It was suggested in 1986 that cue-induced drug craving in cocaine addicts progressively increases over the first several weeks of abstinence and remains high for extended periods. During the last decade, investigators have identified an analogous incubation phenomenon in rodents, in which time-dependent increases in cue-induced drug seeking are observed after withdrawal from intravenous cocaine self-administration. Such an incubation of drug craving is not specific to cocaine, as similar findings have been observed after self-administration of heroin, nicotine, methamphetamine, and alcohol in rats. In this review, we discuss recent results that have identified important brain regions involved in the incubation of drug craving, as well as evidence for the underlying cellular mechanisms. Understanding the neurobiology of the incubation of drug craving in rodents is likely to have significant implications for furthering our understanding of brain mechanisms and circuits that underlie drug craving in human addicts.

In a rat model of context-induced relapse to heroin, we identified sparsely distributed ventral medial prefrontal cortex (mPFC) neurons that were activated by the heroin-associated context. Selective pharmacogenetic inactivation of these neurons inhibited context-induced drug relapse. A small subset of ventral mPFC neurons forms neuronal ensembles that encode the learned associations between heroin reward and heroin-associated contexts; re-activation of these neuronal ensembles by drug-associated contexts during abstinence provokes drug relapse.

In humans, exposure to contexts previously associated with heroin use can provoke relapse. In rats, exposure to heroin-paired contexts after extinction of drug-reinforced responding in different contexts reinstates heroin seeking. This effect is attenuated by inhibition of glutamate or dopamine transmission in nucleus accumbens shell, or inactivation of ventral medial prefrontal cortex (vmPFC). Here, we used an anatomical asymmetrical disconnection procedure to demonstrate that an interaction between glutamatergic projections from vmPFC to accumbens shell and local dopamine D1 postsynaptic receptors contributes to context-induced reinstatement of heroin seeking. We also combined the marker of neuronal activity, Fos, with the retrograde tracer Fluoro-Gold (FG) to assess activation in this pathway during context-induced reinstatement. Rats were trained to self-administer heroin for 12 days; drug infusions were paired with a discrete tone-light cue. Lever-pressing was subsequently extinguished in a non-drug-associated context in the presence of the discrete cue. Rats were then tested in the heroin- or extinction-associated contexts under extinction conditions. Injections of muscimol+baclofen into vmPFC in one hemisphere and D1-family receptor antagonist SCH 23390 into the contralateral or ipsilateral accumbens shell decreased context-induced reinstatement. Unilateral injections of muscimol+baclofen into vmPFC or SCH 23390 into the accumbens shell had no effect. Context-induced reinstatement was associated with increased Fos expression in vmPFC neurons, including those projecting to accumbens shell, with higher double-labeling in the ipsilateral projection than in the contralateral projection. Our results demonstrate that activation of glutamatergic projections from ventral mPFC to accumbens shell, previously implicated in inhibition of cocaine relapse, promotes heroin relapse.

In humans, exposure to cues previously associated with heroin use often provokes relapse after prolonged withdrawal periods. In rats, cue-induced heroin-seeking progressively increases after withdrawal (incubation of heroin craving). Here, we examined the role of orbitofrontal cortex (OFC) neuronal ensembles in the enhanced response to heroin cues after prolonged withdrawal or the expression of incubation of heroin craving. We trained rats to self-administer heroin (6-h/d for 10 d) and assessed cue-induced heroin-seeking in extinction tests after 1 or 14 withdrawal days. Cue-induced heroin-seeking increased from 1 day to 14 days and was accompanied by increased Fos expression in ~12% of OFC neurons. Non-selective inactivation of OFC neurons with the GABA agonists baclofen+muscimol decreased cue-induced heroin-seeking on withdrawal day 14 but not day 1. We then used the Daun02 inactivation procedure to assess a causal role of the minority of selectively activated Fos-expressing OFC neurons (that presumably form cue-encoding neuronal ensembles) in cue-induced heroin-seeking after 14 withdrawal days. We trained cfos-lacZ transgenic rats to self-administer heroin and 11 days later re-exposed them to heroin-associated cues or novel cues for 15 min (induction day) followed by OFC Daun02 or vehicle injections 90 min later; we then tested the rats in extinction tests 3 days later. Daun02 selectively decreased cue-induced heroin-seeking in rats previously re-exposed to the heroin-associated cues on induction day, but not in rats previously exposed to novel cues. Results suggest that heroin-cue-activated OFC neuronal ensembles contribute to the expression of incubation of heroin craving.