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Learned tolerance to the behaviorally disruptive effects of ethanol
BEHAVIORAL AND NEURAL BIOLOGY 28, 418--430 (1980)
Learned Tolerance to the Behaviorally Disruptive Effects of Ethanol JOHN R . W E N G E R , 1 VICKI BERLIN, AND STEPHEN C . WOODS ~
Department of Psychology, University of Washington, Seattle, Washington 98195 Rats were trained to walk on a moving belt in order to avoid footshock. Rats which were then given ethanol each day before behavioral training became tolerant; rats which received equal doses of ethanol after behavioral training did not become tolerant relative to a group of saline controls. This result was interpreted to mean that the acquisition of behavioral tolerance to ethanol was mediated by learning. The methodology of measuring behavioral tolerance was discussed.
"Behavioral tolerance" to a drug is a decrease of the behavioral effects of the drug after it has been administered for a number of times. Behavioral tolerance to most behavior-modifying drugs has been reported to be due at least in part to learning. This has been reported for amphetamine (Campbell, & Seiden, 1973; Carlton & Wolgin, 1971; Schuster, Dockens, & Woods, 1966), marihuana (Carder & Olson, 1973; Olson & Carder, 1974), opiates (Siegel, 1975a, 1975b, 1977, 1978; Siegel, Hinson, & Krank, 1978), phenobarbital (Tang & Falk, 1978), and ethanol (Chen, 1968, 1972). However, recent reports (LeBlanc, Gibbins, & Kalant, 1973; LeBlanc, Kalant, & Gibbins, 1976) have severely criticized the methodology of the earlier ethanol tolerance studies and have concluded that intoxicated practice (i.e., learning) accelerates but is not necessary for the development of behavioral tolerance to ethanol. Recent reviews (Cicero, 1978; Corfield-Sumner & Stolerman, 1978; Kalant, LeBlanc, & Gibbins, 1971; LeBlanc & Cappell, 1977; LeBlanc, Poulos, & Cappell, 1978) have endorsed the interpretation that learning facilitates the development of cellular tolerance but is not a separate tolerance process. This view regarding behavioral tolerance to ethanol seems to be well documented (LeBlanc et al., 1976). For example LeBlanc et al. reported that ethanol-intoxicated practice contributes to a faster acquisition of, but 1 To whom requests for reprints should be addressed: Department of Psychology, NI-25, University of Washington, Seattle, Wash. 98195. Supported by grants from the University of Washington Alcoholism and Drug Abuse Institute and from the University of Washington Graduate Research Fund. We thank Herman Samson for his critical comments. 418 0163-1047/80/040418-13502.00/0 Copyright(~ t980 by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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not a higher final level of, tolerance to ethanol-induced impairment of motor performance. They termed this phenomenon behavioral augmentation of tolerance acquisition (BAT). In their experiments, they trained rats to walk on a moving belt (treadmill) to avoid footshock. In order to measure the rate of development of tolerance, they intraperitoneally (ip) injected animals with ethanol every 4 days and then required them to walk on the treadmill while intoxicated. On the intervening 3 days, some of the animals received ethanol after their daily practice on the treadmill (Ethanol-After-Practice Group), some before their practice (EthanolBefore-Practice Group), and others received saline before their practice (Saline Group). The animals which performed the moving belt task while intoxicated (i.e., which were given ethanol before practice each day) became tolerant sooner than those which received ethanol after practicing the task although these latter animals eventually reached the same level of intoxicated performance. These investigators concluded that learning accelerates but is not necessary for the development of behavioral tolerance to ethanol. There is an alternative interpretation of their results. If one regards each of the tolerance-test days as a potential learning opportunity, (i.e., the days when ethanol was given to determine if tolerance was developing) then the Ethanol-After-Practice Group would also be expected to learn the response, but learning would be relatively slow since this group received only one-fourth of the (relevant) intoxicated practice as did the Ethanol-Before-Practice Group. LeBlanc et al. recognized this possibility and performed another experiment to test it. They found that animals tested while intoxicated every day became tolerant sooner than animals tested every 2 days, and that these animals in turn became tolerant sooner than animals tested every 3 days. Animals tested 4 or more days apart never became tolerant. These findings were interpreted by LeBlanc et al., to mean that testing for ethanol tolerance every 4 days does not in itself lead to tolerance acquisition and that therefore the tolerance tests in the previous study should not be regarded as tolerance-learning trials. However, no ethanol was given to the animals (in the second experiment) on the days between tolerance-testing days. Therefore, the results of this (second) study could not be used to rule out the possibility of an interaction effect (in the first study) between tolerance testing every fourth day and exposure to ethanol on the intertest days. Thus, it is possible that in LeBlanc's first experiment, testing for tolerance every 4 days in combination with exposure to ethanol on the intertest days led to learned tolerance by the Ethanol-After-Practice Group. The questioning of the conclusions of LeBlanc et al. has been necessitated by a more recent series of investigations by Gallaher (1976) that suggests that learning is actually involved in the acquisition of tolerance to ethanol. Using the same treadmill-type apparatus as LeBlanc et al., Gal-
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laher found that rats which were injected with ethanol and tested daily developed nearly maximum tolerance in 30 days, whereas rats injected with ethanol daily but tested behaviorally only weekly attained only half of this asymptotic level of tolerance after 47 days. Giving this latter group of rats subsequent daily intoxicated practice led to their becoming fully tolerant within only 5 additional days. Gallaher interpreted his data to mean that the rate of tolerance acquisition was limited by the rate of practice in the drugged state and that at least some aspects of the tolerance were therefore learned. The low but reliable level of presumably not-learned (and therefore presumably induced by mere exposure to ethanol) tolerance exhibited by Gallaher's animals given daily ethanol but tested only weekly could have been caused by several factors: the daily physiological exposure to the ethanol, or the opportunity to learn during the weekly tolerance-testing sessions, or (more likely) some combination of these two factors. However, an alternative interpretation is possible. His weekly tested animals were not run at all between tests and therefore may have forgotten important components of the treadmill task. Moreover, since the daily tested animals had seven times the amount of practice as the weekly tested animals, the extra practice would be expected to produce overlearning of the treadmill task. Therefore, the greater practice of the daily tested animals would be expected to render their behavior more resistant to disruption by ethanol. To summarize the relevant literature, it is becoming increasingly accepted that behavioral tolerance to many drugs is, at least in part, learned (Chen, 1968, 1972; Campbell & Seiden, 1973; Carder & Olson, 1973; Olson & Carder, 1974; Carlton & Wolgin, 1971; Schuster et al., 1966; Siegel, 1975a, 1975b, 1977, 1978; Siegel et al., 1978; Tang & Falk, 1978). It seems reasonable that learning may also contribute to the behavioral tolerance shown to ethanol by rats. However, for reasons discussed above, the issue remains controversial and unsettled. The social importance of the behaviorally disruptive effects of ethanol and the possibility that learning may mediate its behavioral tolerance dictate a more thorough investigation of the problem. The present experiments were designed to determine in an unambiguous manner whether learning plays a role in the development of behavioral tolerance to ethanol. The goal was to evaluate the relative contributions of learning and (physiological) exposure to ethanol per se to the acquisition of behavioral tolerance to ethanol. Single tolerance-testing sessions were used to eliminate the possibility of learning occurring over test days. No attempt was made to assess the rate of development of tolerance in this experiment, the entire object being to obtain uncontaminated estimates of the effects of drugged practice and drug exposure on the acquisition of behavioral tolerance to ethanol.
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EXPERIMENT 1 Methods
Subjects Subjects were 24 naive male Long-Evans rats obtained from the colony maintained by the University of Washington Department of Psychology. They were 100 days old and had an average initial weight of 270 g. They were housed individually in standard galvanized iron cages. The room was on a 12/12 hr light/dark cycle illuminated from 8 AM to 8 eM. The animals received ad lib food and water throughout the experiment.
Apparatus The apparatus was an automated motor-performance evaluator (hereafter called the treadmill) originally developed by Gibbins, Kalant, & LeBlanc (1%8) and modified by Gallaher (1976). The chamber measured 38 cm long by 32 cm wide by 20.7 cm high. This apparatus required the animals to walk a constantly moving (8.26 crrdsec), 6.35 cm-wide, stainless-steel mesh conveyor belt which ran through the middle of and was horizontally flush with an electrified grid floor. Any contact by the rat with the grid floor caused him to receive continuously a 0.95-mA footshock. The belt was programmed to run on the following trial cycle: a 60-sec belt-moving trial was followed by a 30-sec rest period with the belt stationary but the floor remaining electrified. This cycle was repeated for three 60-sec trials, daily. Animals were not removed or handled during the two intervening rest periods.
Drugs Ethanol injections consisted of 15% (v/v) ethanol in physiological saline (0.9% NaC1 in distilled water, w/v). The control injections consisted of saline (0.9%) in equivalent volumes. Stock solutions were prepared 24 hr in advance and stored in tightly stoppered refrigerated bottles. All injections were intraperitoneal and computed on the basis of the animal's weight measured immediately prior to injection.
Procedure The experiment was conducted in a room separate from the one which housed the animals. The animals were carried in their home cages to the experimental room where they were trained and/or injected. After being injected, they were carried back to their home room until they were needed again. The experiment had three phases. Phase I: Treadmill training. Undrugged animals were given daily a standard practice session on the treadmill (three trials per day) until they reached an overall level of performance of three "criterion" trials in
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succession for 3 consecutive days. A criterion trial was any 60-sec trial wherein the animal received cumulative shock of 1 sec or less. Animals were given one warm-up trial each day before the three criterion measures were taken. When this criterion was attained, animals were randomly assigned to one of three equally sized groups. Phase II began the next day. Phase II: Tolerance acquisition. The basic manipulation was the same as that of the earlier investigators (Chen, 1968, 1972; LeBlanc et al., 1973, 1976; Gallaher, 1976). All animals were first given one daily undrugged trial (prerun) to verify that baseline treadmill performance had not been altered. They were then given additional practice on the treadmill task as follows: Animals in Group 1, the Ethanol-Before-Practice Group, were administered ethanol and subsequently given a standard three-trial session on the treadmill 1 hr later. Tolerance exhibited by these animals (in a subsequent tolerance test session) could be a consequence of their intoxicated practice or of their exposure to ethanol or to some combination of these two factors. Animals in Group 2, the Ethanol-After-Practice Group, were first given a standard three-trial session on the treadmill in the undrugged state and subsequently injected with ethanol 1 hr later. The three-trial session occurred from 15 to 60 min after the prerun trial. The doses of ethanol were the same as those given to Group 1. Any tolerance exhibited by these animals (in a subsequent tolerance test) could be due to the exposure to the ethanol but not to intoxicated practice. Subsequent differences of tolerance between these two groups are attributable to the intoxicated practice given to the Ethanol-Before-Practice Group. Last, animals in Group 3, the Saline-Control Group, were injected with 0.9% saline and then given a standard practice session on the treadmill 1 hr later. The volumes of the saline injections were equivalent to those given the other groups. Differences of subsequent tolerance between the Saline Group and the Ethanol-After-Practice Group are logically attributable to physiological exposure to the ethanol. An escalating series of doses of ethanol was used because we reasoned that if learned tolerance were to occur, it would occur maximally if the difficulty of the task increased relatively slowly. The series of doses was 1.4, 1.6, 1.8, 1.8, 2.0, 2.0, 2.2, and 2.4 g/kg of ethanol. All animals were given only one ip injection per day. This phase therefore lasted 8 days. Phase III: Tolerance testing. On the day following the termination of Phase II, animals in all three groups were tested for tolerance to ethanol. They were injected with 2.4 g/kg of ethanol, returned to their cages for 1 hr, and then given a standard three-trial session on the treadmill. In other words, on this test day, rats in all three groups were treated in the manner of those in Group 1 during tolerance acquisition.
Results The results of the 2.4 g/kg ethanol-tolerance test are depicted in Fig. 1 as a function of treatment group and test trial. Heterogeneity of variance
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I 2 5 I 2 5 ETHANOL BEFORE ETHANOL AFTER PRACTICE
PRACTICE
I 2 :3 SALrNE BEFORE PRACTICE
FIG. 1. G r o u p m e a n treadmill error scores for rats given ethanol (2.4 g/kg ip) 1 h r earlier. Data s h o w n are from three c o n s e c u t i v e tolerance-test trials. The a b s c i s s a indicates the tolerance-induction t r e a t m e n t s administered to the animals on the previous 8 days.
precluded the use of analysis of variance techniques to test for an effect of repeated trials. Therefore, in order to exclude the possibility of intertrial practice effects (Kulig, 1977), we analyzed only the data from the first of the three test trials. Jonckheer's test (Hollander & Wolfe, 1973) indicated that at least one of the groups was significantly different from the others. Planned comparisons using Mann-Whitney U-tests (Siegel, 1956) indicated that the rats in Group 1 made significantly less error (p < .05) than rats in either of the other two groups, and that these other two groups were not significantly different from one another. Discussion
The data suggest that intoxicated practice is necessary to develop behavioral tolerance to ethanol. They do not support the hypothesis that tolerance results from the mere exposure to ethanol. These results contrast with those of LeBlanc et al. and extend those of Gallaher. However, the level of impairment observed was far lower in the present study than in the studies of LeBlanc et al. Furthermore, a number of methodological differences may have accounted for the differences from LeBlanc et al. In the present experiments (and in those of Gallaher), the animals were tested 60 min after injection with ethanol when ethanol absorption is complete; LeBlanc et al. tested their animals during drug absorption. In that study, each animal was given six 2-min trials beginning 2.5 min after the injection of ethanol and separated by 2.5-min rest intervals. Bloodethanol concentrations rise during this period. LeBlanc et al. selected the tolerance test trial with the greatest behavioral impairment for each animal for subsequent analysis. Because we were concerned about the possibility of acute learning occurring across trials within any one session (Kulig, 1977), we elected instead to test the animals 60 min after ethanol injection so that absorption would be complete and the blood ethanol concentration would be stable or decreasing (Czaja and Kalant, 1961).
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This was done in the hope of minimizing individual differences of ethanol absorption and to eliminate the need for numerous repeated tolerance test trials. Animals are known to be less impaired by equivalent blood ethanol concentrations when the blood ethanol concentration is falling than when it is rising (Mellanby, 1919; LeBlanc, Kalant, & Gibbins, 1975). The treadmill belt speed was also higher in the present study. This belt speed was selected empirically as one minimally required to maintain undrugged animals walking at a reasonably constant speed. Slower speeds resulted in the animals running down to the end of the belt where it entered the chamber and then passively riding it through the chamber. The effect of treadmill belt speed upon this measure of behavioral tolerance is unknown. Because all of our animals performed with less error than those of LeBlanc et al., we decided to replicate our experiment with a higher test dose of ethanol so that our animals would be approximately as impaired as were those of LeBlanc et al.
EXPERIMENT 2 The experiment was repeated 3 months later but with a higher test dose of ethanol. A higher test dose of ethanol was used in order to ensure that all of the animals would be substantially impaired when tested on the treadmill.
Methods Subjects The same animals were used. They were 3 months older and had a mean weight of 428 g. The 3-month interval was chosen since it has been reported that behavioral tolerance to ethanol decays with time (LeBlanc, Kalant, Gibbins, & Berman, 1969) and pilot work with other animals suggested that only negligible tolerance would remain from the first experiment. Two of the subjects had been discarded for health reasons. The same three-phase procedure was used with only a few differences. The original group membership was preserved. Phase I. No changes. Phase H. The series of tolerance acquisition doses was 2.0, 2.2, 2.4, 2.6, and 2.8 g/kg of ethanol. This phase lasted only 5 days. Phase III. All animals were tested for behavioral tolerance to a challenge dose of 3.0 g/kg of ethanol.
Results The results of the 3.0 g/kg ethanol tolerance test are depicted in Fig. 2 as a function of treatment group and test trial. Again, in order to exclude the possibility of intrasessional practice effects across test trials within the session (i.e., " a c u t e " tolerance), only the data from the first of the three test trials are of interest here. The effects of acute tolerance gained within
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I 2 5 ETHANOL A F T E R PRACTICE
I 2 3 SALINE BEFORE PRACTICE
FIG. 2. Group m e a n treadmill error scores for rats given ethanol (3.0 g/kg ip) 1 hr earlier. Data s h o w n are from three consecutive tolerance-test trials. The abscissa indicates the tolerance-induction t r e a t m e n t s administered to the animals on the previous 5 days.
sessions is the subject of an upcoming series of reports. These data were analyzed with planned comparisons using analysis of variance techniques. Rats in Group 1 made significantly less error (/9 < .05) (as measured by total trial shock time) than rats in either of the other two groups; and rats from the other two groups were not significantly different from one another.
Discussion The data suggest that intoxicated practice is necessary to develop tolerance to ethanol. They fail to support the hypothesis that mere exposure to ethanol is sufficient to induce tolerance to it.
GENERAL DISCUSSION Both of the present experiments support the conclusions of Gallaher and fail to support those of LeBlanc et al. The use of the equal practice controls in the present studies excludes the interpretation that the results are due to differences of undrugged practice. It now seems likely that they were not important in the investigations of Gallaher either. The data from the 2.4 g/kg tolerance-test trial indicate that intoxicated practice is necessary to develop behavioral tolerance to ethanol. They do not support the hypothesis that mere exposure to ethanol is sufficient to induce behavioral tolerance to ethanol. It seems reasonable that if notlearned tolerance exists, it should have been manifest within the 8 days of ethanol exposure in Experiment 1, although one could argue that exposure-induced tolerance to ethanol takes longer than 8 days to develop. However, it is also possible that the dose of ethanol was too low to differentiate the groups since the rats in Group 1 performed as if they received no ethanol at all. However, the data of the second experiment also fail to support the hypothesis that mere exposure to ethanol is sufficient to induce behavioral tolerance to ethanol. Again, all of the
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observed tolerance was attributable to the effects of intoxicated practice. These data were gathered on the same animals 3 months later, and a considerably higher test dose of ethanol was used (3.0 g/kg). These results extend the findings of Chen (1968, 1972) who used a different apparatus and who did not include a no-drug control group. He was therefore only able to determine that his Ethanol-After-Practice Group was less tolerant than his Ethanol-Before-Practice Group; he was not able to determine whether his Ethanol-After-Practice Group was tolerant at all, i.e., more tolerant than a group of control animals which never received ethanol until the tolerance-test session. The results of the present experiments clearly indicate that animals which receive ethanol after behavioral practice are not reliably more tolerant than nontolerant animals. The data of both experiments can be explained by either of two learning theories of tolerance. The first, which might be called "learning to cope with drug-induced impairments of behavior," is an instance of instrumental learning. According to this model, tolerance results from the animals learning how to perform behaviors (such as walking the treadmill) while intoxicated. Any responses which enable the animal to avoid, escape, or reduce punishment will be learned through the mechanism of negative reinforcement. Thus, responses that enable the animals to remain on the treadmill despite severe intoxication will be learned. This learning can be regarded as the result of overt practice on the treadmill while intoxicated. Such learning may explain the tolerance developed by the EthanolBefore-Practice Group. Similarly, responses that enable animals to obtain, despite severe intoxication, reinforcing substances such as food and water, will be learned through the mechanism of positive reinforcement. Thus, responses that enable the animal to balance and to stand without falling despite severe intoxication will be learned. Such learning could have occurred in both groups of animals which were given ethanol when they were in their home cages while intoxicated. This can be regarded as covert practice while intoxicated. There was no reliable evidence in either experiment for tolerance occurring as a result of covert practice. This can be inferred from the lack of a significant difference between the performances of the Ethanol-After-Practice and Saline-Control Groups. The second learning theory of tolerance is based upon classical conditioning and has been developed by Siegel (1978). According to this model, tolerance results in part from the learning of an association between the environmental cues (CS) that reliably predict the systemic effects (stimulation) induced by the drug, with the actual pharmacological effect constituting the unconditioned stimulus (UCS). Such an association can be shown by presenting the environmental cues (CS) alone without giving the drug (UCS). The consequent response is called a conditioned response (CR) and is often opposite in direction from the unconditioned effects of
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the drug. The CR therefore attenuates the observed effect of the drug. Repeated pairing of the CS with the effect of the drug (UCS) leads to a stronger association and therefore a stronger CR. As the CR becomes stronger, the net observed drug effect decreases and this defines tolerance. Siegel (1975a, 1975b, 1977) has shown that some tolerance to morphine-induced analgesia results from classical conditioning. He found that (i) stimuli associated with morphine became predictive cues for the morphine-induced analgesia, (ii) tolerance to morphine-induced analgesia depends upon the presence of these predictive cues, and (iii) tolerance manifested itself as a classically conditioned compensation to the morphine-induced analgesia, i.e., as a classically conditioned hyperalgesia. More recently, Parker (1978) extended Siegel's procedures to study tolerance to ethanol-induced narcosis in the rat. In parallel with the findings of Siegel, Parker found that (i) stimuli associated with ethanol administration became predictive cues for the onset of ethanol-induced narcosis and (ii) that tolerance to ethanol-induced narcosis depends upon the presence of these predictive cues. In the present experiments, the animals were removed from their rack but kept in their cages, carried into the experimental room, injected with the appropriate injectate, and then returned to their home room until they were needed again. The classical conditioning model of tolerance predicts that the act of being carried into the experimental room, the experimental room itself, the presence of the experimenter, and the smell of ethanol will become predictive cues for the effects of the ethanol and therefore classically conditioned compensatory responses will develop in all of the animals given ethanol. However, there was no reliable evidence of this in either of the present experiments since there was no reliable difference between the performance of the Ethanol-After-Practice Group and that of the Saline Group. It is true that the tolerance shown by the Ethanol-Before-Practice Group can be explained by the classical conditioning theory. The animals in this group always had the treadmill apparatus, handling, etc., paired with the impairing effects of the ethanol upon the behavior of walking the treadmill. Thus the stimuli associated with the treadmill came to predict reliably the impairment of treadmill-walking caused by the ethanol. Therefore, conditioned ethanol-compensatory responses may have developed in this group and therefore these responses may have mediated the tolerance shown by this group. In hindsight, perhaps the Ethanol-After-Practice Group would have shown some tolerance if they had been tested for tolerance on the treadmill while they were in their home room. The present results are somewhat surprising in light of the results of previous classical conditioning
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studies of tolerance to morphine and ethanol. In all instances, cues associated with the room where the animals experienced the effects of the drug, rather than those associated with the behavioral procedure used to measure tolerance, became conditioned stimuli that elicited conditioned compensatory responses. In the present study, the animals were in the tolerance-testing room for only a few minutes per day, and therefore it would be surprising if this brief time were sufficient for conditioning to occur. On the contrary, one might expect the cues associated with the home room to have become conditioned stimuli. Thus, the tolerance shown by the Ethanol-Before-Practice Group can be explained by either of the learning theories of tolerance. The present data do not compel a choice between these two theories. Until such data become available, it seems prudent to consider a two-factor theory of learned tolerance. We propose that both instrumental learning of coping behaviors and classical conditioning of drug-compensatory responses contribute, at least in part, to the development of tolerance to ethanol and to other psychoactive drugs. We further propose that the relative contributions of these two postulated learning processes depend upon both the specific drug involved and the specific behavioral drug effect used operationally to define and measure tolerance. As a final comment, the tolerance reported in this study cannot be the result of state-dependent learning (Overton, 1978) since, by definition (Overton, 1968), it is the phenomenon wherein an organism learns to perform some behavior in one physiological-psychological state (generally drug-induced) and then is able to perform well that behavior again in the same state but not in a different (generally undrugged) state. It is called state-dependent learning because the performance of the learned behavior depends upon the animal being tested in the same state as the one in which he learned the behavior. In contrast, all the animals in the present study were first trained to walk on the treadmill while in the normal (nonintoxicated) state and then some of them (Group 1) were given additional treadmill training while intoxicated. Their daily preruns easily met the training criterion so the rats were not forgetting how to walk the treadmill while unintoxicated. Therefore, their performance (and by inference their learning) was not dependent upon either state; i.e., their performance (learning) was state independent. In contrast, animals in the other two groups were not given any overt opportunity to learn to walk on the treadmill while intoxicated. Thus, their failure to learn to walk the treadmill while intoxicated (i.e., failure to become tolerant) was state dependent by design. In summary, the present experiment demonstrates the powerful contribution of learning to the acquisition of tolerance to the behaviorally disruptive effects of ethanol in the rat. In other words, animals can learn to operate more effectively under the influence of ethanol and this ex-
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plains why they become behaviorally "tolerant" to it. A similar explanation may well prevail for human performance while intoxicated. The implication is that most and perhaps all of behavioral tolerance is a consequence of intoxicated practice; i.e., of some form of learning. Moreover, exposure-induced tolerance to the behaviorally disruptive effects of ethanol does not develop within an 8-day exposure period using the present parameters whereas learned behavioral tolerance does. Therefore, it is useful to conceptualize the phenomenon of behavioral tolerance in terms of a learning model because it affords a unitary explanation of behavioral tolerance. REFERENCES Campbell, J. C., & Selden, L. S. (1973). Performance influence on the development of tolerance to amphetamine. Pharmacology, Biochemistry and Behavior, 1, 703-708. Carder, B., & Olson, J. (1973). Learned behavioral tolerance to marihuana in rats. Pharmacology, Biochemistry and Behavior, 1, 73-76. Carlton, P. L., & Wolgin, D. L. (1971). Contingent tolerance to the anorexigenic effects of amphetamine. Physiology and Behavior, 7, 221-223. Chen, C. S. (1968). A study of the alcohol-tolerance effect and an introduction of a new behavioral technique. Psychopharmacology, 12, 433-440. Chen, C. S, (1972). A further note on studies of acquired behavioral tolerance to alcohol. Psychopharmacology, 27, 265-274. Cicero, T. J. (1978). Tolerance to and physical dependence on alcohol: Behavioral and neurobiological mechanisms. In M. A. Lipton et al. (Eds.), Psychopharmacology: A Generation of Progress, pp. 1613-1615. New York: Raven Press. Corfield-Sumner, P. K., & I. P. Stolerman (1978). Behavioral tolerance. In D. E. Blackman & D. J. Sanger (Eds.), Contemporary Research In Behavioral Pharmacology, p. 401. New York: Plenum. Czaja, C., & Kalant, H. (1961). The effect of acute alcoholic intoxication on adrenal aseorbic acid and cholesterol in the rat. Canadian Journal of Biochemistry and Physiology, 39, 327-334. Gallaher, E. (1976). The Kinetics of Alcohol Tolerance in the Rat: The Effects of Practice. Ph.D. Thesis, Department of Pharmacology, University of Washington. Gibbins, R. J., Kalant, H., & LeBlanc, A. E. (1968). A technique for accurate measurement of moderate degrees of alcohol intoxication in small animals. Journal of Pharmacology and Experimental Therapeutics, 159, 236-242, Hollander, M., & Wolfe, D. A. (1973). Nonparametric Statistical Methods, pp. 120-123. New York: Wiley. Kalant, H., LeBlanc, A. E., & Gibbins, R. J. (1971). Tolerance to, and dependence on, some non-opiate psychotropic drugs. Pharmacological Reviews, 23, 135-191. Kulig, B. M. (1977). Acute tolerance to the motor impairment effects of ethanol. Physiological Psychology, 5, 3-6. LeBlanc, A. E., & Cappell, H. (•977). Tolerance as adaptation: interactions with behavior and parallels to other adaptive processes. In K. Blum (Ed.), Alcohol and Opiates: Neurochemical and Behavioral Mechanisms, pp. 65-77. New York: Academic Press., LeBlanc, A. E., Gibbins, R. J., & Kalant, H. (1973). Behavioral augmentation of tolerance to ethanol in the rat. Psychopharmacology, 30~ 117-122. LeBlanc, A. E., Kalant, H., & Gibbins, R. J. (1975). Acute tolerance to ethanol in the rat. Psychopharmacology, 41, 43-46. LeBlanc, A. E., Kalant, H., & Gibbins, R. J. (1976). Acquisition and loss of behaviorally augmented tolerance tO ethanol in the rat. Psychopharmaeology, 48, 153-158.
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LeBlanc, A. E., Kalant, H., Gibbins, R. J., & Berman, N. D. (1969). Acquisition and loss of tolerance to ethanol by the rat. Journal of Pharmacology and Experimental Therapeutics, 168, 244-250. LeBlanc, A. E., Poulos, C. X., & CappeU, H. D. (1978). Tolerance as a behavioral phenomenon: Evidence from two experimental paradigms. In N. A. Krasnegor (Ed.), Behavioral Tolerance: Research and Treatment Implications, p. 78. National Institute on Drug Abuse Research Monograph No. 18; U.S. Department of Health, Education and Welfare Publication No. (ADM) 78-551. Washington, D.C.: U.S. Govt. Printing Office. Mellanby, E., (1919). Alcohol: Its Absorption into and Disappearance from the Blood Under Different Conditions. Special Report Series, No. 31, Medical Research Committee, London. Olson, J., & Carder, B. (1974). Behavioral tolerance to marihuana as a function of amount of prior training. Pharmacology, Biochemistry and Behavior, 2, 243-247. Overton, D. A. (1968). Dissociated learning in drug states (state-dependent learning). In D. H. Elfr0n et al. (Eds.), Psychopharmacology. A Review of Progress, p. 918. Public Health Service Publication No. 1936. Washington, D.C.: U.S. Govt. Printing Office. Overton, D. A. (1978). Personal communication. Beerse, Belgium. Parker, L. F. (1978). Conditioned Tolerance to the Depressant Effects of Ethanol. Ph.D. Thesis, Department of Physiology/Psychology, University of Washington. Schuster, C. R., Dockens, W. S., & Woods, J. H. (1966). Behavioral variables affecting the development of amphetamine tolerance. Psychopharmacologia, 9, 170-182. Siegel, S. (1975). Morphine Analgesia Tolerance: Its Situation Specificity Supports a Pavlovian Conditioning Model. Science, 193, 323-325. (a) Siegel, S. (1975). Evidence from rats that morphine tolerance is a learned response. Journal of Comparative and Physiological Psychology, 89, 498-506. (b) Siegel, S. (1977). Morphine tolerance acquisition as an associative process. Journal of Experimental Psychology: Animal Behavior Processes, 3, 1-13. Siegel, S. (1978). A Pavlovian conditioning analysis of morphine tolerance. In N. A. Krasnegor (Ed.), Behavioral Tolerance: Research and Treatment Implications. NIDA Research Monograph 18. Siegel, S., Hinson, R. E., & Krank, M. D. (1978). The role of predrug signals in morphine analgesic tolerance: Support for a Pavlovian conditioning model of tolerance. Journal of Experimental Psychology: Animal Behavior Processes, 4, 188-196. Siegel, Sidney (1956). Nonparametric Statistics for the Behavioral Sciences, pp. 116-127. New York: McGraw-Hill. Tang, M., & Falk, J. (1978). Behavioral and pharmacological components of phenobarbital tolerance. In N. A. Krasnegor (Ed.), Behavioral Tolerance: Research and Treatment Implications. NIDA Research Monograph 18.
Learned Tolerance to the Behaviorally Disruptive Effects of Ethanol JOHN R . W E N G E R , 1 VICKI BERLIN, AND STEPHEN C . WOODS ~
Department of Psychology, University of Washington, Seattle, Washington 98195 Rats were trained to walk on a moving belt in order to avoid footshock. Rats which were then given ethanol each day before behavioral training became tolerant; rats which received equal doses of ethanol after behavioral training did not become tolerant relative to a group of saline controls. This result was interpreted to mean that the acquisition of behavioral tolerance to ethanol was mediated by learning. The methodology of measuring behavioral tolerance was discussed.
"Behavioral tolerance" to a drug is a decrease of the behavioral effects of the drug after it has been administered for a number of times. Behavioral tolerance to most behavior-modifying drugs has been reported to be due at least in part to learning. This has been reported for amphetamine (Campbell, & Seiden, 1973; Carlton & Wolgin, 1971; Schuster, Dockens, & Woods, 1966), marihuana (Carder & Olson, 1973; Olson & Carder, 1974), opiates (Siegel, 1975a, 1975b, 1977, 1978; Siegel, Hinson, & Krank, 1978), phenobarbital (Tang & Falk, 1978), and ethanol (Chen, 1968, 1972). However, recent reports (LeBlanc, Gibbins, & Kalant, 1973; LeBlanc, Kalant, & Gibbins, 1976) have severely criticized the methodology of the earlier ethanol tolerance studies and have concluded that intoxicated practice (i.e., learning) accelerates but is not necessary for the development of behavioral tolerance to ethanol. Recent reviews (Cicero, 1978; Corfield-Sumner & Stolerman, 1978; Kalant, LeBlanc, & Gibbins, 1971; LeBlanc & Cappell, 1977; LeBlanc, Poulos, & Cappell, 1978) have endorsed the interpretation that learning facilitates the development of cellular tolerance but is not a separate tolerance process. This view regarding behavioral tolerance to ethanol seems to be well documented (LeBlanc et al., 1976). For example LeBlanc et al. reported that ethanol-intoxicated practice contributes to a faster acquisition of, but 1 To whom requests for reprints should be addressed: Department of Psychology, NI-25, University of Washington, Seattle, Wash. 98195. Supported by grants from the University of Washington Alcoholism and Drug Abuse Institute and from the University of Washington Graduate Research Fund. We thank Herman Samson for his critical comments. 418 0163-1047/80/040418-13502.00/0 Copyright(~ t980 by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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not a higher final level of, tolerance to ethanol-induced impairment of motor performance. They termed this phenomenon behavioral augmentation of tolerance acquisition (BAT). In their experiments, they trained rats to walk on a moving belt (treadmill) to avoid footshock. In order to measure the rate of development of tolerance, they intraperitoneally (ip) injected animals with ethanol every 4 days and then required them to walk on the treadmill while intoxicated. On the intervening 3 days, some of the animals received ethanol after their daily practice on the treadmill (Ethanol-After-Practice Group), some before their practice (EthanolBefore-Practice Group), and others received saline before their practice (Saline Group). The animals which performed the moving belt task while intoxicated (i.e., which were given ethanol before practice each day) became tolerant sooner than those which received ethanol after practicing the task although these latter animals eventually reached the same level of intoxicated performance. These investigators concluded that learning accelerates but is not necessary for the development of behavioral tolerance to ethanol. There is an alternative interpretation of their results. If one regards each of the tolerance-test days as a potential learning opportunity, (i.e., the days when ethanol was given to determine if tolerance was developing) then the Ethanol-After-Practice Group would also be expected to learn the response, but learning would be relatively slow since this group received only one-fourth of the (relevant) intoxicated practice as did the Ethanol-Before-Practice Group. LeBlanc et al. recognized this possibility and performed another experiment to test it. They found that animals tested while intoxicated every day became tolerant sooner than animals tested every 2 days, and that these animals in turn became tolerant sooner than animals tested every 3 days. Animals tested 4 or more days apart never became tolerant. These findings were interpreted by LeBlanc et al., to mean that testing for ethanol tolerance every 4 days does not in itself lead to tolerance acquisition and that therefore the tolerance tests in the previous study should not be regarded as tolerance-learning trials. However, no ethanol was given to the animals (in the second experiment) on the days between tolerance-testing days. Therefore, the results of this (second) study could not be used to rule out the possibility of an interaction effect (in the first study) between tolerance testing every fourth day and exposure to ethanol on the intertest days. Thus, it is possible that in LeBlanc's first experiment, testing for tolerance every 4 days in combination with exposure to ethanol on the intertest days led to learned tolerance by the Ethanol-After-Practice Group. The questioning of the conclusions of LeBlanc et al. has been necessitated by a more recent series of investigations by Gallaher (1976) that suggests that learning is actually involved in the acquisition of tolerance to ethanol. Using the same treadmill-type apparatus as LeBlanc et al., Gal-
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laher found that rats which were injected with ethanol and tested daily developed nearly maximum tolerance in 30 days, whereas rats injected with ethanol daily but tested behaviorally only weekly attained only half of this asymptotic level of tolerance after 47 days. Giving this latter group of rats subsequent daily intoxicated practice led to their becoming fully tolerant within only 5 additional days. Gallaher interpreted his data to mean that the rate of tolerance acquisition was limited by the rate of practice in the drugged state and that at least some aspects of the tolerance were therefore learned. The low but reliable level of presumably not-learned (and therefore presumably induced by mere exposure to ethanol) tolerance exhibited by Gallaher's animals given daily ethanol but tested only weekly could have been caused by several factors: the daily physiological exposure to the ethanol, or the opportunity to learn during the weekly tolerance-testing sessions, or (more likely) some combination of these two factors. However, an alternative interpretation is possible. His weekly tested animals were not run at all between tests and therefore may have forgotten important components of the treadmill task. Moreover, since the daily tested animals had seven times the amount of practice as the weekly tested animals, the extra practice would be expected to produce overlearning of the treadmill task. Therefore, the greater practice of the daily tested animals would be expected to render their behavior more resistant to disruption by ethanol. To summarize the relevant literature, it is becoming increasingly accepted that behavioral tolerance to many drugs is, at least in part, learned (Chen, 1968, 1972; Campbell & Seiden, 1973; Carder & Olson, 1973; Olson & Carder, 1974; Carlton & Wolgin, 1971; Schuster et al., 1966; Siegel, 1975a, 1975b, 1977, 1978; Siegel et al., 1978; Tang & Falk, 1978). It seems reasonable that learning may also contribute to the behavioral tolerance shown to ethanol by rats. However, for reasons discussed above, the issue remains controversial and unsettled. The social importance of the behaviorally disruptive effects of ethanol and the possibility that learning may mediate its behavioral tolerance dictate a more thorough investigation of the problem. The present experiments were designed to determine in an unambiguous manner whether learning plays a role in the development of behavioral tolerance to ethanol. The goal was to evaluate the relative contributions of learning and (physiological) exposure to ethanol per se to the acquisition of behavioral tolerance to ethanol. Single tolerance-testing sessions were used to eliminate the possibility of learning occurring over test days. No attempt was made to assess the rate of development of tolerance in this experiment, the entire object being to obtain uncontaminated estimates of the effects of drugged practice and drug exposure on the acquisition of behavioral tolerance to ethanol.
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EXPERIMENT 1 Methods
Subjects Subjects were 24 naive male Long-Evans rats obtained from the colony maintained by the University of Washington Department of Psychology. They were 100 days old and had an average initial weight of 270 g. They were housed individually in standard galvanized iron cages. The room was on a 12/12 hr light/dark cycle illuminated from 8 AM to 8 eM. The animals received ad lib food and water throughout the experiment.
Apparatus The apparatus was an automated motor-performance evaluator (hereafter called the treadmill) originally developed by Gibbins, Kalant, & LeBlanc (1%8) and modified by Gallaher (1976). The chamber measured 38 cm long by 32 cm wide by 20.7 cm high. This apparatus required the animals to walk a constantly moving (8.26 crrdsec), 6.35 cm-wide, stainless-steel mesh conveyor belt which ran through the middle of and was horizontally flush with an electrified grid floor. Any contact by the rat with the grid floor caused him to receive continuously a 0.95-mA footshock. The belt was programmed to run on the following trial cycle: a 60-sec belt-moving trial was followed by a 30-sec rest period with the belt stationary but the floor remaining electrified. This cycle was repeated for three 60-sec trials, daily. Animals were not removed or handled during the two intervening rest periods.
Drugs Ethanol injections consisted of 15% (v/v) ethanol in physiological saline (0.9% NaC1 in distilled water, w/v). The control injections consisted of saline (0.9%) in equivalent volumes. Stock solutions were prepared 24 hr in advance and stored in tightly stoppered refrigerated bottles. All injections were intraperitoneal and computed on the basis of the animal's weight measured immediately prior to injection.
Procedure The experiment was conducted in a room separate from the one which housed the animals. The animals were carried in their home cages to the experimental room where they were trained and/or injected. After being injected, they were carried back to their home room until they were needed again. The experiment had three phases. Phase I: Treadmill training. Undrugged animals were given daily a standard practice session on the treadmill (three trials per day) until they reached an overall level of performance of three "criterion" trials in
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succession for 3 consecutive days. A criterion trial was any 60-sec trial wherein the animal received cumulative shock of 1 sec or less. Animals were given one warm-up trial each day before the three criterion measures were taken. When this criterion was attained, animals were randomly assigned to one of three equally sized groups. Phase II began the next day. Phase II: Tolerance acquisition. The basic manipulation was the same as that of the earlier investigators (Chen, 1968, 1972; LeBlanc et al., 1973, 1976; Gallaher, 1976). All animals were first given one daily undrugged trial (prerun) to verify that baseline treadmill performance had not been altered. They were then given additional practice on the treadmill task as follows: Animals in Group 1, the Ethanol-Before-Practice Group, were administered ethanol and subsequently given a standard three-trial session on the treadmill 1 hr later. Tolerance exhibited by these animals (in a subsequent tolerance test session) could be a consequence of their intoxicated practice or of their exposure to ethanol or to some combination of these two factors. Animals in Group 2, the Ethanol-After-Practice Group, were first given a standard three-trial session on the treadmill in the undrugged state and subsequently injected with ethanol 1 hr later. The three-trial session occurred from 15 to 60 min after the prerun trial. The doses of ethanol were the same as those given to Group 1. Any tolerance exhibited by these animals (in a subsequent tolerance test) could be due to the exposure to the ethanol but not to intoxicated practice. Subsequent differences of tolerance between these two groups are attributable to the intoxicated practice given to the Ethanol-Before-Practice Group. Last, animals in Group 3, the Saline-Control Group, were injected with 0.9% saline and then given a standard practice session on the treadmill 1 hr later. The volumes of the saline injections were equivalent to those given the other groups. Differences of subsequent tolerance between the Saline Group and the Ethanol-After-Practice Group are logically attributable to physiological exposure to the ethanol. An escalating series of doses of ethanol was used because we reasoned that if learned tolerance were to occur, it would occur maximally if the difficulty of the task increased relatively slowly. The series of doses was 1.4, 1.6, 1.8, 1.8, 2.0, 2.0, 2.2, and 2.4 g/kg of ethanol. All animals were given only one ip injection per day. This phase therefore lasted 8 days. Phase III: Tolerance testing. On the day following the termination of Phase II, animals in all three groups were tested for tolerance to ethanol. They were injected with 2.4 g/kg of ethanol, returned to their cages for 1 hr, and then given a standard three-trial session on the treadmill. In other words, on this test day, rats in all three groups were treated in the manner of those in Group 1 during tolerance acquisition.
Results The results of the 2.4 g/kg ethanol-tolerance test are depicted in Fig. 1 as a function of treatment group and test trial. Heterogeneity of variance
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6o rr 5O 0 n~
~ 4c bA
_> 5c
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I 2 5 I 2 5 ETHANOL BEFORE ETHANOL AFTER PRACTICE
PRACTICE
I 2 :3 SALrNE BEFORE PRACTICE
FIG. 1. G r o u p m e a n treadmill error scores for rats given ethanol (2.4 g/kg ip) 1 h r earlier. Data s h o w n are from three c o n s e c u t i v e tolerance-test trials. The a b s c i s s a indicates the tolerance-induction t r e a t m e n t s administered to the animals on the previous 8 days.
precluded the use of analysis of variance techniques to test for an effect of repeated trials. Therefore, in order to exclude the possibility of intertrial practice effects (Kulig, 1977), we analyzed only the data from the first of the three test trials. Jonckheer's test (Hollander & Wolfe, 1973) indicated that at least one of the groups was significantly different from the others. Planned comparisons using Mann-Whitney U-tests (Siegel, 1956) indicated that the rats in Group 1 made significantly less error (p < .05) than rats in either of the other two groups, and that these other two groups were not significantly different from one another. Discussion
The data suggest that intoxicated practice is necessary to develop behavioral tolerance to ethanol. They do not support the hypothesis that tolerance results from the mere exposure to ethanol. These results contrast with those of LeBlanc et al. and extend those of Gallaher. However, the level of impairment observed was far lower in the present study than in the studies of LeBlanc et al. Furthermore, a number of methodological differences may have accounted for the differences from LeBlanc et al. In the present experiments (and in those of Gallaher), the animals were tested 60 min after injection with ethanol when ethanol absorption is complete; LeBlanc et al. tested their animals during drug absorption. In that study, each animal was given six 2-min trials beginning 2.5 min after the injection of ethanol and separated by 2.5-min rest intervals. Bloodethanol concentrations rise during this period. LeBlanc et al. selected the tolerance test trial with the greatest behavioral impairment for each animal for subsequent analysis. Because we were concerned about the possibility of acute learning occurring across trials within any one session (Kulig, 1977), we elected instead to test the animals 60 min after ethanol injection so that absorption would be complete and the blood ethanol concentration would be stable or decreasing (Czaja and Kalant, 1961).
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This was done in the hope of minimizing individual differences of ethanol absorption and to eliminate the need for numerous repeated tolerance test trials. Animals are known to be less impaired by equivalent blood ethanol concentrations when the blood ethanol concentration is falling than when it is rising (Mellanby, 1919; LeBlanc, Kalant, & Gibbins, 1975). The treadmill belt speed was also higher in the present study. This belt speed was selected empirically as one minimally required to maintain undrugged animals walking at a reasonably constant speed. Slower speeds resulted in the animals running down to the end of the belt where it entered the chamber and then passively riding it through the chamber. The effect of treadmill belt speed upon this measure of behavioral tolerance is unknown. Because all of our animals performed with less error than those of LeBlanc et al., we decided to replicate our experiment with a higher test dose of ethanol so that our animals would be approximately as impaired as were those of LeBlanc et al.
EXPERIMENT 2 The experiment was repeated 3 months later but with a higher test dose of ethanol. A higher test dose of ethanol was used in order to ensure that all of the animals would be substantially impaired when tested on the treadmill.
Methods Subjects The same animals were used. They were 3 months older and had a mean weight of 428 g. The 3-month interval was chosen since it has been reported that behavioral tolerance to ethanol decays with time (LeBlanc, Kalant, Gibbins, & Berman, 1969) and pilot work with other animals suggested that only negligible tolerance would remain from the first experiment. Two of the subjects had been discarded for health reasons. The same three-phase procedure was used with only a few differences. The original group membership was preserved. Phase I. No changes. Phase H. The series of tolerance acquisition doses was 2.0, 2.2, 2.4, 2.6, and 2.8 g/kg of ethanol. This phase lasted only 5 days. Phase III. All animals were tested for behavioral tolerance to a challenge dose of 3.0 g/kg of ethanol.
Results The results of the 3.0 g/kg ethanol tolerance test are depicted in Fig. 2 as a function of treatment group and test trial. Again, in order to exclude the possibility of intrasessional practice effects across test trials within the session (i.e., " a c u t e " tolerance), only the data from the first of the three test trials are of interest here. The effects of acute tolerance gained within
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60
+++
50
4c ,~ 3c ~ 2c z 1C
I+1 r-h I 2 5 ETHANOL BEFORE PRACTICE
I 2 5 ETHANOL A F T E R PRACTICE
I 2 3 SALINE BEFORE PRACTICE
FIG. 2. Group m e a n treadmill error scores for rats given ethanol (3.0 g/kg ip) 1 hr earlier. Data s h o w n are from three consecutive tolerance-test trials. The abscissa indicates the tolerance-induction t r e a t m e n t s administered to the animals on the previous 5 days.
sessions is the subject of an upcoming series of reports. These data were analyzed with planned comparisons using analysis of variance techniques. Rats in Group 1 made significantly less error (/9 < .05) (as measured by total trial shock time) than rats in either of the other two groups; and rats from the other two groups were not significantly different from one another.
Discussion The data suggest that intoxicated practice is necessary to develop tolerance to ethanol. They fail to support the hypothesis that mere exposure to ethanol is sufficient to induce tolerance to it.
GENERAL DISCUSSION Both of the present experiments support the conclusions of Gallaher and fail to support those of LeBlanc et al. The use of the equal practice controls in the present studies excludes the interpretation that the results are due to differences of undrugged practice. It now seems likely that they were not important in the investigations of Gallaher either. The data from the 2.4 g/kg tolerance-test trial indicate that intoxicated practice is necessary to develop behavioral tolerance to ethanol. They do not support the hypothesis that mere exposure to ethanol is sufficient to induce behavioral tolerance to ethanol. It seems reasonable that if notlearned tolerance exists, it should have been manifest within the 8 days of ethanol exposure in Experiment 1, although one could argue that exposure-induced tolerance to ethanol takes longer than 8 days to develop. However, it is also possible that the dose of ethanol was too low to differentiate the groups since the rats in Group 1 performed as if they received no ethanol at all. However, the data of the second experiment also fail to support the hypothesis that mere exposure to ethanol is sufficient to induce behavioral tolerance to ethanol. Again, all of the
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observed tolerance was attributable to the effects of intoxicated practice. These data were gathered on the same animals 3 months later, and a considerably higher test dose of ethanol was used (3.0 g/kg). These results extend the findings of Chen (1968, 1972) who used a different apparatus and who did not include a no-drug control group. He was therefore only able to determine that his Ethanol-After-Practice Group was less tolerant than his Ethanol-Before-Practice Group; he was not able to determine whether his Ethanol-After-Practice Group was tolerant at all, i.e., more tolerant than a group of control animals which never received ethanol until the tolerance-test session. The results of the present experiments clearly indicate that animals which receive ethanol after behavioral practice are not reliably more tolerant than nontolerant animals. The data of both experiments can be explained by either of two learning theories of tolerance. The first, which might be called "learning to cope with drug-induced impairments of behavior," is an instance of instrumental learning. According to this model, tolerance results from the animals learning how to perform behaviors (such as walking the treadmill) while intoxicated. Any responses which enable the animal to avoid, escape, or reduce punishment will be learned through the mechanism of negative reinforcement. Thus, responses that enable the animals to remain on the treadmill despite severe intoxication will be learned. This learning can be regarded as the result of overt practice on the treadmill while intoxicated. Such learning may explain the tolerance developed by the EthanolBefore-Practice Group. Similarly, responses that enable animals to obtain, despite severe intoxication, reinforcing substances such as food and water, will be learned through the mechanism of positive reinforcement. Thus, responses that enable the animal to balance and to stand without falling despite severe intoxication will be learned. Such learning could have occurred in both groups of animals which were given ethanol when they were in their home cages while intoxicated. This can be regarded as covert practice while intoxicated. There was no reliable evidence in either experiment for tolerance occurring as a result of covert practice. This can be inferred from the lack of a significant difference between the performances of the Ethanol-After-Practice and Saline-Control Groups. The second learning theory of tolerance is based upon classical conditioning and has been developed by Siegel (1978). According to this model, tolerance results in part from the learning of an association between the environmental cues (CS) that reliably predict the systemic effects (stimulation) induced by the drug, with the actual pharmacological effect constituting the unconditioned stimulus (UCS). Such an association can be shown by presenting the environmental cues (CS) alone without giving the drug (UCS). The consequent response is called a conditioned response (CR) and is often opposite in direction from the unconditioned effects of
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the drug. The CR therefore attenuates the observed effect of the drug. Repeated pairing of the CS with the effect of the drug (UCS) leads to a stronger association and therefore a stronger CR. As the CR becomes stronger, the net observed drug effect decreases and this defines tolerance. Siegel (1975a, 1975b, 1977) has shown that some tolerance to morphine-induced analgesia results from classical conditioning. He found that (i) stimuli associated with morphine became predictive cues for the morphine-induced analgesia, (ii) tolerance to morphine-induced analgesia depends upon the presence of these predictive cues, and (iii) tolerance manifested itself as a classically conditioned compensation to the morphine-induced analgesia, i.e., as a classically conditioned hyperalgesia. More recently, Parker (1978) extended Siegel's procedures to study tolerance to ethanol-induced narcosis in the rat. In parallel with the findings of Siegel, Parker found that (i) stimuli associated with ethanol administration became predictive cues for the onset of ethanol-induced narcosis and (ii) that tolerance to ethanol-induced narcosis depends upon the presence of these predictive cues. In the present experiments, the animals were removed from their rack but kept in their cages, carried into the experimental room, injected with the appropriate injectate, and then returned to their home room until they were needed again. The classical conditioning model of tolerance predicts that the act of being carried into the experimental room, the experimental room itself, the presence of the experimenter, and the smell of ethanol will become predictive cues for the effects of the ethanol and therefore classically conditioned compensatory responses will develop in all of the animals given ethanol. However, there was no reliable evidence of this in either of the present experiments since there was no reliable difference between the performance of the Ethanol-After-Practice Group and that of the Saline Group. It is true that the tolerance shown by the Ethanol-Before-Practice Group can be explained by the classical conditioning theory. The animals in this group always had the treadmill apparatus, handling, etc., paired with the impairing effects of the ethanol upon the behavior of walking the treadmill. Thus the stimuli associated with the treadmill came to predict reliably the impairment of treadmill-walking caused by the ethanol. Therefore, conditioned ethanol-compensatory responses may have developed in this group and therefore these responses may have mediated the tolerance shown by this group. In hindsight, perhaps the Ethanol-After-Practice Group would have shown some tolerance if they had been tested for tolerance on the treadmill while they were in their home room. The present results are somewhat surprising in light of the results of previous classical conditioning
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studies of tolerance to morphine and ethanol. In all instances, cues associated with the room where the animals experienced the effects of the drug, rather than those associated with the behavioral procedure used to measure tolerance, became conditioned stimuli that elicited conditioned compensatory responses. In the present study, the animals were in the tolerance-testing room for only a few minutes per day, and therefore it would be surprising if this brief time were sufficient for conditioning to occur. On the contrary, one might expect the cues associated with the home room to have become conditioned stimuli. Thus, the tolerance shown by the Ethanol-Before-Practice Group can be explained by either of the learning theories of tolerance. The present data do not compel a choice between these two theories. Until such data become available, it seems prudent to consider a two-factor theory of learned tolerance. We propose that both instrumental learning of coping behaviors and classical conditioning of drug-compensatory responses contribute, at least in part, to the development of tolerance to ethanol and to other psychoactive drugs. We further propose that the relative contributions of these two postulated learning processes depend upon both the specific drug involved and the specific behavioral drug effect used operationally to define and measure tolerance. As a final comment, the tolerance reported in this study cannot be the result of state-dependent learning (Overton, 1978) since, by definition (Overton, 1968), it is the phenomenon wherein an organism learns to perform some behavior in one physiological-psychological state (generally drug-induced) and then is able to perform well that behavior again in the same state but not in a different (generally undrugged) state. It is called state-dependent learning because the performance of the learned behavior depends upon the animal being tested in the same state as the one in which he learned the behavior. In contrast, all the animals in the present study were first trained to walk on the treadmill while in the normal (nonintoxicated) state and then some of them (Group 1) were given additional treadmill training while intoxicated. Their daily preruns easily met the training criterion so the rats were not forgetting how to walk the treadmill while unintoxicated. Therefore, their performance (and by inference their learning) was not dependent upon either state; i.e., their performance (learning) was state independent. In contrast, animals in the other two groups were not given any overt opportunity to learn to walk on the treadmill while intoxicated. Thus, their failure to learn to walk the treadmill while intoxicated (i.e., failure to become tolerant) was state dependent by design. In summary, the present experiment demonstrates the powerful contribution of learning to the acquisition of tolerance to the behaviorally disruptive effects of ethanol in the rat. In other words, animals can learn to operate more effectively under the influence of ethanol and this ex-
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plains why they become behaviorally "tolerant" to it. A similar explanation may well prevail for human performance while intoxicated. The implication is that most and perhaps all of behavioral tolerance is a consequence of intoxicated practice; i.e., of some form of learning. Moreover, exposure-induced tolerance to the behaviorally disruptive effects of ethanol does not develop within an 8-day exposure period using the present parameters whereas learned behavioral tolerance does. Therefore, it is useful to conceptualize the phenomenon of behavioral tolerance in terms of a learning model because it affords a unitary explanation of behavioral tolerance. REFERENCES Campbell, J. C., & Selden, L. S. (1973). Performance influence on the development of tolerance to amphetamine. Pharmacology, Biochemistry and Behavior, 1, 703-708. Carder, B., & Olson, J. (1973). Learned behavioral tolerance to marihuana in rats. Pharmacology, Biochemistry and Behavior, 1, 73-76. Carlton, P. L., & Wolgin, D. L. (1971). Contingent tolerance to the anorexigenic effects of amphetamine. Physiology and Behavior, 7, 221-223. Chen, C. S. (1968). A study of the alcohol-tolerance effect and an introduction of a new behavioral technique. Psychopharmacology, 12, 433-440. Chen, C. S, (1972). A further note on studies of acquired behavioral tolerance to alcohol. Psychopharmacology, 27, 265-274. Cicero, T. J. (1978). Tolerance to and physical dependence on alcohol: Behavioral and neurobiological mechanisms. In M. A. Lipton et al. (Eds.), Psychopharmacology: A Generation of Progress, pp. 1613-1615. New York: Raven Press. Corfield-Sumner, P. K., & I. P. Stolerman (1978). Behavioral tolerance. In D. E. Blackman & D. J. Sanger (Eds.), Contemporary Research In Behavioral Pharmacology, p. 401. New York: Plenum. Czaja, C., & Kalant, H. (1961). The effect of acute alcoholic intoxication on adrenal aseorbic acid and cholesterol in the rat. Canadian Journal of Biochemistry and Physiology, 39, 327-334. Gallaher, E. (1976). The Kinetics of Alcohol Tolerance in the Rat: The Effects of Practice. Ph.D. Thesis, Department of Pharmacology, University of Washington. Gibbins, R. J., Kalant, H., & LeBlanc, A. E. (1968). A technique for accurate measurement of moderate degrees of alcohol intoxication in small animals. Journal of Pharmacology and Experimental Therapeutics, 159, 236-242, Hollander, M., & Wolfe, D. A. (1973). Nonparametric Statistical Methods, pp. 120-123. New York: Wiley. Kalant, H., LeBlanc, A. E., & Gibbins, R. J. (1971). Tolerance to, and dependence on, some non-opiate psychotropic drugs. Pharmacological Reviews, 23, 135-191. Kulig, B. M. (1977). Acute tolerance to the motor impairment effects of ethanol. Physiological Psychology, 5, 3-6. LeBlanc, A. E., & Cappell, H. (•977). Tolerance as adaptation: interactions with behavior and parallels to other adaptive processes. In K. Blum (Ed.), Alcohol and Opiates: Neurochemical and Behavioral Mechanisms, pp. 65-77. New York: Academic Press., LeBlanc, A. E., Gibbins, R. J., & Kalant, H. (1973). Behavioral augmentation of tolerance to ethanol in the rat. Psychopharmacology, 30~ 117-122. LeBlanc, A. E., Kalant, H., & Gibbins, R. J. (1975). Acute tolerance to ethanol in the rat. Psychopharmacology, 41, 43-46. LeBlanc, A. E., Kalant, H., & Gibbins, R. J. (1976). Acquisition and loss of behaviorally augmented tolerance tO ethanol in the rat. Psychopharmaeology, 48, 153-158.
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