Introduction

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Since the original observation that cocaine administration resulted in activation of the hypothalamic-pituitary-adrenal (HPA) axis via a corticotropin-releasing hormone (CRH)-mediated mechanism in the rat (Rivier and Vale, 1987; Moldow and Fischman, 1987), a number of published studies have attempted to address the significance of this effect. Activation of the HPA axis by cocaine is not unique to the rat; the same has been reported in rhesus monkeys (Sarnyai et al., 1996) as well as in human subjects (Mendelson et al., 1989; Vescovi et al., 1992; Heesch et al., 1995). In humans, the rise in plasma adrenocorticotropic hormone (ACTH) subsequent to i.v. administered cocaine has been shown to mirror the time course of cocaine's distribution in the blood (Scholar et al., 1998), both of which precede the slower and more sustained cortisol response (Wilkins et al., 1992). Scholar and coworkers' results also indicate that in experienced cocaine users, the administration of cocaine rapidly triggers the release of CRH, which in turn stimulates the rise in plasma ACTH.

Manipulations that use surgical or pharmacological means to decrease HPA activity in rats also produce a notable increase in both the dose and time needed for the acquisition of cocaine self-administration (Goeders and Guerin, 1994). These manipulations also decrease ongoing self-administration behavior (Goeders and Guerin, 1996a) as well as reduce the likelihood of a resumption in drug-maintained responding after a period of abstinence or saline extinction (Piazza et al., 1994; Piazza and le Moal, 1998). In related studies, rats that were designated as "high responders" after being prescreened for their locomotor and corticosterone responses to either a novel environment or an i.p. amphetamine injection also showed an increased propensity to self-administer amphetamine (Piazza et al., 1989, 1991) or cocaine (Goeders and Guerin, 1996) at smaller doses. Collectively, the results of these studies have led to speculation that individuals whose HPA axis is highly responsive to a stressor may possess a physiological profile that renders them more vulnerable to drug abuse (Piazza and le Moal, 1996; Goeders, 1997).

Although it is generally accepted that passive cocaine administration stimulates activity of the HPA axis, there are very little published data demonstrating the effects of self-administered cocaine on HPA activity. The only exception was in a study done by Goeders and Guerin (1996b) where single samples of blood were obtained from rats before and after self-administration of different doses of cocaine. Mean plasma corticosterone levels after cocaine self-administration were reported to be significantly elevated above levels measured before cocaine exposure for all of the doses of cocaine that were tested. However, there was no indication as to whether this increase was dose-dependent in terms of either the dose of cocaine that was available, or the number of infusions that were taken.

Complex logistics are involved in studying the relationship between cocaine administration and the corresponding HPA response. Environmental factors such as handling of the animals, needle poke in the case of both i.p. cocaine administration and peripheral blood sampling, and relocation to a testing enclosure may each produce substantial HPA activation quite apart from any effect produced by the cocaine (Elvidge et al., 1976; Blank et al., 1983; Reinhardt et al., 1990; Capitanio et al., 1996). These kinds of manipulations may actually blunt the response to cocaine as a prior increase in baseline ACTH and glucocorticoids may reduce HPA reactivity to subsequent stimulation via negative feedback (Keller-Wood and Dallman, 1984; Dallman et al., 1994), as well as obscure differences between cocaine and saline controls. This is not to say that all extra-hypothalamic CRH release is inhibited in the same way; however the apparent sensitivity of the HPA system to extraneous stimuli highlights the need for use of familiar procedures that minimize disturbance to the subject.

In the present study, we initially examined the basal release of ACTH and cortisol and its circadian rhythmicity in male and female monkeys, some of whom were experienced with cocaine self-administration and some of whom had no such experience. Using these data as a reference, we evaluated the HPA response to self-administered cocaine in the morning and afternoon in male rhesus monkeys with extensive histories of cocaine self-administration. To minimize both disturbance to the monkeys as well as experimenter-induced increases in ACTH and cortisol, testing took place in their home cages and blood was sampled from behind each cage via the same indwelling venous catheter as was used for drug infusions.

	Materials and Methods

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Subjects

Seven adult male rhesus monkeys (Macaca mulatta), weighing between 9.0 and 14 kg, and three adult females, weighing between 5.0 and 7.4 kg, were used in this study. These monkeys were individually housed in stainless steel cages measuring 83.3 × 76.2 × 91.4 cm deep (Bryan Research Equipment Corporation, Bryan, TX) located in a laboratory that contained a total of 24 monkeys. The monkeys were fed 8 to 14 Purina Monkey Chow biscuits twice daily to maintain adult body weight and water was available ad libitum. The laboratory was illuminated from 7:00 AM to 10:30 PM. Each monkey had an indwelling venous catheter in a femoral, internal, or external jugular vein. Catheters were inserted during aseptic surgery under ketamine (10 mg/kg) and xylazine (2 mg/kg) anesthesia. After placement in the vein, the catheter was guided s.c. to the mid-scapular region where it exited the monkey. The external portion of the catheter was protected inside the cage by a flexible stainless steel arm, with one end of the arm attached to the double-layer polyester jacket (Lomir, New York, NY) worn by the monkey and the other bolted to the rear of the cage.

Circadian Rhythm Study. Six male and three female monkeys were subjects for this study. All monkeys had been acclimated to the jacket and flexible arm apparatus over a period of months or years. Five monkeys had a history of cocaine self-administration, two monkeys had received cocaine passively on one or more occasions, and two monkeys were cocaine-naive. Ketamine, used for sedation, was the only psychoactive drug noted in the histories of the latter two monkeys. For those monkeys conditioned to cocaine self-administration, testing was suspended in this phase of the study. Blood (1.4 ml) was sampled at 3-h intervals over a 24-h period from the rear of the cage via the venous catheter. Each monkey was exposed to this sampling procedure once only. Sample collection and handling is described below.

Cocaine Self-Administration Study. Seven adult male monkeys (five of whom were in the circadian study) were subjects for this study. Four of the seven monkeys had 1 or more years experience with the self-administration of different doses of cocaine under the fixed-ratio (FR) 30 time-out (TO) 10 min. The remaining three subjects were more recently trained and had 1- to 6-months' experience with cocaine self-administration under the FR 30 TO 10 min schedule. Each monkey had blood samples taken between one and twelve times (four on average) at each dose of cocaine or saline.

Apparatus

Each cage had a 15- × 20-cm panel fixed to the right wall of the cage. Each panel had three stimulus lights, two red and one central green light, placed above two response levers. The red stimulus light over the right lever signaled drug availability. Drug delivery was contingent on the monkey emitting the required response (30 lever presses). The green center light was illuminated for the duration of the drug infusion, 1 ml over 5 s. During each 10-min time-out, all stimulus lights were extinguished and responding had no programmed consequences.

The experiment was controlled by IBM/PS2 computers located in an adjacent room. The computers were programmed using Med Associates software (Georgia, VT).

Procedure

Circadian Rhythm Study. Blood sampling for the study of basal release of ACTH and cortisol took place on two separate occasions. Five monkeys were tested on the first occasion and four on the second. Blood (1.4 ml) was sampled at 3-h intervals, beginning at 9:00 AM and ending with the 9:00 AM sample the following day. Self-administration testing for the monkeys in this study was suspended for the duration of the basal sampling period, although the other monkeys in the room were tested as usual. The blood sample handling procedure is described below.

Self-Administration Study. Drug self-administration sessions were scheduled twice daily for 130 min starting at approximately 10:00 AM and 4:00 PM. There was a maximum of 13 infusions available in each session. The effect of cocaine self-administration on ACTH and cortisol was measured primarily before, during, and after the morning session, although on a few occasions the HPA response to cocaine was measured during the evening session for comparison.

Data Analysis

Circadian Rhythm Study. The plasma cortisol and ACTH levels over the 24-h sampling period were averaged for each sampling time according to gender and then analyzed for gender differences. In the absence of a sex difference, the data for male and female monkeys were averaged and analyzed as described below.

Self-Administration Study. Rate of responding during each session was defined as the number of responses emitted while the red stimulus light was on divided by the number of seconds that the light was illuminated (responses/s). The total dose of cocaine (mg/kg) for each monkey was the number of infusions taken during the session multiplied by the dose per infusion (mg/kg/injection). Plasma cortisol (µg/dl) and ACTH (pg/ml) levels are shown in raw form as well as after normalization to area under curve (AUC) data. AUC values provide an estimate of the cortisol or ACTH release relative to basal levels during the self-administration session. AUC values were calculated according to the trapezoidal rule (e.g., Tallarida and Murray, 1987). The presession plasma level before each experiment was used as the reference for the calculation of the AUC for cortisol (µg · min/dl) or ACTH (pg · min/ml). The AUC was calculated from the six samples taken before, during, and 15 min after the session.

Statistics

All data are presented as mean ± S.E.M. One or two-way ANOVA and post hoc pairwise comparisons using the Tukey HSD test of significance (p < .05) were carried out using Statistica (v.5.0; Statsoft, Tulsa, OK). When experiments were replicated within subjects, the mean response for each subject was used in calculating treatment effects across subjects.

	Results

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Circadian Rhythm. Fig. 1 shows the plasma levels of ACTH and cortisol measured in blood samples taken at 3-h intervals over a 24-h period. Basal plasma cortisol and ACTH levels in both male and female rhesus monkeys demonstrated a circadian pattern of release during the sampling period (Fig. 1A). Both cortisol and ACTH levels differed across the 24-h sampling period (ACTH: df = 8, F = 3.75, p < .001; cortisol: df = 8, F = 4.87, p < .0001). The details from post hoc pairwise comparisons are listed in the legend to Fig. 1. Peak levels for plasma cortisol (19.60 ± 2.16 µg/dl) and ACTH (19.63 ± 2.56 pg/ml) occurred at 6:00 AM. The nadir for plasma ACTH (6.27 ± 0.62 pg/ml) occurred at 6:00 PM, preceding the cortisol nadir (5.55 ± 1.21 µg/dl), which occurred at 9:00 PM. Although the female monkeys in this study appeared to have higher cortisol and ACTH peaks than male rhesus monkeys (Fig. 1, B and C), this difference was not statistically significant. A comparison between monkeys with a cocaine self-administration history (n = 5; all male) versus those with no such history (n = 4; 1 male, 3 female) showed a near-significant difference in basal cortisol levels, with the monkeys lacking self-administration history tending to have higher levels (df = 1, F = 5.00, p < .06), but there was no difference in basal ACTH levels between the two groups (data not shown). Six of the nine monkeys showed an additional cortisol peak at 12:00 PM, whereas eight of the nine monkeys showed an increase in ACTH at this time.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Basal levels of plasma cortisol (µg/dl) and ACTH (pg/ml) exhibiting circadian cyclicity. A, plasma was sampled from unrestrained monkeys (six males, three females) at 3-h intervals from the rear of the cage via indwelling venous catheter. Comparison of basal release of cortisol (µg/dl; B) and ACTH (pg/ml; C) in male and female subjects; there were no significant gender differences. a, cortisol lower than at 09:00 and 00:00 (p < .05), and 00:00 h (p < .005). b, cortisol higher than at 18:00, 09:00, and 00:00 (p < .005). c, ACTH lower than at 18:00 (p < .005) and 00:00 PM (p < .05). d, ACTH higher than at 18:00 and 09:00 (p < .01).

Cocaine Self-Administration. The response rate and cocaine intake from the cocaine self-administration study are shown in Fig. 2. The response rate for the session was lowest when saline was available for self-administration (0.3 ± 0.1 responses/s) and increased as a function of the available dose of cocaine (0.01-0.1 mg/kg/injection). The ascending arm of the dose-response curve was generated, with the peak rate of responding (3.5 ± 0.5 responses/s) maintained by 0.1 mg/kg/injection cocaine. There was a slight, nonsignificant rate decrease at 0.3 mg/kg/injection cocaine (2.6 ± 0.4 responses/s), the largest dose that was tested. Total drug intake (mg/kg) was positively related to the dose of cocaine that was available for self administration. One-way ANOVA was used to examine the effects of cocaine dose on both rate of responding and total drug intake (rate: df = 4, F = 20.52, p < .001; intake: df = 4, F = 5177, p < .001), indicating that increasing the dose of cocaine led to significantly increased rates of responding and cocaine intake. The results of subsequent pairwise comparisons are shown in the legend to Fig. 2.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Comparison of response rate (responses/s) versus total cocaine intake (mg/kg) when saline or cocaine (mg/kg/injection) was available for self-administration (n = 7). Cocaine (or saline) availability was signaled by a red stimulus light that remained illuminated until the requisite number of lever presses had been emitted (FR 30, TO 10 min). Drug or saline delivery was signaled by a green stimulus light. Each session lasted 130 min and a maximum of 13 infusions was possible. The response rates for 0.01 mg/kg/injection cocaine and saline were not significantly different. Cocaine intake for each dose differed across all doses tested (p < .05). a, response rate is lower than for 0.03, 0.1, and 0.3 mg/kg/injection cocaine (saline: p < .001; 0.01 mg/kg/injection cocaine: p < .01).

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Mean plasma cortisol levels (µg/dl, A) and ACTH levels (pg/ml, B) determined from blood sampled before, during, and after saline or cocaine (mg/kg/injection) self-administration (n = 7). Procedural details as for Fig. 2. a, greater than for saline, p < .05. b, greater than for saline and 0.01 mg/kg/injection cocaine, p < .05. c, greater than for saline, 0.01, and 0.03 mg/kg/injection cocaine, p < .05. d, greater than for saline and 0.03 mg/kg/injection cocaine, p < .05.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Effect of self-administered cocaine (0.3 mg/kg/injection) on plasma cortisol (µg/dl) and ACTH (pg/ml) during morning or afternoon self-administration sessions in five subjects. The HPA response to cocaine was more robust in some monkeys during the "AM SESSION", when basal cortisol and ACTH levels were at a peak, than during the "PM SESSION" when basal HPA activity was approaching its nadir (see Fig. 1). Rates of responding as well as cocaine intake during the morning and afternoon sessions were comparable for each monkey. Other details as for Fig. 2.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Cumulative release (AUC) of plasma cortisol (ug · min/dl) versus ACTH (pg · min/ml) above basal (presession) levels during saline or cocaine (mg/kg/injection) self-administration (n = 6). One monkey was not included as there was no within-session sampling done. Details as for Fig. 2. a, greater than the cortisol AUC generated by saline self-administration (p < .005). b, greater than the cortisol AUC for saline, 0.01, and 0.03 mg/kg/injection cocaine (p < .005). c, greater than the ACTH AUC for saline (p < .05).

	Discussion

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The results of the circadian study presented here demonstrate a diurnal rhythmicity in the release of ACTH and cortisol in both male and female rhesus monkeys. This basal release constituted a stable baseline from which the HPA axis response to self-administered cocaine could be measured. Males and females were no different with respect to the timing of the peak, nadir, or magnitude of the plasma ACTH and cortisol levels. Four of the nine monkeys tested had no self-administration history. These four tended to have higher cortisol levels; however, ACTH levels for both groups were the same. This difference may reflect either drug-taking history or the novelty of the experimental procedure, as three of these monkeys had undergone their first catheter placement surgeries within 2 weeks of the circadian study. Comparison of the male and female data is complicated by the fact that all of the female monkeys in our study also belonged to the group that lacked self-administration history.

Our circadian data showed a second, smaller peak at 12:00 PM for the majority of subjects. Additional peaks in ACTH and cortisol have been reported in humans (Slag et al., 1981; Goldman et al., 1985) and were attributed to meal ingestion, particularly high protein lunches. The unexpected rise in ACTH and cortisol seen in our data is unlikely to be due to food ingestion as the monkeys were fed at 8:30 AM and 2:30 PM daily. The only other activity likely to have disrupted basal release of cortisol and ACTH was the 2-h 10-min self-administration session scheduled daily from 10:00 AM until 12:10 PM. Although the monkeys in this study had their self-administration sessions suspended during the sampling period, the remaining monkeys in the room were tested as usual. A recent study by Caggiula and coworkers (1998) reported that rats showed a conditioned elevation of plasma corticosterone in response to environmental cues associated with nicotine delivery. The fact that a self-administration session did take place during the sampling time makes the possibility of a conditioned HPA axis response the most likely explanation. The fact that four of the nine monkeys had no self-administration history suggests that this increase may have resulted from a more general social effect.

A few earlier studies have shown evidence for circadian release of plasma ACTH (Kalin, 1986) and cortisol (Quabbe et al., 1982) in male rhesus monkeys, and cortisol in female rhesus monkeys (Leshner et al., 1978). In these earlier studies, all subjects but one were chronically chair-restrained. It is interesting to note that in the one paper where a caged, freely moving monkey was compared with the chronically chair-restrained monkeys (Quabbe et al., 1982), the freely moving monkey had cortisol levels that were similar to his restrained counterparts, as well as to our monkeys. Although we did not investigate whether hormonal fluctuations in the female monkeys affected cortisol and ACTH release, Leshner and coworkers (1978) sampled throughout the menstrual cycle and found no consistent variations in cortisol levels. Our study was not designed to measure the secretory pulses that accompany the circadian fluctuations, as this has already been studied by Sarnyai and coworkers (1995), who examined the ultradian release of both cortisol and ACTH at 2-min intervals and concluded that the pulse frequency, interpulse interval, and pulse amplitude in male rhesus monkeys was very similar to what has been measured in humans.

Cocaine served as a reinforcer in this study. Both rate of responding and total cocaine intake increased as a function of cocaine dose (Fig. 2, Table 1), similar to what has been described previously (e.g., Winger, 1993). As the cocaine dose available for self-administration was increased, there was a dose-dependent increase in ACTH and cortisol release, although only at the highest doses of cocaine (0.1 and 0.3 mg/kg/injection) were these increases in ACTH and cortisol statistically greater than were measured for saline. When blood was sampled during the afternoon self-administration session, a diminished HPA response to cocaine was noted in some monkeys, perhaps indicating that diurnal fluctuations in the release of ACTH and cortisol were associated with alterations in the sensitivity of the HPA axis to cocaine administration. It is important to note that under these experimental conditions, a dose of cocaine (0.03 mg/kg/injection) that was sufficient to maintain behavior above saline levels did not produce a significant elevation in HPA activity above levels produced by saline self-administration. In fact, under these schedule conditions there was no difference in the rates of responding maintained by 0.03, 0.1, and 0.3 mg/kg/injection cocaine, although the intake of cocaine differed markedly, as did the ACTH and cortisol response. This is evidence for a dissociation between doses of cocaine that clearly maintained responding versus doses that clearly elevated HPA activity. So although HPA activation by cocaine appeared dose-dependent, a dose of cocaine that maintains self-administration behavior does not necessarily produce significant HPA activation. This point is discussed further below.

It is difficult to ascertain from other published work in this field whether a dose of cocaine will produce the same HPA response when it is injected by the investigator or self-administered by the subject. When cocaine was injected i.v. into rats, it produced a dose-dependent increase in plasma ACTH (Rivier and Vale, 1987) and corticosterone (Moldow and Fischman, 1987). The one paper that directly addressed cocaine self-administration and activation of the HPA axis in the rat did not provide an estimate of cocaine intake (Goeders and Guerin, 1996b). This prevents a comparison of the results of these two studies. The only work of this kind in rhesus monkeys examined the plasma ACTH and cortisol response to a single infusion of 0.4 and 0.8 mg/kg cocaine in males (Sarnyai et al., 1996) and females (Sarnyai et al., 1995). Although female monkeys showed no change in HPA function in response to cocaine, increases in ACTH and cortisol levels in male monkeys were proportional to the cocaine-induced level of behavioral activation. It is noteworthy that the preinfusion cortisol and ACTH levels in most of the monkeys in the study by Sarnyai et al. (1996) are far greater (cortisol is 2-fold higher and ACTH is 4-fold higher) than in the monkeys in the present study, perhaps reflecting procedural differences between the studies. A consequence of these high baseline values may have been a blunting of cocaine's effect on HPA activity. Sarnyai gave 0.4 and 0.8 mg/kg i.v. in a single infusion, whereas in the present study, monkeys self-administered cocaine incrementally with the total dose (0.13, 0.39, 1.3, and 3.9 mg/kg) being injected over the course of a 2-h session. Sarnyai found that the subgroup of monkeys that were responsive to cocaine had increases in cortisol and ACTH release after the 0.8 mg/kg but not after the 0.4 mg/kg dose. In the present study, the dose of cocaine that resulted in a cumulative intake of 0.39 mg/kg (0.03 mg/kg/injection) did not lead to a significant increase in ACTH and cortisol release, despite the fact that this dose of cocaine maintained high rates of responding. The earliest indication in the session that cocaine was increasing HPA axis activity beyond saline levels was after the 4th infusion of 0.3 mg/kg/injection (total dose = 1.2 mg/kg over 30 min), and after the 8th infusion of 0.1 mg/kg/injection (total dose = 0.8 mg/kg over 70 min). Thus it is possible that the dose of i.v. cocaine that marks the threshold for activation of the HPA axis lies between 0.4 and 0.8 mg/kg. If this were the case, it could be anticipated that doses of cocaine that, either singly or cumulatively, do not reach this range, would not activate the HPA axis. This casts some doubt on any suggestion that glucocorticoid release may be integral to cocaine's reinforcing effects.

The results presented here are in agreement with previous reports that cocaine administration is correlated with increased HPA activity. However, in contrast to previous studies, the data presented here were collected from awake, minimally disturbed monkeys, behaving in their home cages, and with control over cocaine delivery. Under these conditions, male and female monkeys exhibited similar, normal diurnal variation in basal ACTH and cortisol release. In male monkeys with an extensive cocaine self-administration history, plasma ACTH and cortisol levels increased as a function of cocaine intake, indicating that self-administered cocaine dose-dependently increased HPA activity. Nevertheless, there was evidence that small, but nevertheless reinforcing, doses of cocaine failed to produce significant activation of the HPA axis. Thus, the procedure used in this study for examining cocaine's effect on HPA function demonstrated a low, stable cortisol and ACTH baseline upon which a clear effect of cocaine was measured.