Molecular Mechanisms of Inhibitory Circuit Development

描述（由申请人提供）：在理解皮层网络发育期间如何实现兴奋性和抑制性（E/I）连接的适当平衡以及如何通过突触可塑性进行调整以实现大脑皮层的正常功能方面存在根本性差距。在这个空白被填补之前，理解具有GABA能抑制连接缺陷的神经精神疾病，如精神分裂症和自闭症，将仍然是一个谜。长期目标是确定在前额叶皮层建立E/I平衡的分子机制，这可能会确定这种平衡被改变的疾病的新目标。目的是定义一种新的机制来限制篮状中间神经元与发育中的前额叶皮质锥体神经元体周区之间的抑制性连接。核心假设是神经细胞粘附分子NCAM、酪氨酸激酶EphA 3和ADAM 10金属蛋白酶组成突触后ephrinA 5的突触前受体复合物，该复合物促进消除对适当的前额叶网络组织和功能至关重要的体周突触，例如在工作记忆中。目标1。为了鉴定通过NCAM依赖性ephrinA 5/EphA 3信号传导限制发育中的小鼠前额皮质中的体周篮细胞神经支配的新分子机制，我们将鉴定NCAM/EphA 3结合位点，评估NCAM通过抑制内吞作用和促进ephrinA 5诱导的EphA 3激酶信号传导来稳定细胞表面上的EphA 3的能力，并确定ephrinA 5在小鼠前额叶皮层中的发育和活动依赖性调节。目标2.为了确定突触前和突触后的功能NCAM，ephrinA 5/EphA 3，和ADAM 10金属蛋白酶在体周抑制性突触调节新的条件性NCAM和ADAM 10突变小鼠和细胞特异性表达在脑切片的分析将区分突触前与突触后的功能NCAM，ephrinA 5/EphA 3，和ADAM 10，并测试其相互作用的因果关系在体周突触调节。抑制性突触消除的动力学将在皮层切片培养中通过延时双光子显微镜进行分析。目标3。采用光遗传学作图和工作记忆行为学评估方法，描述NCAM对前额叶皮层网络组织和功能的贡献。 将在来自NCAM无效和条件突变小鼠的脑切片中进行光遗传学作图，所述NCAM无效和条件突变小鼠在中间神经元中从VGAT启动子表达通道视紫红质-2。将通过延迟的非样本匹配T-迷宫任务在活小鼠中测量工作记忆表现。 这些研究的结果预计将产生持续的积极影响，因为它将阐明控制认知功能的神经元间连接的新分子机制，而创新的光遗传学技术将阐明神经发育障碍中靶向的皮质网络。

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how an appropriate balance of excitatory and inhibitory (E/I) connectivity is achieved during development of cortical networks and adjusted through synaptic plasticity for normal functioning of the cerebral cortex. Until this gap is filled, understanding neuropsychiatric disorders with GABAergic inhibitory connection deficits, such as schizophrenia and autism, will remain a mystery. The long term goal is to identify the molecular mechanisms that establish E/I balance in the prefrontal cortex, which may identify new targets for disorders where this balance is altered. The objective is to define a novel mechanism for limiting inhibitory connections between basket interneurons and the perisomatic region of pyramidal neurons in developing prefrontal cortex. The central hypothesis is that neural cell adhesion molecule NCAM, tyrosine kinase EphA3, and ADAM10 metalloprotease comprise a presynaptic receptor complex for postsynaptic ephrinA5 that promotes elimination of perisomatic synapses critical for proper prefrontal network organization and functioning, such as in working memory. Aim 1. To identify a novel molecular mechanism for limiting perisomatic basket cell innervation in the developing mouse prefrontal cortex through NCAM-dependent ephrinA5/EphA3 signaling We will identify NCAM/EphA3 binding sites, assess the ability of NCAM to stabilize EphA3 on the cell surface by inhibiting endocytosis and promoting ephrinA5-induced EphA3 kinase signaling, and define the developmental and activity-dependent regulation of ephrinA5 in mouse prefrontal cortex. Aim 2. To define presynaptic and postsynaptic functions of NCAM, ephrinA5/EphA3, and ADAM10 metalloprotease in perisomatic inhibitory synapse regulation Analysis of new conditional NCAM and ADAM10 mutant mice and cell-specific expression in brain slices will distinguish pre- versus post-synaptic functions for NCAM, ephrinA5/EphA3, and ADAM10, and test causal roles for their interactions in perisomatic synapse regulation. Dynamics of inhibitory synapse elimination will be analyzed by time-lapse two-photon microscopy in cortical slice cultures. Aim 3. To delineate the contribution of NCAM to prefrontal cortical network organization and function using optogenetic mapping and behavioral assessment of working memory. Optogenetic mapping will be performed in brain slices from NCAM null and conditional mutant mice expressing channelrhodopsin-2 from the VGAT promoter in interneurons. Working memory performance will be measured in live mice by the delayed non-match-to-sample T-maze task. The outcome of these studies is expected to have a sustained, positive impact, because it will illuminate novel molecular mechanisms of interneuronal connectivity that control cognitive function, while innovative optogenetic technology will elucidate cortical networks targeted in neurodevelopmental disorders.