Prelimbic cortex is a common brain area activated during cue‐induced reinstatement of cocaine and heroin seeking in a polydrug self‐administration rat model

Many preclinical studies examined cue‐induced relapse to heroin and cocaine seeking in animal models, but most of these studies examined only one drug at a time. In human addicts, however, polydrug use of cocaine and heroin is common. We used a polydrug self‐administration relapse model in rats to determine similarities and differences in brain areas activated during cue‐induced reinstatement of heroin and cocaine seeking. We trained rats to lever press for cocaine (1.0 mg/kg per infusion, 3‐hr/day, 18 day) or heroin (0.03 mg/kg per infusion) on alternating days (9 day for each drug); drug infusions were paired with either intermittent or continuous light cue. Next, the rats underwent extinction training followed by tests for cue‐induced reinstatement where they were exposed to either heroin‐ or cocaine‐associated cues. We observed cue‐selective reinstatement of drug seeking: the heroin cue selectively reinstated heroin seeking and the cocaine cue selectively reinstated cocaine seeking. We used Fos immunohistochemistry to assess cue‐induced neuronal activation in different subregions of the medial prefrontal cortex, dorsal striatum, nucleus accumbens, and amygdala. Fos expression results indicated that only the prelimbic cortex (PL) was activated by both heroin and cocaine cues; in contrast, no significant cue‐induced neuronal activation was observed in other brain areas. RNA in situ hybridization indicated that the proportion of glutamatergic and GABAergic markers in PL Fos‐expressing cells was similar for the heroin and cocaine cue‐activated neurons. Overall, the results indicate that PL may be a common brain area involved in both heroin and cocaine seeking during polydrug use.     

Induction of the learning and plasticity-associated gene Zif268 following exposure to a discrete cocaine-associated stimulus

We investigated whether the expression of the plasticity-associated gene, zif268, was associated with memories retrieved by exposure to a discrete stimulus that had been associated with cocaine, either self-administered or administered noncontingently. In the absence of drug, passive presentation of a cocaine-associated light stimulus induced changes in the expression of zif268 measured by in situ hybridization within a limbic cortical-ventral striatal circuit that was not only regionally selective but related to whether the rats had originally received response-contingent or noncontingent stimulus-drug pairings. In rats that had self-administered drug, the cocaine-conditioned stimulus (CS) increased zif268 expression in neurons of the ventral tegmental area, nucleus accumbens core and shell, and basal nucleus of the amygdala but not hippocampus, prelimbic area of the medial prefrontal cortex or amygdala central nucleus. The same CS that had been associated with cocaine administered noncontingently additionally increased zif268 mRNA levels in area Cg1 of the anterior cingulate cortex, ventral and lateral regions of the orbitofrontal cortex and lateral nucleus of the amygdala. Zif268 induction was related to the predictive relationship between the stimulus and cocaine as no changes were seen in cocaine-experienced rats that had received unpaired light and drug presentations during training. Thus, zif268 expression is increased by appetitively (drug) conditioned stimuli after Pavlovian learning. Zif268 may participate in the molecular mechanisms underlying the reconsolidation or re-encoding of Pavlovian stimulus-drug associations across a distributed limbic cortical-ventral striatal neural network and that may contribute to the basis of the enduring drug-seeking behaviour produced by environmental cues.     

Selective induction of c-Fos immunoreactivity in the prelimbic cortex during reinstatement of heroin seeking induced by acute food deprivation in rats

We previously reported that acute 1-day food deprivation reinstates heroin seeking in rats. The goal of the present study was to begin identifying brain sites potentially involved in this effect. For this purpose, we measured, by immunohistochemistry, the expression of c-Fos following a test for food deprivation-induced reinstatement. Groups of rats (n=9-10 per group) were trained to lever-press for heroin (0.05-0.1mg/kg/infusion) or saline for 10 days (9 h/day); each infusion was paired with a cue light. Rats were then given 10 days of extinction during which the heroin and saline syringes were removed. Next, a test for reinstatement was conducted after exposure to 0 (baseline) or 1-day food deprivation. During training, lever pressing for heroin increased over days, while responding for saline infusions paired with the cue light decreased over time. During extinction, responding on the heroin-paired lever decreased over time, while responding on the saline-paired lever remained low. In heroin-trained rats, food deprivation induced a large increase in responding on the lever associated with drug infusions. Surprisingly, food deprivation also modestly increased responding in the saline-trained rats. Food deprivation selectively increased c-Fos immunoreactivity (IR) in the prelimbic cortex of heroin-trained, but not saline-trained, rats (n=4 per condition). Food deprivation also increased c-Fos IR in both heroin- and saline-trained rats in the basolateral amygdala and the ventrolateral bed nucleus of stria terminalis (BNST), but had no effect on c-Fos expression in the dorsolateral BNST, cingulate cortex, nucleus accumbens, and central amygdala. These results raise the possibility that the prelimbic cortex is involved in food deprivation-induced reinstatement of heroin seeking.     

Role of the prefrontal cortex and nucleus accumbens in reinstating methamphetamine seeking

Although the involvement of the medial prefrontal cortex projection to the nucleus accumbens in the reinstatement of cocaine seeking has been well studied, it is not known if this projection plays a similar role in the reinstatement of cue‐ and methamphetamine‐induced drug seeking in animals extinguished from methamphetamine self‐administration. Accordingly, following extinction from long‐access methamphetamine self‐administration, rats were bilaterally microinjected with either a combination of the GABA agonists baclofen/muscimol or vehicle (artificial cerebrospinal fluid) into the infralimbic or prelimbic subcompartments of the medial prefrontal cortex or into the shell or core subcompartments of the nucleus accumbens. Similar to cocaine seeking, inactivation of either the prelimbic cortex or accumbens core eliminated cue‐ and methamphetamine‐induced reinstatement, and inactivation of neither the infralimbic cortex nor shell subcompartments inhibited methamphetamine‐induced drug seeking. However, in contrast to previous reports with cocaine, cue‐induced reinstatement of methamphetamine seeking was inhibited by inactivation of the infralimbic cortex. In conclusion, although a primary role in reinstated drug seeking by the prelimbic and the accumbens core is similar between cocaine and methamphetamine, the recruitment of the infralimbic cortex by conditioned cues differs between these two psychostimulant drugs.     

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.     

Complementary activation of hippocampal-cortical subregions and immature neurons following chronic training in single and multiple context versions of the water maze

Neurobiological studies of memory typically involve single learning sessions that last minutes or days. In natural settings, however, animals are constantly learning. Here we investigated how several weeks of spatial water maze training influences subsequent activation of neocortical and hippocampal subregions, including adult-born neurons. Mice were either trained in a single context or in a variant of the task in which the spatial cues and platform location changed every 3 days, requiring constant new learning. On the final day, half of the mice in each training group were tested in a novel context and the other half were tested in their previous, familiar context. Two hours later mice were perfused to measure subregion-specific expression of the immediate-early gene zif268, a marker of neuronal activation. None of the training paradigms affected the magnitude of adult neurogenesis. However, different neuronal populations were activated depending on prior training history, final context novelty, or a combination of these 2 factors. The anterior cingulate cortex was more activated by novel context exposure, regardless of the type of prior training. The suprapyramidal blade of the dentate gyrus and region CA3 showed greater activation in mice trained in multiple contexts, primarily after exposure to a familiar context. In immature granule neurons, multiple context training enhanced activation regardless of final context novelty. CA1 showed no significant changes in zif268 expression across any training condition. In naïve control mice, training on the final day increased zif268 expression in CA3, CA1 and the anterior cingulate cortex, but not the dentate gyrus, relative to mice that remained in their cages (transport controls). Unexpectedly, immature granule cells showed a decrease in zif268 expression in naïve learners relative to transport controls. These findings suggest novel and complementary roles for hippocampal, neocortical, and immature neuronal populations in learning and memory.     

Dysregulation of gene induction in corticostriatal circuits after repeated methylphenidate treatment in adolescent rats: differential effects on zif 268 and homer 1a

Psychostimulants and other dopamine agonists produce molecular changes in neurons of cortico-basal ganglia-cortical circuits, and such neuronal changes are implicated in behavioural disorders. Methylphenidate, a psychostimulant that causes dopamine overflow (among other effects), alters gene regulation in neurons of the striatum. The present study compared the effects of acute and repeated methylphenidate treatment on cortical and striatal gene regulation in adolescent rats. Changes in the expression of the immediate-early genes zif 268 and homer 1a were mapped in 23 striatal sectors and 22 cortical areas that provide input to these striatal sectors, in order to determine whether specific corticostriatal circuits were affected by these treatments. Acute administration of methylphenidate (5 mg/kg, i.p.) produced modest zif 268 induction in cortical areas. These cortical zif 268 responses were correlated in magnitude with zif 268 induction in functionally related striatal sectors. In contrast, after repeated methylphenidate treatment (10 mg/kg, 7 days), cortical and striatal gene induction were dissociated. In these animals, the methylphenidate challenge (5 mg/kg) produced significantly greater gene induction (zif 268 and homer 1a) in the cortex. This enhanced response was widespread but regionally selective, as it occurred predominantly in premotor, motor and somatosensory cortical areas. At the same time, striatal gene induction was partly suppressed (zif 268) or unchanged (homer 1a). Thus, repeated methylphenidate treatment disrupted the normally coordinated gene activation patterns in cortical and striatal nodes of corticostriatal circuits. This drug-induced dissociation in cortical and striatal functioning was associated with enhanced levels of behavioural stereotypies, suggesting disrupted motor switching function.     

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.     

Upregulation of Arc mRNA expression in the prefrontal cortex following cue-induced reinstatement of extinguished cocaine-seeking behavior

Cocaine-associated cues acquire incentive motivational effects that manifest as cue-elicited craving in humans and cocaine-seeking behavior in rats. Here we examine the hypothesis that neuronal processes associated with incentive motivational effects of cocaine cues involve increased expression of the plasticity-associated gene, Arc. Rats trained to self-administer cocaine subsequently underwent extinction training, during which cocaine-seeking behavior (i.e., responses without cocaine reinforcement) progressively decreased. Rats were then tested for cocaine-seeking behavior either with or without response-contingent presentations of light/tone cues that had been previously paired with cocaine infusions during self-administration training. Cues elicited reinstatement of cocaine-seeking behavior and were accompanied by increased Arc mRNA levels in the orbitofrontal, prelimbic, and anterior cingulate cortices, suggesting Arc involvement in conditioned plasticity associated with incentive motivational effects of cocaine cues. Additionally, rats with a history of cocaine self-administration and extinction exhibited upregulation of Arc expression in several limbic and cortical regions relative to saline-yoked controls regardless of cue exposure condition, suggesting persistent neuroadaptations involving Arc within these regions.     

Resistance to extinction is associated with impaired immediate early gene induction in medial prefrontal cortex and amygdala

Extinction of classical fear conditioning is thought to involve activity-dependent potentiation of synaptic transmission in the medial prefrontal cortex (mPFC), resulting in the inhibition of amygdala-dependent fear responses. While many studies have addressed the mechanisms underlying extinction learning, it is unclear what determines whether extinction memory is consolidated or whether spontaneous recovery of the fear response occurs. Here we show, using a combined electrophysiological and immunocytochemical approach, that spontaneous recovery of conditioned fear in mice is associated with a prolonged expression of long-term depression of synaptic transmission in the mPFC and the failure of induction of the immediate-early genesc-Fos and zif268 in the mPFC and the basolateral nucleus of the amygdala. This suggests that coordinated activity-dependent changes in gene expression in the mPFC and the amygdala may underlie the formation of long-term fear extinction memory.     

Long-lasting dysregulation of gene expression in corticostriatal circuits after repeated cocaine treatment in adult rats: effects on zif 268 and homer 1a

Human imaging studies show that psychostimulants such as cocaine produce functional changes in several areas of cortex and striatum. These may reflect neuronal changes related to addiction. We employed gene markers ( zif 268 and homer 1a ) that offer a high anatomical resolution to map cocaine-induced changes in 22 cortical areas and 23 functionally related striatal sectors, in order to determine the corticostriatal circuits altered by repeated cocaine exposure (25 mg/kg, 5 days). Effects were investigated 1 day and 21 days after repeated treatment to assess their longevity. Repeated cocaine treatment increased basal expression of zif 268 predominantly in sensorimotor areas of the cortex. This effect endured for 3 weeks in some areas. These changes were accompanied by attenuated gene induction by a cocaine challenge. In the insular cortex, the cocaine challenge produced a decrease in zif 268 expression after the 21-day, but not 1-day, withdrawal period. In the striatum, cocaine also affected mostly sensorimotor sectors. Repeated cocaine resulted in blunted inducibility of both zif 268 and homer 1a , changes that were still very robust 3 weeks later. Thus, our findings demonstrate that cocaine produces robust and long-lasting changes in gene regulation predominantly in sensorimotor corticostriatal circuits. These neuronal changes were associated with behavioral stereotypies, which are thought to reflect dysfunction in sensorimotor corticostriatal circuits. Future studies will have to elucidate the role of such neuronal changes in psychostimulant addiction.     

The mGluR2/3 agonist LY379268 attenuates context- and discrete cue-induced reinstatement of sucrose seeking but not sucrose self-administration in rats

We recently reported that systemic injections of the mGluR(2/3) agonist LY379268, which decreases evoked glutamate release, attenuate context- and discrete cue-induced reinstatement of heroin seeking, but not heroin self-administration. Here, we report that systemic injections of LY379268 attenuate context- and discrete cue-induced reinstatement of sucrose seeking, but not sucrose self-administration. These results suggest that similar neuronal mechanisms mediate cue-induced relapse to opiate- and palatable food seeking.     

Investigation of cortical reorganization in area 17 and nine extrastriate visual areas through the detection of changes in immediate early gene expression as induced by retinal lesions

The effect of binocular central retinal lesions on the expression of the immediate early genes c-fos and zif268 in the dorsal lateral geniculate nucleus (dLGN) and the visual cortex of adult cats was investigated by in situ hybridization and immunocytochemistry. In the deafferented region of the dLGN, the c-fos mRNA level was decreased within 3 days. The dimensions of the geniculate region showing decreased amounts of c-fos mRNA matched the predictions based on the lesion size and the retinotopic maps of Sanderson ([1971] J. Comp. Neurol. 143:101-118). We did not detect zif268 mRNA in the dLGN. At the cortical level, both c-fos and zif268 mRNA expression decreased in the sensory-deprived region of area 17. In addition, the portions of areas 18, 19, 21a, 21b, and 7, as well as the posterior medial lateral suprasylvian area, the posterior lateral lateral suprasylvian area, the ventral lateral suprasylvian area, and the dorsal lateral suprasylvian area corresponding to the retinal lesions also displayed decreased c-fos and zif268 mRNA levels. Immunocytochemistry revealed similar changes for Zif268 and Fos protein. Three days post lesion, the dimensions of the lesion-affected cortical loci exceeded the predictions in relation to the size of the retinal lesions and the available retinotopic maps. Longer postlesion survival times clearly resulted in a time-dependent restoration of immediate early gene expression from the border to the center of the lesion-affected cortical portions. Our findings represent a new approach for investigating the capacity of adult sensory systems to undergo plastic changes following sensory deprivation and for defining the topographic nature of sensory subcortical and cortical structures.     

Reduced expression of conditioned fear in the R6/2 mouse model of Huntington's disease is related to abnormal activity in prelimbic cortex

Prefrontal cortex (PFC) dysfunction is common in patients with Huntington's disease (HD), a dominantly inherited neurological disorder, and has been linked to cognitive disruption. We previously reported alterations in neuronal firing patterns recorded from PFC of the R6/2 mouse model of HD. To determine if PFC dysfunction results in behavioral impairments, we evaluated performance of wild-type (WT) and R6/2 mice in a fear conditioning and extinction behavioral task. Fear conditioning and extinction retrieval were similar in both genotypes, but R6/2s exhibited less fear during extinction by freezing less than WTs. A fear reinstatement test after extinction retrieval indicated that faster extinction was not due to poor memory for conditioning. During initial extinction and extinction retrieval training, neuronal activity was recorded from prelimbic (PL) cortex, a subregion of PFC known to be important for fear expression. In WTs, a large number of neurons were activated by the conditioned stimulus during initial extinction and this activation was significantly impaired in R6/2s. Notably, there was no genotype difference in PFC activity during extinction retrieval. Thus, altered extinction is likely a result of reduced fear expression due to impairments in PL activation. Collectively, our results suggest that PFC dysfunction may play a key role in R6/2 cognitive impairments.     

Novel cues reinstate cocaine-seeking behavior and induce Fos protein expression as effectively as conditioned cues

Cue reinstatement of extinguished cocaine-seeking behavior is a widely used model of cue-elicited craving in abstinent human addicts. This study examined Fos protein expression in response to cocaine cues or to novel cues as a control for activation produced by test novelty. Rats were trained to self-administer cocaine paired with either a light or a tone cue, or received yoked saline and cue presentations, and then underwent daily extinction training. They were then tested for reinstatement of extinguished cocaine-seeking behavior elicited by response-contingent presentations of either the cocaine-paired cue or a novel cue (that is, tone for those trained with a light or vice versa). Surprisingly, conditioned and novel cues both reinstated responding and increased Fos similarly in most brain regions. Exceptions included the anterior cingulate, which was sensitive to test cue modality in saline controls and the dorsomedial caudate-putamen, where Fos was correlated with responding in the novel, but not conditioned, cue groups. In subsequent experiments, we observed a similar pattern of reinstatement in rats trained and tested for sucrose-seeking behavior, whereas rats trained and tested with the cues only reinstated to a novel, and not a familiar, light or tone. The results suggest that novel cues reinstate responding to a similar extent as conditioned cues regardless of whether animals have a reinforcement history with cocaine or sucrose, and that both types of cues activate similar brain circuits. Several explanations as to why converging processes may drive drug and novel cue reinforcement and seeking behavior are discussed.