tolerance with an i n t e r e s t i n g analogy to a well-known perceptual phenomenon. According to these authors, to expect tolerance to develop i n the absence of the c r i t e r i o n response i s " l i k e expecting adaptation to the e f f e c t s 22 of l a t e r a l l y displacing prisms to develop i n an organism maintained i n the dark. Without an adequate i n s t i g a t i n g stimulus to provide the basis for perceptual adaptation, none can occur" (Poulos et a l . , 1981, p. 745). Although t h e i r a l l u s i o n to the "displaced v i s i o n " phenomenon i s i n s i g h t f u l , i t requires a s l i g h t but s i g n i f i c a n t modification. I t i s not l i g h t per se, but the subject's v i s u a l perception of "self-produced movement... with i t s contingent reafferentation stimulation [that] i s the c r i t i c a l f actor i n compensating for displaced v i s u a l images" (Held, 1972, p. 375; see also Rock & Harris, 1972). That i s , adaptation to the d i s r u p t i v e e f f e c t s of v i s u a l displacement on visuomotor responding does not occur unless such responding occurs under the influence of the displaced v i s i o n . In the same way, tolerance to a drug's e f f e c t s does not develop unless the e f f e c t s are expressed. In instances of contingent tolerance, performance of the c r i t e r i o n response during periods of drug exposure f a c i l i t a t e s the expression of the drug's e f f e c t and thus the development of tolerance to that e f f e c t . Generality of Contingent Tolerance. The a c t i v i t y of the drug r e c i p i e n t during periods of drug exposure has been shown to be an important, i f not c r i t i c a l , factor in the development of tolerance to a wide variety of drug e f f e c t s . This subsection presents representative examples of contingent tolerance to the e f f e c t s of the following drugs: 1) amphetamine and other psychostimulants; 2) morphine; 3) delta-9-THC; 4) the barbiturates; 5) the benzodiazepines; and 6) ethanol. A more comprehensive summary of the contingent tolerance l i t e r a t u r e i s avai l a b l e i n an excellent recent review by Wolgin (1989). 1. Contingent Tolerance to the Effects of Psychostimulants. Carlton and Wolgin's (1971) seminal report that the development of tolerance to the anorexigenic effects of amphetamine i s contingent upon the opportunity to eat during periods of drug exposure has since been confirmed by a number of researchers (e.g., Demellweek & Goudie, 1982; 1983a; Emmett-Oglesby, Spencer, Wood, & L a i , 1984; Poulos et a l . , 1981). Contingent tolerance also has been demonstrated to the anorexigenic e f f e c t s of other psychostimulants: cocaine (Woolverton, Kandel, & Schuster, 1978), cathinone ( F o l t i n & Schuster, 1982), methylphenidate (Emmett-Oglesby & Taylor, 1981), and the serotonergic receptor agonist quipazine (Rowland & Carlton, 1983). The pioneering work of Schuster and his colleagues on the importance of the reinforcement schedule during periods of drugged responding to the development of tolerance to the e f f e c t s of psychostimulant drugs on operant responding has been widely supported (e.g., Campbell & Seiden, 1973; Emmett-Oglesby et a l . , 1984; Smith, 1986a; see Wolgin, 1989). In a p a r t i c u l a r l y i n t e r e s t i n g paper, Smith (1986a) found that rats trained to bar press on a schedule of reinforcement that alternated between a random-ratio (RR) schedule and a differential-reinforcement-for-low-rates-of-responding (DRL) schedule developed tolerance to the drug's e f f e c t only on the RR portion of the schedule. When these rats were given additional t r i a l s i n which only the DRL component of the reinforcement schedule was used, they r a p i d l y developed tolerance to amphetamine's effects on t h e i r responding but t h i s tolerance disappeared as soon as the RR component of the reinforcement schedule was reintroduced. Smith (1986a) suggested that t h i s pattern of res u l t s could be accounted for i f the development of tolerance was a response to amphetamine's e f f e c t s on the "global" density of reinforcement and not to the drug's e f f e c t s on each component of the reinforcement schedule. Because the loss of reinforcement was much greater during the RR component than the DRL component of the schedule, the rats were more l i k e l y to demonstrate tolerance during the RR component than the DRL component when both were active. 2. Contingent Tolerance to the Effects of Morphine. M i t c h e l l and his colleagues (e.g., Kayan, Woods, & M i t c h e l l , 1969) were the f i r s t to report that the development of tolerance to morphine's analgesic e f f e c t was f a c i l i t a t e d i f rats received nociceptive stimulation ( i . e . , a hotplate test of nociception) during periods of drug exposure (see also Advokat, 1989; Milne, Gamble, & Holford, 1989; Moore, 1983). A similar e f f e c t has been demonstrated i n human subjects (Ferguson & M i t c h e l l , 1969). Advokat's (1989) study i s p a r t i c u l a r l y noteworthy. She found that nociceptive stimulation during periods of morphine exposure i s c r i t i c a l to the development of tolerance to the drug's analgesic e f f e c t s i n s p i n a l l y transected, but not i n t a c t , r a t s . Based upon these observations, Advokat (1989) suggested that the phy s i o l o g i c a l changes underlying the development of contingent tolerance to morphine's analgesic effects are l o c a l i z e d to spi n a l c i r c u i t s . Because none of these studies used the before-and-after design, they do not provide unequivocal evidence that nociceptive stimulation during periods of drug exposure i s an important factor i n the development of such tolerance to morphine's analgesic e f f e c t . However, a before-and-after design has been used to show that the drugged responding plays an important r o l e i n the development of tolerance to the disruptive e f f e c t s of morphine on operant responding i n the rat (Sannerud & Young, 1986). 3. Contingent Tolerance to the Effects of Barbiturates. Contingent tolerance has been demonstrated to the di s r u p t i v e e f f e c t s of barbiturates on operant behavior (e.g., Branch, 1983; Harris & S n e l l , 1980; Tang & Falk, 1978) and rotorod performance (Commissaris & Rech, 1981). Tang and Falk's (1978) demonstration i s p a r t i c u l a r l y noteworthy. They assessed the development of contingent tolerance to phenobarbital 1s e f f e c t s on operant responding i n terms of the dose-response curves obtained for both the drug-before and the drug-after groups p r i o r to and a f t e r the tolerance-development phase. They found l i t t l e evidence of contingent tolerance when low test doses of the drug were administered; however, as the test dose was increased the drug-before rat s demonstrated progressively greater tolerance to phenobarbital's e f f e c t s than the rats from the drug-after group. 26 4. Contingent Tolerance to the Effects of Delta-9-THC. Contingent tolerance has been demonstrated to the disr u p t i v e e f f e c t s of delta-9-THC on bar-press behavior i n the monkey (Carder & Olson, 1973; Elsmore, 1972), on bar-press and avoidance behavior i n the rat (Manning, 1976a,b), and operant responding i n the pigeon (Smith, 1986b). Manning's (1976a,b) experiments are noteworthy i n that he found that the development of contingent tolerance to delta-9-THC 1s ef f e c t on operant responding was not influenced by p r i o r drug history; rats that had previously received the drug aft e r performing the c r i t e r i o n a c t i v i t y developed tolerance no faster than drug-naive controls when both groups of rats received the drug before performing the task. 5. Contingent Tolerance to the Effects of Benzodiazepines. There have been only two reported attempts to demonstrate contingent tolerance to the eff e c t s of benzodiazepines. G r i f f i t h s and Goudie (1987) found that tolerance r e a d i l y developed to the ef f e c t s of the short-acting benzodiazepine midazolam on a food-reinforced bar-press task; however, rats that received midazolam before the opportunity to bar-press for food did not develop tolerance any faster, or to any greater extent, than rat s that did not receive the drug u n t i l after performing the operant task. G r i f f i t h s and Goudie (1987) concluded that "Tolerance to midazolam cannot therefore be explained i n terms of learned strategies acquitted as a re s u l t of drug-induced loss of rewarding s t i m u l i . " (p. 201). In contrast, Herberg and Montgomery (1987) found that the development of tolerance to the depressant, but not the f a c i l i t a t o r y , e f f e c t s of chlordiazepoxide on i n t r a c r a n i a l s e l f -stimulation was contingent upon t h e i r subjects having the opportunity to self-stimulate during periods of drug exposure (see also Herberg & Williams, 1983). Herberg and Montgomery (1987; 1988) have suggested that the inconsistency between t h e i r data and those reported by G r i f f i t h s and Goudie (1987) may be due to the f a c t that chlordiazepoxide has a much longer h a l f - l i f e than midazolam, thereby allowing more time for the a c q u i s i t i o n of strategies that would reduce the disruptive e f f e c t s of the drug. 6. Contingent Tolerance to the Effects of Ethanol. As described e a r l i e r i n t h i s section, Chen (19 68) provided the f i r s t report of contingent tolerance to ethanol's e f f e c t s . Since then, contingent tolerance has been demonstrated to a variety of ethanol's e f f e c t s . For example, contingent tolerance has been demonstrated to the e f f e c t of ethanol on treadmill running (LeBlanc, Gibbins, & Kalant, 1973; 1975; Wenger, Tiffany, Bombardier, N i c h o l l s , & Woods, 1981) and operant responding (Chen, 1979; Wiggell & Overstreet, 1984) and to the ethanol-induced acceleration i n the decay of postsynaptic potentiation in the abdominal ganglia of the marine mollusc Aplvsia (Traynor, Schlapfer, & Barondes, 198 0). Contingent tolerance has also been demonstrated to ethanol*s analgesic e f f e c t , using the t a i l - f l i c k t e s t of analgesia (J^rgenson & Hole, 1984; J^rgenson, Berge, & Hole, 1985; Jtyrgenson, Farmer, & Hole, 1986), and to ethanol 1s hypothermic e f f e c t (Alkana, Finn, & Malcolm, 1982). This l a t t e r 28 study i s noteworthy because of the novel method used to manipulate alcohol's hypothermic e f f e c t . Alkana and h i s colleagues found that mice that received an i n j e c t i o n of ethanol and were then immediately warmed with microwave rad i a t i o n , so that ethanol's hypothermic e f f e c t could not be experienced, displayed no tolerance on the test t r i a l . In contrast, mice that were injected with ethanol and l e f t at room temperature displayed a substantial amount of tolerance to ethanol*s hypothermic e f f e c t s (N.B., Le, Kalant, & Khanna [1986a] found that warming the environment i n which ethanol's effects are experienced only slowed, rather than prevented, the development of tolerance to i t s hypothermic e f f e c t s ) . F i n a l l y , Pinel and his colleagues have reported that the development of tolerance to ethanol*s disruptive e f f e c t s on sexual behavior i n rats i s contingent upon the rodents having the opportunity to engage i n sexual behavior while they are intoxicated (Pinel, Pfaus, & Christensen, submitted). In the l a s t seven years, Pinel and his colleagues have shown that the development of tolerance to ethanol's anticonvulsant e f f e c t on convulsions e l i c i t e d i n amygdala-kindled rats i s contingent upon the administration of convulsive stimulation during periods of ethanol exposure. Because t h i s work provided the basis for the present thesis, i t i s described i n d e t a i l i n the next section. 29 4. Contingent Tolerance to Ethanol's Anticonvulsant E f f e c t In t h e i r o r i g i n a l report, Pinel and his colleagues (1983) used a before-and-after design to examine the e f f e c t that convulsive stimulation administered during periods of ethanol exposure had on the development of tolerance to ethanol 1s anticonvulsant e f f e c t . After establishing a b i d a i l y (once every 48 hr) convulsive stimulation baseline, kindled rats were assigned to either an ethanol-before-stimulation group or an ethanol-after-stimulation group. The subjects i n both groups were then stimulated six more times on the b i d a i l y schedule. The ethanol-before-stimulation rats received ethanol (4.5 g/kg, by intubation, i n a 3 0% volume/volume solution) 1.5 hr before each stimulation and a comparable volume of isotonic s a l i n e 1.5 hr afterwards. The ethanol-after-stimulation rats received the same intubations but i n the reverse order ( i . e . , the s a l i n e before each stimulation and the ethanol a f t e r ) . On the t e s t t r i a l , the rats from both treatment groups were challenged with an i n j e c t i o n of ethanol (1.5 g/kg, IP, i n a 25% volume/volume solution) 1.5 hr before the t e s t stimulation. As i s evident i n Figure 1, the rats i n the ethanol-before-stimulation group displayed substantial tolerance to ethanol's anticonvulsant e f f e c t on the test t r i a l . In contrast, there was no evidence of tolerance i n any of the subjects from the ethanol-af t e r group. A subsequent analysis of the blood ethanol l e v e l for each r a t immediately after testing revealed no s i g n i f i c a n t difference between the two groups. 30 FIGURE 1. The e f f e c t of the response contingency on the development of tolerance to ethanol's anticonvulsant e f f e c t . During the treatment phase, ethanol (4.5 g/kg) was intubated at 48-hr i n t e r v a l s , either before or after convulsive stimulation. On the t e s t t r i a l , the rats that had received ethanol before stimulation on the treatment days (the ethanol-before-stimulation group) demonstrated substantial tolerance to the anticonvulsant e f f e c t of the t e s t dose of ethanol (1.5 g/kg, IP), whereas there was no evidence of tolerance i n the rats that had received ethanol a f t e r each convulsive stimulation on the treatment days (the ethanol-after-stimulation group). (From Pinel et a l . , 1983) . MEAN DURATION OF FORELIMB CLONUS (sec) _ 4 N> 00 O O O O Aside from the sizable e f f e c t that convulsive stimulation had on the development of tolerance to ethanol 1s anticonvulsant e f f e c t , perhaps the most notable r e s u l t of Pinel et a l . ' s (1983) experiment was the lack of any evidence of tolerance i n the ethanol-after-stimulation group: ethanol exposure alone produced no detectable tolerance. In a follow-up series of experiments, Pin e l et a l . (1985) extended t h i s finding by showing that kindled ra t s that received ethanol 1 hr after each b i d a i l y stimulation f a i l e d to develop tolerance to ethanol's anticonvulsant e f f e c t even when the treatment dose was as high as 5 g/kg (by gavage), or as many as 20 tolerance-development t r i a l s were conducted p r i o r to the t e s t t r i a l . Mana, Le, Kalant, and Pin e l (in preparation) subsequently demonstrated that kindled rats that received a t o t a l of 2 0 ethanol intubations (5 g/kg), on a d a i l y rather than b i d a i l y basis, developed tolerance to ethanol's anticonvulsant e f f e c t i n the absence of concurrent convulsive stimulation. There i s no doubt, however, that the development of tolerance to the anticonvulsant e f f e c t s of ethanol i s greatly f a c i l i t a t e d by the administration of convulsive stimulation during periods of ethanol exposure. P i n e l and Puttaswamaiah (1985) demonstrated that contingent tolerance to ethanol*s anticonvulsant e f f e c t i s not a form of conditional or context-dependent tolerance. There was no evidence that contingent tolerance to ethanol's anticonvulsant e f f e c t i s context s p e c i f i c ; the tolerance that the kindled rats displayed i n an environment that had never been paired with ethanol was just as robust as that displayed by the rats i n the environment that was consistently paired with the ethanol i n j e c t i o n s during the tolerance-development phase. Furthermore, they found no evidence of a conditional compensatory response when tolerant rats received a saline i n j e c t i o n i n a drug-p r e d i c t i v e environment, and preexposure to the contextual cues associated with ethanol administration had no e f f e c t on tolerance development. And Mana and Pinel (unpublished observations) tested the idea that the cue properties of the convulsive stimulation i t s e l f could serve as a CS predictive of ethanol's anticonvulsant e f f e c t . They found that varying the number of stimulations that kindled rats received ( i . e . , the degree of CS preexposure) p r i o r to the st a r t of the tolerance-development phase had no e f f e c t on the rate or magnitude of tolerance that developed. The f a c t that convulsive stimulation played such a c r i t i c a l r o l e i n the development of tolerance to ethanol's anticonvulsant e f f e c t led Mana and Pinel (1987) to investigate the r o l e of convulsive stimulation i n the di s s i p a t i o n of contingent tolerance to ethanol's anticonvulsant e f f e c t . Drug withdrawal i s generally considered to be the necessary and s u f f i c i e n t condition for the d i s s i p a t i o n of tolerance to a drug's e f f e c t s . In contrast, Mana and P i n e l (1987) found that ethanol withdrawal was neither necessary nor s u f f i c i e n t for the di s s i p a t i o n of tolerance to ethanol's anticonvulsant e f f e c t ; instead, the administration of convulsive stimulation in the absence of ethanol was the c r i t i c a l 34 Figure 2. Contingent d i s s i p a t i o n of tolerance to ethanol 1s anticonvulsant e f f e c t s on kindled convulsions. Ethanol withdrawal had no e f f e c t on the di s s i p a t i o n of tolerance, as the rats i n the No Ethanol-No Stim group demonstrated no loss of tolerance even though they were not administered ethanol during the retention i n t e r v a l . Furthermore, the administration of ethanol was not a necessary condition for the maintenance of tolerance, as the rats i n the Ethanol-After-Stimulation group l o s t t h e i r tolerance even though they continued to receive ethanol on the same schedule of administration associated with the development of tolerance. The c r i t i c a l factor i n the d i s s i p a t i o n of tolerance to ethanol anticonvulsant e f f e c t s was the administration of convulsive stimulation i n the absence of ethanol. (from Mana & Pinel, 1987). NOETOH TOLERANCE BASELINE DEVELOPMENT TRIALS 14-DAY noETOH-noSTIM 1 = 3 ETOH-noSTIM ETOH-before-STIM STIM-before-ETOH STIM-noETOH 1 RETENTION RETENTION INTERVAL TEST factor i n the d i s s i p a t i o n of tolerance. Kindled rats were rendered tolerant to ethanol's anticonvulsant e f f e c t i n the usual fashion ( i . e . , b i d a i l y ethanol injections (1.5 g/kg, IP), each followed 1 hr l a t e r by a convulsive amygdala stimulation. They were then assigned to one of six d i f f e r e n t treatment groups; the rats i n each group were treated d i f f e r e n t l y during the ensuing 14-day retention i n t e r v a l . The rats i n two groups received an ethanol i n j e c t i o n either 1 hr before or 1 hr a f t e r each of s i x b i d a i l y stimulations. The rats i n two other groups were treated i n the same manner except that saline rather that ethanol was injected. The rats i n the f i f t h group received the s i x b i d a i l y ethanol i n j e c t i o n s but no convulsive stimulations, whereas those i n the s i x t h and f i n a l group received neither the b i d a i l y stimulations nor the ethanol injections during the retention i n t e r v a l . A single tolerance test t r i a l was administered to a l l subjects at the end of the 14-day retention i n t e r v a l . This t e s t was i d e n t i c a l to the tolerance-development t r i a l s ; a l l subjects were stimulated 1 hr after receiving the standard 1.5 g/kg (IP) ethanol t e s t i n j e c t i o n . I t i s c l e a r i n Figure 2 that the d i s s i p a t i o n of tolerance to ethanol's anticonvulsant e f f e c t over the 14-day retention i n t e r v a l was greatly influenced by the treatment received by the ra t s during t h i s period. In three of the groups—the s a l i n e -before-stimulation and saline-after-stimulation groups (combined i n Figure 2), and the ethanol-after-stimulation group—tolerance to ethanol's anticonvulsant e f f e c t had completely disappeared following the retention i n t e r v a l . In contrast, the magnitude of tolerance i n the other three groups—the no-ethanol-no-stimulation group, the ethanol-before-stimulation group, and the ethanol-no-stimulation group—did not decline. These r e s u l t s c l e a r l y show the inadequacy of the view that the d i s s i p a t i o n of ethanol tolerance i s a r e s u l t of the discontinuation of ethanol exposure: The cessation of ethanol exposure did not lead to a decline i n tolerance i n the rats from the no-ethanol-no-stimulation group, and continued exposure to ethanol did not maintain the tolerance i n the rats from the ethanol-after-stimulation group. Instead, the key factor i n the d i s s i p a t i o n of tolerance to ethanol's anticonvulsant e f f e c t appeared to be the administration of convulsive stimulation i n the absence of ethanol. Tolerance did not decline at a l l i n the two groups that did not receive any stimulations during the retention i n t e r v a l or in the group receiving stimulations following each ethanol i n j e c t i o n , but i t dissipated completely i n groups stimulated i n the absence of ethanol, even when ethanol was s t i l l administered a f t e r each b i d a i l y stimulation. The important r o l e that convulsive stimulation has on the d i s s i p a t i o n of tolerance to ethanol's anticonvulsant e f f e c t can be e a s i l y understood i f one returns to the prism analogy that was introduced e a r l i e r i n t h i s Introduction to i l l u s t r a t e the ro l e that convulsive stimulation has on the development of tolerance to ethanol's anticonvulsant e f f e c t . Just as subjects that have adapted to the eff e c t s of vision-displacing prisms must 38 experience the eff e c t s of t h e i r removal for the adaptation to di s s i p a t e (see Rock, 1966), so too rats tolerant to the anticonvulsant e f f e c t s of ethanol must experience seizures i n the absence of ethanol for tolerance to dissipate (see Poulos & Hinson, 1984, and Poulos, Wilkinson, & Cappell, 1981, for further evidence that the di s s i p a t i o n of contingent tolerance i s influenced by the performance of the c r i t e r i o n response i n an undrugged st a t e ) . P i n e l , Kim, Paul, and Mana (1989) recently demonstrated that the development of cross-tolerance between ethanol and pentobarbital's anticonvulsant effects i s also contingent upon the administration of convulsive stimulation during periods of drug exposure. Only rats exposed to pentobarbital on a b i d a i l y drug-before-stimulation treatment regimen subsequently displayed cross tolerance to the anticonvulsant e f f e c t of ethanol; pentobarbital-after-stimulation rats displayed no cross tolerance. And the same proved true for the transfer of tolerance i n the other d i r e c t i o n : Only rats from the ethanol-bef ore-stimulation group displayed cross-tolerance to pentobarbital's anticonvulsant e f f e c t . Although there i s now strong evidence that the r e l a t i o n between convulsive stimulation and drug exposure has a substantial e f f e c t on development and d i s s i p a t i o n of tolerance to ethanol's anticonvulsant e f f e c t , there are no published reports suggesting that convulsive stimulation, or any type of seizure a c t i v i t y i n the brain, plays a role i n the development or d i s s i p a t i o n of tolerance to the effects of c l i n i c a l l y relevant a n t i e p i l e p t i c drugs. Instead, the research i n t h i s area has been predicated on the t r a d i t i o n a l idea that the development and d i s s i p a t i o n of tolerance i s s t r i c t l y a function of pharmacological variables. Because one purpose of t h i s thesis i s to compare contingent and pharmacologic tolerance to the anticonvulsant drug ef f e c t s , the next section b r i e f l y reviews the e x i s t i n g l i t e r a t u r e on pharmacologic tolerance to the anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs. The f i r s t subsection describes the paradigms that have t r a d i t i o n a l l y been used to study the development of tolerance to anticonvulsant drug e f f e c t s ; the next three subsections focus on reports of tolerance to the drugs studied i n t h i s thesis: carbamazepine, diazepam, and sodium valproate, respectively. 5. Pharmacologic Tolerance to the Anticonvulsant Effects of A n t i e p i l e p t i c Drugs Although anecdotal evidence that tolerance develops to the anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs has existed almost since t h e i r introduction as a treatment for the e p i l e p s i e s i n the early 19 00's (e.g., phenobarbital; Hauptmann, 1912, c i t e d i n Frey, 1985), there was l i t t l e clear experimental evidence of t h i s phenomenon u n t i l Frey and Kampmann published t h e i r landmark study of tolerance to the anticonvulsant effects of phenobarbital and phenytoin i n 1965. Since then, there have been numerous laboratory demonstrations of tolerance to the anticonvulsant 40 e f f e c t s of c l i n i c a l l y prescribed a n t i e p i l e p t i c drugs. In contrast, c l i n i c a l support for the idea that tolerance develops to the anticonvulsant effects of a n t i e p i l e p t i c drugs i n human e p i l e p t i c s remains equivocal (see Frey, 1987). This apparent inconsistency can be attributed to at least three factors. F i r s t , i t i s routine therapeutic procedure for physicians to increase the dosage of an a n t i e p i l e p t i c drug i f i t does not provide adequate protection once steady plasma l e v e l s have been reached (e.g., see Eadie, 1985); thus, the development of tolerance to an a n t i e p i l e p t i c drug's anticonvulsant e f f e c t may be mistakenly attributed to an inadequate i n i t i a l treatment dose (see Butler, Mahaffee, & Waddell, 1954 for a prescient discussion of t h i s problem; see also Frey, 1987). Second, e t h i c a l considerations r e s t r i c t the scope of c l i n i c a l investigations of the development of tolerance to the anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs. Third, the development of tolerance to the anticonvulsant e f f e c t of one drug may often be obscured by the presence of one or more other a n t i e p i l e p t i c drugs (see Koella & Meinardi, 1986a)—polypharmacy (the concurrent administration of more than one type of drug) i s s t i l l the rule rather than the exception i n the c l i n i c a l treatment of epilepsy (Koella & Meinardi, 1986a). In s p i t e of these d i f f i c u l t i e s , i t i s now generally accepted that tolerance does develop to the effects of most a n t i e p i l e p t i c drugs i n c l i n i c a l settings (see Frey, 1987). Accordingly, the next four subsections review the evidence that tolerance develops to the anticonvulsant effects of a n t i e p i l e p t i c drugs. The f i r s t subsection describes the two types of experimental convlsions that have t r a d i t i o n a l l y been used to study the development of tolerance to anticonvulsant drug e f f e c t s : maximal electroshock convulsions and pentylenetetrazol convulsions. The f i n a l three subsections review both experimental and c l i n i c a l reports of tolerance to the anticonvulsant effects of the three a n t i e p i l e p t i c drugs that are the focus of t h i s d i s s e r t a t i o n : 1) carbamazepine (CBZ); 2) diazepam (DZP); and 3) sodium valproate (VPA). Each subsection includes a b r i e f description of the drug's history, pharmacologic p r o f i l e , spectrum of anticonvulsant a c t i v i t y , and history of tolerance. T r a d i t i o n a l Convulsion Paradigms  Maximal Electroshock Convulsions Maximal electroshock (MES) convulsions are commonly used i n drug-development t r i a l s to assess the anticonvulsant e f f i c a c y of drugs (Koella, 1985), as well as to assess the development of tolerance to anticonvulsant drug e f f e c t s . Although the d e t a i l s of the procedure may vary, MES convulsions are usually studied i n rats or mice, with the e l e c t r i c a l stimulation (either 60 Hz, alternating sine-wave current for about 0.2 sec, or 6 Hz, pulsating d i r e c t current for about 3 sec) administered by ear c l i p s or corneal electrodes (Koella, 1985). Maximal electroshock convulsions are produced by administration of high-intensity stimulation (e.g., i n r a t s , 60 Hz a l t e r n a t i n g current at 150 mA for 3 sec); they l a s t approximately 2 0 sec to 2 5 sec and involve tonic f l e x i o n , 42 followed by tonic extension of a l l four limbs, and then a period of clonus involving a l l four limbs (Koella, 1985). Maximal electroshock convulsions are the model of choice i n the assessment of drug e f f i c a c y against primarily generalized and complex-partial epilepsy (Swinyard, 1980). Pentylenetetrazol Convulsions Pentylenetetrazol (PTZ) i s probably used more often than any other chemical convulsant to assess the development of tolerance to anticonvulsant drug e f f e c t s . Pentylenetetrazol i s usually administered i n t r a v e n t r i c u l a r l y or subcutaneously; slow infusion by the IV route i s frequently used when the threshold dose i s of i n t e r e s t (Koella, 1985). Convulsions usually begin 60 sec to 210 sec a f t e r the administration of PTZ; they generally involve several whole-body c l o n i c convulsions which l a s t from 2 min to 3 minutes. The c l o n i c convulsions are often followed by a tonic convulsion; animals e x h i b i t i n g t h i s tonic phase usually die. Drugs e f f e c t i v e at reducing PTZ convulsions are usually also e f f e c t i v e against p e t i t mal and absence seizures i n humans (Swinyard, 1980). Tolerance to the Anticonvulsant Effects of Carbamazepine  History The synthesis of CBZ in 1957 by the laboratories of J.R. Geigy AG was part of a long-term study of the anticonvulsant e f f e c t s of various iminodibenzyl compounds. Experimental and c l i n i c a l t r i a l s proceeded almost in tandem, with the f i r s t reports of i t s anticonvulsant e f f i c a c y i n both laboratory animals and humans being published i n 1963 (see Schmutz, 1985). 43 Approved i n 1974 for use in the United States as a treatment for epilepsy (see Suria & Killam, 1980), CBZ has become the drug of choice for the treatment of p a r t i a l and generalized t o n i c - c l o n i c seizures i n both adults and children (see Engel, 1989; Porter, 1986). I t i s also used i n the treatment of trigeminal neuralgia (Rail & S c h l e i f e r , 1980). Pharmacologic P r o f i l e CBZ i s the only known anticonvulsant possessing a t r i c y c l i c structure, more sim i l a r to that of the neuroleptic chlorpromazine and the antidepressant imipramine than to other a n t i e p i l e p t i c drugs (see Schmutz, 1985). CBZ i s insoluble i n water but soluble in solvents such as alcohol, acetone, and propylene gl y c o l (Schmutz, 1985). The pharmacokinetics of CBZ have been well established. Peak plasma l e v e l s i n the rat are reached within 45 min of IP i n j e c t i o n (Moreselli, 1975; Morton, 1984); absorption a f t e r o r a l administration tends to be slower. CBZ i s evenly d i s t r i b u t e d throughout the body; there i s no p r e f e r e n t i a l a f f i n i t y for any p a r t i c u l a r organs or tissue (Faigle, Brechbiiler, Feldman, & Richter, 1976). The metabolism of CBZ i s q u a l i t a t i v e l y s i m i l a r i n r a t s and man (see Schmutz, 1985); notably, the product of the primary metabolic pathway for CBZ i s an epoxy metabolite with s i g n i f i c a n t anticonvulsant properties of i t s own (Rail & S c h l e i f e r , 1980). The metabolism of CBZ i s accelerated during chronic exposure to the drug (metabolic tolerance) by the autoinduction of hepatic enzymes (Faigle et a l . , 1976; see also M o r s e l l i , Bossi, & Gerna, 1976, for similar r e s u l t s i n human e p i l e p t i c p a t i e n t s ) ; thus, i t i s often necessary to increase the treatment dose several times to maintain an e f f e c t i v e plasma concentration. The mechanism of action for CBZ's anticonvulsant e f f e c t i s unknown. I t i s generally agreed that CBZ's anticonvulsant e f f e c t s are not mediated by a change i n the function of the i n h i b i t o r y neurotransmitter GABA; at therapeutic l e v e l s , CBZ does not appear to a l t e r the release, reuptake, or synthesis of GABA in the brain (see Schmutz, 1985). Similarly, dopaminergic, noradrenergic, and serotonergic systems i n the CNS do not seem to play a r o l e i n CBZ's anticonvulsant e f f e c t (see Schmutz, 1985). CBZ has been shown to s e l e c t i v e l y increase the le v e l s of acetylcholine i n the striatum, but not i n the hippocampus, diencephalon, mesencephalon, or cerebellum (Consolo, Bianchi, & Ladinski, 1976). The fact that many convulsants increase the release and decrease the brain concentration of acetylcholine led Consolo et a l . (1976) to suggest that CBZ's anticonvulsant e f f e c t may be mediated by a cholinergic mechanism. I t has also been suggested that CBZ's anticonvulsant e f f e c t might be due to i t s p a r t i a l agonist action at adenosine receptors (Schmutz, 1985; though see Marangos, Post, Patel, Zander, Parmer, & Weiss, 1983), or by a reduction i n sodium or potassium currents (Schauf, Davis, & Marder, 1974) or synaptosomal potassium-mediated calcium uptake (e.g., F e r e n d e l l i & McQueen, 1982). Anticonvulsant A c t i v i t y CBZ has been shown to reduce the severity of a wide variety of experimental convulsions, including both maximal and minimal electroshock convulsions (Julien & H o l l i s t e r , 1975; Koella, Levin, & Baltzer, 1976); pentylenetetrazol convulsions (Koella, Levin, & Baltzer, 1976) ; audiogenic convulsions (Consroe, Kudray, & Schmitz, 1980); kindled convulsions (Weiss & Post, 1987); p e n i c i l l i n - f o c u s convulsions (Julien & H o l l i s t e r , 1975); and photic convulsions i n the baboon Papio papio (Killam, 1976). In c l i n i c a l p r a ctice, CBZ i s used i n the treatment of complex p a r t i a l and secondarily generalized epilepsy (Honack & Loscher, 1989; Schmutz, 1985; Engel, 19 89). Tolerance to CBZ's Anticonvulsant Effects M o r s e l l i ' s (1975) comment that CBZ's "anticonvulsant e f f e c t i s reduced" i n rats that demonstrate an acceleration of CBZ metabolism following chronic exposure may be the f i r s t report of tolerance to CBZ's anticonvulsant e f f e c t . However, because M o r s e l l i (1975) f a i l e d to provide any data to support t h i s claim, Farghali-Hassan et a l . (1976) are credited with the f i r s t conclusive demonstration that tolerance develops to the anticonvulsant e f f e c t of CBZ. Farghali-Hassan et a l (1976) found that tolerance developed to CBZ's (25 mg/kg, IV) anticonvulsant e f f e c t on maximal electroshock convulsions following twice-daily i n j e c t i o n s of the drug f o r 12 days. They attributed the development of tolerance to an increase i n the rate of metabolism of CBZ, with a corresponding decrease i n brain levels of the drug. Similar r e s u l t s were subsequently reported by Masuda, Utsui, S h i r a i s h i , Karasawa, Yoshida, and Shimizui (1979), who also found that the 46 development of tolerance to CBZ's anticonvulsant e f f e c t (50 mg/kg, p e r i o r a l [PO]) on maximal electroshock convulsions was accompanied by an increase i n the breakdown of the drug. Masuda and his colleagues (1979) also found evidence of a functional component to the tolerance that developed to CBZ 1s anticonvulsant e f f e c t ; rats that had received chronic exposure to CBZ were less sensitive to i t s anticonvulsant e f f e c t s than were drug-naive control rats displaying an i d e n t i c a l plasma concentration of the drug. Similarly, Honack and Loscher (1989) suggested that the development of tolerance to the anticonvulsant e f f e c t of CBZ (30 mg/kg, IP, administered three times per day for 2 weeks) on kindled convulsions i n the rat i s at t r i b u t a b l e , i n part, to a functional mechanism because of t h e i r observation that tolerance developed at s i g n i f i c a n t l y d i f f e r e n t rates to d i f f e r e n t e f f e c t s of the drug. Given the general agreement about the prevalence of d i s p o s i t i o n a l tolerance to CBZ's eff e c t s , i t i s somewhat sur p r i s i n g that Baltzer, Baud, Degen, and Koella (1980) observed no evidence of tolerance to the anticonvulsant e f f e c t of CBZ (at d a i l y doses of 6, 10, or 18 mg/kg, PO) on maximal electroshock convulsions, even afte r 28 da i l y treatment t r i a l s (see also Schmutz, David, Grewal, Bernasconi, & Baltzer, 1986) . This discrepancy might be attributable to the fact that Baltzer et a l . (1980) administered a r e l a t i v e l y low treatment dose to t h e i r subjects (the largest dose was 18 mg/kg/day), whereas r e l a t i v e l y large treatment doses were administered by Farghali-Hassan et a l . (1976; 50 mg/kg/day), Masuda et a l . (1979; 50 mg/kg/day) and Honack and Loscher (1989; 90 mg/kg/day). Tolerance to the Anticonvulsant Ef f e c t of Diazepam  History Although the f i r s t benzodiazepine compounds were synthesized i n the early 1930s by Sternbach (see Killam & Suria, 1980), DZP was not approved for c l i n i c a l use u n t i l 1963 (Killam & Suria, 1980). DZP i s e f f e c t i v e against a wide var i e t y of e p i l e p t i c seizures; however, i t i s not used as a primary form of treatment for epilepsy except for certain forms of myoclonic seizures and for status epilepticus. This lack of acceptance i s p r i m a r i l y due to the fact that tolerance rapidly develops to the anticonvulsant e f f e c t of t h i s drug (see Frey, 1987, Haigh & Feely, 1988) and to the fact that stable therapeutic l e v e l s of DZP are d i f f i c u l t to maintain during chronic administration of the drug (Engel, 1989). Pharmacologic P r o f i l e DZP i s insoluble i n water but e a s i l y dissolves i n solvents such as die t h y l ether, propylene g l y c o l , or ethanol. Its absorption i s rapid, with peak plasma l e v e l s being reached within 12 min of IP administration i n rats (Morton, 1984). DZP i s widely distributed through the body i n s p i t e of considerable protein binding (Killam & Suria, 1980), although i t accumulates p r e f e r e n t i a l l y i n the l i v e r , lungs, and adipose t i s s u e (Caccia & G a r a t t i n i , 1985). DZP e a s i l y crosses the blood-brain b a r r i e r (Killam & Suria, 1980); once i n the brain, i t accumulates f i r s t i n c o r t i c a l gray matter and then i s 48 r e d i s t r i b u t e d to white matter (Harvey, 1980). Chronic exposure to DZP does not seem to a l t e r i t s absorption, d i s t r i b u t i o n , or elimination rates (Kaplan & Jack, 1981). DZP i s largely eliminated by the body following biotransformation by hepatic enzymes (Caccia & G a r r a t i n i , 1985). Chronic exposure to DZP does not appear to accelerate the a c t i v i t y of these hepatic enzymes (Killam & Suria, 1980; though see L i n n o i l a , K o r t t i l a , & Mattila, 1975, c i t e d i n Harvey, 1980); thus, an increase i n metabolism i s not l i k e l y to contribute to the development of tolerance to i t s e f f e c t s . Nordiazepam, one of DZP's major metabolites, possesses s i g n i f i c a n t e f f e c t s of i t s own; Killam & Suria (1980) reported that nordiazepam i s act u a l l y more potent than the parent compound at antagonizing both maximal electroshock and pentylenetetrazol-induced convulsions i n mice. I t i s generally agreed that the majority of DZP's central e f f e c t s are mediated at the GABA-benzodiazepine-chloride ionophore complex (Bruun-Meyer, 1987). DZP binds to a high-a f f i n i t y , saturable, stereospecific receptor (Mohler & Okada, 1977; Squires & Braestrup, 1977) that i s usually associated with the l o w - a f f i n i t y , GABA-A receptor (Haefly, 1989); GABA and DZP a l l o s t e r i c a l l y modulate each others receptors so that the presence of one enhances the binding of the other (see Martin, 1987) . Functionally, DZP acts at the GABA-benzodiazepine-chloride ionophore complex to increase the frequency of opening of the chloride channel when GABA i s also bound to i t s receptor (Martin, 1987); the net ef f e c t of t h i s increase i n chloride conductance i s a decrease i n neuronal e x c i t a b i l i t y (Haefly, 1989). In addition, P h i l l i s and O'Regan (1988) have provided compelling evidence that some of the actions of the benzodiazepines, including DZP, may be att r i b u t a b l e to t h e i r i n h i b i t o r y e f f e c t on the reuptake of adenosine, a putative i n h i b i t o r y neuromodulator (e.g., N i c o l l , Malenka, & Kauer, 1990). Anticonvulsant A c t i v i t y Benzodiazepines are among the most potent anticonvulsants, e f f e c t i v e against a wide var i e t y of experimental convulsions as well as various forms of human epilepsy (see Haefly, P i e r i , Pole, & Schaffner, 1981; Caccia & G a r a t t i n i , 1985). DZP attenuates maximal electroshock convulsions (e.g., Baltzer et a l . , 1980); b i c u c u l l i n e - and penetylenetetrazol-induced convulsions (e.g., Matthews & McCafferty, 1979); p h o t i c a l l y induced convulsions i n the baboon Papio papio (e.g., Killam, Matsuzaki, & Killam, 1973); kindled convulsions (e.g., Albright & Burnham, 1980); picrotoxin-induced convulsions (e.g., Jenner, Marsden, Pratt, & Reynolds, 1979); and p e n i c i l l i n - i n d u c e d convulsions (e.g., Stark, Edmonds, & Keesling, 1974) (see Haefly et a l . , 1981, for a complete review). In general, larger doses of DZP are needed to attenuate electroshock convulsions than are needed to attenuate the e f f e c t s of chemical convulsants such as pentylenetetrazol, b i c u c u l l i n e , or strychnine (e.g., Haefly et a l . , 1981). Tolerance to DZP's Anticonvulsant Effect There are more reports of tolerance to the anticonvulsant effects of the benzodiazepines, including DZP, than to any other a n t i e p i l e p t i c 50 drug. Although anecdotal reports existed e a r l i e r , Killam, Matsuzaki, and Killam (1973) were the f i r s t to unequivocally demonstrate the development of tolerance to the anticonvulsant e f f e c t of DZP (0.2-0.4 mg/kg, SC, administered once per day for up to 16 weeks). Perhaps the most interesting aspect of t h e i r data was the finding that tolerance was less l i k e l y to develop to the anticonvulsant e f f e c t s of DZP on photically-induced convulsions i n Papio i f a r e l a t i v e l y high treatment dose (0.5 mg/kg per day) was administered from the st a r t of the treatment regimen; when Killam et a l . (1973) followed the conventional c l i n i c a l wisdom of st a r t i n g with a "threshold" dose of DZP that was just large enough to suppress convulsive a c t i v i t y , tolerance r a p i d l y developed to the drug's anticonvulsant e f f e c t s . Since Killam et al ' s (1973) seminal report, there have been many reports of tolerance to DZP's anticonvulsant e f f e c t on a varie t y of d i f f e r e n t types of convulsions. For example, Baltzer et a l . (1980) reported tolerance to the anticonvulsant e f f e c t s of DZP (5 mg/kg to 15 mg/kg, PO) on maximal electroshock convulsions i n r a t s a f t e r just 5 days of treatment; these authors noted that tolerance was not expressed in a consistent fashion, but instead waxed and waned on a 10-day cycle. Interestingly, a s i m i l a r pattern i s apparent i n the data reported by Killam et a l . (1973), and t h i s phenomenon i s described i n d e t a i l by Koella (1986). F i l e (1983) reported the development of tolerance to the anticonvulsant e f f e c t of DZP (4 mg/kg, IP) on p i c r o t o x i n - or pentylenetetrazol-induced convulsions in mice a f t e r as few as 10 days. Loscher and Schwark (1985) reported the development of tolerance to DZP's anticonvulsant e f f e c t (5 mg/kg, IP, 3 times per day for 14 days) on kindled convulsions i n rats . Concomitant determination of plasma concentrations of DZP revealed that the le v e l s of DZP actually increased over the 2-week treatment period, suggesting that a functional rather than d i s p o s i t i o n a l change was responsible for the development of tolerance. F i n a l l y , Schneider and Stephens (1988) reported that tolerance develops to DZP's (5 mg/kg or 20 mg/kg, IP, for 9 days) anticonvulsant e f f e c t s on convulsions e l i c i t e d by the benzodiazepine inverse agonist FG 7142, a drug that also binds at the benzodiazepine receptor but e l i c i t s e f f e c t s that are opposite (e.g., proconvulsant; anxiogenic) to those e l i c i t e d by benzodiazepine agonists such as DZP. Tolerance to the Anticonvulsant Effects of Valproic Acid  History Although valproic acid was synthesized i n the 1880s, i t s anticonvulsant e f f e c t s did not become apparent u n t i l Meunier's group (1963; c i t e d i n Kupferberg, 1980) made the serendipitous observation that many compounds demonstrated a pronounced anticonvulsant e f f e c t upon PTZ convulsions when they were administered i n a valproic acid vehicle. Meunier's conclusion that the anticonvulsant e f f e c t was due to the vehicle, rather than to the drugs themselves, has since been confirmed many times and today sodium valproate (VPA), the sodium s a l t of v a l p r o i c acid, i s widely used i n the treatment of absence seizures, p a r t i a l seizures, and generalized t o n i c - c l o n i c seizures (see Loscher, 1985; Engel, 1989). Pharmacologic P r o f i l e VPA i s a hygroscopic white powder that dissolves e a s i l y i n water or ethanol but less r e a d i l y i n solvents such as acetone. I t i s rapidly absorbed following IP or PO administration, with peak plasma levels being reached within 30 min of administration (e.g., Morton, 1984). VPA enters the CNS vi a an active transport mechanism; consequently, brain l e v e l s of VPA may lag behind plasma concentrations (see Nau & Loscher, 1978). Once i t enters the brain, VPA i s homogeneously d i s t r i b u t e d and there i s l i t t l e evidence of drug accumulation following repeated administration (see Loscher, 1985). VPA has a h a l f - l i f e of about 4.5 hr i n the rat; hepatic metabolism i s the major route of elimination. Loscher (1981) has demonstrated that many of VPA's metabolites have anticonvulsant a c t i v i t y of t h e i r own, though only 2-en valproic acid occurs i n s i g n i f i c a n t l e v e l s i n the brain under therapeutic conditions (see Loscher, 1985). VPA i s noted for the pronounced and often unpredictable interactions that i t can have on the effects of other drugs (Henriksen & Johannessen, 1984). For example, the dose of phenobarbital can be cut in half with no loss of plasma concentration of the drug when VPA i s added to therapy because VPA i n t e r f e r e s with the breakdown of phenobarbital. Conversely, i t i s often d i f f i c u l t to establish therapeutic plasma concentrations of VPA when i t i s coadministered with a n t i e p i l e p t i c drugs such as phenytoin or CBZ because these drugs f a c i l i t a t e the breakdown of VPA (Eadie, 1985; Henriksen & Johannessen, 1984). For t h i s reason, monotherapy may be preferable when VPA i s used i n the treatment of epilepsy (Henriksen & Johannessen, 1984). The mechanism of VPA's anticonvulsant action i s unknown. Loscher (1985) suggested that VPA's anticonvulsant e f f e c t i s at t r i b u t a b l e to an increase i n synaptosomal leve l s of the i n h i b i t o r y neurotransmitter GABA, which would subsequently increase the amount available for release from these neurons. More recently, Loscher (1989) reported that l e v e l s of GABA i n GABAergic neurons with terminal f i e l d s i n the substantia nigra are p a r t i c u l a r l y enhanced by VPA. Interestingly, GABAergic ennervation of the substantia nigra has been implicated as a c r i t i c a l factor i n the ontogeny of seizures (e.g.., Gale, 1988; McNamara, Galloway, Rigsbee, & Shin, 1984). I t has also been suggested that VPA mediates i t s anticonvulsant e f f e c t s by a d i r e c t postsynaptic enhancement of GABAergic transmission s i m i l a r to that demonstrated by the benzodiazepines (see Jurna, 1985). However, Loscher (1989) pointed out that a f a c i l i t a t i o n of GABAergic transmission i s only observed at VPA concentrations that are fa r greater than those required for anticonvulsant e f f e c t s (see also McLean & Macdonald, 1986). A l t e r n a t i v e l y , the anticonvulsant e f f e c t of VPA has been attributed to a reduction i n the release of the putative neuromodulator gamma-hydroxybutyrate (e.g., Vayer, Cash, & Maitre, 1988), a product of 54 GABA breakdown that has been shown to e l i c i t seizures i n both cats and rats (see Vayer et a l . , 1988). F i n a l l y , VPA's anticonvulsant e f f e c t may also be related to i t s voltage- and use-dependent i n h i b i t o r y e f f e c t on sodium-dependent, high-frequency r e p e t i t i v e f i r i n g of cultured c o r t i c a l neurons (McLean & Macdonald, 1986). Although the basis for t h i s i n h i b i t o r y e f f e c t i s unknown, McLean and Macdonald (1986) suggested that i t may be related to VPA's a b i l i t y to decrease sodium conductances. A l t e r n a t i v e l y , Morre, Keane, Vernieres, Simiand, and Ronucci (1984) suggested that VPA's anticonvulsant e f f e c t i s due to i t s a b i l i t y to increase potassium currents, although t h i s e f f e c t i s only apparent at VPA concentrations that are at least an order of magnitude greater than those required to produce an anticonvulsant e f f e c t (Johnston, 1984). Anticonvulsant A c t i v i t y VPA displays a broad spectrum of moderate a c t i v i t y against a number of d i f f e r e n t types of convulsions (see Loscher, 1985). For example, Tulloch, Walter, Howe, and Howe (1982) reported that VPA (200 mg/kg to 800 mg/kg, IP) had an anticonvulsant e f f e c t on maximal electroshock, pentylenetetrazol-induced, and kindled convulsions i n r a t s . VPA has also been shown to attenuate audiogenic convulsions i n r a t s (Consroe et a l . , 1980) and mice (e.g., Anlezark, Horton, Meldrum, & Sawaya, 1976). Meldrum, Anlezark, Ashton, Horton, and Sawaya (1977) found that VPA attenuated p h o t i c a l l y induced convulsions i n the baboon Papio papio at doses of 100 mg/kg to 200 mg/kg, IV. And VPA (150 mg/kg to 400 mg/kg, SC) has been shown to reduce the 55 convulsions e l i c i t e d by b i c u c u l l i n e , picrotoxin, and strychnine (see Kupferberg, 1980). C l i n i c a l l y , VPA i s the drug of choice for the treatment of mixed seizure disorders. I t i s most e f f e c t i v e against generalized convulsions but also used to control p a r t i a l seizures (Engel, 1989). VPA i s especially useful when sedative side-e f f e c t s are undesirable becaue i t produces l i t t l e sedation at therapeutic doses (Engel, 1989); however, the remote p o s s i b i l i t y of hepatotoxic side e f f e c t s l i m i t s i t s widespread acceptance as a drug of f i r s t choice i n the treatment of the e p i l e p s i e s (see Eadie, 1985; Engel, 1989). Tolerance to the Anticonvulsant Effects of VPA Although c l i n i c a l reports of tolerance to VPA's anticonvulsant e f f e c t have existed since the early 1970's (see Frey, 1985) the phenomenon has been d i f f i c u l t to demonstrate i n the laboratory. Lockard, Levy, Congdon, DuCharme, and Patel (1977) found that a low dose of VPA (administered IV at a rate that maintained plasma concentrations at between 50 micrograms/ml to 150 micrograms/ml for up to 6 weeks) frequently f a i l e d to provide a sustained anticonvulsant e f f e c t on alumina-gel-induced seizures i n the baboon; however, i t i s not c l e a r whether t h i s loss of e f f i c a c y was due to fluctuations i n plasma levels of the drug or to the development of genuine tolerance. Paule and Killam (1986) subsequently reported the development of tolerance to VPA's anticonvulsant e f f e c t (increasing doses from 7.5 mg/kg to 240 mg/kg per day, PO, spread over three equal doses, for up to 12 weeks) on photic seizures i n P. papio; they found that the expression of tolerance to VPA's anticonvulsant e f f e c t could not be prevented by increasing the treatment dose. There have been several c l i n i c a l reports of tolerance to VPA's anticonvulsant e f f e c t . For example, Bruni and A l b r i g h t (1983) reported that tolerance rea d i l y to VPA's anticonvulsant e f f e c t on complex p a r t i a l seizures; they found that the development of tolerance proceeded most re a d i l y i n patients with the highest frequency of seizures p r i o r to the i n i t i a t i o n of treatment. S i m i l a r l y , Meinardi, Smits, and van den Brink (1986) reported a rapid loss i n VPA's ef f i c a c y i n patients with i n t r a c t a b l e epilepsy; however, these authors questioned whether the loss of e f f i c a c y was actually due to the development of tolerance or to an exacerbation of the patients' condition. These p o s i t i v e demonstrations of tolerance to VPA's anticonvulsant e f f e c t s are tempered by a number of reported f a i l u r e s to observe such tolerance. For example, Young, Lewis, Harris, J a r r o t t , and Vadja (1987) found no evidence of tolerance to VPA's anticonvulsant effects (200 mg/kg, IP, twice a day) on kindled convulsions i n rats after 12 treatment days. S i m i l a r l y , Gent, Bently, Feely, and Haigh (1986) f a i l e d to demonstrate the development of tolerance to VPA's anticonvulsant e f f e c t on pentylenetetrazol-induced convulsions, and Loscher (1986b) found no evidence of tolerance to VPA's anticonvulsant e f f e c t s (600 mg/kg/day, i n drinking water, or continuous administration with osmotic minipumps) on electroshock convulsions i n mice or 57 audiogenic convulsions i n g e r b i l s . Summary: Tolerance to Anticonvulsant Drug E f f e c t s The development of tolerance to the anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs such as CBZ, DZP, and VPA has been c l e a r l y and repeatedly demonstrated. However, t h i s work has been guided by the conventional premise that drug tolerance i s a pharmacologic phenomenon, sensitive only to the manipulation of variables associated with drug administration: for example, the s i z e of the treatment dose (e.g., Killam et a l . , 1973), the interdrug i n t e r v a l (e.g., Frey, 1987), and the length of the treatment period (e.g., Baltzer et a l . , 1980). The p o s s i b i l i t y that convulsive a c t i v i t y during periods of drug exposure could play an important role i n the development of tolerance to the anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs has not been entertained by the researchers i n t h i s area. As described i n the next two sections of t h i s Introduction, t h i s oversight provided the i n i t i a l motivation for the present thesis. 6. General Rationale There were three general reasons for my i n i t i a l i n t e r e s t i n the characterization of contingent and pharmacologic tolerance to the anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs. F i r s t , such int e g r a t i v e work i s necessary to further our understanding of these two phenomenologically d i s t i n c t forms of tolerance. As Goudie and Emmett-Oglesby (1989) noted: 58 " I t i s important to know how the r e l a t i v e s i g n i f i c a n c e of various mechanisms that produce tolerance...may be modified by basic pharmacological variables, on the one hand (such as the drug studied, drug dose, frequency of dosing, duration of dosing, route of dosing, and so on) and on the other hand, by behavioral variables (such as the s p e c i f i c behaviors studied and the environmental and behavioral contexts within which they are studied)." (p. 5). The present thesis was motivated by a s i m i l a r desire for a better understanding of the r e l a t i o n between contingent and pharmacologic drug tolerance. The second reason for my interest i n the phenomena of contingent and pharmacologic tolerance to the anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs stems from the c l i n i c a l implications of such knowledge. For example, Haigh and Feely (1988) have pointed out that the wide therapeutic window, broad spectrum of a c t i v i t y , and remarkable lack of s i d e - e f f e c t s combine to make the benzodiazepines an ideal pharmacologic treatment for the e p i l e p s i e s ; however, these advantages are outweighed by the fa c t that tolerance rapidly develops to the anticonvulsant e f f e c t s of these drugs. Accordingly, a better understanding of the factors involved i n the development of tolerance to a n t i e p i l e p t i c drugs might a s s i s t the development of new a n t i e p i l e p t i c compounds that are less prone to the development of tolerance, as well as the more informed p r e s c r i p t i o n of e x i s t i n g drugs so that the problems associated with the development of tolerance are minimized (see also Baltzer et a l . , 1980; Koella & Meinardi, 1986). The t h i r d reason for my interest i n the r e l a t i o n between contingent and pharmacologic tolerance to the anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs arose from the fact that the ki n d l i n g paradigm that we have developed to study tolerance to anticonvulsant drug effects i s well suited to studying the r e l a t i o n between contingent and pharmacologic t o l e r a n c e — i n fact, my colleagues and I had already used the paradigm to demonstrate both contingent and pharmacologic tolerance to the anticonvulsant e f f e c t s of ethanol (Mana, Pinel, & Le, 1988; Mana, Le, Kalant, & Pi n e l , i n preparation). The next section describes the k i n d l i n g phenomenon i n general as well as the general methodology for a l l f i v e experiments i n the present thesis. 60 II. GENERAL METHODS The Kindling Paradigm Periodic e l e c t r i c a l stimulation of the rat amygdala at i n t e n s i t i e s capable of e l i c i t i n g afterdischarges but no overt convulsions leads to a progressive i n t e n s i f i c a t i o n of the afterdischarges and to t h e i r convulsive e f f e c t s ; a f t e r approximately 15 such stimulations each rat w i l l respond to each stimulation with a f u l l y generalized electrographic and motor seizure (see Goddard, 1967; Goddard, Mclntyre & Leech, 1969; Racine, 1972a; 1972b; McNamara, 1988). This phenomenon i s referred to as k i n d l i n g (Goddard et a l . , 1969); i t has subsequently been shown that kindling can be e l i c i t e d by both chemical and e l e c t r i c a l stimulation of a wide var i e t y of brain s i t e s i n many d i f f e r e n t species (for reviews, see McNamara, 1988; Racine & Burnham, 1984) although the majority of k i n d l i n g experiments have involved e l e c t r i c a l stimulation of the amygdala i n r a t s (Racine & Burnham, 1984). The k i n d l i n g model has emerged as a useful t o o l for assessing the anticonvulsant effects of a wide var i e t y of drugs (e.g., Albertson, Peterson, & Stark, 1980; Albright & Burnham, 1980). I t has been shown that c l i n i c a l l y prescribed a n t i e p i l e p t i c drugs may attenuate the i n t e n s i t y and duration of kindled convulsions and of the underlying afterdischarges. I t i s important to recognize that the various electrographic and behavioral indices of the kindled seizure are not a l l affected i n the same way by every anticonvulsant drug; Racine and Burnham (1984) have argued that the varying s e n s i t i v i t y of d i f f e r e n t aspects of the kindled seizure to anticonvulsants may model the s e n s i t i v i t y of d i f f e r e n t types of human epilepsy to these same drugs. Given the u t i l i t y of the kindling paradigm i n the study of the anticonvulsant effects of a wide variety of drugs (e.g., Al b r i g h t & Burnham, 1980; Ashton & Wauquier, 1979; P i n e l , 1983; Schmidt, 1987), i t i s surprising that i t has only recently been adopted to study the development of tolerance to anticonvulsant drug e f f e c t s . Kindled convulsions have three important advantages over the experimental convulsions that have t r a d i t i o n a l l y been employed to assess the development of tolerance to anticonvulsant d r u g s — s p e c i f i c a l l y , over convulsions induced by maximal electroshock stimulation (MES) or pentylenetetrazol (PTZ). Both MES and PTZ induce convulsions that are variable i n form and duration, are d i f f i c u l t to measure, and i n the case of PTZ, often associated with subject injury or f a t a l i t y (e.g., Swinyard, 1980; Voskuyl et a l , 1986). This l a t t e r problem i s p a r t i c u l a r l y serious i n studies of tolerance i n which anticonvulsant effects are repeatedly assessed i n the same subjects, because any systematic change i n the apparent anticonvulsant action of a drug i s always confounded by the progressive d e b i l i t a t i o n and a t t r i t i o n of those subjects experiencing the most severe convulsions. In contrast, kindled rats remain healthy and easy to handle for the duration of an experiment, and f a t a l i t i e s are rare. Moreover, i n well-kindled r a t s i t i s possible to e l i c i t convulsions that vary l i t t l e from subject to subject i n either form or duration, and baselines can be established i n individual animals that display almost no f l u c t u a t i o n from stimulation to stimulation (see Pi n e l , P h i l l i p s , & MacNeil, 1973). The importance of such r e l i a b i l i t y i n the study of tolerance to the effects of anticonvulsant drugs i s obvious, and the stereotyped nature of kindled motor convulsions makes i t easy to measure t h e i r intensity. The following sections describe the kin d l i n g paradigm that we have developed to study anticonvulsant drug e f f e c t s and other methodology common to each of the experiments i n the present t h e s i s . Any variations to t h i s general methodology are described i n the methods sections of the respective experiments. Subjects. The subjects i n a l l of the experiments were male hooded rats (Charles River, Canada), weighing 350 g to 450 g at the time of surgery. The rats were i n d i v i d u a l l y housed i n wire mesh cages with continuous access to food and water. Each experiment was conducted at approximately the same time (+/- 2 hr) during the l i g h t phase of the 12/12 hr light/dark cycle. Surgical Procedure. A single bipolar electrode ( P l a s t i c Products MS-303-2) was implanted in the l e f t basolateral amygdaloid nucleus of each rat, 2.8 mm posterior to bregma, 5 mm l a t e r a l and 8.5 mm ventral to the s k u l l surface at bregma, with the i n c i s o r bar set at -3.3 (coordinates taken from Paxinos & Watson, 1982). Tetracycline was sprinkled on the i n c i s i o n before suturing, and i t was added to the drinking water for 7 days a f t e r surgery. 63 Kindling. The kindling phase of each study began at least 7 days a f t e r surgery. During the kindling phase, each rat was stimulated (1 sec, 60 Hz, 400 microamps) three times per day, 5 days per week, for 3 weeks. There were at least 2 hr between consecutive stimulations. Prior to each stimulation, the stimulation lead was connected and the subject was placed i n a 58 X 58 X 25 cm opaque p l a s t i c chamber containing a 5-cm layer of San-i-cel bedding material. The stimulation was delivered immediately, and the rat was returned to i t s home cage once the behavioral signs of convulsive a c t i v i t y ceased. As i s usual, the only behavioral e f f e c t of the i n i t i a l stimulations was a b r i e f period of behavioral arrest, but by the end of the k i n d l i n g phase each stimulation e l i c i t e d a generalized c l o n i c convulsion characterized i n sequence by f a c i a l clonus, forelimb clonus, repeated bouts of rearing and a loss of equilibrium (see P i n e l & Rovner, 1978; Racine, 1972b). Stimulation-Baseline Phase. In each study, the stimulation-baseline phase began 48 hr after the completion of the k i n d l i n g phase. During the stimulation-baseline phase, each r a t received four amygdaloid stimulations, one every 48 hr. The duration of forelimb clonus e l i c i t e d by each stimulation was recorded as the dependent measure. Drug-Baseline T r i a l . The drug-baseline t r i a l occurred 48 hr a f t e r the fourth and f i n a l stimulation-baseline t r i a l . On the drug-baseline t r i a l of each experiment, each subject received an IP i n j e c t i o n of one of the drugs (either CBZ, DZP, or VPA) 1 hr 64 p r i o r to the delivery of the convulsive stimulation. The duration of forelimb clonus e l i c i t e d by the stimulation was recorded for each subject. This was done to determine each subject's i n i t i a l s e n s i t i v i t y to the drug's anticonvulsant e f f e c t . Subjects not displaying at least an 80% reduction i n the duration of forelimb clonus from the fourth stimulation-baseline t r i a l to the drug-baseline t r i a l were dropped from the study at t h i s point. After the drug-baseline t r i a l , the remaining rats i n each experiment were assigned to groups i n such a way that the mean durations of forelimb clonus for each group on both the fourth stimulation-baseline t r i a l and the drug-baseline t r i a l were approximately equal. Tolerance-Development T r i a l s . Because the tolerance-development phase varied from experiment to experiment, a description of the tolerance-development phase i s included i n the methodology section of each experiment. Tolerance-Test T r i a l . The methodology for the tolerance-test t r i a l was i d e n t i c a l to that described for the drug-baseline phase; each subject received an IP i n j e c t i o n of the appropriate drug (either CBZ, DZP, or VPA) 1 hr p r i o r to the delivery of the convulsive stimulation. The duration of forelimb clonus e l i c i t e d by the t e s t stimulation was recorded for each subject. Histology. At the end of each experiment, a l l the subjects were s a c r i f i c e d i n a C02 chamber according to Canada Council on Animal Care guidelines and t h e i r brains were removed. A representative sample (20%) of the brains from each experiment were sectioned at 60 microns and stained with luxol blue to permit h i s t o l o g i c a l v e r i f i c a t i o n of electrode s i t e s (see Figure 3). A l l electrode s i t e s were located within the amygdaloid complex, with the majority l y i n g within the basolateral amygdaloid nucleus (see Figure 3). S t a t i s t i c a l Analyses. Unless otherwise noted, the data from the drug-baseline t r i a l and the tolerance-test t r i a l for each experiment were analyzed i n a between-within, repeated-measures analysis of variance (ANOVA). Si g n i f i c a n t interactions were further assessed with tests of simple main e f f e c t s (Kirk, 1968), so that the contributions of the respective between- and within-group factors to the interaction could be determined. When there were more than two factors involved in a signfic a n t t e s t of main e f f e c t s , Neuman-Keuls posthoc comparisons were performed to determine the contribution of each factor to the s i g n i f i c a n c e . Alpha was maintained at the .05 l e v e l for the repeated-measures ANOVAs and the tests of simple main eff e c t s , as well as the Neuman-Keuls post hoc comparisons. 66 Figure 3. Representative electrode placements for the rats in each of the f i v e experiments from the present th e s i s . Although h i s t o l o g i c a l v e r i f i c a t i o n was completed on a t o t a l of 65 subjects (2 0% of the rats that completed one of the f i v e experiments), the placements for only 50 rats are presented so that t h e i r r e l a t i v e placements can be established. The remaining placements were within the boundaries of the placements that are presented, i n the sections between -1.0 mm and -2.0 mm from bregma. o o I CM 7 68 II I . Experiment 1 In Experiment 1, we used the kindling paradigm developed by P i n e l and h i s colleagues to study the development of tolerance to the anticonvulsant e f f e c t s of three widely prescribed a n t i e p i l e p t i c s : CBZ, DZP, and VPA. These drugs were chosen for four reasons. F i r s t , each of the three drugs i s e f f e c t i v e against generalized t o n i c - c l o n i c e p i l e p t i c seizures i n humans (see Schmutz, 1985; Loscher, 1985; Haigh & Feeley, 1988), which are c l o s e l y modelled by generalized kindled convulsions i n rats (see Racine & Burnham, 1984) . Second, each has been shown to exert a r e l i a b l e anticonvulsant e f f e c t on kindled convulsions (e.g., Albertson, Peterson & Stark, 1980; Albright & Burnham, 1980). Third, each drug i s representative of a d i f f e r e n t family of a n t i e p i l e p t i c with d i f f e r e n t putative mechanisms of action. And fourth, there i s a marked difference i n the prevalence of previous reports that tolerance w i l l develop to the anticonvulsant e f f e c t s of these drugs: There have been many experimental and c l i n i c a l reports of tolerance to DZP's anticonvulsant e f f e c t , r e l a t i v e l y fewer reports of tolerance to CBZ's anticonvulsant e f f e c t , and so few reports of tolerance to VPA's anticonvulsant e f f e c t that i t s existence has recently been questioned (Haigh & Feely, 1988). Previous attempts to demonstrate the development of tolerance to the anticonvulsant effects of CBZ, DZP, and VPA on kindled convulsions have produced equivocal r e s u l t s . For example, Loscher and Schwark (1985) reported only a small attenuation i n the anticonvulsant e f f e c t of DZP (5 mg/kg, IP, administered every 8 hr for 10 days); they found that the convulsions e l i c i t e d on the f i n a l test t r i a l were generally l i m i t e d to bouts of f a c i a l clonus and head bobbing ( i . e . , Class 2 convulsions according to Racine's [1972b] scale). S i m i l a r l y , Honack and Loscher's (1988) report of tolerance to CBZ's anticonvulsant e f f e c t i s tempered by a lack of tolerance to CBZ's e f f e c t s on the mean afterdischarge duration and by the f a c t that the drug maintained an inconsistent but obvious anticonvulsant e f f e c t on a l l dependent measures throughout the tolerance-development phase—even on the l a s t t r i a l before the f i n a l t e s t t r i a l . And equally equivocal results provided the basis for Young et a l . ' s (1987) claim that tolerance does not develop to VPA's anticonvulsant e f f e c t on kindled convulsions; although there was l i t t l e evidence of tolerance on the f i n a l t e s t t r i a l there was evidence of a substantial decrease i n VPA's anticonvulsant e f f e c t during the tolerance-development phase. The equivocal nature of the previous reports of tolerance to the anticonvulsant effects of CBZ, DZP, and VPA on kindled convulsions leave unanswered the question "Does tolerance develop to the anticonvulsant effects of these drugs on kindled convulsions?". Given the importance of t h i s question to the remaining experiments i n the present thesis, the purpose of Experiment 1 was to determine whether tolerance develops to the 70 anticonvulsant e f f e c t of CBZ, DZP, and VPA on kindled convulsions i n the r a t . Methods Subjects. The subjects were 74 male Long Evans rats, weighing between 350 g and 400 g at the time of surgery and between 550 g and 650 g at the completion of the experiment. Drugs. A l l drugs were administered in t r a p e r i t o n e a l l y , i n a 2% Tween-80/ i s o s a l i n e vehicle, at a volume of 4 ml/kg. The DZP (2 mg/kg; purchased as Valium, i n ampoule form, from Hoffman-LaRoche) was injected i n solution; both VPA (250 mg/kg; purchased as Depakote from Abbott Laboratories) and CBZ (75 mg/kg; purchased as Tegretol from Geigy) were injected as suspensions. These doses were selected on the basis of p i l o t data which indicated that they represented an ED(90) for suppression of forelimb clonus i n kindled rats following an acute i n j e c t i o n . Stimulation-Baseline Phase. The stimulation-baseline phase began 48 hr a f t e r the completion of the kindling phase. Rats that did not demonstrate at least 20 sec of forelimb clonus on the l a s t stimulation-baseline t r i a l were not studied further (n = 9). Immediately aft e r the stimulation-baseline phase, the subjects were assigned to one of three d i f f e r e n t g r o u p s — e i t h e r a CBZ group, a DZP group, or a VPA g r o u p — i n such a way that the average duration of forelimb clonus for each group on the l a s t stimulation-baseline t r i a l was approximately equal. Drug-Baseline T r i a l . On the drug-baseline t r i a l , the rats from one of the groups received DZP 1 hr before the scheduled convulsive stimulation; those from the second group received VPA and those from the remaining group received CBZ. Rats not showing at least an 80% decrease i n forelimb clonus duration on the drug-baseline t r i a l r e l a t i v e to the l a s t t r i a l of the stimulation-baseline phase were not studied further; two rats r e c e i v i n g VPA, two rats receiving DZP, and one rat receiving CBZ did not meet t h i s c r i t e r i o n for inclusion. Thus, 60 rats remained i n the experiment at the st a r t of the tolerance-development phase. The remaining rats from each of the three drug groups were then assigned to one of two c o n d i t i o n s — e i t h e r a drug condition or a vehi c l e - c o n t r o l c o n d i t i o n — s o that the mean forelimb clonus durations on both the l a s t t r i a l of the stimulation-baseline phase and the drug-baseline t r i a l were approximately equal for the r e s u l t i n g s i x groups. Tolerance-Development Phase. The tolerance-development t r i a l s began 48 hr a f t e r the drug-baseline t r i a l . During each of the ten tolerance-development t r i a l s , each rat was removed from i t s home cage, weighed, and the appropriate dose of drug (DZP, n = 12; VPA, n = 12; or CBZ, n = 12) or vehicle (DZP-Control, n = 8; VPA-Control, n = 8; CBZ-Control, n = 8) was administered 1 hr before the scheduled convulsive stimulation. Tolerance-Test T r i a l . The tolerance-test t r i a l occurred 48 hr af t e r the l a s t tolerance-development t r i a l and was i d e n t i c a l to 72 the drug-baseline t r i a l ; that i s , the rats i n each drug group and i t s respective control group received the appropriate drug 1 hr before a convulsive stimulation. S t a t i s t i c s . Separate 2 (Groups) X 2 (Trials) between-within, repeated-measures analyses of variance were used to analyze the data from the drug-baseline t r i a l and the tolerance-test t r i a l f o r each of the three drugs. Analyses of simple main e f f e c t s were used to assess the significance of the d i f f e r e n t between-and within-groups factors for each s i g n i f i c a n t i n t e r a c t i o n . Results Tolerance developed to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA i n the three drug groups but not i n the three veh i c l e - c o n t r o l groups. As i l l u s t r a t e d i n Figure 4 (CBZ), Figure 5 (DZP), and Figure 6 (VPA), each of the three drugs almost t o t a l l y suppressed forelimb clonus i n each of the groups on the drug-baseline t r i a l . In contrast, on the tolerance-test t r i a l the drugs had l i t t l e anticonvulsant e f f e c t on the rats from each of the three drug groups, whereas the rats from the three vehicle control groups displayed no tolerance whatsoever. The s t a t i s t i c a l analyses confirmed the s i g n i f i c a n c e of the e f f e c t s summarized in Figure 4, Figure 5, and Figure 6. The analyses of variance revealed a s i g n i f i c a n t group X t r i a l i n t e r a c t i o n for each of the three drugs: CBZ, F (1, 18) = 14.83; DZP, F (1,18) = 21.27; VPA, F (1,18) = 11.48; a l l p_'s < .01. Subsequent analysis of each s i g n i f i c a n t i n t e r a c t i o n using 73 Figure 4. Tolerance to the anticonvulsant e f f e c t s of CBZ. On the drug-baseline t r i a l , CBZ exerted a potent anticonvulsant e f f e c t on a l l of the subjects. However, on the tolerance-test t r i a l the rats from the CBZ group displayed substantial tolerance to the anticonvulsant effects of the drug. In contrast, there was no evidence of tolerance in any of the CBZ-Control subjects. MEAN FORELIMB CLONUS DURATION (sec) 75 Figure 5. Tolerance to the anticonvulsant e f f e c t s of DZP. On the drug-baseline t r i a l , DZP exerted a potent anticonvulsant e f f e c t on a l l of the subjects. However, on the tolerance-test t r i a l the rats from the DZP group displayed substantial tolerance to the anticonvulsant effects of the drug. In contrast, there was no evidence of tolerance i n any of the DZP-Control subjects. 60-1 o Z O % on ZD Q (/) Z s o 00 _J LJ or P < UJ "2 DRUG DRUG 1 2 3 4 5 FREE BASE BASE *1 TOLERANCE 6 7 8 9 DEVELOPMENT | — 1 10 TOLERANCE TEST 77 Figure 6. Tolerance to the anticonvulsant e f f e c t s of VPA. On the drug-baseline t r i a l , VPA exerted a potent anticonvulsant e f f e c t on a l l of the subjects. However, on the tolerance-test t r i a l the rats from the VPA group displayed substantial tolerance to the anticonvulsant effects of the drug. In contrast, there was no evidence of tolerance i n any of the VPA-Control subjects. 60-. analyses of simple main effects revealed a s i g n i f i c a n t increase i n the duration of forelimb clonus between the drug-baseline t r i a l and the tolerance-test t r i a l for the rats from the CBZ group (F (1,11) = 21.59, p < .001), the DZP group (F (1,11) = 32, = .71, p_ < .001), and the VPA group (F (1,11) = 17.54, p < .01). In contrast, tests of simple main e f f e c t s revealed no s i g n i f i c a n t differences i n forelimb clonus duration between the drug-baseline t r i a l and the tolerance-test t r i a l for the rats from the three respective control conditions ( a l l p_'s > .40). F i n a l l y , t e sts of simple main effects indicated that the rats from the three drug groups displayed s i g n f i c a n t l y more forelimb clonus on the tolerance-test t r i a l than the rats from the three respective control conditions (CBZ: F (1,18) = 18.53; DZP: F (1,18) = 28.19; VPA: F (1,18) = 15.31; a l l p_'s < .01). Discussion The present demonstrations of tolerance to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA on kindled convulsions i n the rat are important for two reasons. F i r s t , they confirm and strengthen e a r l i e r reports of tolerance to the anticonvulsant e f f e c t s of DZP (Loscher & Schwark, 1985) and CBZ (Honack & Loscher, 1989) on kindled convulsions i n the r a t , and they provide conclusive evidence of tolerance to VPA's anticonvulsant e f f e c t on kindled convulsions. Second, the present r e s u l t s provide further evidence of the u t i l i t y of the k i n d l i n g model as a useful tool in the study of tolerance to the 80 anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs. I t i s not clear from the present experiment whether a metabolic or a functional change underlies the development of tolerance to the anticonvulsant effects of CBZ, DZP, and VPA. HSnack and Loscher (1988) suggested that the development of tolerance to CBZ's anticonvulsant e f f e c t was a t t r i b u t a b l e to both a d i s p o s i t i o n a l and a functional change; rats that had received CBZ on a chronic basis were able to metabolize the drug s i g n i f i c a n t l y faster than drug-naive rats (suggesting a d i s p o s i t i o n a l change), but the drug-experienced rats were also less affected by CBZ than drug-naive rats experiencing equal plasma concentrations of the drug (supporting a functional change). Although the resolution of t h i s question i s beyond the scope of t h i s paper, the results of Experiment 2 support the notion that a functional change underlies the development of tolerance to the anticonvulsant effects of a l l three drugs. Perhaps the most interesting aspect of the present r e s u l t s i s the f a c t that they stand i n sharp contrast to e a r l i e r e f f o r t s to study the development of tolerance to the anticonvulsant e f f e c t s of CBZ (Honack & Loscher, 1988), DZP (Loscher and Schwark, 1985), and VPA (Young et a l . , 1987) on kindled convulsions. The magnitude of tolerance that Loscher and h i s colleagues reported to the anticonvulsant e f f e c t s of CBZ (Honack & Loscher, 1988) and DZP (Loscher & Schwark, 1985) on kindled convulsions was markedly less than that demonstrated i n the 81 present experiment; tolerance developed to the anticonvulsant e f f e c t s of these drugs on only half of the dependent measures they recorded and i t was not substantial even on these measures. Furthermore, Young et a l . (1987) found no evidence of tolerance to the anticonvulsant e f f e c t s of VPA (2 00 mg/kg, IP, administered every 12 hr for 14 days) on kindled convulsions i n r a t s . There are at least three plausible explanations for the differences between the results of the present experiment and those reported by Loscher and Schwark (1985) , Young et a l . (1987), and Honack and Loscher (1988). The f i r s t reason concerns obvious differences i n the drug-treatment regimens employed i n the respective experiments. The doses of CBZ, DZP, or VPA administered i n the present experiments were considerably smaller, and/or administered less frequently, than those administered by Loscher and Schwark (1985; they administered DZP 5 mg/kg, IP, every 8 hr, i n comparison to 2 mg/kg, IP, every 48 hr, i n the present study), Young et a l . (1987; they administered VPA 200 mg/kg, IP, every 12 hr, i n comparison to 250 mg/kg, IP, every 48 hr i n the present study), or Honack and Loscher (1988; they administered CBZ at a dose of 30 mg/kg, IP, every 8 hr, i n comparison to 75 mg/kg, IP, every 48 hr, i n the present study). Although the treatment strategy employed by Loscher and Schwark (1985), Young et a l . (1987), and Honack and Loscher (1988) makes sense from the t r a d i t i o n a l perspective that tolerance i s more l i k e l y to develop when a high treatment dose i s used, i t i s 82 accompanied by a l i a b i l i t y that i s often neglected and may account for the r e l a t i v e l y small amount of tolerance reported i n t h e i r respective papers; the use of a high treatment dose may f a c i l i t a t e the development of tolerance while obscuring i t s detection, because an accumulation of the drug produces higher plasma l e v e l s , and thus a greater drug e f f e c t , than would be produced by an acute i n j e c t i o n of the same dose (see Kalant et a l . , 1971). A second possible explanation for the discrepancy between the present r e s u l t s and those reported by Loscher and Schwark (1985), Young et a l . (1987), and Honack and Loscher (1988), concerns the differences i n the kindling protocols used i n the various studies. In the present experiment, every subject had demonstrated at least 3 0 class 5 convulsions (according to Pinel and Rovner's (1978) modification of Racine's (1972b) r a t i n g scale) before the tolerance-development phase began. In contrast, Loscher and Schwark (1985) and Honack and Loscher (1988) began t h e i r experiments after t h e i r subjects had demonstrated just 10 class 5 kindled convulsions; and Young et a l . (1987) consider 2 consecutive stage 5 convulsions to represent a f u l l y kindled state (although i t i s not cl e a r how many c l a s s 5 convulsions the rats in t h e i r study had demonstrated before t h e i r experiments began). Thus, i t i s possible that the p h y s i o l o g i c a l changes underlying the kindling process were more fir m l y established i n the rats from our experiments than i n the 83 kindled rats used by Loscher and Schwark (1985), Honack and Loscher (1988), or Young et a l . (1987) because they administered fewer c o n v u l s i o n - e l i c i t i n g stimulations before drug treatment began. As a r e s u l t , the convulsions i n the kindled rats employed i n the present experiments may have been more r e s i s t a n t to the anticonvulsant e f f e c t s of DZP or VPA and therefore less l i k e l y to be e f f e c t i v e l y controlled by these drugs. A t h i r d possible reason for the differences between the present experiment and those reported by Loscher and Schwark (1985), Honack and Loscher (1988), and Young et a l . (1987) i s based upon our e a r l i e r observation that the development of tolerance to ethanol*s anticonvulsant e f f e c t on kindled convulsions i s often f a c i l i t a t e d by the administration of convulsive stimulation during periods of drug exposure (e.g., P i n e l et a l . , 1983; Pinel & Mana, 1986). In the present experiment, each drug i n j e c t i o n was followed 1 hr l a t e r by a convulsive stimulation; i n contrast, t h i s condition was present i n only h a l f of the treatment t r i a l s in the work reported by Loscher and Schwark (1985) and Young et a l . (1987). Thus, the differences between the present results and those reported by Loscher and Schwark (1985) and Young et a l . (1987) may r e f l e c t the f a c t that the r e l a t i o n between drug exposure and convulsive stimulation plays an important role i n the development of tolerance to the anticonvulsant effects of c l i n i c a l l y relevant a n t i e p i l e p t i c drugs. This hypothesis was tested i n Experiment 2. 84 IV. Experiment 2 The purpose of Experiment 2 was to determine whether convulsive stimulation during periods of drug exposure would f a c i l i t a t e the development of tolerance to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA. As noted e a r l i e r , we (e.g., Pinel et a l . , 1983; 1985; 1989) have demonstrated that convulsive stimulation during periods of drug exposure plays an important r o l e i n the development of tolerance to ethanol's anticonvulsant e f f e c t . However, there have been no reported attempts to determine whether convulsions have a similar e f f e c t on the development of tolerance to the anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs. Accordingly, the purpose of Experiment 2 was to determine whether the administration of convulsive stimulation during periods of drug exposure can influence the development of tolerance to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA. Methods Subjects. The subjects were 117 male Long Evans rats, weighing 350 g to 400 g at the time of surgery and 550 g to 650 g at the time of the tolerance-development phase. Stimulation-Baseline T r i a l . A t o t a l of 8 rats were removed from the experiment because they did not display at least 20 sec of forelimb clonus on the stimulation-baseline t r i a l . Following the stimulation-baseline t r i a l , the subjects were assigned to one of three drug groups—a CBZ group, a DZP group, or a VPA group—so 85 that the average durations of forelimb clonus for of the three groups on the stimulation-baseline t r i a l were approximately equal. Drugs. The DZP and CBZ were administered IP, i n a 2% Tween-80/isosaline vehicle, at a volume of 4 ml/kg. The DZP (2 mg/kg) was injected i n solution, whereas CBZ (70 mg/kg) was injected as a suspension. In contrast to Experiment 1, VPA (250 mg/kg) was administered by gavage i n a 2% Tween 80/isosaline vehicle, at a volume of 4 ml/kg. This change i n protocol was made because gavage administration caused less d i s t r e s s i n the VPA subjects than did the IP injections used i n Experiment 1. Drug-Baseline T r i a l . On the drug-baseline t r i a l , one group of rats received CBZ; one group received DZP; and the f i n a l group received VPA. The appropriate drug was administered to each rat 1 hr before the convulsive stimulation was delivered, and the duration of forelimb clonus recorded. Rats not showing at least an 80% decrease i n the duration of forelimb clonus duration displayed on the drug-baseline t r i a l as compared to the l a s t t r i a l of the stimulation-baseline phase were rejected from the study (10 rats receiving VPA; 4 rats receiving DZP; and 4 rats receiving CBZ). Thus, 91 rats remained i n the three drug groups at the s t a r t of the tolerance-development phase. The remaining rats from each drug group were then assigned to one of three c o n d i t i o n s — e i t h e r a drug-before-stimulation condition, a drug-after-stimulation condition, or a v e h i c l e -86 control c o n d i t i o n — s o that the mean forelimb clonus durations on both the l a s t t r i a l of the stimulation-baseline phase and the drug-baseline t r i a l were approximately equal for the r e s u l t i n g nine groups. Tolerance-Development T r i a l s . The tolerance-development t r i a l s began 48 hr af t e r the drug-baseline t r i a l . There were a t o t a l of ten b i d a i l y (one every 48 hr) tolerance-development t r i a l s i n each experiment. On each tolerance-development t r i a l , the rats from the drug-before-stimulation condition continued to received CBZ (CBZ-Before-Stimulation, n = 11), DZP (DZP-Before-Stimulation, n = 11), or VPA (VPA-Before-Stimulation, n = 10) 1 hr p r i o r to each stimulation. The rats from the drug-after-stimulation condition (CBZ-After-Stimulation, n = 10; DZP-After-Stimulation, n = 12; or VPA-After-Stimulation, n = 10), received the same dose of the appropriate drug 1 hr afte r each stimulation. And the rats from the vehicle control condition (CBZ-Control, n = 10; DZP-Control, n = 8; or VPA-Control, n = 9) received a vehicle i n j e c t i o n 1 hr before or 1 hr a f t e r each stimulation; because the vehicle injections had no e f f e c t on the duration of forelimb clonus regardless of whether they were administered before or after the convulsive stimulation, these groups were combined to create a single control group for each drug. Tolerance-Test T r i a l . The tolerance-test t r i a l occurred 48 hr af t e r the l a s t tolerance-development t r i a l and followed the same 87 protocol described for the drug-baseline t r i a l ; that i s , each r a t received the appropriate drug 1 hr before convulsive stimulation and the duration of forelimb clonus was recorded. S t a t i s t i c s . Separate 3 (Groups) X 2 (Trials) between-within factor, repeated-measures analysis of variance were used to analyze the data from the drug-baseline t r i a l and the tolerance-t e s t t r i a l for each of the three drugs. Tests of simple main e f f e c t s were used to assess the contribution of the respective between- and within-group factors to each s i g n f i c a n t i n t e r a c t i o n . Results The r e s u l t s c l e a r l y demonstrate that convulsive stimulation can play a key r o l e i n the development of tolerance to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA on amygdaloid-kindled convulsions i n the r a t . The test doses of CBZ (Figure 7), DZP (Figure 8), and VPA (Figure 9) a l l exerted a powerful anticonvulsant e f f e c t on the kindled rats on the drug-baseline t r i a l ; the mean duration of forelimb clonus on t h i s t r i a l was almost zero for each of the nine groups of ra t s . Over the course of the 10 tolerance-development t r i a l s , the rats i n each of the drug-before-stimulation groups gradually developed tolerance to the respective drugs' anticonvulsant e f f e c t s . In contrast, there was l i t t l e evidence of tolerance displayed by the rats i n any of the three drug-after-stimulation groups or the three control groups on the tolerance-test t r i a l ; each of the drugs retained i t s a b i l i t y to block the forelimb clonus of the rats i n these 88 Figure 7. Contingent tolerance to the anticonvulsant e f f e c t s of CBZ on amygdaloid kindled convulsions i n the r a t . On the drug-baseline t r i a l , CBZ exerted a potent anticonvulsant e f f e c t on a l l of the rats. On the tolerance t e s t t r i a l , the rats from the CBZ-Before-Stimulation group displayed substantial tolerance to the anticonvulsant effects of the drug. In contrast, there was no evidence of tolerance demonstrated by the rats from the CBZ-After-Stimulation or the CBZ-Control groups even though the rats i n the former group had received the same amount of drug exposure during the tolerance-development phase as the r a t s from the CBZ-Before-Stimulation group. 90 Figure 8. Contingent tolerance to the anticonvulsant e f f e c t s of DZP on amygdaloid kindled convulsions i n the r a t . On the drug-baseline t r i a l , DZP exerted a potent anticonvulsant e f f e c t on a l l of the ra t s . On the tolerance t e s t t r i a l , the rats from the DZP-Before-Stimulation group displayed substantial tolerance to the anticonvulsant effects of the drug. In contrast, there was no evidence of tolerance demonstrated by the rats from the DZP-After-Stimulation or the DZP-Control groups even though the rats i n the former group had received the same amount of drug exposure during the tolerance-development phase as the r a t s from the DZP-Before-Stimulation group. MEAN FORELIMB CLONUS DURATION (sec) 53 -n o > -X) 70 m m 0 o O I o _ J L _ O I o _ L _ cn o _J m < m 5 m Z —I > z o rn • • U 0 0 0 0 M z -0 T> 1 > 1 CD rn -n • m O TO m T 6 92 Figure 9. Contingent tolerance to the anticonvulsant e f f e c t s of VPA on amygdaloid kindled convulsions i n the r a t . On the drug-baseline t r i a l , VPA exerted a potent anticonvulsant e f f e c t on a l l of the rats. On the tolerance t e s t t r i a l , the rats from the VPA-Before-Stimulation group displayed substantial tolerance to the anticonvulsant effects of the drug. In contrast, there was no evidence of tolerance demonstrated by the rats from the VPA-After-Stimulation or the VPA-Control groups even though the rats i n the former group had received the same amount of drug exposure during the tolerance-development phase as the rats from the VPA-Before-Stimulation group. groups on the tolerance-test t r i a l . The s t a t i s t i c a l analyses support the observations made from Figure 7, Figure 8, and Figure 9. The analyses of variance revealed a s i g n i f i c a n t group X t r i a l i n teraction for each of the three drugs: CBZ, F (2,25) = 30.15; DZP, F (2,27) = 22.82; VPA, F (2,22) = 11.26; a l l p_'s < .001. Subsequent tests of simple main e f f e c t s revealed a s i g n i f i c a n t increase i n the duration of forelimb clonus between the drug-baseline and tolerance-test t r i a l s for each of the three Drug-Before-Stimulation groups (CBZ-Before: F (1,9) = 34.41; DZP-Before, F (1,10) = 35.64; VPA-Before, F (1,8) = 12.47; a l l p_'s < .01). In contrast, t e s t s of simple main e f f e c t s indicated that there were no s i g n f i c a n t differences between the drug-baseline t r i a l and the tolerance t e s t t r i a l for any of the Drug-After-Stimulation or Drug-Control groups ( a l l p_'s > .5). Accordingly, although tests of simple main e f f e c t s for the data from the drug-baseline t r i a l revealed no s i g n i f i c a n t differences between each Drug-Before-Stimulation group and the corresponding Drug-After-Stimulation or Drug-Control groups on the f i n a l t r i a l of the stimulation-baseline phase or on the drug-baseline t r i a l ( a l l p_"s > .5), there was a s i g n i f i c a n t difference between the three groups for each drug on the tolerance-test t r i a l (CBZ: F (2,25) = 38.91; DZP: F (2,27) = 29.05; VPA: F (2,22) = 15.75; a l l p_'s < .001). Further analysis of the data from the tolerance-test t r i a l for each of the three drugs using Neuman-Keuls posthoc comparisons revealed that the r a t s i n each Drug-Before-Stimulation group displayed s i g n i f i c a n t l y more forelimb clonus than the rats i n the corresponding Drug-After-Stimulation or Drug-Control groups ( a l l Neuman-Keuls p's < .05). This pattern of r e s u l t s r e f l e c t s the development of tolerance to the drugs' anticonvulsant e f f e c t s i n the r a t s from the CBZ-Before, DZP-Before, and VPA-Before-Stimulation groups. Discussion The r e s u l t s of the present experiment are important because they represent the f i r s t conclusive evidence that the development of tolerance to the anticonvulsant effects of c l i n i c a l l y prescribed a n t i e p i l e p t i c drugs can be influenced by the occurrence of convulsive a c t i v i t y during periods of drug exposure. Drug exposure was c l e a r l y not a s u f f i c i e n t condition for the development of tolerance to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA because there was no evidence of tolerance i n the kindled rats from the three drug-after-stimulation groups. In contrast, tolerance developed rapidly to the anticonvulsant e f f e c t s of each drug when kindled rats were stimulated during each b i d a i l y period of drug exposure. The f a c t that the development of tolerance to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA on kindled convulsions i n the present experiment was contingent upon convulsive stimulation being delivered during the b i d a i l y periods of drug exposure, and was not simply a consequence of drug exposure, implies that a functional rather than a d i s p o s i t i o n a l change underlies the development of tolerance i n the rats from the respective drug-before-stimulation groups (see also Wolgin, 1989). More importantly, the results of the present experiment provide further support for a drug-effect theory of functional drug tolerance (e.g., Pinel, Kim & Mana, 1990; P i n e l & Mana, i n press), which emphasizes the idea that functional tolerance i s a response to a drug's e f f e c t on the a c t i v i t y of the nervous system or other target tissue (see also Jaffe, 1980; Kalant et a l . , 1971; Kalant, 1985). Consequently, tolerance w i l l develop only to those drug e f f e c t s that manifest themselves during periods of drug exposure. In many instances, a drug w i l l produce a disruption i n the basal a c t i v i t y of the nervous system that i s s u f f i c i e n t to i n i t i a t e the development of tolerance to t h i s e f f e c t ; that i s , the drug ef f e c t i s an inevitable consequence of drug exposure and tolerance w i l l develop without the r e c i p i e n t engaging i n any p a r t i c u l a r pattern of a c t i v i t y . In other instances, however, the expression and/or magnitude of a p a r t i c u l a r drug e f f e c t i s dependent upon the pattern or l e v e l of a c t i v i t y of the nervous system of the drug r e c i p i e n t during periods of drug exposure; in these situations, the development of tolerance to the drug e f f e c t w i l l be contingent upon the occurrence of a p a r t i c u l a r pattern or l e v e l of neural a c t i v i t y during periods of drug exposure. In the present experiments, the 97 development of tolerance to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA administered on a b i d a i l y basis was contingent upon the administration of convulsive stimulation during each b i d a i l y period of drug exposure. The demonstration that the development of tolerance to a given drug e f f e c t i s contingent upon a p a r t i c u l a r pattern or l e v e l of a c t i v i t y i n the nervous system does not imply that tolerance cannot develop to the same drug e f f e c t i n the absence of such neural a c t i v i t y under a d i f f e r e n t set of circumstances. For example, the present demonstration that convulsive stimulation plays a key role in the development of tolerance to the anticonvulsant effects of CBZ, DZP and VPA on kindled convulsions does not imply that such stimulation i s necessary for the development of a l l instances of tolerance to the anticonvulsant e f f e c t s of these or any other drugs. Tolerance to anticonvulsant drug effects has been demonstrated experimentally for nearly every known a n t i e p i l e p t i c drug (Frey, 1987)—including CBZ (e.g., Frey & Loscher, 1980), and DZP (e.g., Rosenberg, Chiu, & T e i t z , 1 9 8 6 ) — i n subjects that never experience convulsions u n t i l the t e s t t r i a l . These posit i v e r e s u l t s may be due to the chronic drug administration schedule that these studies t y p i c a l l y employ; i n contrast, the present experiment involved an intermittent, b i d a i l y schedule of drug administration. As mentioned e a r l i e r , a schedule of chronic drug administration may f a c i l i t a t e the development of tolerance to a drug's e f f e c t s (see 98 Frey, 1987), and i n our own laboratory we have used a chronic administration schedule to demonstrate the development of tolerance to the anticonvulsant effects of ethanol i n the absence of convulsive stimulation (Mana, Pinel, & Le, 1988; Mana, Le, & P i n e l , i n preparation). Accordingly, a s i m i l a r schedule of drug administration was used i n Experiment 3 to study the development of pharmacologic tolerance to the anticonvulsant e f f e c t s of DZP on kindled convulsions. 99 V. GENERAL BACKGROUND FOR EXPERIMENTS 3. 4, AND 5. The three remaining experiments focus upon the comparison of contingent and pharmacologic tolerance to the anticonvulsant e f f e c t s of DZP. The decision to r e s t r i c t the focus of these experiments to a single drug was made for four reasons. The f i r s t and most obvious reason was one of economy. The second reason for focusing upon DZP i s that tolerance to DZP's anticonvulsant e f f e c t i s a much more widely recognized experimental phenomenon and c l i n i c a l problem than i s tolerance to the anticonvulsant e f f e c t s of either CBZ or VPA. Haigh and Feely (1988) noted that the broad spectrum of a c t i v i t y , low t o x i c i t y , and v i r t u a l absence of peripheral side e f f e c t s make the benzodiazepines p a r t i c u l a r l y e f f e c t i v e anticonvulsants—however, these v i r t u e s are negated by the fact that tolerance develops so r a p i d l y to t h e i r anticonvulsant e f f e c t s . Consequently, a considerable amount of attention has been focused upon the phenomenon of tolerance to the anticonvulsant e f f e c t s of the benzodiazepines i n an e f f o r t to resolve t h i s problem. The t h i r d reason for studying contingent and pharmacologic tolerance to the anticonvulsant e f f e c t of DZP, as opposed to CBZ or VPA, had to do with the fact that more i s known about the s i t e and mechanism of action for DZP. The discovery of s p e c i f i c benzodiazepine receptors i n the central nervous system (Mohler & Okada, 1977; Squires & Braestrup, 1977) has provided a focus for the study of the mechanisms involved in the development of tolerance to the ef f e c t s of the benzodiazepines (see Haigh & Feely, 1988); t h i s advantage does not exis t for either CBZ or VPA. Furthermore, the existence of a variety of d i r e c t - a c t i n g antagonists for the benzodiazepine receptor, which are known to a f f e c t the manner i n which the benzodiazepines exert t h e i r anticonvulsant e f f e c t , provides a tool with which to study both pharmacologic and contingent tolerance that i s not av a i l a b l e with eith e r CBZ or VPA. The fourth and f i n a l reason for r e s t r i c t i n g the focus of the remaining experiments to the study of contingent and pharmacologic tolerance to DZP's anticonvulsant e f f e c t i s that more i s known about the physiological changes underlying the development of tolerance to i t s anticonvulsant e f f e c t than to the anticonvulsant e f f e c t of either CBZ or VPA. The GABA/benzodiazepine/chloride ionophore complex has provided the focus for much of the work in t h i s area. Sher (1983) suggested that a downregulation (decrease i n the number) of benzodiazepine receptors following chronic administration of DZP underlies the development of tolerance to i t s anticonvulsant e f f e c t ; however, there have been many contradictory reports (e.g., Gallager, Lakoski, Gonsalves, & Rausch, 1984; Mohler, Okada, & Enna, 1978) and the present consensus i s that the development of tolerance to the anticonvulsant e f f e c t s of DZP and other benzodiazepines cannot be f u l l y explained simply i n terms of a downregulation of benzodiazepine receptors (e.g., Caccia & G a r a t t i n i , 1985; Teitz & 101 Rosenberg, 1988). Several authors have suggested that a decrease i n the s e n s i t i v i t y of the GABA-A receptor following chronic DZP administration can account for the development of tolerance to i t s anticonvulsant e f f e c t s . This hypothesis was f i r s t proposed by Gallager and her collaborators, based upon t h e i r observation that chronic DZP administration decreased the s e n s i t i v i t y of serotonergic neurones i n the dorsal raphe nucleus to iontophoretically applied GABA, but not serotonin (Gallager et a l . , 1984). More importantly, the time course for the emergence and disappearance of GABAergic subsensitivity during chronic administration and withdrawal of DZP was found to roughly correspond to the emergence and di s s i p a t i o n of tolerance to the anticonvulsant e f f e c t s of the drug (Gonsalves & Gallager, 1987; Teitz & Rosenberg, 1988). The decrease i n the functional s e n s i t i v i t y of the GABA receptor following chronic benzodiazepine administration i s not accompanied by a decrease i n i t s binding a f f i n i t y ; i n fact, although chronic benzodiazepine treatment may decrease the number of GABA receptors i n the brain (e.g., Mohler et a l . , 1978), the a f f i n i t y of the remaining GABA receptors has been reported to increase (Gallager, Malcolm, Anderson, & Gonsalves, 1985). The decrease i n the e f f i c a c y of the GABAergic receptor i s also associated with a decrease i n the GABAergic enhancement of benzodiazepine binding at the benzodiazepine receptor (e.g., Gallager et a l . , 1984; Teit z , Rosenberg, & Chiu, 102 1989) . This may also contribute to the development of pharmacologic tolerance to DZP's anticonvulsant e f f e c t . VI. Experiment 3 The purpose of Experiment 3 was to r e p l i c a t e Loscher and Schwark's (1985) report of pharmacologic tolerance to DZP's anticonvulsant e f f e c t on kindled convulsions i n r a t s . Accordingly, the drug administration regimen used i n Experiment 3 was i d e n t i c a l to that used by Loscher and Schwark (1985), with one noteable exception. Loscher and Schwark (1985) reported only a moderate amount of tolerance to DZP's anticonvulsant e f f e c t ; t h i s may be due to the fact that they administered a r e l a t i v e l y high treatment dose of DZP (5 mg/kg, IP), every 8 hr for 10 days, and there was a marked accumulation of the drug and i t s active metabolites over the course of the tolerance-development phase of t h e i r experiment. As noted e a r l i e r , such an accumulation can obscure the detection of tolerance to a drug's e f f e c t because the e f f e c t i v e plasma and brain concentration of the drug and i t s metabolites i s much higher i n rats receiving chronic drug treatment than would be produced by an acute i n j e c t i o n of the same dose of the drug. To reduce the p o s s i b i l i t y of t h i s occurring i n the present experiment, a smaller dose of DZP than that used by Loscher and Schwark (1985) was administered i n Experiment 3. 103 Methods Subjects. The subjects were 38 male, Long-Evans rats, weighing between 350 g and 400 g at the time of surgery and between 500 g and 600 g at the completion of the experiment. Drugs. DZP (2 mg/kg, IP) was injected at a volume of 2 ml/kg i n a 2% Tween-80/isosaline vehicle. Stimulation-Baseline Phase. The stimulation-baseline phase began 48 hr a f t e r the completion of the kindling phase. A single r a t did not demonstrate at least 20 sec of forelimb clonus on the l a s t stimulation-baseline t r i a l and was not studied further. Drug-Baseline T r i a l . On the drug-baseline t r i a l , each r a t received DZP 1 hr before the scheduled convulsive stimulation. Rats not showing at least an 80% decrease i n forelimb clonus duration on the drug-baseline t r i a l r e l a t i v e to the l a s t t r i a l of the stimulation-baseline phase were not studied further (n=7). The remaining 30 rats were assigned to one of two groups—-a Pharmacologic-Tolerance group (n = 21) or a Control group (n = 9) — s o that the mean forelimb clonus durations for the two groups on both the l a s t stimulation-baseline t r i a l and the drug-baseline t r i a l s were approximately equal. Tolerance-Development Phase. The tolerance-development phase began 24 hr afte r the drug-baseline t r i a l . During the 10-day tolerance-development phase, the rats i n the two groups were not stimulated. The rats i n the Pharmacologic-Tolerance group received DZP every 8 hr for 10 days, whereas the rats from the 104 Control group received vehicle injections on the same schedule. Tolerance-Test T r i a l . The tolerance-test t r i a l occurred 8 hr a f t e r the l a s t i n j e c t i o n of the tolerance-development phase. Each r a t received an i n j e c t i o n of DZP 1 hr before the delivery of convulsive stimulation. The duration of forelimb clonus e l i c i t e d by the stimulation was recorded for each rat. S t a t i s t i c s . A single 2 (Groups) X 2 (Trials) between-within factor, repeated measures analysis of variance was used to analyze the data from the l a s t t r i a l of the drug-baseline t r i a l and the tolerance-test t r i a l . Tests of simple main e f f e c t s were used to assess the contribution of the respective between- and within-group factors to the s i g n i f i c a n t i n t e r a c t i o n . Results As can be seen i n Figure 10, pharmacologic tolerance to DZP's anticonvulsant e f f e c t developed i n the rats that received DZP every 8 hr during the 10-day tolerance-development phase. The t e s t dose of DZP almost completely suppressed the forelimb clonus of each rat on the drug-baseline t r i a l . On the tolerance-t e s t t r i a l , however, the rats from the Pharmacologic-Tolerance group displayed a small amount of forelimb clonus; i n contrast, the t e s t dose of DZP continued to suppress forelimb clonus i n almost a l l of the rats from the Control group. The s t a t i s t i c a l analyses support the observations made from Figure 10. The analysis of variance indicated revealed 105 Figure 10. Pharmacologic tolerance to the anticonvulsant e f f e c t s of DZP. On the drug-baseline t r i a l , DZP exerted a potent anticonvulsant e f f e c t on a l l of the subjects. On the tolerance-t e s t t r i a l the rats from the Pharmacologic-Tolerance group displayed tolerance to the anticonvulsant e f f e c t s of the drug. There was no evidence of tolerance i n the rats from the Control group on the tolerance-test t r i a l . 107 s i g n i f i c a n t group X t r i a l interaction (F (1,28) = 6.16, p = .05). Subsequent analysis of t h i s interaction using tests of simple main e f f e c t s revealed a s i g n i f i c a n t increase i n the duration of forelimb clonus between the drug-baseline and tolerance-test t r i a l s f o r the rats from the Pharmacologic-Tolerance group (F (1,20) = 6.72, p < .05), but not for the rats from the Control group (F (1,8) = 0.3, p_ > .90. Discussion The r e s u l t s of Experiment 3 support the many previous demonstrations of pharmacologic tolerance to DZP's anticonvulsant e f f e c t , including the report by Loscher and Schwark (1985) of pharmacologic tolerance to DZP's anticonvulsant e f f e c t on kindled convulsions i n the r a t . It i s interesting to note that Loscher and Schwark (1985) reported only a modest amount of tolerance i n the r a t s from t h e i r experiment, an observation that was supported by the i n i t i a l analysis of the data from the present experiment. Thus, a comparison between the magnitude of pharmacologic tolerance to DZP's anticonvulsant e f f e c t presented i n Figure 10 i n the present experiment and the magnitude of contingent tolerance to DZP's anticonvulsant e f f e c t presented i n Figure 8 from Experiment 2 leads to the conclusion that the development of tolerance i s more complete i n kindled rats that receive convulsive stimulation during periods of drug exposure. However, t h i s conclusion cannot be supported by the data from these two 108 experiments; when the data for the rats from the Pharmacologic-Tolerance group that actually developed tolerance to DZP's anticonvulsant e f f e c t were compared to the data from the rats from the Contingent-Tolerance group from Experiment 2, i t i s cle a r that the magnitude of tolerance i s almost equal for the two groups (Mean = 35.3 for the Contingent-Tolerance rats from Experiment 2 compared to Mean = 39.9 for the Pharmacologic-Tolerance rats i n the present experiment). The apparent difference i n magnitude between contingent tolerance and pharmacologic tolerance i s actually due to the fa c t that fewer rats i n a given group develop pharmacologic tolerance than contingent tolerance to DZP's anticonvulsant e f f e c t ; that i s , although half of the rats (11/21) in the Pharmacologic-Tolerance group displayed a substantial degree of tolerance to DZP's anticonvulsant e f f e c t , the other half of the rats i n t h i s group displayed no tolerance at a l l . This dispersion i n the degree of tolerance development i n the rats from the Pharmacologic-Tolerance group i n Experiment 3 i s i l l u s t r a t e d i n Figure 11. The remaining experiments explore the question of whether pharmacological and contingent tolerance to DZP's anticonvulsant e f f e c t are attr i b u t a b l e to a single set of phy s i o l o g i c a l changes or instead are expressions of independent changes i n the nervous system. In the next experiment, the rate of d i s s i p a t i o n of these two phenomenologically d i s t i n c t forms of tolerance was examined to determine whether there was any difference i n the reversal of Figure 11. D i s t r i b u t i o n of data for the rats from the Pharmacologic-Tolerance group on the tolerance-test t r i a l . DZP continued to exert an anticonvulsant e f f e c t on 10 rats from the Pharmacologic-Tolerance group (as noted, 9 of the rats i n t h i s group displayed no forelimb clonus on the tolerance-test t r i a l ) , but tolerance had c l e a r l y developed i n the other 11 rat s i n t h i s group. NON-TOLERANT TOLERANT G R O U P S I l l the p h y s i o l o g i c a l changes responsible for t h e i r development and expression. VII. Experiment 4 A difference i n the rate at which tolerance to a drug's e f f e c t s develops or dissipates has been assumed to r e f l e c t a difference i n the physiological changes that underlie the development of tolerance (e.g., F i l e , 1985; LeBlanc et a l . , 1976; Okamoto, 1984; Teitz & Rosenberg, 1988). For example, LeBlanc et a l (1976) argued that the fact that contingent tolerance and pharmacologic tolerance to ethanol's ataxic e f f e c t dissipated at the same rate indicated that these two forms of tolerance were separate manifestations of a single set of underlying p h y s i o l o g i c a l changes. And Teitz and Rosenberg (1988), using a s i m i l a r form of l o g i c , proposed that the fact that the development and d i s s i p a t i o n of tolerance to the anticonvulsant e f f e c t s of the benzodiazepine flurazepam occurs much more slowly than that for the drug's locomotor effects r e f l e c t e d a difference i n the p h y s i o l o g i c a l bases of these two forms of tolerance. In Experiment 4, t h i s comparative-rate approach was used i n an e f f o r t to determine whether pharmacologic and contingent tolerance to DZP's anticonvulsant e f f e c t are a t t r i b u t a b l e to a si n g l e p h y s i o l o g i c a l change or instead to unique p h y s i o l o g i c a l changes i n the nervous system. Experiment 4 focused upon the rate of d i s s i p a t i o n of pharmacologic and contingent tolerance to DZP's anticonvulsant e f f e c t . Methods Subjects. The subjects were 151 male, Long-Evans rats (Charles River, Canada), weighing between 350 g and 400 g at the time of surgery and between 500 g and 650 g at the completion of the experiment. Drugs. DZP (2 mg/kg, IP) was injected at a volume of 2 ml/kg i n a 2% Tween-80/ is o s a l i n e vehicle. Kindling Phase. A t o t a l of 16 rats were l o s t during the ki n d l i n g phase; 10 rats did not meet the c r i t e r i o n for kindling and the electrode assemblies became detached in 6 others. Stimulation-Baseline Phase. The stimulation-baseline phase began 48 hr a f t e r the completion of the kindling phase. Rats that did not demonstrate at least 2 0 sec of forelimb clonus on the l a s t stimulation-baseline t r i a l were not studied further (n=4). Drug-Baseline T r i a l . The drug-baseline t r i a l occurred 48 hr af t e r the fourth and l a s t stimulation-baseline t r i a l . On the drug-baseline t r i a l , each rat received DZP 1 hr before the convulsive stimulation. Rats not showing at least an 80% decrease i n forelimb clonus duration on the drug-baseline t r i a l r e l a t i v e to the l a s t t r i a l of the stimulation-baseline phase were not studied further (n=21). The remaining 110 rats were assigned to one of four groups—a Pharmacologic-Tolerance group (n=57), a PharmacolOgic-Control group (n=9), a Contingent-Tolerance group (n=36), or a Contingent-Control group (n=8)—so that the mean forelimb clonus durations on both the l a s t stimulation-baseline t r i a l and the drug-baseline t r i a l were approximately equal for each group. Tolerance-Development Phase. The tolerance-development phase for the rat s from the Pharmacologic-Tolerance and the Pharmacologic-Control groups began 24 hr after the drug-baseline t r i a l . During the 10-day tolerance-development phase, the rats were not stimulated. The rats i n the Pharmacologic-Tolerance group received DZP every 8 hr for 10 days; the rats from the Pharmacologic-Control group received vehicle i n j e c t i o n s on the same schedule. The tolerance-development phase for the rats from the Contingent- Tolerance and the Contingent-Control groups began 48 hr a f t e r the drug-baseline t r i a l . The rats i n both groups continued to received a convulsive stimulation every 48 hr during the 2 0-day tolerance-development phase. Each rat received an i n j e c t i o n 1 hr before each stimulation; the rats from the Contingent-Tolerance group received DZP, whereas the rat s from the Contingent-Control group received an equal volume of the vehicle. Tolerance-Test T r i a l . The tolerance-test t r i a l occurred 8 hr a f t e r the l a s t i n j e c t i o n of the tolerance-development phase for the rat s from the Pharmacologic-Tolerance and the Pharmacologic-Control groups and 48 hr after the l a s t tolerance-development t r i a l s for the rats from the Contingent-Tolerance and the 114 Contingent-Control groups. The tolerance-test t r i a l was i d e n t i c a l for a l l of the rats; an i n j e c t i o n of DZP was administered 1 hr before convulsive stimulation and the duration of forelimb clonus e l i c i t e d by the stimulation was recorded. Following the tolerance-test t r i a l , rats from the Pharmacologic-Tolerance and the Contingent-Tolerance groups that did not demonstrate at least 2 0 sec of forelimb clonus on the tolerance-test t r i a l were removed from the experiment (n = 21 for the Chronic-Tolerance group; n = 4 for the Contingent-Tolerance group). Accordingly, by the end of the tolerance-test t r i a l the 85 rats that remained i n the experiment (Pharmacologic-Tolerance group n = 36; Pharmacologic-Control n = 9; Contingent-Tolerance group n = 32; Contingent-Control n = 8) were a l l tolerant to DZP's anticonvulsant e f f e c t . The tolerant rats i n both the Pharmacologic-Tolerance and Contingent-Tolerance groups were then assigned to one of four tolerance-retention groups, which determined whether they were retested 2 days, 4 days, 8 days, or 16 days a f t e r the tolerance-t e s t t r i a l . The rats from the Pharmacologic-Control and Contingent-Control groups were retested only at the 16-Day i n t e r v a l . Thus, there were 10 groups of rats i n Experiment 4: These were the Pharmacologic-Tolerance 2-Day, 4-Day, 8-Day, 16-Day, and Control groups; and the Contingent-Tolerance 2-Day, 4-Day, 8-Day, 16-Day, and Control groups. Tolerance-Retention Interval. During the tolerance-retention i n t e r v a l , each r at was remained in i t s home cage u n t i l i t was scheduled to be retested. Tolerance-Retention T e s t - T r i a l . The protocol followed on the tolerance-retention t e s t - t r i a l for the rats from the Pharmacologic-Tolerance and the Contingent-Tolerance groups was i d e n t i c a l to the drug-baseline t r i a l and the tolerance-test t r i a l ; on the appropriate day after the tolerance-test t r i a l , DZP was administered 1 hr before a convulsive stimulation was delivered and the duration of forelimb clonus e l i c i t e d by the stimulation was recorded. The rats from the two Control groups were not retested u n t i l 16 days a f t e r the tolerance-test t r i a l . The nature of the treatment that the rats from the two Control groups received on the tolerance-retention t e s t - t r i a l was determined by the performance of the corresponding Pharmacologic-Tolerance and Contingent-Tolerance groups on the 16-Day Retention t r i a l . The rats from the Pharmacologic-Tolerance 16-Day group showed a substantial loss of tolerance to DZP's anticonvulsant e f f e c t ; consequently, the rats from the Pharmacologic-Control group received a vehicle i n j e c t i o n 1 hr before convulsive stimulation was delivered on the tolerance-retention test t r i a l . The purpose of stimulating these control rats in an undrugged state was to control for the p o s s i b i l i t y that the loss of tolerance to DZP's anticonvulsant e f f e c t i n the rats from the Pharmacologic-Tolerance 16-Day group was due to a decline i n the s e n s i t i v i t y of 116 the rat s to the kindling stimulation rather than to a genuine d i s s i p a t i o n of tolerance. In contrast, there was no loss of tolerance i n the rats from the Contingent-Tolerance 16-Day group. Consequently, the rats from the Contingent-Control group were administered DZP 1 hr before they were stimulated on the tolerance-retention t e s t t r i a l . The purpose of stimulating these rats i n a drugged state was to determine whether the retention of tolerance to DZP's anticonvulsant e f f e c t displayed by the rats from Contingent-Tolerance group was genuine, or instead a t t r i b u t a b l e to an increase i n the s e n s i t i v i t y of the rats to the convulsive stimulation over the 16-day retention i n t e r v a l . S t a t i s t i c s . A single 10 (Groups) X 2 ( T r i a l s ) , between-within factor, repeated-measures analysis of variance was used to analyze the data from the tolerance-test t r i a l and the tolerance-r e t e n t i o n - t r i a l for the f i v e Pharmacologic-Tolerance groups and the f i v e Contingent-Tolerance groups i n Experiment 4. Tests of simple main e f f e c t s were performed on the respective between- and within group factors involved i n the s i g n i f i c a n t i n t e r a c t i o n ; where necessary, Neuman-Keuls posthoc comparisons were used to further analyze the data from s i g n i f i c a n t tests of simple main e f f e c t s . Results. The retention of pharmacologic and contingent tolerance to DZP's anticonvulsant e f f e c t over the 16-day retention i n t e r v a l i s presented i n Figure 12 (Pharmacologic Tolerance) and Figure 13 117 (Contingent Tolerance). As i n the previous experiments, the t e s t dose of DZP almost completely suppressed the forelimb clonus i n the rats from each of the 10 treatment groups on the drug-b a s e l i n e - t r i a l . In contrast, on the tolerance-test t r i a l the rats from the four Pharmacologic-Tolerance and four Contingent-Tolerance groups that were included i n the f i n a l analysis displayed a substantial degree of tolerance to the anticonvulsant e f f e c t of DZP during the tolerance-development phase. Over the 16-day retention i n t e r v a l , pharmacologic tolerance to DZP's anticonvulsant e f f e c t gradually dissipated; the t e s t dose of DZP completely suppressed the forelimb clonus of almost every r a t i n the Pharmacologic-Tolerance 16-day group (see Figure 12). In contrast, there was no evidence of a decline i n contingent tolerance to DZP's anticonvulsant e f f e c t over the 16-day retention i n t e r v a l ; there was l i t t l e difference i n the mean duration of forelimb clonus between the tolerance-test t r i a l and the tolerance-retention-test t r i a l for the rats from the Contingent-Tolerance 16-Day group (see Figure 13). The s t a t i s t i c a l analyses support the observations made from Figure 12 and Figure 13. The analysis of variance revealed a s i g n i f i c a n t group X t r i a l interaction (F (9,75) = 7.37, p <.001). The analyses of simple main effects for each group across the tolerance-test and r e t e s t - t r i a l s revealed a s i g n i f i c a n t decrease i n forelimb clonus for the rats from the Pharmacologic-Tolerance 16-Day group (F (1,7) = 81.86, p < .001), but not for any of the 118 Figure 12. Dissipation of pharmacologic tolerance to DZP's anticonvulsant e f f e c t . DZP had a potent anticonvulsant e f f e c t on a l l of the rats on the drug-baseline t r i a l ; i n contrast, on the tolerance-test t r i a l the drug's anticonvulsant e f f e c t had almost disappeared for the four Pharmacologic-Tolerance groups. There was a steady loss of tolerance to DZP's anticonvulsant e f f e c t over the retention i n t e r v a l , although there was a s i g n i f i c a n t difference between the tolerance-test and retest t r i a l only for the rats from the 16-Day group. This loss of tolerance cannot be att r i b u t e d to a loss of s e n s i t i v i t y to the stimulation over the retention i n t e r v a l because the rats from the Control group displayed as much forelimb clonus on the retention-test t r i a l as they had on the l a s t t r i a l of the stimulation-baseline phase. : : : 5 L (••••A* ••••>%% " • • • • • • •••••TO Legend • I P H A R M - 2 IZ2 PHARM-4 a PHARM-8 Gffl PHARM-16 BB PHARM-CONTROL DRUG-FREE DRUG-BASE TOL-TEST TRIALS L MisaQ. RETEST 120 Figure 13. Dissipation of contingent tolerance to DZP's anticonvulsant e f f e c t . DZP had a potent anticonvulsant e f f e c t on a l l of the rats on the drug-baseline t r i a l ; i n contrast, on the tolerance-test t r i a l the drug's anticonvulsant e f f e c t had almost disappeared i n the four Contingent-Tolerance groups. There i s no evidence of a loss of tolerance to DZP's anticonvulsant e f f e c t over the retention i n t e r v a l ; there was no s i g n i f i c a n t difference between the tolerance-test t r i a l and the retention-test t r i a l for any of the four Contingent-Tolerance groups. This cannot be attri b u t e d to an increase i n the s e n s i t i v i t y of these r a t s to the stimulation over the retention i n t e r v a l as DZP continued to suppress the convulsions of the rats from the Contingent-Control group on the rete s t t r i a l . 60-i < Ul 50-40-O <D z o ZD o ZD Z 3 O CD 2 £ 20-30 -10-• Legend •I CONT-2 EZ3 CONT-4 O CONT-8 CD CONT-16 G53 C0NT-C0NTR0L !•••»» )•••»>: •  • " ii DRUG-FREE DRUG-BASE T0L-TEST TRIALS •J M RETEST to other Pharmacologic-Tolerance groups or any of the Contingent-Tolerance groups ( a l l p's > .10). The analyses for simple main e f f e c t s across groups within a t r i a l revealed a s i g n i f i c a n t e f f e c t for both the tolerance-test t r i a l (F (9,75) = 7.55, p < .001) and the tolerance-retest t r i a l (F (9,75) = 6.48, p < .001). Further analysis of the data from the tolerance-test t r i a l with Neuman-Keuls posthoc comparisons indicated that the rats from the two Control groups displayed s i g n i f i c a n t l y less tolerance than any of the other groups on the tolerance-test t r i a l ( a l l Neuman-Keuls p's < .05), and there were no differences between the d i f f e r e n t Pharmacologic-Tolerance and Contingent-Tolerance groups on t h i s t r i a l ( a l l Neuman-Keuls p's > .10). Neuman-Keuls posthoc analysis of the data from t e s t of simple main e f f e c t s for the tolerance-retest t r i a l indicated that the rats from the Pharmacologic-Tolerance 4-Day, 8-Day, and 16-Day groups, and the Contingent-Control group, displayed s i g n f i c a n t l y less forelimb clonus than the rats from the Pharmacologic-Tolerance 2-Day group and a l l four of the Contingent-Tolerance groups ( a l l Neuman-Keuls p's < .05). Discussion The difference i n the rate of d i s s i p a t i o n between pharmacologic tolerance and contingent tolerance to DZP's anticonvulsant e f f e c t supports the idea that these phenomenologically d i s t i n c t forms of tolerance also represent independent physiological changes. It i s p a r t i c u l a r l y i n t e r e s t i n g to note that the time course for the d i s s i p a t i o n of pharmacologic tolerance (8 days) i n the present experiments i s si m i l a r to that reported by Teitz and Rosenberg (1988) for the d i s s i p a t i o n of tolerance to DZP's anticonvulsant e f f e c t s on PTZ convulsions (between 4 and 7 days). This finding gains further signficance when considered i n l i g h t of the observation by Gonsalves and Gallager (1987) that the impairment of GABAergic function produced by chronic DZP administration also d i s s i p a t e s within 8 days of the cessation of drug exposure. Based upon these observations, Gonsalves and Gallager (1988) and Tei t z and Rosenberg (1988) have suggested that a downregulation i n the number of benzodiazepine receptors (which occurs r a p i d l y when benzodiazepines are administered on a chronic basis and disappears r a p i d l y when the drug i s withdrawn) underlies the development of tolerance to DZP's locomotor e f f e c t s , whereas a decrease i n the s e n s i t i v i t y of GABA-A receptors associated with benzodiazepine receptors (which occurs slowly when benzodiazepines are administered on a chronic basis and disappears slowly when the drug i s withdrawn) underlies the development of tolerance to the drug's anticonvulsant e f f e c t s . If t h i s hypothesis i s correct, the fact that the time course for the d i s s i p a t i o n of contingent tolerance to DZP's anticonvulsant e f f e c t i s substantially d i f f e r e n t from that for the d i s s i p a t i o n of pharmacologic tolerance to DZP's anticonvulsant e f f e c t and the di s s i p a t i o n of the GABAergic 124 su b s e n s i t i v i t y that r e s u l t s from chronic DZP exposure r a i s e s the p o s s i b i l i t y that the development of contingent tolerance to DZP's anticonvulsant e f f e c t i s not attributable to a decrease i n the s e n s i t i v i t y of GABA-A receptors associated with benzodiazepine receptors. This p o s s i b i l i t y i s examined i n Experiment 5. VIII. Experiment 5 The discovery of the benzodiazepine receptor antagonist RO 15-1788 by Hunkeler et a l . (1981) provided an important pharmacologic t o o l for studying the structure and function of the benzodiazepine receptor. S i g n i f i c a n t l y less toxic than most of the c l a s s i c benzodiazepine agonists (e.g., LD50 of 1,360 mg/kg, IP, i n the r a t ; Hunkeler et a l . , 1981), RO 15-1788 was o r i g i n a l l y reported to be devoid of any of the behavioral e f f e c t s associated with the c l a s s i c benzodiazepines such as DZP; Hunkeler et a l . (1981) reported no evidence of s i g n i f i c a n t sedative, anticonvulsant, muscle relaxant, or a n x i o l y t i c e f f e c t s i n either mice, r a t s , cats or dogs—even at near-toxic doses. In addition, RO 15-1788 did not appear to be a proconvulsant or to produce stimulatory e f f e c t s i n these species (see Hunkeler et a l . , 1981). Since the seminal report by Hunkeler and his colleagues, i t has become apparent that RO 15-1788 i s not behaviorally or p h y s i o l o g i c a l l y i n e r t . RO 15-1788 has a variety of i n t r i n s i c e f f e c t s that can complement or oppose those of the c l a s s i c a l benzodiazepine agonists i n a dose- and test-dependent manner (see F i l e & Pellow, 1986, for a review of t h i s l i t e r a t u r e ) . Of p a r t i c u l a r s i g n i f i c a n c e to the present experiments, Gonsalves and Gallager (1988) found that a single i n j e c t i o n of RO 15-1788, administered 2 4 hr pr i o r to testing, reverses both the su b s e n s i t i v i t y of benzodiazepine-linked GABA-A receptors that i s produced by chronic benzodiazepine treatment and the expression of pharmacologic tolerance to the anticonvulsant e f f e c t s of DZP on bicuculline-induced convulsions in rats. The r e s u l t s of Experiment 4 suggested that a decrease i n the s e n s i t i v i t y of the GABA-A receptor might be responsible for the development of pharmacologic tolerance, but not contingent tolerance, to DZP's anticonvulsant e f f e c t on kindled convulsions. If t h i s i s true, then a singl e i n j e c t i o n of RO 15-1788 should subsequently reduce the expression of pharmacologic tolerance, but not contingent tolerance, to DZP's anticonvulsant e f f e c t on kindled convulsions. The purpose of Experiment 5 was to test t h i s hypothesis. Methods Subjects. The subjects were 51 male, Long-Evans rats, weighing between 350 g and 400 g at the time of surgery and between 500 g and 650 g at the completion of the experiment. Drugs. DZP (2 mg/kg) was injected at a volume of 2 ml/kg i n a 2% Tween-80/isosaline vehicle. Kindling Phase. Four rats that did not meet the c r i t e r i o n f or ki n d l i n g were removed from the experiment at the end of t h i s phase. 126 Stimulation-Baseline Phase. The stimulation-baseline phase began 48 hr a f t e r the completion of the kindling phase. A l l of the remaining rats successfully completed the stimulation-baseline phase. Drug-Baseline T r i a l . The drug-baseline t r i a l occurred 4 8 hr af t e r the fourth and l a s t stimulation-baseline t r i a l . On the drug-baseline t r i a l , each rat received DZP 1 hr before the convulsive stimulation. Rats not showing at least an 8 0% decrease i n forelimb clonus duration on the drug-baseline t r i a l r e l a t i v e to the l a s t t r i a l of the stimulation-baseline phase were not studied further (n = 4). The remaining 43 rats were assigned to one of four groups—a Pharmacologic-Tolerance group (n = 14), a Pharmacologic-Control group (n = 10), a Contingent-Tolerance group (n = 10), and a Contingent-Control group (n = 9 ) — s o that the mean forelimb clonus durations on both the l a s t stimulation-baseline t r i a l and the drug-baseline t r i a l were approximately equal for the four groups. Tolerance-Development Phase. The tolerance-development phase for the rats from the Pharmacologic-Tolerance and the Pharmacologic-Control groups began 24 hr after the drug-baseline t r i a l . During the 10-day tolerance-development phase, the rats were not stimulated. The rats i n the Pharmacologic-Tolerance group received DZP every 8 hr for 10 days; the rats from the Pharmacologic-Control group received vehicle i n j e c t i o n s on the same schedule. 127 The tolerance-development phase for the rats from the Contingent- Tolerance and the Contingent-Control groups began 4 8 hr a f t e r the drug-baseline t r i a l . The rats i n both groups continued to received convulsive stimulation every 48 hr during the 2 0-day tolerance-development phase. Each r a t received an i n j e c t i o n 1 hr before each stimulation; the rats from the Contingent-Tolerance group received DZP, whereas the rats from the Contingent-Control group received an equal volume of the vehicle. Tolerance-Test T r i a l . The tolerance-test t r i a l occurred 8 hr a f t e r the l a s t i n j e c t i o n of the tolerance-development phase for the rats from the Pharmacologic-Tolerance and the Pharmacologic-Control groups and 4 8 hr after the l a s t tolerance-development t r i a l for the rats from the Contingent-Tolerance and the Contingent-Control groups. The tolerance-test t r i a l was i d e n t i c a l for a l l of the rats; an i n j e c t i o n of DZP was administered 1 hr before convulsive stimulation and the duration of forelimb clonus e l i c i t e d by the stimulation was recorded. Following the tolerance-test t r i a l , rats from the Pharmacologic- Tolerance and the Contingent-Tolerance groups that did not demonstrate at least 20 sec of forelimb clonus on the tolerance-test t r i a l were removed from the experiment (n = 5 for the Chronic-Tolerance group; n = 2 for the Contingent-Tolerance group). Accordingly, by the end of the tolerance-test t r i a l 36 rats that remained i n the experiment (Pharmacologic-Tolerance 128 group n = 9; Pharmacologic-Control group, n = 10; Contingent-Tolerance group n = 8; Contingent-Control group, n = 9 ) . RO 15-1788 Administration. A single i n j e c t i o n of RO 15-1788 (5 mg/kg, IP, i n a 2% Tween 80/isosaline solution at a volume of 2 ml/kg) was administered to each rat 24 hr a f t e r the tolerance-t e s t t r i a l . Tolerance-Retest T r i a l . The tolerance-retest t r i a l occurred 24 hr a f t e r the adminstration of RO 15-1788. The protocol followed on the tolerance-retest t r i a l for the rats from the Pharmacologic-Tolerance and Contingent-Tolerance groups was i d e n t i c a l to the drug-baseline t r i a l and the tolerance-test t r i a l ; the t e s t dose of DZP was administered 1 hr before convulsive stimulation was delivered and the duration of forelimb clonus e l i c i t e d by the stimulation was recorded. In contrast, the rats from the two Control groups received an i n j e c t i o n of the vehicle 1 hr before they were stimulated; the purpose of t h i s procedure was to control for the p o s s i b i l i t y that RO 15-1788 might a f f e c t the expression of tolerance to DZP's anticonvulsant e f f e c t by a f f e c t i n g the s e n s i t i v i t y of the kindled r a t s to the convulsive stimulation. S t a t i s t i c s . A single 4 (Groups) X 2 (Trials) between-within factors, repeated-measures analysis of variance was used to analyze the data from the tolerance-test t r i a l and the tolerance-r e t e s t t r i a l of Experiment 5. Tests of simple main e f f e c t s were used to assess the contribution of the respective between- and 129 within-groups factors to the s i g n i f i c a n t i n t e r a c t i o n ; where necessary, Neuman-Keuls posthoc comparisons were used to further analyze the data from s i g n i f i c a n t tests of simple main e f f e c t s . F i n a l l y , separate correlated t-tests were used to analyze the data from the l a s t stimulation-baseline t r i a l and the re t e s t t r i a l f o r the rats from the two control groups. Results The e f f e c t s of RO 15-1788 on the expression of pharmacologic and contingent tolerance to DZP's anticonvulsant e f f e c t on kindled convulsions are i l l u s t r a t e d i n Figure 14. The t e s t dose of DZP had a potent anticonvulsant e f f e c t on almost a l l of the rats on the drug-baseline t r i a l . On the tolerance-test t r i a l , the rats from the Pharmacologic-Tolerance and Contingent-Tolerance groups displayed a substantial amount of tolerance to DZP's anticonvulsant e f f e c t , whereas there was no evidence of tolerance i n the rats from the two Control groups. I t i s cle a r that RO 15-1788 had l i t t l e e f f e c t on the expression of contingent tolerance to DZP's anticonvulsant e f f e c t ; there was no difference i n the mean duration of forelimb clonus between the tolerance-t e s t t r i a l and the tolerance-retest t r i a l for the rats from the Contingent-Tolerance group. In contrast, RO 15-1788 had a s i g n i f i c a n t e f f e c t on the expression of tolerance i n the rats from the Pharmacologic-Tolerance group on the tolerance-retest t r i a l ; the mean duration of forelimb clonus for the rats from 130 Figure 14. Effects of RO 15-1788 on the d i s s i p a t i o n of pharmacologic and contingent tolerance to DZP's anticonvulsant e f f e c t . DZP almost completely suppressed forelimb clonus i n each rat on the drug-baseline t r i a l . In contrast, on the tolerance-t e s t t r i a l the rats from the Pharmacologic-Tolerance and Contingent-Tolerance groups displayed a considerable amount of tolerance to DZP's anticonvulsant e f f e c t . The RO 15-1788 was administered to each r at between the tolerance-test t r i a l and the tolerance-retest t r i a l ; i t reduced the expression of tolerance i n the rats from the Pharmacologic-Tolerance group but had no e f f e c t on the tolerance i n the Contingent-Tolerance group or on the s e n s i t i v i t y of the two control groups to the convulsive stimulation. 60-. 50-40-30-C£ 20-10- I Leg«nd ' WB P H A R M — T O L E R A N C E • 3 P H A R M - C O N T R O L O C O N T I N O E N T - T O L E R A N C E C O C O N T I N G E N T - C O N T R O L DRUG-FREE DRUG-BASE TOL-TEST TRIALS POST-RO t h i s group decreased over 2 6 sec (52%) between the tolerance-t e s t t r i a l and tolerance-retest t r i a l . This e f f e c t could not be a t t r i b u t e d to an RO 15-1788 induced decrease i n the s e n s i t i v i t y of the r a t s from the Pharmacologic-Tolerance group to the convulsive stimulation because the drug had no e f f e c t on the duration of forelimb clonus of the rats from either the Pharmacologic-Control group or the Contingent-Control group on the tolerance-retest t r i a l . The s t a t i s t i c a l analysis of the data from Experiment 5 confirms the observations made from Figure 14. There was a s i g n i f i c a n t group X t r i a l interaction (F (3,32) = 72.01, p < .01). The tests of simple main effects revealed a s i g n i f i c a n t decrease on the tolerance-retest t r i a l compared to the tolerance-t e s t t r i a l for the rats from the Pharmacologic-Tolerance group (F (1,8) = 16.96, p < .01), but not for the rats from the Contingent-Tolerance group (F (1,7) = 0.25, p > .50). Tests of simple main e f f e c t s revealed a s i g n i f i c a n t difference between groups on both the tolerance-test t r i a l (F (3,32) = 39.7, p < .0001) and the tolerance-retest t r i a l (F (3,32) = 7.12, p < .01). Neuman-Keuls posthoc analysis of t h i s t e s t of simple main e f f e c t s across groups on the tolerance-test t r i a l indicated that the rats from the two control groups displayed less forelimb clonus than the rats from the Pharmacologic-Tolerance and Contingent-Tolerance groups ( a l l Neuman-Keuls p's <.05); there were no differences between the two control groups (Neuman-Keuls p > .05) 133 or the Pharmacologic-Tolerance and the Contingent-Tolerance groups (Neuman-Keuls p_ > .05). Neuman-Keuls analysis of the t e s t of simple main e f f e c t s across groups on the tolerance-retest t r i a l indicated that the rats from the Pharmacologic-Tolerance group displayed s i g n i f i c a n t l y less forelimb clonus than the r a t s from the Contingent-Tolerance group or the two control groups ( a l l p_'s < .05), and there were no other differences between these l a t t e r 3 groups ( a l l p_'s > -05). Correlated t - t e s t s revealed no differences between the l a s t stimulation-baseline t r i a l and the retest t r i a l for either of the control groups, both p_xs > .10. Discussion The reduction of pharmacologic tolerance to DZP's anticonvulsant e f f e c t on kindled convulsions by a single i n j e c t i o n of RO 15-1788 extends Gonsalves and Gallager's e a r l i e r (1987) fi n d i n g that RO 15-1788 reduces the expression of pharmacologic tolerance to DZP's anticonvulsant e f f e c t on bicuculline-induced convulsions i n mice. Although RO 15-1788 has been shown to possess limited anticonvulsant properties (see F i l e & Pellow, 1986), i t i s unlikely that t h i s property was responsible for i t s e f f e c t s on the expression of pharmacologic tolerance to DZP's anticonvulsant e f f e c t because the dose of RO 15-1788 had no anticonvulsant e f f e c t on the convulsions of the rats i n the two control groups. In addition, L i s t e r , Greenblatt, Abernethy, and F i l e (1984) have shown that RO 15-1788 has a half l i f e i n the CNS of about 16 min following an IP i n j e c t i o n ( at a dose of 10 mg/kg, IP); i t i s therefore u n l i k e l y that RO 15-1788 would even be present i n the brain 2 4 hr afte r i t s administration, which i s when the convulsive stimulation for the tolerance-retest t r i a l was delivered i n Experiment 5. The demonstration that RO 15-1788 reverses pharmacologic tolerance to DZP's anticonvulsant e f f e c t on kindled convulsions provides further support for the hypothesis that t h i s form of tolerance i s attributable to a decrease i n the s e n s i t i v i t y of GABA-A receptors (see also Gonsalves & Gallager, 1985; 1987). Furthermore, the fact that an i d e n t i c a l dose of RO 15-1788 had no e f f e c t on the expression of contingent tolerance to DZP's anticonvulsant e f f e c t strengthens the claim that the phys i o l o g i c a l changes which underlie the development of contingent tolerance are not the same as those responsible for the development of pharmacologic tolerance to DZP's anticonvulsant e f f e c t . Meldrum and Chapman (1986) have previously suggested that d i f f e r e n t physiological changes underlie the development of tolerance to DZP's anticonvulsant e f f e c t on maximal electroshock convulsions and pentylenetetrazol convulsions, respectively. However, the present r e s u l t s are unique because they indicate that d i f f e r e n t p h y s i o l o g i c a l changes may underlie the development of tolerance to the same e f f e c t , of the same drug, on the same type of convulsion—with the nature of the p h y s i o l o g i c a l change dependent upon the schedule of drug administration and/or the administration of convulsive stimulation during periods of drug exposure. Although the present r e s u l t s support the idea that separate p h y s i o l o g i c a l changes are responsible for pharmacologic and contingent tolerance to DZP's anticonvulsant e f f e c t , they do not provide incontrovertible evidence for t h i s conclusion. For example, RO 15-1788 may have antagonized contingent tolerance to DZP's anticonvulsant e f f e c t i f a larger dose was used i n the present experiment. Alternatively, contingent tolerance to DZP's anticonvulsant e f f e c t may re s u l t from a physiological change completely independent of the GABA-A/benzodiazepine receptor complex. If t h i s i s the case, the administration of RO 15-1788 would not a f f e c t the expression of contingent tolerance to DZP's anticonvulsant e f f e c t , regardless of the dose of the antagonist that was administered. This issue i s examined more f u l l y i n the general discussion. 136 IX. GENERAL DISCUSSION. The r e s u l t s of the f i v e experiments reported i n the present d i s s e r t a t i o n lead to two general conclusions. F i r s t , they c l e a r l y indicate that the occurrence of convulsive stimulation during periods of drug exposure can play a key r o l e i n the development of tolerance to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA. And second, they support the idea that contingent tolerance and pharmacologic tolerance, at least to the anticonvulsant e f f e c t of DZP, are ph y s i o l o g i c a l l y d i s t i n c t . These two general conclusions are discussed i n more d e t a i l i n the following two major sections of t h i s general discussion. 1. The Role of Convulsive Stimulation i n the Development of Contingent Tolerance to the Anticonvulsant E f f e c t s of CBZ, DZP, and VPA. The most notable feature of the res u l t s from Experiment 1 was the magnitude of the tolerance that developed to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA on kindled convulsions i n the ra t . Previous attempts to study the development of tolerance to the anticonvulsant e f f e c t s of these drugs on kindled convulsions had reported only a moderate l e v e l of tolerance for CBZ (Honack & Loscher, 1988) and DZP (Loscher & Schwark, 1985), and a complete lack of tolerance to VPA's anticonvulsant e f f e c t s on kindled convulsions (Young et a l . , 1987) of tolerance. In contrast, the magnitude of the tolerance that developed to the anticonvulsant effects of CBZ, DZP, and VPA on kindled convulsions i n Experiment 1 was considerably greater; i n f a c t , tolerance developed to such an extent that there was l i t t l e difference i n the durations of forelimb clonus between the tolerance-test t r i a l and the l a s t t r i a l of the no-drug baseline phase for any of the three drug groups i n t h i s experiment. Several possible explanations for the magnitude of the tolerance reported i n Experiment 1 were considered. However, previous work from our laboratory (e.g., Pinel et a l . , 1983; 1985; 1989) indicated that the convulsive stimulation that the rats i n each of the three drug groups i n Experiment 1 received on each tolerance-development t r i a l 1 hr af t e r the appropriate drug was administered was l i k e l y an important factor i n the development of tolerance to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA. The r e s u l t s of Experiment 2 c l e a r l y supported t h i s idea. In Experiment 2, tolerance developed rapi d l y to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA i n the kindled rats from each of the three drug-before-stimulation groups; i n contrast, there was l i t t l e evidence of tolerance development i n the kindled rats from the three drug-after-stimulation groups or the three vehicle-control groups. The f a c t that there have been no previous reports that seizure a c t i v i t y during periods of drug exposure can influence the development of tolerance to the anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs i s especially interesting given the existence of several anecdotal reports that support such an idea. For example, Killam et a l . (1973) reported that the development of 138 tolerance to diazepam's anticonvulsant e f f e c t on p h o t i c a l l y induced seizures i n the baboon P. papio could be prevented i f a r e l a t i v e l y high treatment dose was used from the s t a r t of treatment. This observation was interesting for two reasons. F i r s t , the fac t that a high treatment dose retarded the development of tolerance contradicts the t r a d i t i o n a l assumption that the development of tolerance i s f a c i l i t a t e d by the administration of high treatment doses (e.g., Kalant et a l . , 1971). Second, the use of a high treatment dose c o n f l i c t s with the normal c l i n i c a l procedure, noted e a r l i e r , to i n i t i a t e pharmacologic treatment of epilepsy with as low a dose as possible and to increase i t only when a loss of e f f i c a c y i s noted (e.g., Eadie, 1985; note that t h i s treatment strategy, though advocated as a way to reduce the incidence of unwanted side e f f e c t s , also r e f l e c t s the t r a d i t i o n a l assumption that the development of tolerance i s f a c i l i t a t e d by the administration of a high treatment dose). Although the data reported by Killam et a l . (1973) are puzzling when viewed from the t r a d i t i o n a l perspective of pharmacologic tolerance, they can be explained from the perspective of contingent t o l e r a n c e — t h e use of a high treatment dose would reduce the incidence of seizure a c t i v i t y i n P. papio, thereby reducing the f a c i l i t a t o r y e f f e c t that the occurrence of such a c t i v i t y during periods of drug exposure could have on the development of tolerance. The idea that convulsive stimulation during periods of drug exposure might f a c i l i t a t e the development of tolerance to the anticonvulsant e f f e c t of a n t i e p i l e p t i c drugs i s also supported by Voskuyl et a l . (1986), who found that tolerance developed to the anticonvulsant e f f e c t s of diazepam (2.5 mg/kg, IP) on maximal electroshock seizures only when the rats received convulsive stimulation 3 0 min af t e r each DZP i n j e c t i o n . There was no evidence of tolerance i n rats that received a "minimum" number (no other d e t a i l s given) of convulsive stimulations during the tolerance-development period. Voskuyl et a l . (1986) suggested that the loss of DZP's anticonvulsant e f f e c t i n rats that were re g u l a r l y stimulated i n the presence of the drug was a t t r i b u t a b l e to an exacerbation of the seizures due to a " k i n d l i n g - l i k e e f f e c t " caused by the stimulation schedule used. However, the fa c t that the authors f a i l e d to note a s i m i l a r k i n d l i n g e f f e c t i n control rats given the same number of stimulations r a i s e s the p o s s i b i l i t y that Voskuyl and his colleagues had unknowingly demonstrated contingent tolerance to DZP's anticonvulsant e f f e c t s — t h a t i s , that the development of tolerance was contingent upon the rats receiving convulsive stimulation during periods of diazepam exposure. Several c l i n i c a l reports also support the idea that the occurrence of convulsions during periods of drug exposure might influence the development of tolerance to the anticonvulsant e f f e c t s of a n t i e p i l e p t i c drugs. Bruni and Albright (1983) reported that tolerance to VPA's anticonvulsant e f f e c t developed most r a p i d l y i n e p i l e p t i c s that demonstrated the highest incidence of seizures p r i o r to the i n i t i a t i o n of drug treatment. 140 S i m i l a r l y , Meinardi et a l . (1986) found that the development of tolerance to VPA's anticonvulsant e f f e c t emerged r a p i d l y a f t e r the f i r s t instance i n which the drug l o s t i t s e f f i c a c y . These data can be attributed to an exacerbation of the seizure disorder i t s e l f (as Meinardi et a l . , [1986] suggest), or to the f a c t that e p i l e p t i c s experiencing the most severe seizures are the l e a s t l i k e l y to respond to the effects of a n t i e p i l e p t i c drugs ( i . e . , VPA was never t r u l y e f f e c t i v e i n c o n t r o l l i n g the seizures i n these p a t i e n t s ) . Alternatively, the same pattern of data would be expected i f the development of tolerance to VPA's anticonvulsant e f f e c t i s f a c i l i t a t e d by the occurrence of convulsive a c t i v i t y during periods of drug exposure. The patients with the most severe seizures p r i o r to drug treatment would be most l i k e l y to experience convulsive a c t i v i t y once drug treatment was i n i t i a t e d ; t h i s would accelerate the development of tolerance, so that i t would appear to emerge ra p i d l y once the f i r s t seizure was experienced. 2. Theories of Contingent Drug Tolerance: The Importance of A c t i v i t y to the Development of Tolerance Why should convulsive stimulation play such an important r o l e i n the development of tolerance to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA? A more general question has been the focus of considerable attention i n the area of contingent tolerance: Why does the a c t i v i t y of the drug r e c i p i e n t during periods of drug exposure play such an important r o l e i n the 141 development of tolerance to many drug effects? Three models have been proposed to account for the phenomenon of contingent tolerance: i) the reinforcement-density model; i i ) the state-dependency model; and i i i ) the homeostatic-conditioning model of contingent tolerance. These models w i l l be b r i e f l y reviewed i n the next three subsections, and t h e i r u t i l i t y as an explanation for contingent tolerance to anticonvulsant drug e f f e c t s w i l l be discussed. i . The Reinforcement-Density Model of Contingent Tolerance The reinforcement-density model of contingent tolerance (Corfield-Sumner & Stolerman, 1978; see also Demellweek & Goudie, 1983a,b; Schuster et a l . , 1966; Wolgin, 1989) i s based upon the observation that tolerance to a drug's behavioral e f f e c t s often develops only "when the i n i t i a l e f f e c t of the drug causes a loss of reinforcement; when the drug has no ef f e c t on reinforcement or when i t increases the frequency of reinforcement, no tolerance occurs." (Wolgin, 1989; p. 19). Based upon the p r i n c i p l e s of operant conditioning, the central idea i n the reinforcement-density hypothesis of contingent tolerance i s that tolerance to a drug's e f f e c t s emerges as the drug recipient develops behavioral strategies that compensate for the drug e f f e c t s that are responsible for the loss of reinforcement. The a c t i v i t y of the drug r e c i p i e n t during periods of drug exposure i s important because i t allows the drug recipient to interact with the reinforcement schedule that i s i n place; accordingly, only the 142 subjects i n the drug-before-responding condition develop tolerance to the drug's effects on the c r i t e r i o n response because only these subjects experience the loss of reinforcement that r e s u l t s when the c r i t e r i o n response i s performed while they are under the influence of the drug. The reinforcement-density hypothesis provides a p l a u s i b l e explanation of those examples of contingent drug tolerance i n which operant reinforcement p r i n c i p l e s are involved. For example, the reinforcement-density hypothesis provides a reasonable account of contingent tolerance to amphetamine•s anorexigenic e f f e c t s ; the development of tolerance coincides with the a c q u i s i t i o n of responses that compensate for the drug-induced stereotypy that interferes with the consummatory behavior (e.g., Salisbury & Wolgin, 1985; Wolgin, Thompson, & Oslan, 1987) . Accordingly, t h i s behavioral compensation can only occur i f the rats are allowed to engage i n consummatory behaviors while they are drugged (though see Wolgin, 1989, for a l t e r n a t i v e explanations for these data). However, the generality of the reinforcement-density hypothesis i s limited by at least f i v e shortcomings. F i r s t , there have been few attempts to define the behavioral changes that compensate for the disruptive effects of the drug (Demellweek & Goudie, 1983b; Goudie, 1988; Wolgin, 1989; though see Wolgin & Salisbury, 1985; Wolgin et a l . , 1987); as Goudie has pointed out, "the empirical observation of contingent behavioral tolerance does not allow the conclusion that the mechanism by 143 which tolerance developed necessarily involved instrumental learning." (Goudie, 1988, p. 546). Second, Wolgin (1989) has noted instances i n which contingent tolerance f a i l s to develop to a drug e f f e c t that produces an obvious loss of reinforcement and other instances i n which contingent tolerance develops to drug e f f e c t s that increase the amount of reinforcement that a subject receives (see also Demellweek & Goudie, 1983b) . The remaining three weaknesses of the reinforcement-density model of contingent tolerance are p a r t i c u l a r l y relevant to the present demonstrations of contingent tolerance to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA. The t h i r d shortcoming of the reinforcement-density hypothesis stems from i t s i n a b i l i t y to account for instances of contingent tolerance that do not seem to involve a reinforcement process. For example, the reinforcement-density hypothesis cannot r e a d i l y account for the development of tolerance to the anticonvulsant e f f e c t s of the drugs; I am aware of no evidence that kindled seizures can serve as either a p o s i t i v e or a negative r e i n f o r c e r and anticonvulsant drug effects would at least s u p e r f i c i a l l y appear to be b e n e f i c i a l to the drug re c i p i e n t . The reinforcement-density hypothesis has s i m i l a r d i f f i c u l t i e s accounting for contingent tolerance to the analgesic e f f e c t s of ethanol (e.g., J0rgenson et a l . , 1985; 1986) or morphine (e.g., Advokat, 1989) i n s p i n a l l y transected rats, or to the e f f e c t s of ethanol on the decay of posttetanic potentiation i n the abdominal ganglia of Aplysia (e.g. Traynor et a l . , 1980). 144 The fourth shortcoming of the reinforcement-density hypothesis of contingent tolerance i s i t s i n a b i l i t y to account for the r o l e that the drug dose and schedule of administration plays i n the development of contingent and pharmacologic tolerance to a drug's e f f e c t . Pharmacologic tolerance develops regardless of whether or not the c r i t e r i o n response i s performed during periods of drug exposure—and therefore i n the absence of a loss of reinforcement. The f i f t h , and f i n a l , shortcoming of the reinforcement-density hypothesis of contingent tolerance i s that i t cannot r e a d i l y account for the e f f e c t that performance of the c r i t e r i o n response i n the absence of drug exposure has on the d i s s i p a t i o n of contingent tolerance to the anticonvulsant e f f e c t s of ethanol (Mana & P i n e l , 1987). Although the role of the c r i t e r i o n response i n the d i s s i p a t i o n of contingent tolerance has not been widely studied, i t has been shown to be an important factor i n the d i s s i p a t i o n of contingent tolerance to amphetamine's anorexigenic e f f e c t (Poulos et a l . , 1981) and scopolamine's adipsic e f f e c t (Poulos & Hinson, 1984). A possible exception may be the report by LeBlanc et a l . , (1976), who found that contingent and pharmacologic tolerance to ethanol's e f f e c t s on a moving-belt task dissipate at the same rate. However, these r e s u l t s may r e f l e c t the influence of behaviors common to both the c r i t e r i o n response (a moving belt task) and normal locomotion (e.g., ataxia; sedation) on the d i s s i p a t i o n of tolerance, rather than a lack of e f f e c t of the c r i t e r i o n response. 145 i i . The State-Dependency Model of Contingent Tolerance The term state-dependency refers to situations i n which the e f f i c i e n t performance of a response i s dependent upon a subject being tested i n the same psychological state that existed when the response was acquired (Overton, 1966; 1984). According to the state-dependency hypothesis of tolerance (Chen, 1972; Cicero, 1980; Feldman & Quenzer, 1984; Wolgin, 1989), a response that was acquired by a subject in a drug-free state i s poorly performed during periods of drug exposure because the drug-induced change i n psychological state impairs the subject's a b i l i t y to r e t r i e v e the information necessary to perform the task. The development of tolerance to t h i s drug-induced impairment i s presumed to r e f l e c t the a c q u i s i t i o n of the response i n the drugged state. Thus, instances of contingent tolerance are attributed to the fact that only the subjects i n the drug-before group get the opportunity to acquire the c r i t e r i o n response while under the influence of the drug. The u t i l i t y of the state-dependency model as an explanation for contingent tolerance to the anticonvulsant e f f e c t s of CBZ, DZP, and VPA i s limited i n at least four ways. F i r s t , i t makes an e x p l i c i t prediction that the convulsions of rats that are tole r a n t to the ef f e c t s of an anticonvulsant drug should be impaired when the drug i s withdrawn u n t i l the rats can reacquire the convulsive response i n a drug-free state. Although the data i n the present experiments do not address t h i s issue, the r e s u l t s of e a r l i e r experiments from our lab provide no support f o r the idea that drug withdrawal has any ef f e c t on the convulsions of kindled rats that are tolerant to anticonvulsant drug e f f e c t s (Mana & Pi n e l , unpublished observations). Second, the state-dependency model of contingent tolerance cannot account for the development of pharmacologic tolerance to a drug's e f f e c t s . Although t h i s does not appear to be a serious l i m i t a t i o n for a model of contingent drug tolerance, i t i s es s e n t i a l to an understanding of the phenomena of contingent and drug pharmacologic tolerance unless the two are completely independent e n t i t i e s . Third, the state-dependency model of contingent tolerance cannot account for the ef f e c t that undrugged performance of the c r i t e r i o n response has on the d i s s i p a t i o n of tolerance to a drug's ef f e c t s . And fourth, the r o l e of cognitive processes i n the state-dependency hypothesis of contingent tolerance l i m i t s i t s usefulness to reports of contingent tolerance i n which a change i n the psychological state of the subject might influence the r e t r i e v a l of information required for the e f f i c i e n t performance of a task. For example, a state-dependency model i s more capable of accounting for contingent tolerance to ethanol's effects on a subject's performance of a maze task than to i t s effects on more r e f l e x i v e responses such as convulsions (e.g., Pinel et a l . , 1983), spinal reflexes (e.g., the t a i l - f l i c k response; J0rgenson & Hole, 1985), or responses i n reduced preparations (e.g., posttetanic potentiation i n the i s o l a t e d abdominal ganglion of Aplysia; Traynor et a l . , 1980). 147 i i i . The Homeostatic-Conditioning Model of Contingent Tolerance The homeostatic-conditioning model of drug tolerance was developed to describe the development and d i s s i p a t i o n of f contingent tolerance to the anorexigenic e f f e c t of amphetamine (Poulos et a l . , 1981) and to the adipsic e f f e c t s of scopolamine (Poulos & Hinson, 1984). I t represented an important advance i n the study of behavioral influences on drug tolerance because i t integrated the phenomena of context-specific tolerance and contingent tolerance into a single theory. According to Poulos and h i s associates, the development of contingent tolerance represents a homeostatic adaptation to a drug's e f f e c t s on the c r i t e r i o n response. That i s , the performance of the c r i t e r i o n response during periods of drug exposure i s important to the development of tolerance because a hypothetical homeostatic regulator compares the subject's actual l e v e l of behavior to a set-point l e v e l appropriate to the organism's needs; "the l e v e l of consummatory behavior i t s e l f rrather than the need state;  present author's note] constitutes a d i r e c t l y monitored b i o l o g i c a l system." (Poulos & Hinson, 1984, pp. 87). The subsequent manifestation of the homeostatic changes responsible for the development of tolerance i s context-specific; that i s , the manifestation of the tolerance i s dependent upon the drug being administered i n the same context that the subject previously experienced the drug's e f f e c t s . Poulos and h i s colleagues argued that t h i s contextual s p e c i f i c i t y i s the product of Pavlovian conditioning. F i n a l l y , performance of the c r i t e r i o n 148 response i n the absence of the drug leads to a d i s s i p a t i o n of the p h y s i o l o g i c a l changes responsible for the development of tolerance as the drug recipient's homeostatic mechanisms re e s t a b l i s h a balance between the subject's needs and the consummatory response. Although the synthesis of the areas of contingent and context-specific tolerance offered by the homeostatic-conditioning model i s appealing, i t s u t i l i t y as an explanation for contingent tolerance to anticonvulsant drug e f f e c t s i s l i m i t e d i n three ways. F i r s t , i t i s limited by the concepts of homeostasis' and Pavlovian conditioning. Many instances of contingent tolerance do not involve an obvious homeostatic regulation of the c r i t e r i o n response involved or a dependence upon contextual s t i m u l i ; these would include instances of contingent tolerance to the anticonvulsant e f f e c t s of drugs (e.g., P i n e l et a l . , 1983; 1989), as well as contingent tolerance to the analgesic e f f e c t s of ethanol (e.g., J0rgenson et a l . , 1985; 1986) or morphine (e.g., Advokat, 1989) i n s p i n a l l y transected rats and contingent tolerance to the e f f e c t s of ethanol on the decay of posttetanic potentiation i n the abdominal ganglia of Aplysia (e.g. Traynor et a l . , 1980). Second, the homeostatic-conditioning theory of contingent tolerance makes no p r e d i c t i o n about the conditions that would lead to pharmacologic, as opposed to contingent, tolerance to a drug's e f f e c t s . And t h i r d , the homeostatic-conditioning theory would predict that the c r i t e r i o n response should change following the cessation of drug 149 treatment u n t i l i t can be homeostatically regulated to a l e v e l s u i t a b l e for drug-free conditions. As mentioned e a r l i e r , drug withdrawal does not appear to a f f e c t the convulsions of kindled rat s that have been rendered tolerant to the anticonvulsant e f f e c t s of a drug. Summary The contingent tolerance phenomenon cannot be e n t i r e l y accounted for by any one of the three e x i s t i n g theories (see also Wolgin, 1989; Goudie & G r i f f i t h s , 1986). One possible reason for t h i s i s the d i v e r s i t y of the phenomenon. Contingent tolerance has been demonstrated to the effects of a variety of pharmacologically disparate drugs: 1) psychostimulants (e.g., amphetamine, Carlton & Wolgin, 1971; Demellweek & Goudie, 1982; cocaine, Woolverton et a l . , 1979); 2) cannabinoids (e.g., d e l t a -9-tetrahydrocannabinol, Manning, 1976) ; 3) sedative-hypnotics (e.g., Tizzano et a l . , 1986) and ethanol (e.g., Alkana et a l . , 1982; Chen, 1968; Pinel et a l . , 1983; 1989); 4) and opioids (e.g., morphine; Advokat, 1989; Kayan & M i t c h e l l , 1969; Smith, 1979) . The c r i t e r i o n response used to" study contingent drug tolerance has also varied widely: 1) barpress responding (e.g., Branch, 1979; Woolverton et a l . , 1979); 2) drinking (Poulos & Hinson, 1984) and 3) feeding (e.g., Carlton & Wolgin, 1971); 4) nociception (e.g., J0rgenson & Hole, 1984); 5) posttetanic potentiation (Traynor et a l . , 1982); 6) thermoregulation (e.g., Alkana et a l . 1982) ; 7) maze-running (Chen, 1968); 8) treadmill 150 running (LeBlanc et a l . , 1972); 9) mental rehearsal of a pursuit-rotor task (e.g., Sdao-Jarvie & Vogel-Sprott, 1986); and 10) convulsions (e.g., Pinel et a l . , 1983; 1989). The generality of the phenomenon and the magnitude of the e f f e c t s generated by manipulating the occurrence of the c r i t e r i o n response during periods of drug exposure c l e a r l y indicate that the a c t i v i t y of the drug r e c i p i e n t during periods of drug exposure i s a key factor i n the development of many forms of drug tolerance; however, i t i s not clear whether any single theory of contingent tolerance could explain each instance of the phenomenon. In p a r t i c u l a r , the ex i s t i n g theories of contingent tolerance cannot e a s i l y account for the phenomenon of contingent tolerance to anticonvulsant drug effects or i t s r e l a t i o n to the phenomenon of pharmacologic tolerance to the same drug e f f e c t s . Accordingly, an a l t e r n a t i v e conceptualization of contingent tolerance i s presented i n the next section. 3. An Activity-Dependent Analysis of Contingent and Pharmacologic Drug Tolerance The development and diss i p a t i o n of contingent tolerance can be summarized as follows: A change in neural a c t i v i t y during periods of drug exposure can influence the response of the neural system to the drug such that the drug subsequently has less of an e f f e c t on the a c t i v i t y . Conversely, when the same pattern or i n t e n s i t y of neural a c t i v i t y occurs i n the absence of the drug, there i s a functional change in these neural c i r c u i t s such that the e f f e c t of the drug on the a c t i v i t y i s restored. This axiom describing the development and d i s s i p a t i o n of contingent tolerance to many drug effects bears a s t r i k i n g s i m i l a r i t y to the more widely-recognized idea that concurrent a c t i v i t y of connected elements in a neural system has a key r o l e i n many forms of neural p l a s t i c i t y . Hebb (1949) i s often acknowledged as the f i r s t to succinctly express the importance of t h i s r e l a t i o n s h i p , i n what has become known as Hebb's Postulate: "When an axon of c e l l A i s near enough to excite a c e l l B and repeatedly and p e r s i s t e n t l y takes part i n f i r i n g i t , some growth or metabolic change takes place i n one or both of the c e l l s such that A's e f f i c i e n c y , as one of the c e l l s f i r i n g B, i s increased." (Hebb, 1949, pp. 62) . Stent's (1973) addendum to Hebb's Postulate captures the e f f e c t that asynchronous a c t i v i t y between the elements of a neural c i r c u i t has on the weakening of the connections between these elements: "When the presynaptic axon of c e l l A repeatedly and p e r s i s t e n t l y f a i l s to excite the postsynaptic c e l l B while c e l l B i s f i r i n g under the influence of other presynaptic axons, some metabolic change takes place i n one or both of the c e l l s such that A's e f f i c i e n c y , as one of the c e l l s f i r i n g B, i s decreased." (Stent, 1973; pp. 997) . In the l a s t decade, Hebb's Postulate has been expanded to 152 include synchrony between e l e c t r i c a l and/or chemical changes i n the postsynaptic neuron as a mechanism of Hebbian changes i n synaptic transmission (e.g., Changeux & Heidmann, 1987). In the next three sections of the discussion, the r o l e of concurrent a c t i v i t y i n three d i f f e r e n t forms of neural p l a s t i c i t y — i n the development of the neuromuscular junction, i n the development of ocular dominance columns i n the v i s u a l system, and i n the development of long-term potentiation i n the hippocampus—is reviewed. The purpose of t h i s review i s to i l l u s t r a t e the general importance of concurrent a c t i v i t y between the elements of a neural c i r c u i t to the p l a s t i c i t y of that c i r c u i t , and to provide a conceptual framework for an activity-dependent analysis of contingent and pharmacologic drug tolerance. i . Concurrent A c t i v i t y and the Neuromuscular Junction The neuromuscular junction i s one of the most widely studied synaptic connections i n the nervous system. The outgrowth of the motor neuron from the spinal cord to the muscle appears to be a function of the neuron following a preferred substrate as well as the concentration gradient for some type of trophic factor secreted by the appropriate target tissue (e.g., Landmesser, 1980; see also Kandel, 1985a). However, once the motor neuron has reached the muscle tissue concurrent a c t i v i t y between the neuron and the muscle plays a c r i t i c a l r ole i n the development of the appropriate synaptic connections. Prio r to the muscle's ennervation, the n i c o t i n i c acetylcholine (n-ACh) receptor i s widely d i s t r i b u t e d over the muscle. Upon ennervation by the motor neuron, the d i s t r i b u t i o n of t h i s receptor (and hence the ennervation of the muscle by the motor neuron) becomes r e s t r i c t e d to the motor end plate (see Kandel, 1985a). This change i s not simply a r e s u l t of the motor neuron ennervating the appropriate target zone but instead i s a re s u l t of the int e r a c t i o n between the a c t i v i t y of the motor neuron and the muscle that i t ennervates (e.g., Landmesser, 1980; L0mo & Rosenthal, 1972). When the motor neuron arrives at the muscle, the ACh that i t releases stimulates the muscle to contract. These contractions lead to the degeneration of n-ACh receptors except i n the area of the end plates; consequently, the muscle f i b r e i s ennervated by a single motor neuron and only in the area of the motor end-plate. The importance of the a c t i v i t y of both the muscle and the motor neuron to the proper development of the n-ACh synapse between them i s i l l u s t r a t e d by the fact that the formation of t h i s connection can be prevented, or established connections weakened, by the application of tetrodotoxin (a drug that blocks the voltage-sensitive sodium channels necessary for nerve impulse a c t i v i t y ) to the motor neuron (see L0mo & Rosenthal, 1972), or by the a p p l i c a t i o n of d-tubocurare (a n-ACh receptor antagonist that prevents the muscle from responding to the ACh released by the motor neuron) to the muscle (e.g., Landmesser, 1980). Although i t i s not clear that synchronous a c t i v i t y of the motor neuron and the muscle f i b r e i s necessary for the establishment of the n-ACh synapse between the two ( i . e . , i t i s possible that the proper 154 synaptic connections would form i f the motor neuron was simply close to a muscle f i b r e that was contracting because of exogenous stimulation), under normal circumstances the development of the n-ACh synapse between motor neurons and muscle f i b r e s can be described as follows: When a motor neuron i s near enough to activate a muscle f i b r e , and repeatedly and pe r s i s t e n t l y takes part i n i t s ac t i v a t i o n , some growth or metabolic change takes place i n the muscle f i b r e that r e s t r i c t s the n-ACh receptor, and thus the cholinergic ennervation of the muscle, to the muscle's end plates. When the muscle i s no longer stimulated by the neuron, the d i s t r i b u t i o n of the n-ACh receptor spreads along the length of the muscle f i b r e u n t i l i t i s reinnervated. i i . Concurrent A c t i v i t y and the Development of  Ocular Dominance Columns Synaptic connections i n the vi s u a l system undergo extensive reorganization during the course of normal development. A p a r t i c u l a r l y s t r i k i n g example of t h i s reorganization i s the development of ocular dominance columns (e.g., Hubel, Weisel, & LeVay, 1977; see also Kandel, 1985b,c). The v i s u a l cortex i s divided into s i x anatomically d i s t i n c t layers; v i s u a l information t r a v e l s v i a the retino-geniculo-striate path to neurons i n layer IVc of the v i s u a l cortex, and from there to the remaining layers. 155 Early i n development the c e l l s i n layer IVc respond to input from both eyes (at least i n the cat; Singer, 1987); over time, the gen i c u l o - s t r i a t e connections change in such a way that c e l l s i n t h i s layer of the v i s u a l cortex w i l l respond to input from only one of the two eyes. This monocular preference alternates i n a regular fashion across layer IVc; the neurons i n these monocular patches project into the layers of v i s u a l cortex above and below them so that many of these neurons w i l l also respond p r e f e r e n t i a l l y to input from one or the other of the two eyes (although there i s considerably less preference i n these layers because of the convergence of information from the d i f f e r e n t areas of layer IVc). These alternating columns of v i s u a l cortex, which respond p r e f e r e n t i a l l y to input from one or the other of the eyes, are referred to as ocular dominance columns. The normal development of ocular dominance columns i s believed to be the r e s u l t of a Hebbian competition between the inputs from the two eyes for connections with c o r t i c a l neurons i n layer IVc of the s t r i a t e cortex. The idea i s that geniculo-s t r i a t e synaptic connections are strengthened when converging input from one of the eyes to a given c o r t i c a l neuron i s s u f f i c i e n t to depolarize the c e l l and e l i c i t an action p o t e n t i a l ; synapses that are not involved i n the action p o t e n t i a l are weakened. This hypothesis i s supported by experiments showing that monocular, but not binocular, deprivation early i n l i f e a l t e r s the development of ocular dominance columns so that a l l c o r t i c a l c e l l s respond to input only from the nondeprived eye (e.g., Weisel & Hubel, 1965). The lack of e f f e c t following binocular deprivation i s attributed to the lack of competition between the eyes; neither eye can e l i c i t an action p o t e n t i a l from the c o r t i c a l neurons so there i s no change i n synaptic strength. The importance of concurrence between the a c t i v i t y of the genicular input and the s t r i a t e neurons to the development of ocular dominance columns was i l l u s t r a t e d by Shaw and Cynader (1984), who found that chronic infusion of glutamate (an excitatory amino acid neurotransmitter that would increase the f i r i n g of c e l l s i n the area of the infusion which normally receive glutaminergic ennervation) into s t r i a t e cortex during periods of monocular deprivation prevented the s h i f t i n ocular dominance that normally occurs. Presumably, the glutamate infusion prevented consistent concurrence between a given set of genicular inputs and the a c t i v i t y of the s t r i a t e cortex neurons, thereby blocking the strengthening and/or weakening of geniculo-s t r i a t e connections by a form of Hebbian competition. This idea was recently extended by Bear, Kleinschmidt, Gu, and Singer (1990), who showed that chronic infusion of the n-methyl-d-aspartate (NMDA) antagonist APV (which blocks the NMDA subtype of glutamate receptor that appears to play a key r o l e i n many forms of activity-dependent neural p l a s t i c i t y ) into s t r i a t e cortex also prevents the ocular dominance s h i f t that i s normally produced by monocular deprivation. Similar r e s u l t s were reported by Reiter and Stryker (1988) following the microinjection of the GABA-A agonist muscimol into s t r i a t e cortex during periods of monocular deprivation. Clearly, normal postsynaptic a c t i v i t y plays a c r i t i c a l r o l e i n the development of ocular dominance columns; more int e r e s t i n g l y , these data extend Stent's (1973) addendum to Hebb's Postulate; i n some cases, synaptic weakening can occur when the postsynaptic element i n a neural c i r c u i t i s inac t i v e and the presynaptic element i s active, as well as when the postsynaptic neurons are active during periods when the presynaptic elements are not. F i n a l l y , the contribution of normal presynaptic a c t i v i t y ( i . e . , the a c t i v i t y of genicular input from the eyes) to the development of ocular dominance columns was i l l u s t r a t e d by Stryker and Harris (1986), who found that binocular blockade of r e t i n a l transmission i n kittens by i n t r a v i t r e a l i n j e c t i o n of tetrodotoxin into the eyes (which would block the voltage-dependent sodium channels necessary for impulse conduction from the r e t i n a to the dorso-lateral geniculate) prevented the development of ocular dominance columns. Interestingly, Stryker and Harris (1986) found that i n t r a v i t r e a l tetrodotoxin was more e f f e c t i v e that dark-rearing or binocular suturing at preventing the development of ocular dominance columns; they argued that the spontaneous a c t i v i t y of r e t i n a l c e l l s i s s u f f i c i e n t to produce some segregation of genicular inputs in kittens that are deprived of normal v i s u a l experience. In summary, the development of ocular dominance columns can be summarized by the following adaptation of Hebb's Postulate: When genicular input to a s t r i a t e neuron repeatedly and 158 p e r s i s t e n t l y takes part i n the f i r i n g of that neuron, some growth or metabolic change takes place to strengthen the common synaptic connections and weaken those from genicular inputs that did not p a r t i c i p a t e i n the a c t i v i t y of the s t r i a t e neuron. When there i s asynchrony between the a c t i v i t y of genicular inputs to s t r i a t e cortex and the c o r t i c a l neurons, there i s a weakening of synaptic strength between these c e l l s . Concurrent a c t i v i t y s u f f i c i e n t to depolarize the s t r i a t e neurons i n an given area of layer IVc i s more l i k e l y to come from genicular input from one eye than the other; as a r e s u l t , the connections from the less well-connected eye eventually weaken to the point that the s t r i a t e c e l l s i n a given area of layer IVc respond p r e f e r e n t i a l l y to input from one eye only. i i i . Concurrent A c t i v i t y and Long-term Potentiation Perhaps the most well-studied example of the r o l e that concurrent a c t i v i t y between the pre- and postsynaptic elements of a neural c i r c u i t can have i n the p l a s t i c i t y of that c i r c u i t i s the phenomenon of long-term potentiation (LTP; e.g., B l i s s & L0mo, 1973; B l i s s & Lynch, 1989; Gustafsson & Wigstrom, 1988). Long-term potentiation involves the prolonged enhancement of synaptic transmission following tetanic stimulation of the afferent neurons; although i t has been most extensively studied i n the c i r c u i t r y of the hippocampus, LTP has been demonstrated 159 elsewhere i n the central nervous system (e.g., A r t o l a & Singer, 1987) . Several c h a r a c t e r i s t i c s of LTP support a Hebbian model of neural p l a s t i c i t y . F i r s t , concurrent a c t i v i t y of both the pre-and postsynaptic neural elements i s c r i t i c a l to the development of LTP. Hyperpolarization of the postsynaptic c e l l (so that i t cannot respond to afferent input) blocks the development of LTP i n s p i t e of tetanic stimulation of the afferent neurons (e.g., Gustafsson, Wigstr6m, Abraham, & Huang, 1987), and LTP can be induced by single afferent volleys (instead of tetanic stimulation of the afferent path) i f each afferent stimulation i s paired with a depolarizing current i n j e c t i o n to the postsynaptic neuron (e.g., Wigstrom, Gustafsson, Huang & Abraham, 1986; see also Freidlander, Sayer, & Redman, 1990). The depolarizaton of the postsynaptic neuron alone, or the occurrence of s i n g l e afferent v o l l e y s alone, are not s u f f i c i e n t to produce LTP (Wigstrom et a l . , 1986; see also B l i s s & Lynch, 1988). Support for Stent's (1973) addendum to Hebb's Postulate comes from studies by Steward and his colleagues (see Steward, White, Korol, & Levy, 1989, for a review) who have found that LTP established at connections between neurons i n the entorhinal cortex and the c o n t r a l a t e r a l dentate gyrus can be reversed i f the neurons i n the dentate gyrus subsequently receive t e t a n i c stimulation from the i p s i l a t e r a l entorhinal cortex i n the absence of concurrent input from the c e l l s i n the c o n t r a l a t e r a l entorhinal cortex. The authors noted, "Synapses that are 160 coactive with those inducing the modifiable state undergo potentiation; synapses that are s i l e n t during the modifiable state exhibit a long-term depression..." (Steward et a l . , 1989; pp. 139); an adequate summary of both Hebb's Postulate and Stent's addendum to that postulate. i v . Summary It i s c l e a r that concurrent a c t i v i t y between the pre- and postsynaptic elements i n a neural c i r c u i t i s an important factor i n the p l a s t i c i t y at many d i f f e r e n t neural systems. Bear (1987) has suggested that "coincidence of a c t i v i t y may be the basic algorithm of activity-dependent changes in excitatory c i r c u i t r y . " (pp. 290) ; i t s r o l e i n the p l a s t i c i t y of neural c i r c u i t r y as diverse as the hippocampus and the neuromuscular junction suggests that such a claim i s not u n r e a l i s t i c (see also Changeux & Heidmann, 1987; Frank, 1987; Merzenich, 1987; Singer, 1987). The next section w i l l extend the idea of activity-dependent neural p l a s t i c i t y to an analysis of contingent and pharmacologic tolerance to anticonvulsant drug e f f e c t s . v. The Role of Neural A c t i v i t y in the Development of  Functional Drug Tolerance: A Dissociation of Contingent and  Pharmacologic Tolerance to DZP's Anticonvulsant E f f e c t Contingent tolerance to anticonvulsant drug e f f e c t s can be distinguished from pharmacologic tolerance to the same drug e f f e c t s i n the following three ways: 1) Contingent tolerance requires less frequent administration, and/or smaller doses, of an anticonvulsant drug than pharmacologic tolerance (see Experiment 2 and Experiment 3, present thesis; Mana, Le, Kalant, & P i n e l , i n preparation; see also J^rgenson et a l . , (1986), and Le et a l . , (1986), for si m i l a r evidence from other types of contingent tolerance). 2) The development of contingent tolerance to anticonvulsant drug e f f e c t s requires that convulsive stimulation i s administered during periods of drug exposure; pharmacologic tolerance does not (Mana, Le, et a l . , i n preparation; Experiment 2 and Experiment 3, present t h e s i s ) . 3) The d i s s i p a t i o n of contingent tolerance to anticonvulsant drug e f f e c t s i s slower than that of pharmacologic tolerance (Experiment 4, present thesis) and may be influenced by the administration of convulsive stimulation i n the absence of the drug (Mana & Pinel, 1987; see also Poulos et a l . , 1981; 1984; for similar evidence from other types of contingent tolerance). In the text to follow, i t i s argued that these differences between contingent and pharmacologic tolerance can be accounted for by an analysis of functional drug tolerance that i s based upon the l e v e l or pattern of neural a c t i v i t y occurring during periods of drug exposure. The key assumption of t h i s activity-dependent model of contingent and pharmacologic tolerance i s that the expression of a drug's e f f e c t i s a function not just of factors associated with drug exposure (e.g., dose; schedule of administration), but also of the a c t i v i t y of the nervous system during periods of drug exposure. A c o r o l l a r y to t h i s assumption i s that the development of tolerance (T) w i l l also be a function of both pharmacologic factors (P) and the a c t i v i t y of the neural c i r c u i t s (N) during the periods of drug e x p o s u r e — r e c a l l that functional drug tolerance develops not to drug exposure per se, but to the drug's e f f e c t s on neural function (e.g., Kalant, 1985; Kalant et a l . , 1971; see also Pinel et a l . , i n press). That i s , the r e l a t i o n s h i p between pharmacologic factors and neural a c t i v i t y to the development of drug tolerance can be represented by the following expression: T = P X N. When an anticonvulsant drug i s administered c h r o n i c a l l y , or at r e l a t i v e l y high doses, the a c t i v i t y of the nervous system i s less important to the development of tolerance to the drug's e f f e c t s because the pharmacologic stimuli are of s u f f i c i e n t magnitude or duration that they disrupt the basal a c t i v i t y of the CNS to a degree s u f f i c i e n t to e l i c i t the appropriate changes. Thus, pharmacologic tolerance can be represented by a s l i g h t change i n the expression used to describe the development of tolerance; s p e c i f i c a l l y : T = P X n (where the case of the l e t t e r s i n the r i g h t part of the expression denotes the r e l a t i v e contribution of the pharmacologic and a c t i v i t y - r e l a t e d factors to the development of tolerance). I t must be stressed that a basal amount of neural a c t i v i t y i s assumed to be necessary for the drug e f f e c t s to express themselves and functional tolerance to develop, even under optimal pharmacologic conditions; i f the a c t i v i t y of the appropriate neural c i r c u i t s could be eliminated during the periods of drug exposure then tolerance would not develop, regardless of the amount of pharmacologic stimulation that was available. In contrast, the a c t i v i t y of the nervous system i s more important to the development of tolerance when an anticonvulsant drug i s given on a subchronic basis because the l i m i t e d pharmacologic stimulation does not disrupt the basal a c t i v i t y of the nervous system enough to e l i c i t the physiological changes responsible for the development of tolerance. In t h i s s i t u a t i o n , the development of tolerance to a drug's anticonvulsant e f f e c t i s contingent upon convulsive stimulation a f f e c t i n g the pattern or i n t e n s i t y of neural f i r i n g during each period of drug exposure, so that the drug's anticonvulsant e f f e c t can be maximally expressed; i n terms of the expression used e a r l i e r , contingent tolerance can be represented as: T = p X N. The application of these ideas to the development of contingent and pharmacologic tolerance to DZP's anticonvulsant e f f e c t i s presented by the model neural c i r c u i t i n Figure 15. In t h i s simple system, there a population of excitatory neurons, designated E, that receive convulsive stimulation (either d i r e c t l y or from inputs from the primary s i t e of seizure a c t i v i t y ) . C o l l a t e r a l axons from these 164 Figure 15. Model system i l l u s t r a t i n g several possible s i t e s of physiologic adaptation that could serve as the physiologic basis for the activity-dependent d i s t i n c t i o n between pharmacologic tolerance and contingent tolerance to DZP's anticonvulsant e f f e c t . Pharmacologic tolerance may be due to a decrease i n the s e n s i t i v i t y of the GABA-A receptors associated with the GABA/benzodiazepine complex (Gb) found on E-type excitatory neurons. Contingent tolerance may be due to a more widespread s u b s e n s i t i v i t y at these GABA-A receptors, to a more permanent expression of these changes, or to other changes i n the GABA/benzodiazepine complex. Alternatively, i t may be due to a change distant from the GABA/benzodiazepine complex—for example, i n the connections between the excitatory neurons and t h e i r target neurons. See text for further d e t a i l s . FEEDBACK INHIBITION + < 6 b E CONVULSIVE STIMULATION 9 ui 166 neurons synapse on in h i b i t o r y GABA-containing neurons, designated I. These i n h i b i t o r y interneurons project back to synapse upon the GABA-A/benzodiazepine receptor complex, designated Gb, that are located on the soma and dendrites of the excitatory neurons. Thus, there i s an in h i b i t o r y feedback loop that reduces the e x c i t a b i l i t y of the excitatory neurons that i s f a c i l i t a t e d when DZP i s present. The excitatory neurons also propogate the seizure a c t i v i t y to other structures in the brain, designated T or target neurons. Even i n t h i s simple c i r c u i t , there are a number of d i f f e r e n t ways that an activity-dependent d i s s o c i a t i o n between contingent and pharmacologic tolerance to DZP's anticonvulsant e f f e c t could be expressed. For example, one possible mechanism i s i s based on the assumption that the s e n s i t i v i t y of the GABA-A receptor i s a function of the concurrence of the occupation of the GABA-A receptor and the benzodiazepine receptor by t h e i r respective agonist ligands. S p e c i f i c a l l y , there i s a decrease i n the s e n s i t i v i t y of the GABA-A receptor when the GABA-A receptor and the benzodiazepine receptor are repeatedly occupied at the same time. Under basal conditions the concurrent occupation of both the GABA-A receptor and the benzodiazepine receptor i s r e l a t i v e l y infrequent because the basal a c t i v i t y of the c i r c u i t , and therefore the basal release of GABA, i s low. Accordingly, DZP must be administered on a chronic basis for GABAergic s u b s e n s i t i v i t y to develop. When convulsive stimulation i s administered, there i s an increase in the release of GABA due to 167 an increase i n the activation of the c i r c u i t ; consequently, DZP can be administered less frequently and s t i l l e l i c i t a s i g n i f i c a n t decrease i n GABAergic subsensitivity. The robustness of contingent tolerance to DZP's anticonvulsant e f f e c t could be due to a decrease i n the s e n s i t i v i t y of a greater number of GABA-A receptors because convulsive stimulation e l i c i t s an increase i n GABA release i n the presence of DZP. Al t e r n a t i v e l y , there could be a more permanent change e l i c i t e d at the GABA-A receptor when convulsive stimulation produces an increase i n GABA release i n the presence of DZP—perhaps because the occupation of both the GABA-A receptor and the benzodiazepine binding s i t e during a convulsive stimulation results in a prolonged i n f l u x of chloride ions (because of the DZP-facilitated e f f e c t of GABA at the chloride channel associated with the GABA/benzodiazepine receptor complex), or because the a l l o s t e r i c modulation of the GABA receptor by DZP i s concurrent with a tremendous change i n membrane po t e n t i a l (due to the convulsive stimulation). This l a t t e r p o s s i b i l i t y could be extended to account for the possible r o l e of convulsive stimulation in the d i s s i p a t i o n of contingent tolerance as well—perhaps there i s a return i n the s e n s i t i v i t y of the GABA-A receptor and a di s s i p a t i o n of tolerance to DZP's anticonvulsant e f f e c t when there i s a prolonged membrane depolarization (produced by convulsive stimulation) while the GABA receptor i s unoccupied, or at least not occupied at the same time as the benzodiazepine binding s i t e . Although r e l a t i v e l y simple, the proposed model accounts for 168 an activity-dependent d i s s o c i a t i o n of pharmacologic and contingent tolerance to DZP's anticonvulsant e f f e c t , both i n terms of the development and the di s s i p a t i o n of these two forms of tolerance. However, i t i s pos s i b l e — a n d l i k e l y — t h a t a completely d i f f e r e n t type of physiological change could be responsible at least i n part for the development and expression of contingent tolerance to DZP's anticonvulsant e f f e c t . For example, contingent tolerance to DZP's anticonvulsant e f f e c t could be due to an activity-dependent change i n the synaptic connections between the excitatory neurons and t h e i r targets. A noteworthy feature of t h i s possible mechanism of contingent tolerance to DZP's anticonvulsant e f f e c t i s i t s emphasis on the idea that the physiologic changes that underlie such tolerance do not have to occur at the GABA/benzodiazepine complex; the fact that there i s a binding s i t e for the benzodiazepines i n the CNS does not imply that changes i n the ef f i c a c y of these drugs has to occur at t h i s s i t e . In pa r t i c u l a r , i f t h i s type of change i s responsible for the development of contingent tolerance to DZP's anticonvulsant e f f e c t then such tolerance would not be affected by the administration of RO 15-1788 because the GABA/benzodiazepine receptor complex i s not involved. In summary, i n an activity-dependent model of pharmacologic and contingent tolerance to anticonvulsant drug e f f e c t s the occurrence of convulsive stimulation during periods of drug exposure can influence the development of tolerance i n two ways. F i r s t , concurrence between convulsive stimulation and drug 169 exposure may f a c i l i t a t e the development of tolerance by f a c i l i t a t i n g the expression of the drug's anticonvulsant e f f e c t . This i s e s p e c i a l l y important when a low, intermittent treatment dose i s administered; t h i s i s recognized as contingent tolerance. Second, convulsive stimulation during periods of drug exposure may e l i c i t a more robust set of, or even a d i f f e r e n t set of, physiologic adaptations to the drug's anticonvulsant e f f e c t ; these are expressed as contingent tolerance. Caveats and Fi n a l Comments The activity-dependent dissoc i a t i o n between pharmacologic and contingent tolerance to DZP's anticonvulsant e f f e c t that was presented i n the preceding section could be generalized to accommodate many of the various instances of contingent and pharmacologic tolerance that have been reported. For example, the idea of an activity-dependent change i n synaptic transmission as a basis for drug tolerance could provide a useful s t a r t i n g point for examining the neural basis of instances of contingent tolerance involving r e l a t i v e l y simple neural c i r c u i t s (e.g., spi n a l reflexes such as the t a i l - f l i c k response; reduced preparations such as the abdominal ganglia of Aplysia). I t i s less l i k e l y that such an approach could provide an immediate ins i g h t into the mechanism responsible for contingent tolerance involving more cognitively-mediated c r i t e r i o n responses (e.g., maze strategies; mental rehearsal of a task). As Kalant (1985) has pointed out, such examples of drug tolerance may prove to be 170 as d i f f i c u l t to understand as the phenomenon of learning i t s e l f — and although Hebbian modification of synaptic e f f i c a c y i s widely regarded as a basic feature of the neural p l a s t i c i t y that underlies many forms of learning (e.g., Bear, 1987; Changeux & Heidmann, 1987), our understanding of the neural basis of learning i s far from complete. The hypothetical example of activity-dependent tolerance to DZP's anticonvulsant e f f e c t that was presented i n the preceding section i s pure speculation; to my knowledge, there i s no evidence to support the idea of an activity-dependent modulation of the GABA-benzodiazepine receptor complex, or a r o l e for some form of Hebbian modulation of either excitatory or i n h i b i t o r y synapses, i n the development of tolerance to the anticonvulsant e f f e c t s of DZP or any other drug. Accordingly, the proposed mechanisms should be considered only as h e u r i s t i c devices useful for conceptualizing the types of changes that could account for the phenomenological differences between pharmacologic and contingent drug—and perhaps as the basis for future studies into the p h y s i o l o g i c a l basis of tolerance to DZP's anticonvulsant e f f e c t . 3. Contingent and Pharmacologic Tolerance to DZP's Anticonvulsant E f f e c t : Common or Independent Physiological Bases? The f i r s t researchers to demonstrate the phenomenon of contingent tolerance frequently argued that i t was not 171 explainable i n terms of the physiological changes believed to underlie pharmacologic tolerance. Schuster et a l . (1966) argued, "Clearly, the common physiological mechanisms responsible for drug tolerance cannot be appealed to as an explanation" (of contingent tolerance to amphetamine's ef f e c t s on an operant task; p. 177); s i m i l a r l y , Chen (1968) believed that "In order to explain t h i s type of tolerance effect, physico-chemical mechanisms are not s u f f i c i e n t . . . " (p. 439). However, t h i s view has been impossible to support empirically; "The problem therefore remains whether the tolerance produced under these d i f f e r i n g conditions i s of d i f f e r e n t types, or i s of a single type to which the additive effects of separate s t i m u l i have contributed." (Kalant, 1989). The r e s u l t s of the f i n a l two experiments i n the present thesis r e f l e c t e d marked differences i n both the spontaneous and the RO 15-1788-induced reduction of contingent tolerance and pharmacologic tolerance to DZP's anticonvulsant e f f e c t . In Experiment 4, pharmacologic tolerance to DZP's anticonvulsant e f f e c t dissipated over the 16-day retention i n t e r v a l ; i n contrast, there was no evidence of a decline i n contingent tolerance to DZP's anticonvulsant e f f e c t over the same time period. The time course for the d i s s i p a t i o n of pharmacologic tolerance to DZP's anticonvulsant e f f e c t was s i m i l a r to that reported e a r l i e r by Rosenberg's group (e.g., Rosenberg et a l . , 1985; Rosenberg et a l . , 1986; Rosenberg et a l . , 1989) and by Gonsalves and Gallager (1987). Both of these groups have suggested that pharmacologic tolerance to DZP's anticonvulsant e f f e c t i s the r e s u l t of a decrease i n the s e n s i t i v i t y of the GABA-A receptor associated with the benzodiazepine binding s i t e (e.g., Gallager et a l . , 1984; 1985; Gonsalves & Gallager, 1987; 1988; Rosenberg et a l . , 1985; Teitz & Rosenberg, 1988), which reduces the i n h i b i t o r y e f f e c t of GABAergic transmission as well as the GABAergic f a c i l i t a t i o n of DZP binding that normally occurs at the GABA/benzodiazepine receptor complex. Accordingly, the difference i n the rate of di s s i p a t i o n between pharmacologic and contingent tolerance to DZP's anticonvulsant e f f e c t s on kindled seizures i n Experiment 4 suggests that contingent tolerance to DZP's anticonvulsant e f f e c t i s not attributable to the same decrease i n GABAergic s e n s i t i v i t y . The r e s u l t s of Experiment 5 support t h i s hypothesis. In Experiment 5, a single i n j e c t i o n of RO 15-1788 produced a s i g n i f i c a n t attenuation i n the expression of pharmacologic tolerance, but not contingent tolerance, to DZP's anticonvulsant e f f e c t . The reduction of pharmacologic tolerance to DZP's anticonvulsant e f f e c t by a single i n j e c t i o n of RO 15-1788 extends e a r l i e r work by Gallager and Gonsalves (1988; see also Gonsalves & Gallager, 1985), and supports the idea that pharmacologic tolerance, but not contingent tolerance, to DZP's anticonvulsant e f f e c t i s attr i b u t a b l e to a decrease in the s e n s i t i v i t y of GABA-A receptors associated with the benzodiazepine receptor. The mechanism by which RO 15-1788 reverses the decrease i n GABAergic s e n s i t i v i t y i s unknown; however, Gonsalves & Gallager (1985; 1988; see Gallager, Rauch, & Malcolm, 1984) have suggested that the h i g h - a f f i n i t y , low-sensitivity state of the GABA-A receptor that i s produced by chronic DZP treatment i s a l l o s t e r i c a l l y reversed when the benzodiazepine receptor i s occupied by an antagonist such as RO 15-1788. The f a c t that RO 15-1788 had no e f f e c t on contingent tolerance to DZP's anticonvulsant e f f e c t supports the idea that GABAergic s u b s e n s i t i v i t y does not play a role i n the expression of t h i s form of tolerance. However, as noted i n the discussion for Experiment 5 and i n the preceding section of the General Discussion, the r e l a t i v e i n s e n s i t i v i t y of contingent tolerance to DZP's anticonvulsant e f f e c t to pharmacologic reversal by RO 15-1788 could also be due to an enhancement of an a l l o s t e r i c decrease i n the s e n s i t i v i t y of the GABA-A receptor i s presumed to undergo during the development of pharmacologic tolerance to DZP's anticonvulsant e f f e c t — s u c h an enhancement could also be responsible for the fact that contingent tolerance to DZP's anticonvulsant e f f e c t did not dissipate over the 16-day retention i n t e r v a l used i n Experiment 4, or following an i n j e c t i o n of RO 15-1788. Thus, both pharmacologic tolerance and contingent tolerance to DZP's anticonvulsant e f f e c t could be due to a change i n the s e n s i t i v i t y of GABA-A receptors, with contingent tolerance representing nothing more than a more extreme manifestation of t h i s change. A resolution to t h i s problem i s beyond the scope of t h i s t h e s i s ; however, the marked differences i n spontaneous and pharmacologic d i s s i p a t i o n of contingent and pharmacologic 174 tolerance to DZP's anticonvulsant e f f e c t suggest that t h i s phenomenon could provide f e r t i l e ground for further analysis of the p h y s i o l o g i c a l bases of these phenomenologically d i s t i n c t forms of tolerance. At the very least, the phenomenon of contingent tolerance may provide a more stable and e a s i l y detected set of physiological changes with which to study the phenomenon of drug tolerance at a more molecular l e v e l . X. IMPLICATIONS, i . C l i n i c a l Implications The data from the f i v e experiments contained i n the present thesis are c l i n i c a l l y relevant i n at least two ways. F i r s t , the data from Experiment 2 c l e a r l y indicate that the occurrence of convulsive stimulation during periods of drug exposure can influence the development of tolerance to the anticonvulsant e f f e c t of a n t i e p i l e p t i c drugs. These data are s i m i l a r to those reported e a r l i e r by Killam et a l . (1973), who found that tolerance to DZP's anticonvulsant effects developed fa s t e r i n P.  papio when the treatment dose of DZP was low enough that the baboons could experience infrequent convulsions. Killam et a l . (1973) noted that the development of tolerance could be blocked by the administration of a high treatment dose of DZP; s i m i l a r data have recently been noted by Kim and Pinel (unpublished observations), who found that the development of tolerance to pentobarbital's anticonvulsant e f f e c t could be retarded i f a very high treatment dose was administered. Considered together, these 175 data support the idea that the development of tolerance to anticonvulsant drug effects could be reduced i f the treatment dose used from the outset of treatment i s high enough to e f f e c t i v e l y suppress a l l seizure a c t i v i t y and not just the convulsions produced by such a c t i v i t y (see also Koella & Meinardi, 1986b). There are two obvious problems with such a treatment strategy. F i r s t , the use of a high treatment dose could reduce the l i k e l i h o o d of contingent tolerance developing to a drug's anticonvulsant e f f e c t at the same time that i t promotes the development of pharmacologic tolerance to the same e f f e c t s . This problem w i l l be discussed further i n the next paragraph. The second problem with such a strategy i s that undesirable side-e f f e c t s are also more l i k e l y with an increase i n drug dose; accordingly, the treatment dose would have to compromise between one high enough to prevent the development of tolerance and one low enough to maintain an acceptable therapeutic index for the drug. The second c l i n i c a l implication of the present data concerns the marked reduction of tolerance to DZP's anticonvulsant e f f e c t that was produced by a single i n j e c t i o n of the benzodiazepine antagonist RO 15-1788. The development of tolerance to the anticonvulsant e f f e c t s of the benzodiazepines has been widely acknowledged as the major reason why t h i s class of a n t i e p i l e p t i c drug i s not widely employed i n the treatment of the ep i l e p s i e s (see Engel, 1989; Haigh & Feely, 1988; Robertson, 1986), and 176 considerable attention has been devoted to the development of new benzodiazepine ligands that are less prone to the development of tolerance (e.g., Haigh & Feely, 1988). The r e s u l t s of Experiment 5 suggest that periodic administration of RO 15-1788 (coadministered with a nonbenzodiazepine a n t i e p i l e p t i c to control seizures while the benzodiazepine receptor i s occupied by the RO compound) might prevent the development of pharmacologic tolerance to the anticonvulsant effects of e x i s t i n g benzodiazepine a n t i e p i l e p t i c drugs. Although RO 15-1788 was i n e f f e c t i v e i n reversing contingent tolerance to DZP's anticonvulsant e f f e c t , t h i s problem might be reduced i f a high treatment dose of DZP was used (to reduce the l i k e l i h o o d of contingent tolerance) i n conjunction with periodic administration of RO 15-1788 and a nonbenzodiazepine anticonvulsant (to reduce the development of pharmacologic tolerance). i i . The Activity-Dependent Model of Drug Tolerance Perhaps the most interesting feature of t h i s thesis i s the introduction of a model of activity-dependent drug tolerance. This model has several advantages over e a r l i e r models of contingent drug tolerance. F i r s t , i t integrates the phenomenon of contingent tolerance into the larger l i t e r a t u r e of pharmacologic drug tolerance; the relationship between these two phenomenologically d i s t i n c t processes has not been e x p l i c i t l y dealt with i n e a r l i e r models of contingent tolerance. Second, an activity-dependent model of drug tolerance provides some s p e c i f i c predictions about the factors that determine which type of tolerance w i l l develop and why they are important, as well as some predictions about the types of physiological change that could underlie the development of pharmacologic or contingent tolerance. F i n a l l y , an activity-dependent model of drug tolerance integrates the study of pharmacologic and contingent drug tolerance into the much larger l i t e r a t u r e on a c t i v i t y -dependent change i n the nervous system that has developed i n other areas of the neurosciences. This type of integration i s c r i t i c a l to a better understanding of the r e l a t i o n s h i p between contingent and pharmacologic tolerance and the phenomenon of drug tolerance i n general. As Kalant (1989) recently observed: "From the b i o l o g i s t s point of view, a purely behavioral explanation of drug tolerance... i s a description, rather than an explanation, unless i t attempts to l i n k the behavioral influences with c e l l u l a r mechanisms. S i m i l a r l y , a c e l l u l a r explanation i s not an explanation i f i t does not take into account the very important influences that behavioral and environmental factors can exert upon the development of tolerance....Unless both approaches are employed in an integrated manner, i t w i l l be impossible to answer the question as to whether the various behavioral and pharmacological factors e l i c i t the same type of tolerance or act through e n t i r e l y d i f f e r e n t mechanisms." (pp. 572). The purpose of t h i s thesis was to contribute to such an integrative approach to the study of drug tolerance. 178 XI. References Advokat, C. (1989). 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