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调节可卡因诱导回路中的作用。 促使可卡因寻求和复发的变化。本R01项目首个资助期的成果 证明可卡因自我给药(SA)激活星形胶质细胞介导的突触生成机制 核内主要中棘神经元(MSN)产生AMPAR-无声突触 伏隔壳(NAcSh)。在戒断可卡因SA的过程中，这些可卡因产生的NAcSh的子集 通过招募AMPAR，突触变得成熟和强大。实验性地转换和锁定可卡因- 戒毒期间在沉默状态下产生的NAcSh突触大大减少了提示- 诱导性可卡因寻觅。通过测量细胞内钙的活性，初步研究表明NAcSh 给予可卡因后，星形胶质细胞的活性上调，并且这种上调在5-d后增强。 可卡因SA，表明星形胶质细胞的“敏化”过程。此外，在可卡因SA之后，NAcSh星形胶质细胞 通过增加活动和实验获得对可卡因相关线索做出反应的能力 增加星形胶质细胞的活动，重新沉默可卡因产生的NAcSh突触。这些和其他初步的 结果导致了目前的假设：NAcSh星形胶质细胞通过可卡因经验获得独特的特性 调节可卡因产生的突触并形成驱动线索诱导的特定神经元群 戒毒后寻找可卡因。这一假设将通过三条线的实验来检验。第一, 利用星形胶质细胞特有的分子工具，拟议的实验将检验星形胶质细胞的特定假设 可卡因SA上调mGluR5水平，进而介导体内致敏反应 可卡因戒断后NAcSh星形胶质细胞对可卡因及其相关线索的反应。同时，选择性RNA- NAC星形胶质细胞的SEQ将揭示可卡因在这种细胞类型中作用的新的分子底物。第二，使用 在活体双光子显微镜与切片电生理学相结合，拟议的实验将测试 NAcSh星形胶质细胞活性增加是线索重现的充分和必要的具体假设 暴露诱导可卡因产生的NAcSh突触重新沉默，因此可用于减少提示- 戒毒后诱发可卡因寻觅。第三，利用GCaMP介导的体内钙成像，提出了 实验将检验特定的假设，即神经元团部分由星形胶质细胞形成- 介导对可卡因反应的突触发生，然后驱动线索诱导的可卡因追药 戒烟。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，与成瘾相关的记忆可以被操纵以达到治疗的效果。

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.