Conditioned Withdrawal Drives Heroin Consumption and Decreases Reward Sensitivity

Introduction

Drug-seeking behavior has long been linked to craving from the perspective of conditioned positive reinforcement, where previously neutral stimuli linked to the positive reinforcing or rewarding effects of addictive drugs acquire motivational significance (Stewart et al., 1984; Shaham et al., 1994). However, as addiction develops, negative reinforcement mechanisms have been hypothesized to emerge that drive the compulsivity of drug addiction (Koob and Le Moal, 2005). Conditioned withdrawal, in which previously neutral stimuli repeatedly paired with drug withdrawal alone precipitate a withdrawal-like state, has been observed in both animal (Baldwin and Koob, 1993; Schulteis et al., 2000; Kenny and Markou, 2005) and human laboratory situations (O'Brien et al., 1977). Essentially unexplored, however, is the hypothesis that stimuli paired with negative reinforcement can acquire motivating properties for drug seeking.

Withdrawal from opiates and other addictive drugs decreases the sensitivity of brain reward systems, measured as elevated intracranial self-stimulation (ICSS) thresholds in rats, and can occur even when physical signs of withdrawal are not manifested (Epping-Jordan et al., 1998; Kenny and Markou, 2005). Decreases in reward sensitivity revealed during withdrawal are hypothesized to reflect compensatory (homeostatic) adaptations in brain reward circuitries that arise to counteract prolonged overstimulation of these systems by drugs of abuse (Solomon and Corbit, 1974; Koob and Le Moal, 2005). Recently, we found that reward deficits observed during withdrawal may be conditioned to environmental stimuli through classical (pavlovian) conditioning processes (Kenny and Markou, 2005). It was hypothesized that such cue-induced reward deficits may serve as a powerful source of craving, motivating drug intake in human opiate addicts (Kenny and Markou, 2005). However, although anecdotal evidence is compelling (Wikler, 1973; Khantzian, 1985; Bradley et al., 1989; Unnithan et al., 1992), little empirical evidence exists supporting a role for withdrawal or conditioned withdrawal in motivating drug intake in humans or animals (Shaham et al., 2003). Indeed, the role of withdrawal in perpetuating the drug habit has remained a fundamental, yet unresolved, question in drug addiction research (Wikler, 1973; Shaham and Stewart, 1995; Hutcheson et al., 2001; Ahmed et al., 2002; Robinson and Berridge, 2003).

Here, we explored the role of conditioned withdrawal in motivating heroin intake in rats. First, we investigated whether the quantities of heroin consumed by rats during unlimited (23 h) daily self-administration sessions were sufficient to induce withdrawal-like deficits in brain reward systems. Recently, we have shown that rats with this schedule of unlimited daily access to heroin demonstrated behavioral alterations, including physical signs of opiate withdrawal and motivational signs of withdrawal such as reduced food intake, reminiscent of human heroin addicts (Chen et al., 2006). Next, we tested the hypothesis that cues paired with precipitated opiate withdrawal would alter heroin intake. Finally, we examined the state of brain reward systems, reflected in alterations of ICSS thresholds, during cue-induced motivational states.

Materials and Methods

Results

Experiment 1: development of heroin dependence and brain reward function

Heroin intake remained stable across self-administration sessions in 1 h (nondependent) rats (F_(4,18) = 1.1; not significant (NS)] (Fig. 1a). Similarly, baseline reward thresholds in 1 h rats, assessed before each self-administration session, remained stable and unaltered across days (F_(4,18) = 1.0; NS) (Fig. 1c). However, post-heroin reward thresholds were significantly lowered compared with daily preheroin thresholds in 1 h rats (F_(4,18) = 2.1; p < 0.01) (Fig. 1c). Reward thresholds remained stable and unaltered for the duration of the experiment in control rats (F_(4,23) = 0.5; NS) (Fig. 1b). In contrast, thresholds became gradually elevated across sessions in 23 h (dependent) rats (F_(4,24) = 1.6; p < 0.05) (Fig. 1d). Similarly, heroin intake in 23 h rats also progressively escalated across self-administration sessions (F_(4,23) = 11.9; p < 0.001) (Fig. 1a).

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Figure 1.

Unlimited daily access to heroin escalated heroin intake and decreased the excitability of brain reward systems. a, Heroin intake (±SEM; 20 μg per infusion) in rats during limited (1 h) or unlimited (23 h) self-administration sessions. ∗∗∗p < 0.001, main effect of access (1 or 23 h), two-way repeated measures ANOVA. b, Percentage change from baseline ICSS thresholds (±SEM) in control rats, which remained heroin naive for the duration of the experiment and had ICSS thresholds assessed at the same time points as the 23 h rats. c, Percentage change from baseline ICSS thresholds (±SEM) in 1 h rats. Daily post-thresholds (±SEM), assessed immediately after each heroin self-administration session, were lowered compared with prethresholds (±SEM), assessed immediately before each self-administration session in 1 h rats. ∗p < 0.05, main effect of heroin on reward thresholds, two-way repeated measures ANOVA. d, Percentage change from baseline ICSS thresholds (±SEM) in 23 h rats. Reward thresholds, assessed immediately after each daily 23 h self-administration session, became progressively more elevated as exposure to self-administered heroin increased across sessions. ∗p < 0.05, main effect of heroin on reward thresholds, two-way repeated measures ANOVA.

Experiment 2: conditioned withdrawal and heroin intake

As seen in Figure 2a, naloxone increased heroin intake in 1 h rats on each of the four conditioning days (F_(3,4) = 4.1; p < 0.05). Importantly, cue presentation with vehicle (saline) did not alter drug intake on the test day in 1 h rats (Fig. 2a), indicating that this action of naloxone was not susceptible to classical conditioning processes. In 23 h rats, naloxone increased heroin intake during the four conditioning days (F_(4,44) = 24.7; p < 0.001) (Fig. 2b). The magnitude by which naloxone increased heroin intake was greater in 23 h rats than in 1 h rats (F_(1,14) = 8.5; p < 0.05, main effect of access, two-way ANOVA). Most importantly, cue presentation with vehicle significantly increased heroin intake in 23 h rats on the test day compared with heroin intake on the preconditioning day (p < 0.05, paired t test) (Fig. 2b). Importantly, a cue-induced increase in heroin intake was not observed in the unpaired 23 h rats that received saline injections instead of naloxone during the conditioning sessions (Fig. 2c). Finally, cue presentation with vehicle significantly increased the percentage of baseline heroin intake in paired 23 h rats on the test day compared with percentage of baseline heroin intake in unpaired 23 h rats (p < 0.05, t test) (Fig. 2d).

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Figure 2.

Conditioned heroin withdrawal motivated heroin intake. a, Heroin intake in 1 h rats for 30 min after saline injection on the preconditioning day (saline), after naloxone (30 μg/kg) injection and cue presentation during the four conditioning sessions (naloxone + cues), and on the test day (saline + cues). Naloxone injection and cue presentation significantly increased heroin intake in 1 h rats. ∗p < 0.05, main effect of naloxone/cues compared with intake observed after saline injection. However, cue presentation and vehicle (saline) administration on the test day did not alter heroin intake in 1 h rats. b, Heroin intake in unpaired 23 h rats injected with saline instead of naloxone during conditioning sessions. Cue presentation and vehicle did not alter heroin intake in unpaired 23 h rats during the conditioning sessions, or on the test day. c, Heroin intake in paired 23 h rats. Naloxone injection and cue presentation significantly increased heroin intake in 23 h rats during the naloxone/cue pairings. ∗∗∗p < 0.001, main effect of naloxone/cues compared with intake observed after saline injection. Importantly, cue presentation and vehicle on the test day significantly increased heroin intake in paired 23 h rats. ∗p < 0.05, paired t test compared with intake on the saline day. d, Percentage change from baseline heroin intake on the test day was significantly increased in paired 23 h rats compared with unpaired 23 h rats. ∗p < 0.05, t test compared with percentage change in intake on the saline day. Data are mean ± SEM.

Experiment 3: conditioned withdrawal and brain reward function

In control rats, postinjection reward thresholds remained stable and unaltered compared with baseline thresholds on the preconditioning day, during the 4 d of cue-injection pairings and on the test day (Fig. 3a). In 1 h rats, saline injection had no effects on heroin-induced lowering of reward thresholds on the preconditioning day. However, during the four conditioning sessions, naloxone reversed heroin-induced lowering of reward thresholds (F_(4,4) = 4.4; p < 0.01) but did not further elevate thresholds above baseline (before heroin) levels (Fig. 3b). On the test day, cue presentation with vehicle did not alter heroin-induced lowering of reward thresholds in 1 h rats. In 23 h rats, postsaline thresholds were unchanged compared with their already elevated baseline levels on the preconditioning day (Fig. 3c). During the conditioning sessions, naloxone elevated postinjection reward thresholds in 23 h rats (i.e., precipitated withdrawal) above their elevated levels (F_(4,4) = 3.6; p < 0.05) (Fig. 3c). Interestingly, there was a nonsignificant trend for the magnitude by which naloxone elevated reward thresholds to progressively increase over the four conditioning days (Fig. 3c). Most importantly, on the test day withdrawal-associated cues and vehicle significantly elevated reward thresholds in 23 h rats (p < 0.05) (Fig. 3c).

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Figure 3.

Conditioned heroin withdrawal decreased the excitability of brain reward systems. a, Percentage change from baseline ICSS thresholds (±SEM) in control rats after saline injection on the preconditioning day (saline), during the four cue/injection pairings (naloxone + cues), and on the test day (saline + cues). b, Percentage change from baseline ICSS thresholds (±SEM) in 1 h rats. Saline did not alter the lowering of reward thresholds observed after heroin intake during 1 h self-administration sessions. However, naloxone (30 μg/kg) administration and cue presentation reversed heroin-induced lowering of reward thresholds. ∗p < 0.05, main effect of naloxone/cues compared with thresholds observed after saline injection, one-way repeated measures ANOVA. On the test day, cue presentation and vehicle did not alter the lowering of reward thresholds observed after heroin intake during 1 h self-administration sessions. c, Percentage change from baseline ICSS thresholds (±SEM) in 23 h rats. Saline did not alter the already elevated baseline reward thresholds in 23 h rats. However, naloxone (30 μg/kg) administration and cue presentation raised reward thresholds above their already elevated baseline levels in 23 h rats. ∗p < 0.05, main effect of naloxone/cues compared with thresholds observed after saline injection, one-way repeated measures ANOVA. On the test day, cue presentation and vehicle significantly elevated reward thresholds in 23 h rats. ∗p < 0.05, paired t test compared with thresholds on the saline day.

Discussion

Development of compulsive opiate intake may depend not only on the positive reinforcing and hedonic actions of opiates, but also on avoidance and escape from the aversive consequences of opiate withdrawal (Ahmed et al., 2002; Kenny et al., 2003b; Koob and Le Moal, 2005). However, the motivational significance of opiate withdrawal in general, and in driving opiate consumption in particular, has remained a controversial issue for many years. Indeed, it has been argued that withdrawal powerfully motivates drug intake (Wikler and Pescor, 1967; Koob and Le Moal, 1997, 2001; Kenny and Markou, 2005), perhaps by increasing the appetitive value of drugs (Hutcheson et al., 2001), whereas others have argued that withdrawal plays no role whatsoever in modulating drug intake (Stewart and Wise, 1992; Robinson and Berridge, 2003). These contrasting views likely arise from the fact that, in most previous studies investigating the role of withdrawal in motivating drug intake, animals were made opiate dependent through nonvolitional (experimenter-administered) delivery of opiates, or had limited histories of opiate self-administration that may not have resulted in dependence (Stewart and Wise, 1992). In contrast, rats in the present study became opiate dependent through excessive volitional opiate consumption during unlimited (23 h) daily self-administration sessions, with potentially many opportunities to associate the effects of volitional heroin intake on the activity in brain reward systems. Furthermore, in the present study, withdrawal was precipitated with a relatively low naloxone dose (0.03 mg/kg) unlikely to otherwise disrupt operant performance. This contrasts with previous studies in which withdrawal has been precipitated with relatively high doses of opioid receptor antagonists (e.g., Stewart and Wise, 1992). With low naloxone doses, it was anticipated that rats could more readily overcome precipitated withdrawal through further drug consumption and therefore better associate drug intake with alleviation of withdrawal (Carrera et al., 1999; Hutcheson et al., 2001).

In 1 h rats, baseline ICSS thresholds assessed before each daily self-administration session remained stable and unaltered across days, whereas heroin self-administration significantly lowered ICSS thresholds. Importantly, in a very recent study we demonstrated that ICSS thresholds remained stable and unaltered in control rats without access to heroin or any drug of abuse when thresholds were assessed at the same time-points as the 1 h rats in the present study (Kenny and Markou, 2006). Indeed, a major advantage of the ICSS procedure is that rats demonstrate little satiation to ICSS during extended testing sessions (Olds, 1958; Annau et al., 1974), and ICSS thresholds can be assessed multiple times each day without significant alterations in thresholds across daily sessions (Markou and Koob, 1991; Kenny et al., 2003a). Furthermore, similar lowering of ICSS thresholds has been observed across a broad range of experimenter-administered heroin doses (Hubner and Kornetsky, 1992). Thus, the daily lowering of reward thresholds observed in 1 h rats most likely reflects transiently increased reward sensitivity through heroin-induced amplification of reward signals in the brain. Importantly, this limited daily access to heroin did not induce persistent alterations in the baseline sensitivity of brain reward systems. In 23 h rats, excessive heroin consumption induced a progressive elevation in daily ICSS thresholds. Thus, excessive heroin consumption in 23 h rats decreased baseline sensitivity of brain reward systems, and this evolving reward deficit motivated an escalating pattern of heroin intake, further decreasing reward sensitivity. Consistent with this hypothesis, we have shown previously that extended (6 h) daily access to cocaine self-administration also persistently decreased brain reward function and propelled an escalation of cocaine intake in rats (Ahmed et al., 2002). Overall, the present data demonstrate that the quantities of heroin consumed by rats with unlimited daily access to the drug induced profound decreases in the excitability of reward systems, likely a crucial factor that drives escalation of heroin intake and the transition to heroin dependence. Furthermore, these data suggest that as opiate dependence develops, a new source of motivation emerges in which heroin is consumed not only for its acute reward-facilitating effects, but also to counter the persistent deficits in the sensitivity of reward systems. Hence, the emergence of a negative reinforcement dimension to heroin self-administration, in conjunction with the positive reinforcing effects of heroin, likely contributes to the compulsive drug intake so characteristic of human addicts.

In 1 h rats, naloxone significantly increased heroin intake and reversed the stimulatory effects of heroin on brain reward systems, but did not reveal any long-lasting adaptations in reward pathways (i.e., did not precipitate withdrawal). Interestingly, naloxone-induced increases in heroin intake and reversal of heroin-induced lowering of reward thresholds were not susceptible to classical conditioning in 1 h rats. Based on these observations, it is likely that naloxone-induced increases in heroin self-administration in 1 h rats represents a compensatory response to overcome reversal of heroin-induced stimulation of brain reward systems. Indeed, similar compensatory increases in drug intake were observed in cocaine or amphetamine self-administering rats treated with doses of dopamine receptor antagonists that attenuated the reward-facilitating effects of these drugs (Yokel and Wise, 1975; Ettenberg et al., 1982). In 23 h rats, naloxone increased heroin intake, and the magnitude by which intake was increase was far greater than that observed in 1 h rats. In addition, naloxone also elevated postinjection reward thresholds in 23 h rats (i.e., precipitated withdrawal) above their already elevated levels. Interestingly, there was a nonsignificant trend for the magnitude by which naloxone elevated reward thresholds to progressively increase over the four conditioning days. This observation likely reflects a progressive increase in the magnitude of the conditioned withdrawal effect with repeated cue-withdrawal pairings (Schulteis et al., 2004; Kenny and Markou, 2005). Most importantly, on the test day, withdrawal-associated cues elevated reward thresholds and provoked significant increases in heroin consumption in 23 h rats. Recently, we demonstrated that in unpaired opiate-dependent rats, cue presentation and saline administration had no effects on ICSS thresholds during conditioning sessions or on the test day (Kenny and Markou, 2005). Thus, the cue-induced elevations of ICSS thresholds observed in paired 23 h rats in the present study were most likely related to cue-withdrawal pairings and were not secondary to intrinsic actions of conditioning stimuli on ICSS thresholds in opiate-dependent rats per se. Thus, naloxone-paired conditioned stimuli attain negative affective valence in heroin-dependent 23 h rats and thereby induce a decrease in the sensitivity of brain reward systems mimicking that observed during unconditioned withdrawal. Moreover, conditioned reward deficits served as a source of motivation that triggered heroin consumption in 23 h rats. These data suggest that naloxone induced two distinct sources of motivation to consume heroin: in nondependent 1 h rats, heroin intake was increased to counter the antagonism of the pharmacological effects of heroin by naloxone. In dependent 23 h rats, heroin consumption may have been triggered to alleviate withdrawal-associated reward deficits. Importantly, only this second source of motivation was susceptible to classical conditioning. Together, these findings provide compelling evidence that conditioned stimuli predicting the onset of withdrawal-associated reward deficits motivate drug intake. These data are also consistent with previous observations demonstrating that withdrawal-associated cues produced an aversive conditioned withdrawal state in opiate-dependent rats (Wikler and Pescor, 1967; Kenny and Markou, 2005), nonhuman primates (Goldberg et al., 1969), and human opiate addicts (O'Brien et al., 1977), and with anecdotal reports that addicts experience withdrawal symptoms and drug craving when encountering drug-related environmental stimuli (Wikler, 1973).

The demonstration that previously neutral stimuli, when repeatedly paired with opiate withdrawal, can produce drug seeking in dependent but not nondependent rats, and that the state elicited by these conditioned stimuli is a negative emotional state as reflected in decreased reward function, is relevant to our current views on the etiology of addiction. Addiction has long been hypothesized to be a neuroadaptive process. This neuroadaptation was originally thought to be reflected in the development of physical drug dependence (Himmelsbach, 1943). However, this position fell out of favor with the discovery that classes of drugs such as psychomotor stimulants could serve as positive reinforcers without development of physical dependence, culminating in the subsequent focus on the role of positive reinforcing effects of drugs of abuse (Wise and Bozarth, 1987) and the sensitization of the neural mechanisms guiding positive reinforcement (Robinson and Berridge, 1993) in addiction. However, accumulating evidence suggests that there is indeed a “dark side” of addiction, in which increases in drug self-administration are motivated by negative emotional states (Koob and Le Moal, 2005). This view is supported by data demonstrating that all drugs of abuse, including the psychomotor stimulants, elicit deficits in brain reward sensitivity during withdrawal distinct from physical withdrawal signs (Markou and Koob, 1991; Schulteis et al., 1995; Epping-Jordan et al., 1998; Ahmed et al., 2002; Kenny et al., 2003b). The present study extends this conceptual framework by showing that such negative emotional states can impart motivational power for drug seeking to previously neutral stimuli, and supports the hypothesis that craving for drugs is elicited not only by the memory of the pleasurable effects of drugs (de Wit and Stewart, 1981; Arroyo et al., 1998; Shaham et al., 2003), but also by the memory of the aversive effects of drug abstinence (Wikler, 1973).