Glial-mediated synaptic remodeling in drug addiction

摘要 这项拨款申请旨在了解星形胶质细胞在介导可卡因诱导的回路中的作用 这些改变促使可卡因寻求和复发。R 01项目第一个供资期的成果 证明可卡因自我给药（SA）激活星形胶质细胞介导的突触发生机制， 在核内的主要中型棘神经元（MSNs）中产生AMPA受体（AMPAR）-沉默突触 贝壳（NAcSh）。在从可卡因SA戒断期间，这些可卡因产生的NAcSh的子集 通过招募AMPAR使突触成熟和加强。实验性地转换和锁定可卡因- 产生的NAcSh突触在他们的沉默状态下在药物戒断大大减少线索， 可卡因的诱惑通过测定细胞内Ca 2+活性，初步研究表明，NAcSh 星形胶质细胞活性被可卡因给药上调，并且这种上调在5-d后增加， 可卡因SA，表明星形胶质细胞中的“致敏”过程。此外，可卡因SA、NAcSh星形胶质细胞 通过增加他们的活动来获得对可卡因相关线索做出反应的能力， 增加星形胶质细胞活性使可卡因产生的NAcSh突触沉默。这些和其他初步 结果导致了目前的假设：NAcSh星形胶质细胞通过可卡因的经验获得独特的特性， 调节可卡因产生的突触，并形成特定的神经元集合， 戒毒后寻找可卡因这一假设将通过三条实验线进行检验。第一、 使用星形胶质细胞特异性分子工具，拟议的实验将测试星形胶质细胞的特异性假设， 可卡因SA上调mGluR 5的水平，可卡因SA反过来介导了 可卡因戒断后NAcSh星形胶质细胞对可卡因和可卡因相关线索的反应与此同时，选择性RNA- NAc星形胶质细胞的测序将揭示可卡因在这种细胞类型中作用的新分子底物。二是利用 在体内双光子显微镜结合切片电生理学，拟议的实验将测试 NAcSh星形胶质细胞活性的增加对于提示重新激活是足够的和必要的。 可卡因引起的NAcSh突触的再沉默，因此可以用来减少cue。 在戒毒后寻求可卡因第三，使用GCaMP介导的体内Ca 2+成像，提出了 实验将检验特定的假设，即神经元集合体部分地由星形胶质细胞形成， 介导突触发生，然后驱动线索诱导的可卡因寻求药物 戒断这些拟议的实验将表征几种新的星形胶质细胞相关底物 通过它可以操纵与成瘾有关的记忆以获得治疗益处。

Abstract This grant application proposes to understand the role of astrocytes in mediating cocaine-induced circuit alterations that drive cocaine seeking and relapse. Outcomes of the first funding period for this R01 project demonstrate that cocaine self-administration (SA) activates an astrocyte-mediated synaptogenic mechanism to generate AMPA receptor (AMPAR)-silent synapses in principal medium spiny neurons (MSNs) in nucleus accumbens shell (NAcSh). During withdrawal from cocaine SA, a subset of these cocaine-generated NAcSh synapses mature and strengthen by recruiting AMPARs. Experimentally converting and locking cocaine- generated NAcSh synapses within their silent state during drug withdrawal substantially decreases cue- induced cocaine seeking. By measuring intracellular Ca2+ activities, preliminary studies reveal that NAcSh astrocyte activities are upregulated by cocaine administration, and that this upregulation is increased after 5-d cocaine SA, indicating a ‘sensitization’ process in astrocytes. Furthermore, after cocaine SA, NAcSh astrocytes acquire the ability to respond to cocaine-associated cues by increasing their activities, and experimentally increasing astrocyte activities re-silences cocaine-generated NAcSh synapses. These and other preliminary results lead to the current hypothesis: NAcSh astrocytes gain unique properties through cocaine experience to regulate cocaine-generated synapses and formulate specific neuronal ensembles that drive cue-induced cocaine seeking after drug withdrawal. This hypothesis will be tested by three lines of experimentation. First, using astrocyte-specific molecular tools, proposed experiments will test the specific hypothesis that astrocytic levels of mGluR5 are upregulated by cocaine SA, which, in turn, mediates sensitized in vivo responses of NAcSh astrocytes to cocaine and cocaine-associated cues after cocaine withdrawal. In parallel, selective RNA- seq of NAc astrocytes will reveal novel molecular substrates for cocaine action in this cell type. Second, using in vivo two-photon microscopy combined with slice electrophysiology, proposed experiments will test the specific hypothesis that increased activities of NAcSh astrocytes is both sufficient and necessary for cue re- exposure-induced re-silencing of cocaine-generated NAcSh synapses, and thus can be used to reduce cue- induced cocaine seeking after drug withdrawal. Third, using GCaMP-mediated in vivo Ca2+ imaging, proposed experiments will test the specific hypothesis that the neuronal ensembles are formed, in part, by astrocyte- mediated synaptogenesis in response to cocaine, and then drive cue-induced cocaine seeking after drug withdrawal. These proposed experiments will characterize several novel astrocyte-associated substrates through which addiction-related memories can be manipulated for therapeutic benefits.