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，这一过程在自闭症和精神分裂症中是失调的。这种突触修剪的机制尚不清楚，但可能与NMDA受体激活的减少有关。我们最近发现，在青春期（~PND 35 ~ 45），突触外α4βδ GABAA受体（gabar）出现在小鼠CA1海马锥体细胞的树突棘上约10d。此外，NMDA受体的激活在+/+小鼠中受损，而在α4或δ-/-小鼠中没有受损，暗示α4βδ参与了青春期NMDA功能的降低。在本研究中，我们将验证这一假设，即青春期突触修剪确实是由于α4βδ gabar的表达增加，我们将探索这种影响的细胞、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，双光子解锁，电生理，药理学，解剖学和免疫细胞化学分析，以直接评估这种新型抑制对青春期脊柱的功能结果。最初，RuBiGABA（一种新型笼型GABA化合物）的光化学技术将用于绘制青春期脊柱中的GABA能电流。这项技术的初步数据表明，脊椎具有gaba能电流。然后，这些受体对NMDA受体诱导的来自个体脊柱的Ca2+内流的影响将使用双光子Ca2+成像和局部释放Rubi-GABA来检查。这些实验还将利用α4βδ的急性药理学（激动剂、调节剂）和基因操作（敲除、腺病毒- shrna敲除）来探索这些受体在调节NMDA受体功能中的作用。同时，为了确定青春期α4βδ介导的抑制对青春期后脊柱密度的影响，我们将在α4βδ受体升高（~PND 35-45）的10天内长期操纵α4βδ功能（如上所述），并在8周龄时量化脊柱密度。初步数据显示，+/+小鼠的脊柱密度确实在青春期后下降，而α4-/-小鼠的脊柱密度则没有下降，这暗示α4βδ GABARs参与了青春期突触修剪。将检查脊柱密度变化的免疫细胞化学和电生理相关因素。最后，为了探讨这种修剪对回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（长期增强（LTP）、长期抑制（LTD）、LTP逆转）的影响，以及对两个海马依赖的空间学习任务的行为灵活性的影响。这些研究结果将直接解决青春期gaba能抑制对树突棘的功能作用，并将提供青春期突触修剪过程的机制，以及探索该过程改变的功能结果。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α4基因的单核苷酸多态性有关，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 α4βδ 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 α4 or δ-/- mice, implicating α4βδ 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 α4βδ 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 α4βδ to explore the role of these receptors in regulating NMDA receptor function. In parallel, to determine the impact of α4βδ-mediated inhibition during puberty on spine density post-pubertally, we will chronically manipulate α4βδ function (as above) over the 10 days when α4βδ 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 α4-/- mice, implicating α4βδ 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 α4 gene, suggesting a genetic link.