大脑皮层枝状吊灯细胞的命运决定与出生后发育

A fundamental issue in understanding neocortical neural circuitry concerns the nature of the identity and diversity of neuron types. Although a numerical minority compared to projection neurons, GABAergic interneurons contribute to a large part of neocortical cellular diversity. Chandelier cells are one of the most stereotyped and distinct classes of cortical interneurons. They are also called axo-axonic cells as their axons selectively synapse onto axon initial segment (AIS) of pyramidal neurons, the site of action potential initiation. Their axon arbor resembles chandelier lamp, with multiple presynaptic terminals aligning AIS of each postsynaptic pyramidal cell, forming vertical array of candlestick like structure (axon cartridge). One chandelier cell could innervate hundreds to thousands of pyramidal cells, likely exerting decisive control over pyramidal neuron output and neuronal network activity. Morphological abnormalities of chandelier cells, especially those related to axon cartridges, have been observed in epilepsy, schizophrenia and Alzheimer diseases. Despite their importance, current knowledge on chandelier cells is exceedingly poor. The main obstacles are their rarity and the lack of unique biochemical marker for reliable identification. Most previous studies relied on GAT1 and parvalbumin immunostaining to examine axon cartridges and blind patch-clamp recordings followed by whole cell reconstruction to study physiological feature and full cell morphology. It was impossible to follow developmental trajectory of chandelier cells or to functionally manipulate them until a recent discovery of their spatial and temporal origin in ventral germinal zone at late embryonic stage using genetic fate mapping in Nkx2.1-CreER mouse model. In this proposal, we will not only utilize the published mouse line but also develop new genetic mouse models to label and manipulate chandelier cells. Incorporating intersection of two recombinases, genetic inducible fate mapping and gene knockout, we will investigate the development origin of chandelier cells in more details and characterize their postnatal maturation. We will also examine their changes under conditions such as cell death blocking, decreased neuronal activity, sensory deprivation and in mouse model of Rett syndrome. We aim to characterize earlier production of chandelier cells, to search for additional transcriptional factors regulating chandelier cell fate, to investigate if and how genetic factors and neuronal activity interplay to influence chandelier cell maturation and circuit integration, and to explore their involvement in Rett syndrome. This study will be an important step towards a better understanding of chandelier cell development and function, laying foundation for future study on their connectivity, plasticity and pathology. It will also be an essential component in understanding the assembly of cortical neural circuits by diverse neurons types.

枝状吊灯细胞（Chandelier cell）是大脑皮层中的一种特殊中间神经元亚型，只在投射神经元轴突起始部建立GABA能突触，并通过这种高度特异的连接模式有效调控投射神经元的电活动和神经网络输出。虽然这类神经元对实现正常脑功能非常重要，其异常在多种神经疾病中也有所报道，但由于数量稀少且缺乏有效标记手段，对其发育、功能、疾病中作用的研究仍十分匮乏。最新研究通过在小鼠胚胎晚期端脑腹侧发生区中诱导Nkx2.1-CreER活性实现对其遗传标记，为深入研究前述科学问题提供了契机。申请人计划应用和拓展遗传小鼠模型，结合遗传诱导命运分析、双重组酶逻辑取交、基因敲除、神经元活性抑制、感觉剥夺等方法，进一步分析枝状吊灯细胞的胚胎来源和产生时间，鉴定其成熟过程中经历的主要变化，揭示调控其命运决定和发育的遗传与环境因素，探索其在雷特综合症中的异常，为深入研究其环路功能和其在神经疾病中的作用奠定基础。

枝状吊灯细胞是大脑皮层中的一种特殊中间神经元亚型，只在投射神经元轴突起始部建立GABA能突触，并通过这种高度特异的连接模式有效调控投射神经元的电活动和神经网络输出。虽然这类神经元对实现正常脑功能非常重要，其异常在多种神经疾病中也有所报道，但由于数量稀少且缺乏有效标记手段，对其发育、功能、疾病中作用的研究仍十分匮乏。最新研究通过在小鼠胚胎晚期端脑腹侧发生区中诱导Nkx2.1-CreER活性实现对其遗传标记，为深入研究前述科学问题提供了契机。我们应用和拓展遗传小鼠模型，结合遗传诱导命运分析、双重组酶逻辑组合、基因敲除、过表达等技术方法，得到以下研究结果：1）发现在胚胎发育较早期，位于内侧神经节隆起的背侧和腹侧的神经干细胞均能产生少量枝状吊灯细胞，所产生细胞的分层分布与胚胎来源有一定相关性，但与出生日期无关；2）利用标记基因取交或取补，可分别标记表达和不表达PV的枝状吊灯细胞亚型；3）建立了出生后诱导、逻辑组合标记枝状吊灯细胞的新策略；4）发现产生较晚的枝状吊灯细胞凋亡也较晚，凋亡的高峰在P9-11，不同皮层分区中凋亡的比例不同；发现增强兴奋性输入可改变不同皮层分区中细胞分布的差异；揭示出神经活性可能通过调控凋亡对皮层不同分区微环路中枝状吊灯细胞的比例进行调控；5）发现阻断凋亡后，仍然不是全部的枝状吊灯细胞都能正常成熟，说明构成皮层微环路的中间神经元存在亚型特异性的精密调控；6）原实验方案未能成功特异敲除枝状吊灯细胞中的Mecp2基因，正在建立和测试新的模型开展研究。上述结果为理解枝状吊灯细胞的发育提供了重要信息，也为深入研究枝状吊灯细胞的功能提供了技术手段。