Molecular Mechanisms of Inhibitory Circuit Development

描述（由申请人提供）：在皮层网络发育过程中，兴奋性和抑制性（E/I）连接的适当平衡是如何实现的，并通过突触可塑性调节大脑皮层的正常功能，在理解这一点上存在根本性的差距。在这一空白被填补之前，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皮质中建立E/I平衡的分子机制，这可能会确定这种平衡改变的疾病的新靶点。目的是确定一种新的机制来限制篮状中间神经元和发育中的前额叶皮层锥体神经元之间的抑制性连接。核心假设是神经细胞粘附分子NCAM、酪氨酸激酶EphA3和ADAM10金属蛋白酶组成突触前受体复合物，用于突触后ephrinA5，促进消除对正常的前额叶网络组织和功能至关重要的突触，例如在工作记忆中。目的1。我们将确定NCAM/EphA3结合位点，评估NCAM通过抑制胞内吞噬和促进ephrinA5诱导的EphA3激酶信号传导来稳定EphA3在细胞表面的能力，并确定ephrinA5在小鼠前额皮质的发育和活性依赖性调节。目标2。研究NCAM、ephrinA5/EphA3和ADAM10金属蛋白酶的突触前和突触后功能，分析新的条件型NCAM和ADAM10突变小鼠和脑片细胞特异性表达，将区分NCAM、ephrinA5/EphA3和ADAM10的突触前和突触后功能，并测试它们在细胞周围突触调节中的相互作用的因果关系。抑制性突触消除的动力学将通过延时双光子显微镜在皮层切片培养分析。目标3。利用光遗传作图和工作记忆行为评估来描述NCAM对前额皮质网络组织和功能的贡献。将在NCAM缺失和条件突变小鼠的脑切片中进行光遗传定位，这些小鼠在中间神经元中表达来自VGAT启动子的通道视紫红质-2。工作记忆表现将通过延迟非匹配-样本t -迷宫任务来测量。这些研究的结果有望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针对神经发育障碍的皮层网络。

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.