Functional consequences of GABAergic inhibition of dendritic spines at puberty

描述(申请人提供)：树突棘在青春期经历突触修剪，这一过程在自闭症和精神分裂症中是调节失调的。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)，突触外突触？小鼠海马CA1区锥体细胞树突棘上可见GABAA受体(GABAR)，持续约10d。此外，NMDA受体的激活在+/+小鼠中受损，而不是？4或？-/-小鼠，这意味着？4？在青春期NMDAR功能降低。在这项提议中，我们将检验这一假设，即青春期突触修剪确实是由于这种增加的表达？GABAR，我们将探索这种效应的细胞、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、双光子去除、电生理学、药理学、解剖学和免疫细胞化学分析，以直接评估这种新形式的抑制在青春期对脊柱的功能结果。最初， RuBiGABA是一种新型的笼式GABA化合物，它的光化学技术将被用来绘制青春期脊柱中的GABA能电流。这项技术的初步数据显示，脊椎有GABA能电流。然后，将使用双光子钙成像和Rubi-GABA的局部去势来研究这些受体对NMDA受体诱导的单个脊柱的钙内流的影响。这些实验还将使用急性药理作用(激动剂、调节剂)和基因操作(敲除、腺病毒shRNA敲除)。探讨这些受体在调节NMDA受体功能中的作用。同时，为了确定青春期期间介导的抑制对青春期后脊柱密度的影响，我们将进行慢性操作。功能(如上所述)在10天内？受体升高(~PND 35-45)，并在8周龄时量化脊柱密度。初步数据显示，在+/+小鼠中，脊柱密度确实在青春期后下降，但不是4-/-小鼠，这意味着4？青春期突触修剪中的GABA。将检查脊柱密度变化的免疫细胞化学和电生理学相关性。最后，为了探讨这种修剪对电路和行为可塑性的影响，我们还将确定这些变化对突触可塑性(长时程增强(LTP)、长时程抑制(LTD)、反转LTP)的影响，以及在两个海马区依赖的空间学习任务中对行为灵活性的影响。来自拟议研究的发现将直接涉及到 GABA对青春期树突棘的抑制作用，也将为青春期突触修剪过程提供机制，以及探索这一过程中变化的功能结果。这些结果将与自闭症等认知障碍相关， 其中，突触修剪减少和行为灵活性降低与？4基因的单核苷酸多态有关，这表明存在遗传联系。

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.