Amphetamine-Induced Conditioned Activity Is Insensitive to Perturbations Known to Affect Pavlovian Conditioned Responses in Rats

Transcription

1 Behavioral Neuroscience Vol. 112, No, 5, Copyright 1998 by the American Psychological Association, Inc /98/$3.00 Amphetamine-Induced Conditioned Activity Is Insensitive to Perturbations Known to Affect Pavlovian Conditioned Responses in Rats Serge H. Ahmed, Luis Stinus, and Martine Cador University of Victor Segalen Bordeaux II Psychostimulant-induced conditioned activity is characterized by the presence of a hyperactivity in drug-free rats exposed to an environment previously paired with the effects of a psychostimulant. This phenomenon is thought to result from a Pavlovian conditioning process. This hypothesis predicts that conditioned activity will he sensitive to perturbations known to affect classical conditioned responses. In direct contrast with this prediction, the authors report here that conditioned activity is insensitive to (a) the temporal order between the stimulant injection and the exposure to the environment, (b) unsignaled stimulant injections between drug-environment pairings, and (c) drug preexposures before the start of drug-environment pairings. It is concluded that the stimulant effects responsible for the establishment of conditioned activity may not be amenable to a Pavlovian associative process. Drug-free rats exposed to an environment previously paired wilh a psychostimulant are hyperactive as compared with rats for which this environment has been previously paired with the vehicle (Barr et al., 1983; Beninger & Harm, 1983; Gold, Swerdlow, & Koob, 1988; Pickens & Crowder, 1967; Stewart & Vezina, 1991). It is thought that this phenomenon, called conditioned activity, depends on a Pavlovian conditioning process whereby some of the drug effects act as an unconditioned stimulus (US) and the environment acts as a conditioned stimulus (CS). During repeated environment-stimulant pairings, an association forms between the relevant drug US and the environmental CS. This associative account of conditioned activity has provided an explanatory basis for the behavioral sensitization observed after repeated stimulant and opiate administration (Ahmed, Stinus, Le Moal, & Cador, 1993; Hinson & Poulos, 1981; Post, Lockfeld, Squillace, & Contel, 1981; Stewart & Vezina, 1988; Tilson & Rech, 1973) and plays a significant role in some current theories of drug addiction (Robinson & Berridge, 1993; Stewart, de Wit, & Eikelboom, 1984; Wise & Bozarth, 1987). The Pavlovian account mainly relics on the fact that conditioned activity has features similar to a classical conditioned response (CR; i.e., stimulus specificity and extinction-like phenomena; for a critical review, see Ahmed, Serge H. Ahmed, Luis Stinus, and Marline Cador, University of Victor Segalen Bordeaux II, Bordeaux, France. This research was supported by grants from the Institut National de la Sante et de la Recherche Medicale, Paris, France and Centre National de la Recherche Scientifique, Paris, France and by the Conseil Regional d'aquitaine, Bordeaux, France. We would like to thank Charles Heyser, John Walker, and Pilar Flores for their comments on a draft of the article, Cecile Spielevoy for her help in conducting some of the experiments reported, and Megan Beschen for proofreading the manuscript. Correspondence concerning this article should be addressed to Serge H. Ahmed, who is now at the Department of Neuropharmacology, Scripps Research Institute, North Torrey Pines Road, La Jolla, California Electronic mail may be sent to aserge@sage.scripps.edu. 1995). Note, however, that these similarities are not sufficient to establish the associative status of conditioned activity. First, they are also compatible with nonassociative mechanisms, involving, for example, a deficit of habituation to the would-be paired environment (e.g., Ahmed, Stinus, Le Moal, & Cador, 1995). Second, data exist that do not support the view of conditioned activity as a Pavlovian CR. There is no evidence that conditioned activity grows with the number of pairings (Ahmed, Stinus, et al., 1995; Hoffman & Wise, 1992; Mazurski & Beninger, 1991; Pickens & Crowder, 1967), which is surprising for a putative Pavlovian learning process. Indeed, even the magnitude of conditioned taste aversion, which is well-known to develop after a single conditioning trial, continues to increase with repeated trials (e.g., Figure 3 in Grigson. 1997). Moreover, conditioned activity does not vary with the dose of the psychostimulant, although the unconditioned drug effect does (Herz & Beninger, 1987; Martin-Iverson & McManus, 1990; Schiff, 1982; but see Tilson & Rech, 1973). Finally, it has been shown recently that the form of conditioned activity differs substantially from the unconditioned behaviors induced by stimulant drugs (Martin-Iverson & Fawcett, 1996). In view of the equivocal status of conditioned activity, the present series of experiments is intended to probe directly its putative associative basis. For this, it is necessary to verify whether perturbations known to interfere with the formation and expression of a CS-US association affect conditioned activity. We test whether conditioned activity is sensitive to the CS-US temporal order (Experiment 1), to unsignaled occurrences of the drug US between CS-US trials (Experiment 2), and to the US preexposure effect (Experiment 3). All of these perturbations are known to affect Pavlovian CRs by deteriorating the predictive relationship between the CS and the US (e.g., Durlach, 1989; Mackintosh, 1983; Rescorla, 1988). Subjects General Method Two-month-old male Wistar rats (Tffa Credo, Lyon, France), weighing g on arrival, were used in all experiments. The 1167

2 1168 AHMED, ST1NUS, AND CADOR rats were housed in groups of 5 and maintained in a 12-hr light-dark cycle, beginning at 6 a.m., and temperature-controlled environment. Food and water were freely available. Before the start of the experiment, each rat was handled every other day for 30 s (spread over 8 days after their arrival in the laboratory). From the beginning of the experiment, they were weighed in the afternoon of the days before the amphetamine-injection days. Apparatus The experiment involved two distinct environments: A and B. They were located in the same sound- and light-attenuated experimental room, which was distinct from the colony room. Environment A was an activity cage. Each cage was built of metal wire mesh and measured 20 X 25 X 36 cm. Two photocell beams were located across the long axis 2 cm above the floor, to enable measurement of general activity. The activity cages were linked to a computer, which recorded each photocell beam break. For Environment B, Plexiglas boxes with transparent walls were used. These boxes, slightly smaller than the activity cages, were inserted into the activity cages to allow the measurement of activity. This was done so that the photocell beams were located 2 cm above the floor of the box equidistant from the middle of the box, as in the activity cages. These two environments featured olfactory, tactile, and visual cues that were different from one another and from those of the home cage. In A, olfactory cues were provided by a diluted solution of Aniseed (pure anise extract; Schilling, Hunt Valley, MD); in B, there were no explicit odor cues. Different textures of the floor represented the tactile cues: In A, the floor was rough; in B, it was wire mesh. The visual cues were represented by different patterns on the walls: In A, there was no explicit pattern; in B, there were horizontal black and white adhesive strips. Drugs d-amphetamine sulfate was dissolved in an isotonic 0.9% (wt/wt) NaCl solution. The dose used was 1.25 mg/ml/kg, expressed as the weight of the salt (see Ahmed et al., 1993, for the choice of the dose). All injections were given subcutaneously. General Experimental Design All experiments involved at least two phases: a drugenvironment pairing phase and a test phase. The pairing phase consisted of successive 2-day sessions. On the 1st day of each session, all rats received an amphetamine injection, but half of them were placed in A and the other half in B. On the 2nd day of each session, rats received a vehicle injection and were placed in the environment that had not been paired with the amphetamine on the 1st day. Thus, for each rat there was an amphetamine (CS + ) and a vehicle (CS )-paired environment. The test phase followed the last session of the drug-environment pairing phase and lasted 2 days. During the 1st test day, rats received a vehicle injection and were placed in the CS +. During the 2nd test day, the procedure was identical to the 1 st test day, except that rats were exposed to the CS. We have previously shown that conditioned activity is not affected by the order of testing (see Experiment 1 in Ahmed, Stinus, etal., 1995). Experiment 1: Effect of the Drug US-CS+ Delay on Conditioned Activity In a typical Pavlovian conditioning trial, the US follows in time the onset of the CS. When the CS-US temporal relationship is arranged differently, with the US presented simultaneously or before the CS, the acquisition of the CR is either abolished or, in a few cases, strongly attenuated (Heth, 1976; Heth & Rescorla, 1973; Mahoney & Ayres, 1976). Therefore, if conditioned activity develops because the environment predicts the drug US, then its acquisition should be affected by presenting the drug US before exposing rats to the would-be CS+. Experiment 1 was intended to test this prediction by using amphetamine as the drug US and different drug US-CS+ delays. Method The experiment involved three groups of rats (n 10-11). During the 1st day of each drug-pairing session, all groups were transported to the experimental room and then received amphetamine. Depending on the group, they were placed in the CS + at different times after the drug injection: 0 min (corresponding to a time preceding the onset of the drug effect), 25 min (corresponding to a time close to the peak of the drug effect), or 50 min (corresponding to the start of the decline of the drug effect). These delays were based on a pilot study of the time course of amphetamine-induced hyperactivity. Thus, the stimulant effect occurred after and at different times before exposure to the CS+ in the 0-min, 25-min, and 50-min groups, respectively. During the delay period, rats remained in their home cage. The duration of exposure to CS+ was 120, 95, and 70 min for the 0-min, 25-min, and 50-min groups, respectively. The duration of exposure was reduced in the 25-min and 50-min groups (a) to ensure that all three groups were removed from the CS+ exactly 120 min after the amphetamine injection and (b) to avoid keeping rats of the 25-min and 50-min groups in the presence of the CS+ while no longer under the drug effects. During the 2nd day of each session, the procedure was identical except that rats received a vehicle injection. After six successive pairing sessions, the rats were tested as described in the General Method section. Results and Discussion Responses to amphetamine and vehicle during the drugpairing phase. The upper part of Figure 1 shows the mean total activity recorded during the first 60 min after each amphetamine injection in the CS +. A two-way analysis of variance (ANOVA) revealed a slight but significant increase of amphetamine-induced hyperactivity across injections, F(5, 135) = 9.82, p <.0001, but the magnitude of this sensitization was not influenced by the drug US-CS + delay, Group X Injections interaction, F(10, 135) = Although there was a trend that amphetamine-induced hyperactivity was lower in the 25-min and 50-min groups than in the 0-min group, it did not reach statistical significance, group, F(2, 27) = The lower part of Figure 1 represents the level of activity recorded after each vehicle injection in the CS -. A two-way ANOVA revealed a significant decrease of activity across injections, F(5, 135) = 51.37, p <.0001, but the magnitude of this habituation did not differ between groups: group, F(2. 27) = 1.97; Group X Injections interaction, F(10, 135) = Activity in the CS+ and the CS during the test phase. Figure 2 shows the mean total activity during the first 60 min after exposure to the CS + and CS- during the test phase for

3 AMPHETAMINE-INDUCED CONDITIONED ACTIVITY t; 2000 amph ACQUISITION SESSIONS Figure I. Mean (±SEM) total activity counts recorded during the 1st hr after each amphetamine (amph)- and each vehicle-injection session. Depending on the experimental group (n = per group), rats were exposed to the conditioned stimulus (CS+) 0, 25, or 50 min after each amphetamine injection (1.25 mg/kg, sc). The same procedure was used for vehicle injections given in the CS. each experimental group. A two-way ANOVA revealed that the level of activity in the CS + was greater than in the CS -, F(l, 27) = 29.33, p < However, the magnitude of this difference was similar in the three experimental groups group, F(2, 27) = 1.66; Group X Environment interaction, F(1, 27) = 0.52 suggesting that the development of conditioned activity was not influenced by the drug US- CS + delay. To assess possible differences in the time course of conditioned activity between the three experimental groups, we performed a three-way ANOVA with one between-subjects factor (experimental groups) and two withinsubject factors (time: six time bins of 10 min each; test: CS + vs. CS-). Figure 3 contains the results. This analysis revealed a significant effect of environment, F(l, 27) = 29.33, p <.0001, and a significant Environment X Time interaction, F(5, 135) = 7.79, p <.0001, suggesting that conditioned activity in the CS+ decreased with time. However, this time course was not significantly influenced by the drug US-CS+ delay, Group X Environment X Time interaction, F(10, 135) = Several issues need to be emphasized. First, the habituation pattern observed after repeated exposures to the CS was not influenced by the duration of exposure. For example, the 50-min group, which was allowed to explore the CS- for a period of 70 min, showed virtually the same rate of habituation across sessions compared with the 0-min group, which was allowed a 120-min period of exploration. This result was not surprising, however, because in grouphoused rats, most of the exploratory activity in a novel environment occurs within thelsthourof exposure (Ahmed, Cador, Le Moal, & Stinus, 1995). Second, as expected, amphetamine-induced hyperactivity in the CS+ tended to be more pronounced in the 0-min group, for which no delay was interposed between the drug injection and the exposure to the CS +. Although not significant, this difference was more pronounced between the 0-min and 50-min groups. With repeated injections, a slight sensitization to this effect of amphetamine was observed, but its magnitude was not influenced by the drug US-CS+ delay. Finally and most important, the drug US-CS+ delay had no significant impact on the acquisition or expression of conditioned activity. Rats exposed to the CS+ 25 or 50 min after the amphetamine injections exhibited a level of conditioned activity comparable to the control group. Moreover, the time course of conditioned activity was unchanged by the drug US-CS+ delay. 600 «500 z O 400 o min 25 min 50 min < min 25 min 50 min DEIAY US-CS+ Figure 2. Effect of the drug unconditioned stimulus-conditioned stimulus (US-CS+) delay on amphetamine-induced conditioned activity. Each drug US-CS + delay (0, 25, or 50 min) represents a different group of rats (n = per group). Each group was exposed after a vehicle injection to the CS + on Day 1 and to the CS - on Day 2 of the test phase. Bars represent mean (± SEM) total activity counts registered during the 1st hr after a vehicle injection TIME (10-min bins) Figure 3. Effect of the drug unconditioned stimulus-conditioned stimulus (US-CS+) delay on the time course of conditioned activity. Each drug US-CS + delay (0, 25, or 50 min) represents a different group of rats (n = per group). Each group was exposed after a vehicle injection to the CS + on Day 1 and to the CS on Day 2 of the test phase. Points represent mean (± SEM) activity counts measured every 10 min during the 1st hour of exposure to both the CS+ and the CS-.

4 1170 AHMED, STINUS, AND CADOR Experiment 2: Effect of Uasignaled Drug US on Conditioned Activity It has been established that to be a reliable predictor, the CS should signal to the subject a period in which the likelihood of US occurrences is increased compared with a condition in which the CS is absent (Dickinson, 1980; Durlach, 1989; Rescorla, 1988). For example, if the experimenter makes the probability of a shock US in the absence of the CS (i.e., p[us/no CS]) equal to that in the presence of the CS (i.e., pfus/csl), the development of a fear CR to the CS is abolished (Rescorla, 1968). The manipulation of p[us/no CS] is achieved by presenting the US alone (i.e., unsignaled presentation) between CS-US pairings. If conditioned activity depends on the predictive value of the CS+, then it would be affected by giving unsignaled amphetamine injections between CS+-drug US pairings. Experiment 2 was intended to test this prediction. Method This experiment involved three groups of rats (n = 10). Each group received six successive drug-pairing sessions (S1 to S6), but the CS+ was paired with amphetamine only during SI, S3, S4. and S6; during the remaining sessions (S2 and S5), the CS+ was paired with a vehicle injection (see Table 1). By reducing Ihe likelihood of amphetamine effects in the presence of the CS+, this partial conditioning procedure was intended to facilitate the detrimental effect amphetamine injections given in the absence of the CS + have on conditioned activity (for an explanation on the use of this procedure, see Durlach, 1983; Rescorla, 1968). The groups only differed from each other by the number of amphetamine injections received in the absence of the CS+ (unsignaled injections). The procedure used to administer unsignaled injections was as follows. Every day, 3 to 6 hr after having been removed from the CS + or the CS and returned to the colony room, rats were brought back to the experimental room, where they received a second injection of either vehicle or amphetamine. After the injection, rats remained in their home cages for 2 hr before being returned to the colony room. The 4-0 group received only vehicle injections (the first number stands for amphetamine injections given in the CS +, and the second number stands for unsignaled amphetamine injections). In contrast, Ihe 4-4 and 4-8 groups received four and eight unsignaled amphetamine injections, respectively, semirandomly interspersed with vehicle injections. Thus, in the 4-0 group, receiving the drug was entirely contingent on the presence of the CS+. In contrast, in the 4-4 and 4-8 groups, receiving the drug in the absence of the CS+ was equally or more likely than in its presence, respectively. Results and Discussion Responses to amphetamine and vehicle during the drugpairing phase. The upper part of Figure 4 shows the mean total activity recorded during the first 60 min after the four amphetamine injections given in the CS +. A two-way ANOVA revealed a slight but significant increase of amphetamine-induced hypcractivity across injections, F(3, 81) = Table 1 Regimen of Unsignaled Amphetamine Injections Used in Experiment 2 Amphetamine injections Unsignaled (3-6 hr later) Paired Trials Environment withcs+ group group group Session 1 Day 1 CS Day 2 cs- CS Session 2 Day 3 CS + Day 4 CS- cs Session 3 DayS CS Day 6 cs- Session 4 Day 7 CSt + DayS cs- Session 5 Day 9 CS+ Day 10 cs- CS Session 6 Day 11 CS Day 12 cs- Note. Each drug-environment pairing session lasted 2 days, for a total of six sessions. On even days, rats were exposed to the amphetamine-paired (CS + ) environment but received amphetamine (indicated by +) only during Days 1, 5, 7, and 11; the remaining days, they received vehicle (indicated by ). On odd days, rats were exposed to the vehicle-paired environment (CS ) after having been given vehicle. In addition to amphetamine injections paired with the CS +, rats received 0 (4-0 group). 4 (4-^ group), or 8 (4-8 group) unsignaled amphetamine injections, interspersed semirandomly with vehicle injections. These injections were given in the experimental context 3 to 6 hr after the exposure to the CS+ or the CS-. The number of unsignaled amphetamine injections given the days after exposure to the CS + was equal to that following exposure to the CS.

5 AMPHETAMINE-INDUCED CONDITIONED ACTIVITY , p <.005, and the magnitude of this sensitization was influenced by the number of amphetamine injections received in the absence of the CS+, Group X Injections interaction, F(6, 81) = 2.74, p <.02. A one-way ANOVA performed for each amphetamine injection revealed only a marginally significant difference between groups at the fourth amphetamine injection, F(2, 27) = 3.22, p =.056. During this injection, the activity of the 4-4 group was greater than that of the 4 0 group (p <.05; Newman-Keuls test), which was similar to that of the 4-8 group. The lower part of Figure 4 represents the level of activity recorded after each vehicle injection in the CS-. A two-way ANOVA revealed a significant decrease of activity across injections, F(5, 135) = 55.44, p <.0001, but the magnitude of this habituation was not influenced by the number of unsignaled amphetamine injections: group, F(2, 27) = 0.21; Group X Injections interaction, F(10, 135) = Activity in the CS+ and the CS during the test phase. Figure 5 shows the mean total activity during the first 60 min after exposure to the CS+andCS during the test phase for each experimental group. A two-way ANOVA revealed that the level of activity in the CS+ was greater than in the CS, F(\, 27) = 14.70, p <.001. However, the magnitude of this difference was similar in the three experimental groups, group F(2, 27) = 1.66, Group X Environment interaction, F(2, 27) = 0.52, suggesting that the development of conditioned activity was not influenced by the number of unsignaled amphetamine injections. Three important findings emerged from Experiment 2. First, unsignaled amphetamine injections did not affect the level of activity in the CS, suggesting that the relatively heavy drug treatment used in Experiment 2 did not induce amph ACQUISITION SESSIONS Figure 4. Mean (±SEM) total activity counts recorded during the 1st hr after each amphetamine injection paired with the amphetamine (amph) environment conditioned stimulus (CS+; i.e., during Sessions 1, 3,4, and 6 of the drug-environment pairing phase) and each vehicle injection paired with the vehicle environment CS (from Session 1 to Session 6). Depending on the experimental group (n = 10 per group), and in addition to the four amphetamine injections paired with the CS +, rats received zero (4-0 group), four (4-4 group), or eight (4-8 group) unsignaled amphetamine injections distributed pseudorandomly between the drug-environment pairing sessions o UNSIGNALED AMPHETAMINE Figure 5. Effect of unsignaled amphetamine injections on amphetamine-induced conditioned activity. Unsignaled amphetamine injections were pseudorandomly distributed between the drugenvironment pairing sessions. Different numbers of unsignaled amphetamine injections (0, 4, and 8) were administered to different groups of rats («= 10 per group). During the test phase, each group was exposed after a vehicle injection to the amphetaminepaired environment (CS+) on Day 1 and to the vehicle-paired environment (CS ) on Day 2. Bars represent mean (±SEM) total activity counts registered during the 1 st hr after a vehicle injection. any general behavioral hypo- or hyperactivity. Second, unsignaled amphetamine injections slightly sensitized the rats to amphetamine when injected in the CS+. However, the relationship between the number of unsignaled injections and the magnitude of this sensitization was not linear. Indeed, although the 4-4 group received fewer unsignaled amphetamine injections than the 4-8 group, it appeared more sensitized than the latter group. This interpretation should be tempered, however, because the relative decrease in locomotor activity observed in the 4-8 group may also reflect a sensitization in some competing behavioral stereotypies (Segal & Mandell, 1974). Finally, the data obtained during the test phase revealed that the partial conditioning procedure used in Experiment 2 had a detrimental effect on conditioned activity (compare the difference in activity in the CS+ and the CS- observed in the 4-0 group with that observed in Experiments 1 and 3; see also Ahmed, Stinus, et al., 1995). As already noted, partial conditioning was used to increase the effect of unsignaled USs (Durlach, 1983; Rescorla, 1968). However, despite the use of this procedure, unsignaled amphetamine injections did not prevent the development of conditioned activity. Rather, those injections seemed to prevent the detrimental effect of the partial conditioning procedure. Experiment 3: Effect of Drug US Preexposures on the Development of Conditioned Activity Another way to reduce the predictive value of the CS consists of preexposing the subject to the US alone before the start of the conditioning. US preexposures usually have a detrimental effect on the acquisition of a CR (for a review, see Randich & LoLordo, 1979). This detrimental effect is

6 1172 AHMED, STINUS, AND CADOR amph ACQUISITION SESSIONS Figure 6. Mean (±5 M) total activity counts recorded during the 1st hr after each amphetamine (amph)-injection session and each vehicle-injection session. Depending of the experimental group (n = 10 per group), rats were preexposed 0 (O^PREE group), 5 (5-PREE group) or 10 (10-PREE group) times to amphetamine (1.25 mg/kg, sc) before the start of the drug-environment pairing phase. thought to result from a blocking mechanism (see Kamin, 1969), wherehy the experimental context of the US preexposures plays the role of the blocking stimulus (Durlach, 1989; Randich & Ross, 1985; Tomie, 1976). Experiment 3 was intended to assess whether repeated preexposures to amphetamine before conditioning block the establishment of conditioned activity. Method The experiment involved three groups of rats (n 10). Before the start of the drug-pairing phase, all rats were transported each day for 10 consecutive days to the experimental room. From Day 1 to Day 10, the 0-PREE group received one daily vehicle injection and then were left in their home cage for 2 hr. The two other groups were preexposed to the training dose of amphetamine: The 5-PREE group received 1 daily vehicle injection on the first 5 days and 1 daily amphetamine injection on the last 5 days; the 10-PREE group received 10 daily amphetamine injections. Note that during the preexposure phase, all the details of the injection procedure (i.e., transport and handling) were similar to those used during the drug-pairing phase. After a 24-hr rest period after the last injection, all rats received three successive drug-pairing sessions, after which they were tested, as described in the General Method section. The relatively small number of pairings used in this experiment was intended to increase the sensitivity of our procedure to the detrimental effect of US preexposures. Results and Discussion Responses to amphetamine and vehicle during the drugpairing phase. The upper part of Figure 6 shows the mean total activity recorded during the first 60 min after each amphetamine injection in the CS +. A two-way ANOVA revealed a significant difference between groups, F(2, 27) = 3.97, p <.04. This difference was due to an increased response to amphetamine in the amphetamine-preexposed groups compared with the vehicle-preexposed group (p <.03; Newman-Keuls test). Moreover, there was a slight but significant increase of amphetamine-induced hyperactivity across injections, F(2, 54) = 6.37, p <.005, but the magnitude of this sensitization was not influenced by the number of amphetamine preexposures, Group X Injections interaction, F(4, 54) = The lower part of Figure 6 represents the level of activity recorded after each vehicle injection in the CS-. A two-way ANOVA revealed a significant decrease of activity across injections, F(2, 54) = 74.47, p <.0001, but the magnitude of this habituation was not influenced by the number of amphetamine preexposures, group, F(2, 27) = 0.61; Group X Injections interaction, f (4, 54) = Activity in the CS+ and the CS during the test phase. Figure 7 shows the mean total activity during the first 60 min after exposure to the CS + andcs during the test phase for each experimental group. A two-way ANOVA revealed that the level of activity in the CS + was greater than in the CS -, F(l, 27) = 30.76, p < However, the magnitude of this difference was similar in the three experimental groups, group, F(2, 27) = 2.46, Group X Environment interaction, F(2, 27) = 0.28, suggesting that the development of conditioned activity was not influenced by preexposure to amphetamine. In Experiment 3, several points deserve attention. First, repeated preexposure to amphetamine did not affect the activity recorded in the CS- during the drug-environment pairing phase, suggesting that amphetamine pretreated rats respond to environmental novelty and habituate to it in a manner similar to drug-naive rats. Second, amphetamine preexposure sensitized rats to subsequent amphetamine injections in the CS +. In addition to showing that our preexposure regimen was effective, this result also suggests AMPHETAMINE PREEXPOSURES Figure 7. Effect of amphetamine preexposures on amphetamineinduced conditioned activity. Amphetamine preexposures were given before the start of the drug-environment pairing phase. Different numbers of amphetamine preexposures (0, 5, and 10) were administered to different groups of rats (n = 10 per group). During the test phase, each group was exposed, after a vehicle injection, to the amphetamine-paired environment (CS+) on Day 1 and to the vehicle-paired environment (CS ) on Day 2. Bars represent mean (± SEM) total activity counts registered during the 1st hr after a vehicle injection.

7 AMPHETAMINE-INDUCED CONDITIONED ACTIVITY 1173 that sensitization can be expressed in an environment that has never been directly paired with the drug (see also Ahmed et a!., 1993; Anagnostaras & Robinson, 1996). Finally, and most important, in contrary to the known deleterious effect of US preexposure on the acquisition of a classical CR, repeated preexposure to amphetamine had no detrimental effect on conditioned activity. This negative outcome is also surprising for another reason. Although amphetaminepreexposed rats exhibited a higher UR to amphetamine, this was not paralleled by a higher level of conditioned activity. This result is congruent with data showing that in sharp contrast to the UR to the drug, conditioned activity does not increase with the training dose (see introduction). As we have suggested (Ahmed et al., 1993), this latter finding could indicate that once a critical level of stimulation is reached, conditioned activity fully develops in an all-or-nothing manner. General Discussion The present study was designed to verify whether stimulant-induced conditioned activity results from an association between the drug-paired environment and some drug US. It was hypothesized that this association developed through a Pavlovian conditioning process, by which a previously neutral environment became a reliable predictor of the drug US. The present data, however, do not support this account. First, amphetamine-induced conditioned activity can manifest in an environment that follows in time the drug US (Experiment 1). Second, conditioned activity appears in an environment that is not a reliable predictor of the drug US (Experiments 2 and 3). The following sections arc intended to discuss possible limitations of the present data and to draw some implications for future studies. Is Conditioned Activity Anticipatory in Nature? In Experiment 1, conditioned activity appeared almost completely insensitive to the drug US-CS+ delay. This finding can be interpreted in several ways. First, it may incidentally be considered as one of the seldom instances of a successful backward conditioning (see Mackintosh, 1983). This is unlikely, however, because CRs obtained in backward conditioning are usually lower in magnitude than CRs obtained in traditional forward conditioning (e.g., Heth, 1976; Heth & Rescorla, 1973). Second, that conditioned activity manifested despite a 50-min drug US-CS+ delay could simply indicate that the relevant drug US begins at 50 min after the drug injection. Although plausible, this is not supported by the time course of conditioned activity observed in the 0-min group. Indeed, if the drug US was really delayed in time, conditioned activity would have been more apparent at a time point close to the putative onset of the US, a phenomenon called inhibition of delay (Pavlov, 1927). Third, Experiment 1 shows also that pairing either the ascending or the descending component of the stimulant effect with the CS+ makes no detectable difference. Because the descending part may partly reflect the intervention of opponent processes (Solomon, 1977; Solomon & Corbit, 1974), these data could suggest that these processes play no significant role in conditioned activity. In conclusion, the negative outcome of Experiment 1 confirms a previous study (Pickens & Crowder, 1967) and indicates that in contrast to a Pavlovian CR (Hollis, 1982; Siegel, 1985), stimulantinduced conditioned activity is not likely an anticipatory response to a forthcoming drug US. Does Conditioned Activity Depend on a Predictive CS-US Relationship? Both Experiments 2 and 3 suggest that conditioned activity is insensitive to the predictive value of the CS +. In Experiment 2, increasing the likelihood to receive the drug US in the absence of the CS+ did not have any detrimental influence on conditioned activity. In Experiment 3, preexposing rats to the drug US alone before pairing it with the CS + had no effect on the acquisition or expression of conditioned activity. This latter finding confirms an earlier study that also failed to document an effect of amphetamine preexposures (5 in total) on conditioned rotations (Drew & Click. 1988). To interpret these data, one should be reminded that the detrimental effect of both unsignaled USs and US preexposures has been shown to depend on an association between the US and the training context (i.e., the background external stimuli present during CS-US pairings). Such a context-us association is thought either to block the acquisition of the CS-US association (Durlach, 1989; Kamin, 1969; Rescorla & Wagner, 1972) or to interfere with its behavioral expression (e.g., Miller & Matzel, 1988). In this light, the negative outcome of Experiments 2 and 3 could be attributed to a lack of contextual conditioning. This is unlikely because caution was taken to administer the unsignaled amphetamine injections or to preexpose rats to amphetamine in the presence of the experimental context in which the CS+ was embedded. This context represents a rich set of stimuli originating from (a) the transport of the rats from the colony room to the experimental room by using a metal cart; (b) the distinctive features of the experimental room (e.g., its continuous white noise, its illumination, and its particular smells); (c) the distinctive stimuli of the injection ritual (handling and subcutaneous injections). This whole experimental context was maintained throughout the experiment to facilitate, if any, its association with the drug US. Moreover, similar contextual cues have been shown to control the expression of opiate-induced tolerance phenomena (for a review, see Siegel, 1989). Although contextual conditioning was likely to occur in our experimental conditions, it still remains possible that the relative strength of the context-us association was too low to interfere with the formation or expression of the CS+/ drug US association. However, there are reasons to believe that this was not the case. First, in Experiment 2, in the 4-8 group, which received the highest number of unsignaled injections, amphetamine was paired 66.6% of the time with the context alone (8 out of 12 occasions). This probability is equal to the probability of receiving amphetamine in the presence of the CS+ (4 out of 6 occasions). This equiprobability is known to completely prevent the acquisition or

8 1174 AHMED, STINUS, AND CADOR expression of a Pavlovian CR (Dickinson, 1980; Durlach, 1989; Rescorla, 1968). Second, in Experiment 3, it would be difficult to argue that amphetamine preexposures were insufficient because in the 10-PREE group, for example, amphetamine was paired to the experimental context alone three times more than to the CS +. In summary, the apparent insensitivity of conditioned activity to unsignaled USs or US preexposures observed in Experiments 2 and 3 is not likely to be the result of a lack of contextual conditioning and may suggest that conditioned activity does not depend on a predictive relationship between the drug US and the environment. Is the Relevant Drug US Malleable? There is another possible limitation of the present study. In the three experiments performed, the environment where rats experienced amphetamine effects when not in the CS + was always the home cage. There, rats had the opportunity to interact with their habitual cage mates. There is evidence that some of the behavioral effects of stimulant drugs can be strongly influenced by the environmental setting (e.g., Beck, Chow, & Cooper, 1986; Ellinwood & Kilbey, 1975). For example, it has been recently shown that the stimulant effect of amphetamine is much higher in a novel environment than in a familiar environment (Badiani, Anagnostaras, & Robinson, 1995). Although the mechanism of this effect is still unknown, it is possible that the US generated by amphetamine in one environmental setting is quantitatively or qualitatively different from the one generated in another setting. Therefore, our failure to affect conditioned activity could depend on the fact that the amphetamine US perceived in the CS+ was different from the amphetamine US experienced in the home cage. First, in Experiment 1, the drug US-CS+ delay could have been ineffective simply because by definition the US related to the CS+ could not occur before exposure to this environment. Second, US preexposures or unsignaled USs were ineffective because what was paired with the experimental context during preexposure or between CS+/US pairings was a drug US different from the drug US experienced in the CS +. This explanation surely warrants further studies. What Is the Nature of Conditioned Activity? To date, two major hypotheses of conditioned activity have been proposed. According to the first, conditioned activity develops because stimulants induce a deficit in habituation to the novelty of the would-be paired environment (Ahmed, Stinus, et al., 1995; Damianopoulos & Carey, 1992; Gold et al., 1988). This hypothesis seems to be supported by two lines of evidence: (a) The magnitude and time course of conditioned activity are similar to those of novelty-induced activity (Ahmed, Stinus, et al., 1995), and (b) the extinction of conditioned activity nicely parallels the behavioral habituation observed in a novel environment (Ahmed, Stinus, et al., 1995; see also Damianopoulos & Carey, 1992). However, the fact that conditioned activity could fully develop in a previously well familiarized environment contradicts this hypothesis (Ahmed, Oberling, Di Scala, & Sandner, 1996). According to the second hypothesis, the Pavlovian hypothesis tested here, conditioned activity develops because of a conditioned association between the drug-paired environment and some drug US. However, as indicated by the present findings, this associative account may not be valid. This conclusion is consistent with the insensitivity of conditioned activity to increases in drug doses and drug-pairing sessions (see introduction). Moreover, it could help researchers understand why conditioned activity is unaffected by lesioning the basolateral amygdala (Ahmed, Stinus, et al., 1995; Brown & Fibiger, 1993), a brain region known to mediate stimulus reward association in rodents (Cador, Robbins, & Everitt, 1989; Everitt, Cador, & Robbins, 1989). What then is the nature of conditioned activity? At present, this question is difficult to answer because the nature of the so-called stimulant US remains unknown. Indeed, although it is widely held that it is interoceptive, one does not know which sensory modalities it affects, what its exact temporal characteristics are, and, if any, what its spatial features are. Consequently, in the case of so-called stimulant conditioning, it is not possible to specify either the content of what is supposedly learned or the form of the particular drug US representation. This situation contrasts with conditioning involving prototypical Pavlovian USs, which are usually well-identified phasic and localized changes in the environment (e.g., Dickinson, 1980; Holland, 1990; Mackintosh, 1983; Miller & Barnet, 1993). One common way to solve this indeterminacy has been to rely on the observed effects of a drug to guess some of the properties of drug USs. Unfortunately, this strategy is limited by the fact that observed drug effects can reflect primary or secondary physiological processes that do not necessarily parallel the drug US of interest (for a related discussion, see Eikelboom & Stewart, 1982). In this general context, the present failure to observe any significant effect of perturbations known to affect classical CRs on conditioned activity could indicate that due to some special features of stimulant USs, they may not be amenable to a Pavlovian associative process. This conclusion could have important implications for the understanding of related phenomena, such as behavioral sensitization and drug addiction. Clearly, more studies are needed to resolve this important issue. In conclusion, the present study shows that conditioned activity is insensitive to experimental perturbations known to affect the acquisition or expression of a Pavlovian CR. Although these data suggest that conditioned activity is not likely to be mediated by a Pavlovian associative process, such a conclusion will not be firmly established until more is known about the nature of so-called stimulant USs. 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