Michael D. Scofield - US grants

? DESCRIPTION (provided by applicant): Cocaine addiction produces long-lasting alterations in neuroplasticity within the nucleus accumbens core (NAcore) linked to an enduring vulnerability to relapse. Following extinction of cocaine self-administration, cocaine-conditioned cues produce a transient synaptic potentiation (t-SP) of NAcore medium spiny neurons (MSNs), characterized by increased dendritic spine head diameter and AMPA:NMDA ratios. This t-SP also extends to the extracellular matrix, where exposure to cocaine-conditioned cues causes a rapid enhancement of matrix metalloproteinase (MMP) activity, which is required for dendritic spine head enlargement. To date, the mechanism of MMP activation following exposure to cocaine-conditioned cues has yet to be elucidated. MMPs are secreted as an inactive pro-form which must be cleaved or modified prior to activation, and one mechanism for this activation is S-nitrosylation of the MMP pro-domain by nitric oxide (NO). The gaseous transmitter NO is produced by a small population of interneurons that express the enzyme neuronal nitric oxide synthase (nNOS), deemed nitrergic interneurons. Preliminary data presented here demonstrates that cocaine exposure enhanced NAcore nNOS and MMP activity and that inhibition of nNOS activity in the NAcore inhibited reinstated cocaine seeking and the cue-induced induction of MMP activity. I also show that selective activation of Gq signaling in NAcore nitrergic interneurons (with Gq-coupled designer receptors exclusively activated by designer drugs (DREADD)) enhanced MMP activity in-vivo and that nitrergic interneurons receive input from the prelimbic cortex (PL) dorsal raphe (DRN) and the ventral tegmental area (VTA). In the K99 aims, I propose to characterize glutamate and DREADD-evoked NO release in the NAcore following cocaine experience using anesthetized NO electrochemical recordings in rats and transgenic mice, which I have gained experience with during my NIDA T32. I will also selectively modulate the activity NAcore nitrergic interneurons with the DREADD receptor technology in order to determine the impact of activation/deactivation of these neurons on cued cocaine reinstatement. To do this, I will learn mouse self-administration techniques. In the R00 aims, I will perform electrochemical recordings in behaving rats to observe NO dynamics during self-administration, extinction and reinstatement. Training for these experiments will be completed in year 2 of the K99 phase. Finally, I will use NOS1cre knock-in mice and a cre-dependent AAV helper / rabies virus system to express Gq- DREADD in the monosynaptic afferents of NAcore nitrergic interneurons. Following DREADD-mediated activation of neurons in the PL, DRN or VTA I will measure NAcore NO levels in vivo. These experiments have the potential to reveal undiscovered neurobiological mechanisms of cocaine addiction, and could contribute to the development of novel therapeutic options aimed at reversing cocaine-induced neurobiological alterations.

PROJECT SUMMARY - Overall The personal, social and criminal consequences of opioid and cocaine abuse are enormous problems in North America. This is most tragically seen in rising morbidity due to heroin, prescription opioids and fentanyl overdose in the USA. Addiction to drugs typically cycles between three phases, active drug use, withdrawal from drug use and relapse to drug use. A point in the cycle of addiction where pharmacological intervention can be particularly beneficial is to interfere with the overwhelming motivation by addicts to relapse to drug use, even after extended periods of abstinence when acute withdrawal symptoms have dissipated. However, the enduring state of relapse vulnerability arises from interdependent brain adaptations produced during all three phases of addiction. Thus, in order to develop biological rationales for treating relapse, it is necessary to understand not only the neurobiology of relapse itself, but to determine which changes produced by drug administration and drug withdrawal contribute to the final enduring state of relapse vulnerability. The overarching goal of the Center for Opioid and Cocaine Addiction (COCA) is to create and maintain mechanisms of scientific synergy that will facilitate discovering the neuropathologies that underpin the enduring and uncontrollable drive to seek opioids and cocaine, and thereby advance biological rationales needed to efficiently generate pharmacotherapies that inhibit drug relapse. This goal will be achieved through a bidirectional translational strategy that involves 3 Cores and 4 research Projects. In addition to the Administrative and Pilot Cores, the Animal & Validation Core makes available transgenic rodents that have been trained to self-administer heroin or cocaine, and have been instrumented with intracranial cannulae, fiber optics or GRIN lens. This Core will also validate all viral reagents and transgenic animals shared by the COCA Cores and Projects. The 4 Projects range from determining the epigenetic substrates of long- lasting drug-induced alterations to understanding the molecular and brain circuit mechanisms of cue-induced drug seeking in rodents and humans. The Projects are designed to be highly integrated and form a bidirectional translation strategy for providing biological rationales for new therapeutic approaches to relapse prevention.

PROJECT SUMMARY Vulnerability to relapse remains a significant clinical hurdle in the treatment of addiction. Data from operant rodent models of addiction and relapse indicate that cocaine-conditioned cue exposure evokes a pronounced glutamate release in the nucleus accumbens core (NAcore). Cued glutamate release engages a cell type-specific transient synaptic potentiation in NAcore medium spiny neurons (MSNs), which does not occur during cued sucrose seeking. This transient synaptic potentiation consists of increased synaptic and structural plasticity in MSNs. Importantly, the magnitude of this plasticity positively correlates with relapse behavior. We posit that both the structural component (dendritic spine head expansion) and the synaptic component (increased insertion of glutamate receptors) of this plasticity are engaged by nitric oxide (NO); a gaseous transmitter produced by interneurons that express neuronal nitric oxide synthase (nNOS). Consistent with a role for these neurons in cued relapse, we have recently demonstrated that elevated NO release in the NAcore also occurs in parallel with glutamate release during cued cocaine seeking. The objectives of the proposed study are to reveal the inputs to the NAcore required for NO release during relapse, to determine which receptors on NAcore nNOS neurons regulate NO release and cued cocaine seeking, and to reveal how nNOS participates in the induction of the transient structural and synaptic plasticity in MSNs that drives cued cocaine seeking. In Aim 1, we will use chemogenetic inhibition of inputs to the NAcore while recording glutamate and NO release during cued cocaine seeking. We predict that inputs to the NAcore carrying discreet aspects of cue and contextual salience will differentially regulate NO release and cued relapse. In Aim 2, we will elucidate how glutamate and dopamine receptors, specifically expressed on NAcore nNOS interneurons, act in concert to regulate NO production and if they are required for cued cocaine seeking. To do this we will use transgenic mice that express Cre in nNOS neurons, Cre-dependent shRNA viral vectors, as well as cocaine self-administration and cued cocaine seeking trials. We predict that glutamate and dopamine receptor systems in nNOS neurons cooperatively regulate NO release and cued cocaine seeking. In Aim 3, we will determine if loss of nNOS in the NAcore will prevent cue- mediated synaptic and structural plasticity in MSNs. To do this we will use an shRNA vector to knockdown nNOS and concomitant viral labeling of D1 or D2 receptor expressing MSNs in the NAcore. This will be done using separate cohorts of D1-and D2-promoter driven Cre rats. In Aim 3, we will measure morphological and electrophysiological readouts synaptic plasticity induced by cues. We expect that loss of nNOS will prevent both forms of plasticity linked to relapse, predominantly in D1 MSNs. In conclusion, findings from these investigations will reveal the mechanistic aspects of how nNOS neurons translate cocaine cue exposure to relapse behavior. Accordingly, preventing this relapse signal transduction by preventing cued activation of nNOS neurons represents a potential therapeutic strategy to reduce drug craving and prevent relapse.