Proc. Natl. Acad. Sci. USA Vol. 88, pp.

2088-2092, March 1991 Neurobiology

Corticosterone levels determine individual vulnerability to amphetamine self-administration

(addiction/individual differences/hypothalamic-pituitary-adrenal axis/psychostimulants/dopamine)

PIER VINCENZO PIAZZA, STEFANIA MACCARI, JEAN-MARIE DEMINItRE, MICHEL LE MOAL, PIERRE MORMtDE, AND HERVI SIMON
Psychobiologie des Comportements Adaptatifs, Institut National de la Sante et de la Recherche MWdicale U. 259, Universitd de Bordeaux II, Domaine de Carreire, rue Camille Saint-Safns, 33077 Bordeaux Cedex, France

Communicated by Floyd E. Bloom, December 6, 1990

ABSTRACT Individual vulnerability to the reinforcing properties of drugs appears to be an essential characteristic predisposing humans to addiction. In animals, a greater behavioral reactivity to a mild stress, such as exposure to a novel environment, is an index of the vulnerability to acquire amphetamine self-administration. Biological responses to stress as well as behavioral reactivity may predict such a vulnerability. In the present study, rats with a longer duration of corticosterone secretion after exposure to novelty showed facilitation of acquisition of amphetamine self-administration. Furthermore, corticosterone administration in nonpredisposed individuals increased the reinforcing value of the drug and facilitated the acquisition of amphetamine self-administration. These results indicate that the stress-related activity of the hypothalamicpituitary-adrenal axis may play a role in the pathogenesis of psychostimulant addiction.

SA vulnerability? (ii) Could corticosterone levels modify individual sensitivity to amphetamine? To explore these questions, the secretion of corticosterone after exposure to novelty was measured in rats tested for sensitivity to amphetamine SA, and the effects of an experimentally induced increase of corticosterone levels on amphetamine-induced SA and locomotor activity were studied.

Individual differences to the reinforcing effects of the drugs (1) are considered by clinicians to be central to the etiology ofaddiction (2, 3). However, this aspect of addiction has been largely neglected in experimental studies using intravenous self-administration (SA) as a model of drug-taking behavior in animals. Nevertheless, when SA is studied during the period of acquisition and low doses of drug are used, individual differences to the drug's reinforcing effects are readily observed (4, 5). The ability to predict individual vulnerability and select populations of rats with different risks for developing drug SA may represent a valuable methodology for investigation of the biological basis of predisposition to drug-seeking behavior. Recently, it has been reported (6) that a predisposition to develop amphetamine SA in animals may be predicted by the behavioral reactivity of the individual animal to exposure to a novel environment. Thus, rats with an higher noveltyinduced locomotor activity readily acquired amphetamine SA, whereas rats with the lower response did not. These results prompted a search for the biological substrate of SA vulnerability in the activity of the hypothalamic-pituitaryadrenal (HPA) axis, such as the secretion of corticosterone during stress. This choice was based on three main observations. First, corticosteroids are central to the control of the homeostatic disturbances induced by environmental stimulations and stress (7-9). Second, this hormone modifies the activity of the central nervous system (10-13). Third, dopaminergic (DA) neurons, whose activity influences SA behavior (14-15), have corticosterone receptors on the cell bodies (16). This study addressed two specific questions. (i) Could corticosterone secretion during stress predict amphetamine
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact.

METHODS AND MATERIALS General Methods. Male Sprague-Dawley rats (Iffa-Credo, St. Germain sur l'Arbresle, France; 280-300 g body weight at the start of the experiment) were used. The animals were housed individually with ad libitum access to food and water. A constant light/dark cycle (on at 0800, off at 2000) was maintained in the animal house, and temperature (220C) and humidity were kept constant. For all experiments, d-amphetamine sulfate was dissolved in saline solution (0.9%o NaCl), which was utilized as the control injection. Corticosterone-21 hemisuccinate was dissolved in 0.9% NaCl, and the pH was adjusted to 7.4 with 6 M HCl before the infusion. SA Procedure. For the study of amphetamine SA behavior, animals were implanted with intracardiac catheters under chloral hydrate anesthesia (150 mg/kg of body weight, i.p.). The catheter (Silastic) was inserted through the external jugular vein, passed under the skin, and fixed in the mild scapular region. SA sessions were carried out during the nocturnal period (at 2100) and lasted 30 min. Before the start of each session the external end of the catheter was connected to a syringe-driven pump. The amphetamine SA cage (35 x 75 cm floor area, 50 cm high) had one hole in each of the short sides. When an animal introduced his nose ("nose poke") in one of the holes (defined as active), it switched on the infusion pump for 2 sec and injected 20 p1 of amphetamine solution. A nose poke in the other hole (defined as inactive) had no effect. During each session, the number of nose pokes in both the active and the inactive holes and the number of effective injections were recorded. The inactive device was used to provide an index of activity level. An animal is considered to perform an active SA only when the number of responses in the active device is significantly higher than that in the inactive one (95% confidence limits). Study of Corticosterone Secretion in Animals Vulnerable and Resistant to Developing Amphetamine SA. Sixteen subjects were used for this experiment. Animals were implanted with intracardiac catheters, and after 4 days of recovery, they were tested for both behavioral (locomotor response) and HPA axis reactivity to a novel environment. The novel environment was a circular corridor (170 cm long and 10 cm wide). Four photoelectric cells placed at the perpendicular
Abbreviations: HPA, hypothalamic-pituitary-adrenal axis; SA, selfadministration; HR, high responders; LR, low responders; DA, dopaminergic; ANOVA, analysis of variance.

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axis recorded locomotor activity every 10 min. Corticosterone was assayed from three blood samples (500 gl each) collected immediately before the exposure to novelty and at 30 and 120 min afterwards. Samples were collected through the catheter. Plasma corticosterone was measured by radiocompetitive binding (17). According to their level of locomotor response to novelty measured during 2 hr in the circular corridor, animals were divided into two equal groups: high responders (HR), which included all rats with an activity score above the median of the whole group, and low responders (LR), which were the remainder of the group. Two days after the novel-environment test, acquisition of amphetamine SA was studied for 10 days at two different doses. Each injection for the first 5 days delivered 10 ,ug of amphetamine and for the following S days delivered 30 .ug of the same drug. Study of the Influence of Corticosterone Administration on Amphetamine SA. In a first experiment, 15 rats were divided into two groups on the basis of their locomotor response in the novel environment. Animals were implanted with intracardiac catheters and, after 4 days of recovery, were tested for amphetamine SA. After 8 days of SA, animals were injected through the cardiac catheter with 3 mg of corticosterone-21 hemisuccinate in bovine serum albumin (Sigma) per kg of body weight (corresponding to 0.36 mg of corticosterone per kg) or saline solution (0.9% NaCl) 10 min before the start of the SA session and over 2 days according to a Latin square design. In a second experiment, 40 animals were first categorized according to their locomotor response to novelty and then were tested for acquisition of amphetamine SA for 6 days. Rats in HR and LR groups were tested either for SA of a solution containing amphetamine (10 pug/20 Al) or of a solution containing both amphetamine (10 gg/20 ;J) and corticosterone-21 hemisuccinate (Agrar) (50 ,g/20 1l). Study of the Relationships Between Corticosterone Levels and Amphetamine-Induced Locomotor Activity. For this experiment 60 rats were used. Animals were first categorized for their locomotor response to novelty and then were implanted with intracardiac catheters. After 2 weeks, animals in HR and LR groups were tested in the circular corridor for their locomotor response to an intravenous infusion either of amphetamine (0.3 mg/kg) or of saline (0.9o NaCI). Amphetamine was injected either after 30 min or after 120 min of exposure to the environment, and the locomotor response to the drug was recorded for 90 min. Saline was injected with the same schedule. Thus, the six experimental groups were HR and LR saline, HR and LR amphetamine after 30 min, and HR and LR amphetamine after 120 min. The response to amphetamine of HR and LR animals was evaluated as the percentage of the response to saline of the equivalent group at that time. Statistics. Analysis of variance (ANOVA) for repeated measures was used to compare the scores of HR and LR animals for the different variable studied. A logarithmic transformation was applied to the data of locomotor activity, corticosterone, and amphetamine SA to normalize the distribution. Pearson's test was used for the correlation studies.

Proc. Natl. Acad. Sci. USA 88 (1991)

2089

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FIG. 1. Behavioral (Upper) and hormonal (Lower) responses to novelty of rats in the HR (n = 8) and LR (n = 8) groups. The two groups differed [F(1,14) = 8.83, P < 0.011 in total locomotor activity in the novel environment. Plasma corticosterone changed in the two groups differently over the time.

RESULTS
Experiment 1: Study of Corticosterone Secretion in Animals Vulnerable and Resistant to the Development of Amphetamine SA. The LR and HR groups of animals could be distinguished not only by their locomotor response to novelty (Fig. 1 Upper) but also by their novelty-induced levels of plasma corticosterone (Fig. 1 Lower). HR rats had an enhanced secretion of corticosterone in response to the novel environment [F(1,14) = 4.93, P < 0.05]. After 120 min, corticosterone levels remained high in this group of animals (around 180 ng/ml), whereas levels in the LR rats returned to preexposure values (around 60 ng/ml). Regardless of group classifi-

cation, after 120 min of exposure to a novel environment there was a linear relation between corticosterone levels and the total locomotor response within this environment (Pearson's correlation test, r = 0.59, P = 0.03).* HR rats acquired and maintained amphetamine SA, whereas the LR animals did not (Fig. 2). Thus, HR animals showed a higher number of nose pokes in the active hole [F(1,14) = 10.15, P < 0.011, while no difference was found between the two groups in the number of nose pokes in the inactive hole. Both noveltyinduced locomotor activity and corticosterone release were positively correlated with the total amphetamine intake during SA (corticosterone/SA: r = 0.66, P < 0.01; Locomotion/ SA: r = 0.56, P < 0.05).t Experiment 2: Effects of Corticosterone Administration on Amphetamine SA. The effects of an acute pretreatment with corticosterone on amphetamine SA of HR and LR animals was examined after a period of 8 days of SA testing. During the 8 days of testing for amphetamine SA, HR animals rapidly acquired and maintained amphetamine SA, while the LR group did not [F(1,13) = 8.35, P < 0.01]. However, corticosterone infusion induced amphetamine SA in LR rats. Thus, this group of animals selectively increased the number of nose pokes in the active hole after the infusion of this hormone (Fig. 3 Left). In contrast, the HR group that had
*We found a linear relation between the two variables also in two other experiments: (i) r = 0.54, P = 0.02 (n = 16) and (ii) r = 0.53, P = 0.01 (n = 20). tA linear relation (r = 0.62, P = 0.002) between corticosterone release during exposure to the novel environment and amphetamine intake during SA was also found in a larger group of animals that contained not only naive subjects (n = 14) but also rats (n = 25) whose vulnerability to amphetamine SA was experimentally increased by repeated exposure to a stress procedure such as the pinching of the tail (18).

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Proc. Natl. Acad. Sci. USA 88 (1991)

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FIG. 2. Amphetamine SA in HR (n = 8) and LR (n = 8) animals. The two groups differed significantly in the number of nose pokes in the active (act.) hole over the 10 days. No difference was found in the number of nose pokes in the inactive (inact.) hole.

already acquired amphetamine SA decreased drug intake after corticosterone administration [ANOVA group X treatment interaction F(1,13) = 7.13, P = 0.01] (Fig. 3 Right). In contrast to nose pokes in the active hole, the number of nose pokes in the inactive hole did not change after corticosterone administration in either group. In a different experiment, we tested the effects of a daily corticosterone administration on amphetamine SA of HR and LR animals. Corticosterone was dissolved in the solution self-administered by the rats together with amphetamine. Corticosterone concentration was calculated so that, for 10 self-injections, the animals received -1.5 mg/kg. HR and LR animals responded differently to amphetamine or to amphetamine/corticosterone SA [ANOVA group x drug interaction F(1,36) = 4.94, P = 0.03] (Fig. 4). Indeed, when HR and LR rats of this experiment were tested for amphetamine SA, HR
EFFECT OF CORTICOSTERONE ON AMPHETAMINE SELF-ADMINISTRATION
50

animals acquired and maintained SA, while LR animals did not [F(1,18) = 13.24, P < 0.01] (Fig. 4 Right). In contrast, the coadministration of corticosterone and amphetamine eliminated the differences between the two groups (Fig. 4 Left). LR animals tested for amphetamine/corticosterone SA showed a longer maintenance of this behavior when compared with LR animals tested for amphetamine alone [ANOVA group x drug interaction F(5,90) = 2.4, P < 0.05]. HR animals tested for corticosterone/amphetamine SA reduced their SA rate when compared to HR rats tested for amphetamine alone [F(1,18) = 5.7, P = 0.02]. Experiment 3: Relationships Between Corticosterone Levels and AmphetamineInduced Locomotor Activity. The previous results suggest that corticosterone may increase the reinforcing effects of amphetamine. To further extend these findings, we have studied the influence of the levels of this hormone on the locomotor activity induced by amphetamine. The difference in corticosterone secretion between HR and LR animals during exposure to a novel environment may provide a clue to the role of this hormone on the intensity of an individual's locomotor response to amphetamine. Although HR animals have higher levels of corticosterone than LR rats after 120 min of exposure to novelty, there was no difference in corticosterone secretion between the two groups in the first 30 min of exposure to the circular corridor. Thus, if corticosterone levels influence the sensitivity to this drug, the difference in amphetamine-induced locomotion between the two groups should be greater when amphetamine is injected after 120 min of exposure to novelty. Indeed, after 30 min of exposure to novelty, the increase in locomotor activity induced by amphetamine was not different in the two groups (Fig. 5). However, after 120 min of exposure to the circular corridor, HR animals showed a higher increase of amphetamine-induced locomotion (Fig. 5) [ANOVA group x time of treatment interaction F(1,37) = 8.03, P < 0.01].
DISCUSSION These results show that the animals with a higher tendency to acquire amphetamine SA (HR group) have a higher novelty-induced corticosterone secretion and, as previously shown (6), a higher locomotor response to novelty. In contrast LR animals, which show a low behavioral and hormonal reactivity to the environment, will self-administer the drug

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FIG. 3. Effects of intravenous infusion of corticosterone-21 hemisuccinate (3 mg/kg) on amphetamine SA of LR (n - 8) and HR (n = 7) groups of animals. Corticosterone increased the nose pokes of LR animals in the active hole while it decreased the nose pokes of HR rats in the same hole. The number of nose pokes in the inactive hole did not change after corticosterone in either group. **, P < 0.01

(ANOVA).

FIG. 4. Amphetamine/corticosterone and amphetamine only SA in HR (n = 10 for each condition) and LR (n = 10 for each condition) rats. LR rats acquired amphetamine/corticosterone SA while progressively stopping SA of amphetamine alone. HR rats selfadministered both solutions but self-administered amphetamine/ corticosterone at a lower rate.

Neurobiology: Piazza et al.

Proc. Natl. Acad. Sci. USA 88 (1991)

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FIG. 5. Amphetamine-induced locomotor activity in HR (n = 10 for each condition) and LR (n = 10 for each condition) animals. When amphetamine was administered after 30 min of exposure to a novel environment, the increase in locomotor activity induced by the drug did not differ in the two groups. Conversely, when amphetamine was administered after 120 min of exposure to novelty, HR animals showed a higher increase of the locomotor activity induced by the drug. The activity scores of the two groups of animals are expressed as a percentage increase of the corresponding group treated with saline. **, P < 0.01 (ANOVA).

during the first days of testing but will not maintain this behavior afterwards. Thus, stress-induced corticosterone secretion can be used as a predictive index of the probability of individual animals to develop psychostimulant SA. This result is strengthened by the finding that corticosterone is positively related to the intake of amphetamine during SA in subjects whose predisposition to administer amphetamine have been experimentally increased by a repeated stress procedure (18). In this paper we showed also a direct influence of corticosterone on SA behavior. SA is facilitated in the naturally resistant animals (LR group) by raising their plasma corticosterone levels in two ways: (i) when this hormone is injected before a self-administration session, LR animals selfadminister amphetamine again after a period of 7 days of testing for SA, during which there was a progressive disappearance of SA in this group of animals; and (ii) when corticosterone is provided daily in the SA solution with amphetamine, LR animals maintain SA for all the days of testing. However, the experimental increase of corticosterone levels in the HR rats led to a decrease in amphetamine intake. This apparent opposite effect of corticosterone in the two groups of rats is similar to that phenomenon observed when the amount of drug delivered for each injection is increased: there is a faster acquisition of this behavior (5) and a parallel decrease of the number of drug injections during SA (19). Thus, it can be hypothesized that corticosterone acts on SA by increasing the reinforcing properties of amphetamine. Corticosterone levels also may influence individual responses to the psychomotor action of amphetamine. Thus, when HR and LR animals do not differ in corticosterone levels (i.e., after 30 min of exposure to novelty), amphetamine induces in the two groups the same increase in locomotor activity. Conversely, after 120 min of exposure to novelty when HR animals show higher corticosterone levels, amphetamine induces in this group of rats a greater increase

of the locomotor response. These results further suggest a facilitatory action of corticosterone on the reinforcing effect of amphetamine. Indeed, it has been hypothesized (20) that reinforcing effect of psychostimulants may be linked to their psychomotor properties. The direct action of corticosterone on SA suggests that differences in novelty-induced corticosterone secretion may explain the differences in amphetamine SA exhibited by the HR and LR rats (Figs. 2 and 4). During the first 2 days of the test, when the novelty of the situation is high, both groups self-administer amphetamine. Afterwards LR animals, which habituate faster to novelty, stop amphetamine SA, while the more reactive HR rats self-administer the drug for all of the days of testing. However, increasing corticosterone levels of LR animals also reestablished their amphetamine SA. High circulating levels of corticosterone may sensitize an animal's response to amphetamine by an action on the DA system because (i) DA transmission has been implicated in psychostimulant SA (14, 15); (ii) DA cell bodies possess corticosterone receptors (16), and corticosterone appears to stimulate DA neurons (21, 22); and (iii) amphetamine or stress-induced behavioral sensitization (23, 24), which affects the reactivity of DA cells (25) and increases the vulnerability to amphetamine SA (6, 18), has been shown to be dependent on corticosteroid receptor activation (26) and to modify the activity of the HPA axis (27). In conclusion, our results show that a behavioral response (novelty-induced locomotor activity) and a biological trait (corticosterone levels) may predict a subject's inclination to self-administer amphetamine. Moreover, deregulation of corticosterone secretion, via an action on central catecholaminergic functions, may be a factor in the enhanced susceptibility to drug-taking behavior. This is why it will be important in the future to test if the relationship between response to novelty, corticosterone secretion, and amphetamine SA can be extended to other addictive drugs such as cocaine or heroin. Finally, since both DA (28) and HPA axis deregulation (29, 30) are thought to be involved in psychopathological processes, the demonstration of a functional relationship between them has implications for our understanding of the biological bases of deviant behavior and may have clinical applications.
We thank George F. Koob for helpful comments on the manuscript. This research was supported by Institut National de la Sante et de la Recherche Mddicale (INSERM) and Universitd de Bordeaux

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