Michel C. Van den Oever

Prefrontal cortex AMPA receptor plasticity is crucial for cue-induced relapse to heroin-seeking

Associative learning processes have an important role in the initiation and persistence of heroin-seeking. Here we show in a rat self-administration model that reexposure to cues previously associated with heroin results in downregulation of AMPA receptor subunit GluR2 and concomitant upregulation of clathrin-coat assembly protein AP2m1 in synaptic membranes of the medial prefrontal cortex (mPFC). Reduced AMPA receptor expression in synaptic membranes was associated with a decreased AMPA/NMDA current ratio and increased rectification index in mPFC pyramidal neurons. Systemic or ventral (but not dorsal) mPFC injections of a peptide inhibiting GluR2 endocytosis attenuated both the rectification index and cue-induced relapse to heroin-seeking, without affecting sucrose-seeking. We conclude that GluR2 receptor endocytosis and the resulting synaptic depression in ventral mPFC are crucial for cue-induced relapse to heroin-seeking. As reexposure to conditioned stimuli is a major cause for heroin relapse, inhibition of GluR2 endocytosis may provide a new target for the treatment of heroin addiction.

Prefrontal cortex plasticity mechanisms in drug seeking and relapse.

Development of pharmacotherapy to reduce relapse rates is one of the biggest challenges in drug addiction research. The enduring nature of relapse suggests that it is maintained by long-lasting molecular and cellular adaptations in the neuronal circuitry that mediates learning and processing of motivationally relevant stimuli. Studies employing the reinstatement model of drug relapse in rodents point to an important role of the medial prefrontal cortex (mPFC), with distinct contributions of the dorsal and ventral regions of the mPFC to drug-, stress- and cue-induced drug seeking. Whereas drug-induced neuroadaptations in the dorsal mPFC function to enhance excitatory output and drive expression of drug seeking, recent evidence suggests that plasticity in the ventral mPFC leads to reduced glutamatergic transmission in this region, thereby impairing response inhibition upon exposure to drug-conditioned stimuli. Treatments aimed at restoring drug-induced neuroadaptations in the mPFC may help to reduce cue-reactivity and relapse susceptibility.

Optogenetic dissection of medial prefrontal cortex circuitry

The medial prefrontal cortex (mPFC) is critically involved in numerous cognitive functions, including attention, inhibitory control, habit formation, working memory and long-term memory. Moreover, through its dense interconnectivity with subcortical regions (e.g., thalamus, striatum, amygdala and hippocampus), the mPFC is thought to exert top-down executive control over the processing of aversive and appetitive stimuli. Because the mPFC has been implicated in the processing of a wide range of cognitive and emotional stimuli, it is thought to function as a central hub in the brain circuitry mediating symptoms of psychiatric disorders. New optogenetics technology enables anatomical and functional dissection of mPFC circuitry with unprecedented spatial and temporal resolution. This provides important novel insights in the contribution of specific neuronal subpopulations and their connectivity to mPFC function in health and disease states. In this review, we present the current knowledge obtained with optogenetic methods concerning mPFC function and dysfunction and integrate this with findings from traditional intervention approaches used to investigate the mPFC circuitry in animal models of cognitive processing and psychiatric disorders.

Extracellular matrix plasticity and GABAergic inhibition of prefrontal cortex pyramidal cells facilitates relapse to heroin seeking

Successful treatment of drug addiction is hampered by high relapse rates during periods of abstinence. Neuroadaptation in the medial prefrontal cortex (mPFC) is thought to have a crucial role in vulnerability to relapse to drug seeking, but the molecular and cellular mechanisms remain largely unknown. To identify protein changes that contribute to relapse susceptibility, we investigated synaptic membrane fractions from the mPFC of rats that underwent 21 days of forced abstinence following heroin self-administration. Quantitative proteomics revealed that long-term abstinence from heroin self-administration was associated with reduced levels of extracellular matrix (ECM) proteins. After extinction of heroin self-administration, downregulation of ECM proteins was also present in the mPFC, as well as nucleus accumbens (NAc), and these adaptations were partially restored following cue-induced reinstatement of heroin seeking. In the mPFC, these ECM proteins are condensed in the perineuronal nets that exclusively surround GABAergic interneurons, indicating that ECM adaptation might alter the activity of GABAergic interneurons. In support of this, we observed an increase in the inhibitory GABAergic synaptic inputs received by the mPFC pyramidal cells after the re-exposure to heroin-conditioned cues. Recovering levels of ECM constituents by metalloproteinase inhibitor treatment (FN-439; i.c.v.) prior to a reinstatement test attenuated subsequent heroin seeking, suggesting that the reduced synaptic ECM levels during heroin abstinence enhanced sensitivity to respond to heroin-conditioned cues. We provide evidence for a novel neuroadaptive mechanism, in which heroin self-administration-induced adaptation of the ECM increased relapse vulnerability, potentially by augmenting the responsivity of mPFC GABAergic interneurons to heroin-associated stimuli.

Ventromedial Prefrontal Cortex Pyramidal Cells Have a Temporal Dynamic Role in Recall and Extinction of Cocaine-Associated Memory

In addicts, associative memories related to the rewarding effects of drugs of abuse can evoke powerful craving and drug seeking urges, but effective treatment to suppress these memories is not available. Detailed insight into the neural circuitry that mediates expression of drug-associated memory is therefore of crucial importance. Substantial evidence from rodent models of addictive behavior points to the involvement of the ventromedial prefrontal cortex (vmPFC) in conditioned drug seeking, but specific knowledge of the temporal role of vmPFC pyramidal cells is lacking. To this end, we used an optogenetics approach to probe the involvement of vmPFC pyramidal cells in expression of a recent and remote conditioned cocaine memory. In mice, we expressed Channelrhodopsin-2 (ChR2) or Halorhodopsin (eNpHR3.0) in pyramidal cells of the vmPFC and studied the effect of activation or inhibition of these cells during expression of a cocaine-contextual memory on days 1–2 (recent) and ∼3 weeks (remote) after conditioning. Whereas optical activation of pyramidal cells facilitated extinction of remote memory, without affecting recent memory, inhibition of pyramidal cells acutely impaired recall of recent cocaine memory, without affecting recall of remote memory. In addition, we found that silencing pyramidal cells blocked extinction learning at the remote memory time-point. We provide causal evidence of a critical time-dependent switch in the contribution of vmPFC pyramidal cells to recall and extinction of cocaine-associated memory, indicating that the circuitry that controls expression of cocaine memories reorganizes over time.

The synaptic pathology of drug addiction.

A hallmark of drug addiction is the uncontrollable desire to consume drugs at the expense of severe negative consequences. Moreover, addicts that successfully refrain from drug use have a high vulnerability to relapse even after months or years of abstinence. In this chapter, we will discuss the current understanding of drug-induced neuroplasticity within the mesocorticolimbic brain system that contributes to the development of addiction and the persistence of relapse to drug seeking. I particular, we will focus at animal models that can be translated to human addiction. Although dopaminergic transmission is important for the acute effects of drug intake, the long-lived behavioral abnormalities associated with addiction are thought to arise from pathological plasticity in glutamatergic neurotransmission. The nature of changes in excitatory synaptic plasticity depends on several factors, including the type of drug, the brain area, and the time-point studied in the transition of drug exposure to withdrawal and relapse to drug seeking. Identification of drug-induced neuroplasticity is crucial to understand how molecular and cellular adaptations contribute to the end stage of addiction, which from a clinical perspective, is a time-point where pharmacotherapy may be most effectively employed.

Tracing tools to resolve neural circuits

In this paper we summarize neuroanatomical tracing methods, in particular combinations of methods designed to achieve the combined goals of tracing connectivity and extracting extra information from the projection or the target neurons. These combinations include techniques that identify projection fibres together with the neurons from which they originate, methods which establish the morphological or chemical identity of the target neurons and techniques to verify the presence of contacts between the terminal boutons on fibres and prospective target neurons. In the second part of this paper we describe a method recently developed in our laboratory, which consists of fluorescence tracing (introducing marker #1) combined with immunofluorescence with different fluorochromes (markers #2 and #3). This method enables us to observe the distribution of terminals of a particular set of projection fibres in contact with neurons belonging to two chemically different populations of interneuron. Multifluorescence confocal laser scanning is used for image acquisition of fibres and processes of presumed target neurons, and follow-up is by three-dimensional computer reconstruction. These reconstructions are used to determine that the contacts between the differentially labelled structures are real, i.e. with no optically empty space in between regardless of the angle of inspection. The specific experiments reported in this paper comprise the tracing with biotinylated dextran amine of fibres in the rat brain running from the presubiculum to layer III of the medial division of the entorhinal cortex. We studied the possibility of contacts in the latter area between the terminal boutons of these projecting fibres in association with presumed target neurons, i.e. parvalbumin and calretinin expressing interneurons.

The Extracellular Matrix Protein Brevican Limits Time-Dependent Enhancement of Cocaine Conditioned Place Preference

Cocaine-associated environmental cues sustain relapse vulnerability by reactivating long-lasting memories of cocaine reward. During periods of abstinence, responding to cocaine cues can time-dependently intensify a phenomenon referred to as ‘incubation of cocaine craving’. Here, we investigated the role of the extracellular matrix protein brevican in recent (1 day after training) and remote (3 weeks after training) expression of cocaine conditioned place preference (CPP). Wild-type and Brevican heterozygous knock-out mice, which express brevican at ~50% of wild-type levels, received three cocaine–context pairings using a relatively low dose of cocaine (5 mg/kg). In a drug-free CPP test, heterozygous mice showed enhanced preference for the cocaine-associated context at the remote time point compared with the recent time point. This progressive increase was not observed in wild-type mice and it did not generalize to contextual-fear memory. Virally mediated overexpression of brevican levels in the hippocampus, but not medial prefrontal cortex, of heterozygous mice prevented the progressive increase in cocaine CPP, but only when overexpression was induced before conditioning. Post-conditioning overexpression of brevican did not affect remote cocaine CPP, suggesting that brevican limited the increase in remote CPP by altering neuro-adaptive mechanisms during cocaine conditioning. We provide causal evidence that hippocampal brevican levels control time-dependent enhancement of cocaine CPP during abstinence, pointing to a novel substrate that regulates incubation of responding to cocaine-associated cues.