Unique gene alterations are induced in FACS-purified Fos-positive neurons activated during cue-induced relapse to heroin seeking.

Cue-induced heroin seeking after prolonged withdrawal is associated with neuronal activation and altered gene expression in prefrontal cortex (PFC). However, these previous studies assessed gene expression in all neurons regardless of their activity state during heroin seeking. Using Fos as a marker of neural activity, we describe distinct molecular alterations induced in activated versus non-activated neurons during cue-induced heroin seeking after prolonged withdrawal. We trained rats to self-administer heroin for 10 days (6 h/day) and assessed cue-induced heroin seeking in extinction tests after 14 or 30 days. We used fluorescent-activated cell sorting (FACS) to purify Fos-positive and Fos-negative neurons from PFC 90 min after extinction testing. Flow cytometry showed that Fos-immunoreactivity was increased in less than 10% of sparsely distributed PFC neurons. mRNA levels of the immediate early genes fosB, arc, egr1, and egr2, as well as npy and map2k6, were increased in Fos-positive, but not Fos-negative, neurons. In support of these findings, double-label immunohistochemistry indicated substantial coexpression of neuropeptide Y (NPY)- and Arc-immunoreactivity in Fos-positive neurons. Our data indicate that cue-induced relapse to heroin seeking after prolonged withdrawal induces unique molecular alterations within activated PFC neurons that are distinct from those observed in the surrounding majority of non-activated neurons.

Role of orbitofrontal cortex neuronal ensembles in the expression of incubation of heroin craving.

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 to 14 d and was accompanied by increased Fos expression in ∼12% of OFC neurons. Nonselective 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 c-fos-lacZ transgenic rats to self-administer heroin and 11 d later reexposed 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 d later. Daun02 selectively decreased cue-induced heroin seeking in rats previously reexposed to the heroin-associated cues on induction day but not in rats exposed previously to novel cues. Results suggest that heroin-cue-activated OFC neuronal ensembles contribute to the expression of incubation of heroin craving.

Association of time-dependent changes in mu opioid receptor mRNA, but not BDNF, TrkB, or MeCP2 mRNA and protein expression in the rat nucleus accumbens with incubation of heroin craving.

RATIONALE AND OBJECTIVES: Responding to heroin cues progressively increases after cessation of heroin self-administration (incubation of heroin craving). We investigated whether this incubation is associated with time-dependent changes in brain-derived neurotrophic factor (BDNF) and methyl-CpG binding protein 2 (MeCP2) signaling and mu opioid receptor (MOR) expression in nucleus accumbens (NAc), dorsal striatum (DS), and medial prefrontal cortex (mPFC). We also investigated the effect of the preferential MOR antagonist naloxone on cue-induced heroin seeking during abstinence. METHODS: We trained rats to self-administer heroin or saline for 9-10 days and then dissected the NAc, DS, and mPFC at different abstinence days and measured mRNA and protein levels of BDNF, TrkB, and MeCP2, as well as MOR mRNA (Oprm1). In other groups, we assessed cue-induced heroin seeking in extinction tests after 1, 11, and 30 abstinence days, and naloxone's (0-1.0 mg/kg) effect on extinction responding after 1 and 15 days. RESULTS: Cue-induced heroin seeking progressively increased or incubated during abstinence. This incubation was not associated with changes in BDNF, TrkB, or MeCP2 mRNA or protein levels in NAc, DS, or mPFC; additionally, no molecular changes were observed after extinction tests on day 11. In NAc, but not DS or mPFC, MOR mRNA decreased on abstinence day 1 and returned to basal levels over time. Naloxone significantly decreased cue-induced heroin seeking after 15 abstinence days but not 1 day. CONCLUSIONS: Results suggest a role of MOR in incubation of heroin craving. As previous studies implicated NAc BDNF in incubation of cocaine craving, our data suggest that different mechanisms contribute to incubation of heroin versus cocaine craving.

Role of projections from ventral medial prefrontal cortex to nucleus accumbens shell in context-induced reinstatement of heroin seeking.

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 (mPFC). Here, we used an anatomical asymmetrical disconnection procedure to demonstrate that an interaction between glutamatergic projections from ventral mPFC to accumbens shell and local dopamine D(1) 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 to assess activation in this pathway during context-induced reinstatement. Rats were trained to self-administer heroin for 12 d; drug infusions were paired with a discrete tone-light cue. Lever pressing was subsequently extinguished in a nondrug-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 ventral mPFC in one hemisphere and D(1)-family receptor antagonist SCH 23390 into the contralateral or ipsilateral accumbens shell decreased context-induced reinstatement. Unilateral injections of muscimol + baclofen into ventral mPFC or SCH 23390 into the accumbens shell had no effect. Context-induced reinstatement was associated with increased Fos expression in ventral mPFC 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.

Ventral medial prefrontal cortex neuronal ensembles mediate context-induced relapse to heroin.

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 formed 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 provoked drug relapse.

The basolateral amygdala and nucleus accumbens core mediate dissociable aspects of drug memory reconsolidation.

A distributed limbic-corticostriatal circuitry is implicated in cue-induced drug craving and relapse. Exposure to drug-paired cues not only precipitates relapse, but also triggers the reactivation and reconsolidation of the cue-drug memory. However, the limbic cortical-striatal circuitry underlying drug memory reconsolidation is unclear. The aim of this study was to investigate the involvement of the nucleus accumbens core and the basolateral amygdala in the reconsolidation of a cocaine-conditioned stimulus-evoked memory. Antisense oligodeoxynucleotides (ASO) were infused into each structure to knock down the expression of the immediate-early gene zif268, which is known to be required for memory reconsolidation. Control infusions used missense oligodeoxynucleotides (MSO). The effects of zif268 knockdown were measured in two complementary paradigms widely used to assess the impact of drug-paired CSs upon drug seeking: the acquisition of a new instrumental response with conditioned reinforcement and conditioned place preference. The results show that both intranucleus accumbens core and intrabasolateral amygdala zif268 ASO infusions at memory reactivation impaired the reconsolidation of the memory underlying a cocaine-conditioned place preference. However, knockdown of zif268 in the nucleus accumbens at memory reactivation had no effect on the memory underlying the conditioned reinforcing properties of the cocaine-paired CS measured subsequently, and this is in contrast to the marked impairment observed previously following intrabasolateral amygdala zif268 ASO infusions. These results suggest that both the basolateral amygdala and nucleus accumbens core are key structures within limbic cortical-striatal circuitry where reconsolidation of a cue-drug memory occurs. However reconsolidation of memory representations formed during Pavlovian conditioning are differentially localized in each site.