Functional consequences of GABAergic inhibition of dendritic spines at puberty

描述（由申请人提供）：树突棘在青春期经历突触修剪，这是自闭症和精神分裂症中失调的一个过程。这种突触修剪的机制尚不清楚，但可能涉及NMDA受体激活的减少。我们最近发现，在青春期（~PND 35-45），突触外？GABAA受体（GABARs）出现在小鼠海马CA 1区锥体细胞树突棘上，持续时间约10 d。此外，NMDA受体的激活在+/+中受损，但在<$4或？-/-中不受损。老鼠，牵连到？在青春期NMDAR功能下降。在这个建议中，我们将测试的假设，即突触修剪在青春期确实是由于这种增加的表达？GABARs，我们将探讨这种效应的细胞，电路和行为后果。拟议的实验将使用广泛的技术组合，包括双光子成像，双光子uncaging，电生理学，药理学，解剖学和免疫细胞化学测定，以直接评估这种新形式的抑制在青春期的脊柱的功能结果。起初， 将使用RuBiGABA（一种新型笼状GABA化合物）的光化学技术来绘制青春期脊髓中的GABA能电流。该技术的初步数据显示，棘具有GABA能电流。然后，将使用双光子Ca 2+成像沿着Rubi-GABA的局部释放来检查这些受体对NMDA受体诱导的来自个体棘的Ca 2+内流的影响。这些实验还将使用急性药理学（激动剂，调节剂）和遗传操作（敲除，腺病毒shRNA敲低）的？探讨这些受体在调节NMDA受体功能中的作用。与此同时，要确定的影响<$4 <$？-介导的抑制在青春期对脊柱密度青春期后，我们将长期操纵？功能（如上所述）超过10天，当<$4 <$？受体升高（~PND 35-45），并在8周龄时定量脊柱密度。初步数据显示，在+/+小鼠中，脊柱密度确实在青春期后降低，但在<$4-/-小鼠中没有，这意味着<$4 <$？GABARs在青春期突触修剪中的作用将检查棘密度变化的免疫细胞化学和电生理相关性。最后，为了探索这种修剪对电路和行为可塑性的影响，我们还将确定这些改变对突触可塑性（长时程增强（LTP），长时程抑制（LTD），LTP逆转）的影响，以及在两个依赖于大脑的空间学习任务中的行为灵活性。拟议研究的结果将直接涉及 GABA能抑制青春期树突棘，也将提供在青春期突触修剪过程中的机制，以及探索在这个过程中的改变功能的结果。这些结果将与自闭症等认知障碍有关， 其中减少的突触修剪和减少的行为灵活性与p4基因的单核苷酸多态性相关，表明存在遗传联系。

DESCRIPTION (provided by applicant): Dendritic spines undergo synaptic pruning during adolescence, a process which is dysregulated in autism and schizophrenia. The mechanism underlying this synaptic pruning is not known, but is likely to involve reductions in NMDA receptor activation. We recently found that, during the pubertal period (~PND 35-45), extrasynaptic ¿4¿? GABAA receptors (GABARs) emerge on dendritic spines of CA1 hippocampal pyramidal cells of mice for ~10d. Moreover, activation of NMDA receptors is impaired in +/+ but not ¿4 or ?-/- mice, implicating ¿4¿? in the reduction of NMDAR function during puberty. In this proposal, we will test the hypothesis that synaptic pruning in adolescence is indeed due to this increased expression of ¿4¿? GABARs, and we will explore the cellular, circuit and behavioral consequences of this effect. The proposed experiments will use a wide combination of techniques, including two-photon imaging, two-photon uncaging, electrophysiological, pharmacological, anatomical and immunocytochemical assays to directly assess the functional outcome of this novel form of inhibition on the spines at puberty. Initially, optochemical techniques with RuBiGABA, a novel caged GABA compound, will be used to map GABAergic currents in spines at puberty. Preliminary data with this technique show that spines have GABAergic currents. Then, the impact of these receptors on NMDA receptor-induced Ca2+ influx from individual spines will be examined using two-photon Ca2+ imaging along with local uncaging of Rubi-GABA. These experiments will also use acute pharmacological (agonists, modulators) and genetic manipulation (knock-out, adenovirus-shRNA knock-down) of ¿4¿? to explore the role of these receptors in regulating NMDA receptor function. In parallel, to determine the impact of ¿4¿?-mediated inhibition during puberty on spine density post-pubertally, we will chronically manipulate ¿4¿? function (as above) over the 10 days when ¿4¿? receptors are elevated (~PND 35-45) and quantify spine density at 8-wks of age. Preliminary data shows that spine density is indeed decreased post-pubertally in +/+ but not ¿4-/- mice, implicating ¿4¿? GABARs in pubertal synaptic pruning. Both immunocytochemical and electrophysiological correlates of spine density changes will be examined. Finally, to explore the effect of this pruning on circuit and behavioral plasticity, we will also determine the impact of these alterations on synaptic plasticity (long-term potentiation (LTP), long-term depression (LTD), reversal of LTP), and on behaviorally flexibility in two hippocampal-dependent spatial learning tasks. The findings from the proposed studies will directly address the functional role of GABAergic inhibition on dendritic spines at puberty, and will also provide mechanisms for the process of synaptic pruning in adolescence, as well as explore functional outcomes of alterations in this process. These results will be relevant for cognitive disorders such as autism, where reduced synaptic pruning and reduced behavioral flexibility are associated with single nucleotide polymorphisms of the ¿4 gene, suggesting a genetic link.