Contributions of Nitrergic Interneurons and NO Signaling in Cocaine Relapse

 描述(申请人提供)：可卡因成瘾会导致伏隔核核心(NAcore)内神经可塑性的长期变化，与持久的复发易感性有关。在可卡因自身给药消失后，可卡因条件线索对NAcore中棘神经元(MSN)产生一过性突触增强(t-SP)，其特征是树突棘头部直径和AMPA/NMDA比率增加。这种t-SP还延伸到细胞外基质，在那里暴露于可卡因条件下的线索会导致基质金属蛋白酶(MMP)活性迅速增强，这是树突状脊柱头部增大所必需的。到目前为止，暴露于可卡因条件线索后的基质金属蛋白酶的激活机制还没有被阐明。MMPs以一种非活性前体的形式分泌，在激活之前必须被切割或修饰，这种激活的一个机制是S-一氧化氮(NO)对基质金属蛋白酶原结构域的亚硝化。气体递质NO是由一小部分中间神经元产生的，这些中间神经元表达神经元型一氧化氮合酶(NNOS)，被认为是氮能中间神经元。初步数据显示，暴露于可卡因可增强NAcore nNOS和MMP的活性，抑制NAcore中的nNOS活性可抑制恢复的可卡因寻找和线索诱导的MMP活性的诱导。在体内，选择性激活NAcore硝基能中间神经元的Gq信号(设计药物(DREADD)独有地激活Gq偶联的设计者受体)增强了MMP的活性，并且NAcore能中间神经元接受来自前皮质(PL)、背侧中缝(DRN)和腹侧被盖区(VTA)的输入。在K99 AIMS中，我建议用麻醉的大鼠和转基因小鼠的NO电化学记录来表征谷氨酸和DREADD诱导的NAcore中的NO释放，这是我在NIDA T32期间获得的经验。我还将利用DREADD受体技术选择性地调节NAcore氮能中间神经元的活动，以确定这些神经元的激活/去激活对提示的可卡因恢复的影响。为此，我将学习鼠标自我管理技术。在R00 AIMS中，我将对行为正常的大鼠进行电化学记录，以观察在自我给药、灭绝和恢复期间没有动态变化。这些实验的培训将在K99阶段的第二年完成。最后，我将使用NOS1cre基因敲除小鼠和依赖cre的AAV助手/狂犬病病毒系统在NAcore能中间神经元的单突触传入中表达GQ-DREADD。在DREADD介导的PL、DRN或VTA神经元激活后，我将在体内测量NAcore NO水平。这些实验有可能揭示尚未发现的可卡因成瘾的神经生物学机制，并可能有助于开发旨在逆转可卡因引起的神经生物学改变的新的治疗方案。

 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.