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Investigating the Discriminative Effects of Morphine and D9-Tetrahydrocannabinol Using Blocking, Overshadowing and Sensory Preconditioning Procedures: A Model

For the Gateway Drug Hypothesis?

Abstract

This research proposal presents three separate experimental procedures designed to study the discriminative effects of morphine and 9-tetrahydrocannabinol (THC) in rats. Discrimination training procedures will attempt to demonstrate the psychological phenomena blocking, overshadowing and sensory preconditiong in order to investigate the effects of behavioral history and contextual conditioning. Experiment one will investigate blocking using the group of 10 rats that have already been trained to discriminate morphine from saline plus a new group using a two-choice task. This experiment will be described in detail as it will serve as the pilot to determine if morphine and 9-THC can serve as distinct discriminative stimuli and to systematize experimental parameters such as route of administration, training dose, and time course of drug effect. Experiments two and three will

investigate overshadowing and sensory preconditioning, respectively, in new groups of rats using twochoice tasks and will not be described in this proposal. A discussion of the results from each of the experiments will focus on an animal model of the gateway drug hypothesis.

Investigating the Discriminative Effects of Morphine and 9-Tetrahydrocannabinol Using Blocking, Overshadowing and Sensory Preconditioning Procedures:

A Model For the Gateway Drug Hypothesis?

Investigations of the behavioral mechanisms of action whereby stimulus events come to control responding have focused primarily on the control of responding by stimuli that follow the response (e.g., reinforcers) although control by those stimuli which precede the response (e.g., discriminative stimuli) may play a much more significant role in everyday life (Dinsmoor, 1995). For example, the compound contextual elements of a bar (e.g., the neon light sign, the odor, the bottles arranged behind the bar, etc.) are likely to become conditional stimuli capable of eliciting certain conditional responses similar to those elicited by alcohol (the unconditional stimulus) due to their consistent pairing. In the example just mentioned, the physical or structural characteristics of the exteroceptive and interoceptive stimuli are of interest, but it is the function of these stimuli and their functional relationship to behavior that determines their significance (Schuster & Balster, 1977). When consequential stimulus events are introduced into the above example, the schedule of reinforcement is defined. For example, if

consequential events occur which increase the probability that drinking will occur, conditional stimuli now serve as discriminative stimuli to evoke or, set the occasion for behavior. The functional relations between discriminative stimuli (antecedents), responses (behavior) and reinforcers (consequences) define the discriminated operant (Young ,1991). Young refers to the discriminated operant as the unit of stimulus control. While a thorough behavioral analysis of stimulus control requires identification of both antecedent and consequent stimulus events, the focus of this study is on investigations of antecedent function altering processes and conditions. The ability of interoceptive antecedent stimuli such as drugs to both engender discriminative effects and control the behavior of humans and nonhumans is studied

by behavioral pharmacologists using the drug discrimination procedure. procedure is a powerful biobehavioral assay for drug classes

The drug discrimination (Holtzman, 1983) and

neuropharmacological effects (Young, Masaki, & Geula, 1992) and involves the differential reinforcement of specific responses (e.g., left lever associated with DrugA and the right lever associated with Not DrugA) correlated with the availability of reinforcement contingent upon response emission (Schuster & Balster, 1977). Tests of stimulus control (discrimination) and stimulus generalization (transfer of control to stimuli which differ from the training stimulus) using choice tasks are carried out under a variety of conditions such as the same drug or drug mixtures and doses used in training, different drug(s) and doses, different contextual conditions, and different schedules of reinforcement. Thus, the actual role that a specific drug or drug mixture plays in shaping or guiding behavior depends upon the training history, the context within which training occurs, and the test conditions (Young & Sannerud, 1989).

The discriminative properties of drugs are explained by, among other things, the psychological processes of operant (instrumental) and respondent (classical) conditioning. Investigations of

respondent conditioning to compound stimuli extend the experimental analysis of behavior to the complex situations of everyday life (Pierce & Epling, 1999) because the terminology and procedures of respondent conditioning offer an explanatory framework to account for drug-behavior-environment learned associations. Three respondent conditioning procedures in particular, blocking, overshadowing, and sensory preconditioning have been investigated with both exteroceptive stimuli (e.g., lights, tones and light/tone combinations) and interoceptive stimuli (drugs and drug mixtures) in order to describe the behavioral mechanisms thought to underlie conditioning to compound stimuli (e.g., Green & Grant,1998; Jrbe, Hiltunen, & Swedberg, 1989; Jrbe, Svensson, & Laaksonen,1983; Klein & Mowrer, 1989; Mariathasan & Stolerman, 1993) (see Figure 1). Similar relations are found with compound discriminative stimuli in the case of operant stimulus control (Michael, 1993).

An early study by Jrbe et al. (1983) utilized the procedures of both blocking and overshadowing to investigate if exteroceptive stimuli can compete with interoceptive drug stimuli for associative strengthan important concern in determining the degree to which control exerted by a compound stimulus is due to the relative saliencies of the component stimuli. The authors trained rats to a differential pairing between the exteroceptive element and the interoceptive element from the outset of the discrimination training. Results from this study demonstrated that not only could drugs be used as stimuli to demonstrate the phenomena of blocking and overshadowing, but also demonstrated that the specific training procedure used, i.e., blocking or overshadowing, had an effect on the degree to which stimulus control was observed, with the drug (pentobarbital) exerting more control over behavior than the exteroceptive stimulus dimension (lightdark). Investigations of blocking and overshadowing in animals are socially relevant to complex human problems especially when seeking to account for the behavioral repertoire of humans who abuse drugs from different pharmacological classes, i.e., polydrug abuse. For example, the concomitant use of a narcotic and a psychomotor stimulant (e.g., heroin + cocaine), known in street slang as a speedball, is a popular occurrence among drug abusers (Jaffe, 1985). The ability of the speedball to control behavior raises experimental questions regarding the behavioral mechanisms of action. For example, is the acquisition of stimulus control related to the discriminative effects of the heterogeneous compound drug mixture or the homogenous drugs that comprise the mixture; and to what degree is stimulus control related to the conditions under which the discriminations are learned? In a study investigating the discriminative stimulus effects of opioid-amphetamine combinations (morphine sulfate and amphetamine), Gauvin and Young (1989) found the discriminative stimulus effects of the drugs were dependent on the training drug and dose such that the training drug masked the stimulus effects of the test drug. Procedurally, this study approximated the respondent conditioning phenomenon of blocking whereby two drugs, DrugA and DrugB were used as separate training stimuli, and subsequent tests

revealed either DrugA as capable of blocking the stimulus effects of DrugB, or DrugB as capable of blocking the stimulus effects of DrugA. However, neither investigations of blocking nor investigations of the combination of DrugA + DrugB as the training compound stimulus (i.e., overshadowing) were the authors prime concern (but see Olufsen & Stolerman, 1996, and White & Stolerman, 1994, for brief reports directly studying blocking of drug discrimination in rats). On the other hand, a study by Mariathasan and Stolerman (1993), investigating overshadowing by training different groups of rats with a mixture of nicotine and midazolam in a two-choice discrimination procedure, found that the stimulus effects of one drug can almost completely overshadow those of another substance that is normally well discriminated. For example, the authors found that while the discriminative effects of nicotine were strong after training occurred with nicotine alone, a mixture of nicotine + midazolam as the training compound stimulus weakened the discriminative stimulus effect of nicotine in a dosedependent manner. Thus, the discriminative effect of midazolam overshadowed that of nicotine.

Procedures for studying the stimulus control exerted by drug mixtures have been conceptualized by Jrbe and Swedberg (1982). One modification of the traditional two-choice assay is the two-choice DrugA vs. DrugB wherein, for example, the rate of acquiring stimulus control by two different drugs is used to indicate whether the discrimination is more dependent upon the qualitative differences between the drugs or the pharmacological properties of the drugs (e.g., Boja & Schechter, 1990). Another modification of the Drug vs. Not Drug paradigm is the two-choice DrugA + DrugB vs. Drug
A

or DrugB or Not Drug. For example, McMillan and Snodgrass (1993) trained pigeons to

discriminate a mixture of d-amphetamine and morphine from saline. On the other hand, Mariathasan & Stolerman (1994) trained rats to discriminate either a mixture of d-amphetamine and pentobarbitone or the component drugs from either saline or the component drugs. Results from these two studies suggest the drug mixture generated a stimulus complex different from the component drugs (i.e., the third state hypothesis conceptualized by Jrbe and Swedberg, 1982).

Finally, the two-choice Drug vs. Not Drug assay has been modified to a three-choice assay (e.g., DrugA vs. DrugB vs. Not Drug). While Callahan and Appel (1990) and Evans, Zacny, and Johanson (1990) used the three-choice procedure to study the in vivo effects of pharmacologically similar drugs, Gauvin and Young (1989), Makhay, Young and Poling (1998) and Preston and Bigelow (1994), for example, used the three-choice procedure to study pharmacologically dissimilar drugs. Four pertinent findings from these studies emerge: 1) The three-choice discrimination procedure can produce a reliable discrimination between two pharmacologically similar drugs (amphetamine and fenfluramine) that is not present when discriminations are established using a two-choice procedure (Evans et al., 1990); 2) The three-choice discrimination procedure may have advantages over the two-choice procedure when investigating the effects of different training drugs with selective antagonists (Callahan & Appel, 1990; Makhay et al., 1998); 3) The three-choice procedure can be used to study drug-drug interactions to determine if a novel third state is created (Gauvin & Young, 1989); and 3) Three-choice procedures may permit a greater characterization of the profile of human receptor activity as compared to twochoice procedures (Preston & Bigelow, 1994).

Two popular psychoactive drugs which have received relatively minimal discrimination investigation as a mixture, but have been implicated in discussions of the gateway drug hypothesis and drug addiction are morphine and marijuana. The primary psychoactive ingredient of marijuana is 9-THC (Gaoni & Mechoulam, 1964). 9-THC discrimination procedures in animals have been used as a model to demonstrate cannabis intoxication and to show pharmacological specificity for the unique cannabinoid receptor CB1 (Balster & Prescott, 1992). Devane et al. (1992) isolated the putative endogenous ligand arachidonyl ethanolamide (anandamide) which has been found to produce discriminative stimulus effects similar to 9-THC (Wiley, Balster, & Martin, 1995). In drug discrimination studies, tolerance to the stimulus effects of 9-THC when administered during drug discrimination training have not been reported (Jrbe & Mathis, 1993). Morphine is a compound which demonstrates unique pharmacological specificity. As Holtzman (1983) says, the discriminative stimulus effects of morphine-like agonists represent a uniform property shared by all members of this pharmacologic class (p. 10). Additionally, tolerance to the discriminative stimulus effects of morphine and other opiates is minimal if regular discrimination training and its correlated drug exposure continues throughout testing sessions (Young & Sannerud, 1989). While opioids and cannabinoids do share some common pharmacological properties such as catalepsy, hypothermia, sedation, hypotension, inhibition of intestinal movement, motor depression and antinociception (Barrett, Wiley, Balster, & Martin, 1995), they do exhibit unique pharmacological specificity. For example, drug discrimination research has shown that various compounds, including

midazolam (Barrett et al., 1995) and morphine (Browne & Weissman, 1981; Jrbe et al., 1998) fail to substitute for 9-THC. Prescott, Gold, and Martin (1992) found the effects of 9-THC on the behavioral measure catalepsy, a behavior produced by both morphine and 9-THC, more closely resembled that of haloperidol than of morphine, suggesting the cataleptogenic effect of 9-THC involved a distinctive mechanism of action. Taken as a whole, these data suggest experiments investigating the behavioral

and pharmacological effects of 9-THC and morphine should yield stable results. However, it should be noted that neurobehavioral interpretations of experimental results will require special attention. For example, both opiates and cannabinoids stimulate dopamine release in the nucleus accumbens (Abood & Martin, 1992; Chiara & North, 1992; Manzanares et al., 1999) suggesting possible similarities in their reinforcing value.

The purpose of the present pilot study is to investigate the discriminate effects of a drug mixture and its component drugs, as well as the effects of contextual conditions and behavioral history in rats. The independent variables, morphine, 9-THC, various agonists and antagonists, contextual conditions and behavioral training history will be systematically manipulated in a two-choice discrimination assay. Because Mariathasan and Stolerman (1994) note that full characterization of discriminations between drug mixtures vs. the separate drugs vs. no drug may require multi-choice procedures arranging these discriminations, future studies will propose to study these independent variables using three- and fourchoice assays. Multi-choice procedures will not be described in this research proposal. Specific experimental questions, derived from those described by Stolerman, Mariathasan, and Garcha (1991), can be addressed by this study: 1) Are the discriminative effects of drug mixtures distinct from the component drugs, or do they resemble new entities? 2) What are the effects of altering training doses and mixture ratios? 3) What are the effects of antagonist drugs that selectively block the stimulus effects of the component drugs? 4) Will utilizing the procedures of overshadowing, blocking and sensory preconditioning contribute to an understanding of the behavioral mechanisms involved in the acquisition of stimulus control by drug mixtures?

The latter experimental question, in particular, may contribute to a behavioral analysis of the gateway drug hypothesis. The gateway drug hypothesis proposes that drug use occurs along a continuum such that the use of a drug with relatively minimal abuse liability is the gateway to the use of a drug with relatively greater abuse liability. Research into the gateway drug hypothesis has typically involved self-report data collected from surveys and questionnaires (e.g., Mackesy-Amiti, Fendrich, & Goldstein, 1997), however, predicting which drugs will provide the gateway to harder drugs is an empirical question and may be dependent on an individuals behavioral history (e.g., blocking, overshadowing and sensory preconditioning). For example, whereas a certain dose of DrugA (the gateway drug) may block the stimulus effects of a certain dose of DrugB (the harder drug) when both are administered in tandem (the situation where the gateway hypothesis is disproved), the gateway hypothesis may emerge if higher doses of DrugB are administered with DrugA. In another hypothetical example of the gateway hypothesis, DrugA is reliably consumed in a certain context. Because of this preconditioning, either the context or DrugA evokes, or sets the occasion for consuming a novel harder drug. As Catania (1998) says, stimuli have varied properties, and there are no guarantees that the organism will respond to just those properties we select (p. 132). The current pilot study, while elucidating a behavioral explanation of the gateway drug hypothesis with nonhumans, will also contribute to an understanding of the behavioral mechanism of action of morphine and 9-THC mixtures.

Method Subjects Three groups of Male Sprague-Dawley rats (N = 30) will be required for this two-choice study (one group of 10 rats has already been procured for discrimination studies). Subjects will be housed in a temperature and humidity controlled vivarium maintained on a 12L:12D cycle by the WSU Department of Laboratory Animal Resources. All animals will be housed in the colony for at least a week before initiation of experiments, and all experiments will be conducted during the light phase of the cycle. Rats will be housed individually, with water freely available. Rats will be housed with water and Purina rat chow freely available. Subjects will be handled daily and weighed before all experimental sessions. All behavioral tests will be conducted in rooms outside the vivarium. Before each test, subjects will be transported to the room housing the test apparatus and allowed to habituate to the testing room for 15 min. Apparatus Two-choice drug discrimination experiments will be performed in MED-PC chambers housed in ventilated, sound-attentuating cubicles. Chambers contain two stimulus lamps mounted on one wall of each chamber above two response levers mounted 7-8 cm above the floor. A recessed food receptacle is located between the two levers. A downward force of .30 N (30 grams) will be required to complete the operant response. Food pellets will be delivered by a pellet dispenser mounted outside the chamber into the food receptacle. Chambers also contain 4 rows of photobeam sensors which will be used to measure locomotor activity. Experimental contingencies will be arranged and data recorded by an IBM-compatible computer equipped with MED-PC software (MED Associates, St. Albans, VT).

Shaping Subjects will be trained by the method of successive approximations first to eat from the food receptacle, and then to press the response lever under a fixed-ratio (FR 1) schedule of reinforcement. During shaping sessions, only one operandum will be operative while the other will be made inoperable; the operandum will change daily. The FR 1 will be in effect until each subject has attained 50 reinforcers via each operanda. Discrimination Training Procedure Each of the three groups of rats will undergo a blocking training (see Table 1). Blocking training will occur in 2 phases (see Table 1)(cf. Olufsen & Stolerman, 1996; White & Stolerman, 1994). During discrimination training, vehicle and either 3.2 mg/kg morphine (morphine group) or 2 mg/kg 9-THC (THC group) will be established as discriminative stimuli in rats. The following training procedures have been used in our laboratory (e.g., Young, Kapitsopoulos, & Makhay, 1991). Training sessions will be divided into three to six discrete trials, each consisting of a 15 minute timeout component followed by a 10 minute ratio component (these parameters have been used to train group 2 rats). At the start of each training trial, the rat will be administered an injection of vehicle or drug and placed in the darkened apparatus. At the end of this timeout component, during which responses on the operanda have no programmed consequences, stimulus lamps will be illuminated and lever-press responses will be reinforced under a fixed ratio schedule of food delivery. After drug injections, responses on one lever will be reinforced while responses on the other lever will be reinforced after injections of saline. Each response on the incorrect lever will reset the ratio counter to 0. For half of the rats in both groups, operanda will be counterbalanced. A random sequence will be used to determine the sequence of injections with the requirements that no more than two consecutive days of drug training will occur. Over successive training sessions, the FR value will be increased gradually until all rats respond reliably under a FR 30 schedule after which the FR value will be backed down to an FR 20. By shaping an FR value higher than the terminal value, break-and-run responding is reduced and the terminal FR sequence

becomes a behavioral unit in its own right (Catania, 1999).

Discrimination training will be conducted 5-7 days/week until the following performance criteria are met for five consecutive sessions: 1) the total number of responses emitted before the first reinforcer is attained will be < 40 and 2) at least 90% of the total session responses will be emitted on the injectionappropriate lever. After these criteria are met, one dose-response function for the training drug (either morphine or 9-THC) will be determined before Phase II training is begun. Tests of Stimulus Control by the Training Substances After training phases I and II, cumulative dose-effect determinations will be conducted. Test doses will be given in a random sequence of doses and drugs. Test sessions will be identical to training sessions except that 20 consecutive responses on any operandum will result in the deliver of food. Similar to training sessions, the FR value will be reset if a new operandum is selected before the FR value has been attained. Once dose-effect determinations have been conducted with the component drugs and the drug mixture after phase II training, generalization gradients with substitution agonists and antagonists may be determined. Continued training with the morphine and 9-THC mixture (phase II) will be interspersed with test sessions to evaluate the persistence of blocking (assuming it was observed).

Drugs Previous research studying the discriminative effects of 9-THC has primarily used intraperitoneal administration 30 minutes prior to training. For the purposes of this study, morphine and 9-THC will be administered to subjects subcutaneously 15 minutes prior to training. Barry and Krimmer (1976) have reported the discriminative stimulus effects of 9-THC (2mg/kg in a vehicle of 10% propylene and 1% polysorbate 80 in isotonic saline) in rats were strongest after 30 minutes i.p. injection but were only slightly weaker as early as 7.5 minutes after i.p. injection, thus, the rapid onset of the drug stimulus indicates rapid absorption and distribution. Wiley (personal communication) doubts changing the route of administration would have much impact. Wiley et al. (1995) studied 9-THC administered i.p. in a 1:1:18 suspension vehicle of absolute ethanol, Emulphor-620 and saline to form a stock solution of 10 mg/ml. Burkey and Nation (1997) studied 9-THC administered i.p. in a preparation that first evaporated the ethanol under positive nitrogen pressure, then diluted it with a vehicle of 92% saline, 5% propylene glycol, 2% Tween-80, and 1% ethanol. Doses of 9-THC studied during testing have ranged from .3 to 56 mg/kg. Dr. Hrapkiewicz from Wayne State Universitys Division of Laboratory Animal Resources suggested balancing the pH level of the 9-THC will help reduce the risk of necrosis. Morphine will be prepared in physiological saline. To control for bias and other differences in vehicles, group 1 rats will be administered each vehicle during phase I discrimination training.

Additional drugs which can be investigated include the opiate reversible antagonist naltrexone HCL and the cannabinoid reversible antagonist SR141716A. Naltrexone HCL will be prepared in physiological saline, while SR141716A will be prepared in a 1:1:18 suspension vehicle of absolute ethanol, Emulphor-620 and saline. Dose-dependent reversible antagonism of the discriminative stimulus effects of 9-THC has been demonstrated by the antagonist SR141716A administered in doses ranging from 0.3 to 5.6 mg/kg (administered 40 minute prior to sessions) (Wiley et al., 1995). Data analysis Discrimination data, response rate and locomotor activity will be analyzed separately for each drug and drug mixture. Discriminative performance, analyzed only if 15 or more responses were emitted during a test component, will be presented as the percentage of total responses made on the drug appropriate lever. Rates of responding on either lever will be expressed as a percentage of the average rate during the 10 saline training sessions conducted immediately prior to the first dose-response determination (the test after phase I training). Locomotor activity will be expressed as a percentage of the average activity during the 10 saline training sessions conducted immediately prior to the first dose-response determination. Dose-response functions will be constructed by pooling measures of discriminative performance or response rate or locomotor activity for all subjects in a group and plotting the resulting mean values as a function of dose. Calculations of ED50s and estimates of variance will be made using regression analysis, and analysis of variance with repeated measures as needed (Tallarida & Murray, 1986), followed by a Tukey h.s.d. test of differences among treatment means. Statistical significance will be set at p < .05.

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