未定带脑区GABA能长投射神经元在皮层局部癫痫中的作用

Since first described in 1877 as a “zone of uncertain function”, the zona incerta (ZI) and its role in the brain remains incompletely understood. Our recent work demonstrated the direct evidence that ZI GABAergic neurons project widely to cortical layer 1 and establish depolarizing GABAergic synapses onto pyramidal neurons in multiple layers of the developing neocortex, thus forming one of the earliest cortical circuits. Blocking ZI GABAergic synaptic activity resulted in reduced apical, but not basal, dendritic branching and reduced synaptic activity of cortical pyramidal neurons; The ZI GABAergic projection becomes inhibitory with maturation and can block epileptiform activity in the adult brain. In this proposed study, we will examine the diversity and function of ZI GABAergic projection neurons in cortical development and disease. We will combine state-of-art technologies including single-cell transcriptomics, optogenetic-based circuit manipulation, in vivo optical and electrical recordings, and behavioral assays to decipher the neural circuits that are defective in brain disorders. The potential results will lead to new insights in understanding the cell types and neural circuits that are targeted by human disease mutations. These results hold the potential to enable the development of circuit-based neural therapies targeting brain diseases.

自从1877年首次被发现并被描述为“功能不明的脑区”，未定带脑区（Zona Incerta）在大脑中的功能尚未完全阐明，申请人的近期工作揭示：腹侧丘脑的未定带脑区有一类特殊的GABA能神经元，其轴突能够远距离投射至大脑皮层，而被称为GABA能长投射神经元。未定带脑区GABA能长投射神经元能够与皮层谷氨酸能神经元建立突触联系，并在发育早期调节皮层神经元顶树突和突触发育；而在成年大脑能够抑制皮层癫痫状放电，提示未定带可能是治疗癫痫等神经发育疾病的重要潜在靶点。本项目拟针对未定带脑区GABA能长投射神经元的分子亚类及对脑功能的调控，结合单细胞测序、神经环路示踪、在体光遗传、多道电极记录和动物行为学，研究未定带脑区GABA能长投射神经元多样性和发育起源，以及如何实时调控大脑功能。预期成果将揭示神经发育和精神类疾病如癫痫和自闭症相关的环路机制，并为发展以神经环路为依据的疾病诊疗寻找新靶点。

丘脑下核团未定带脑区的神经元异质性高且与全脑形成广泛的投射连接。此前的研究表明未定带脑区神经元能够参与多种行为学及生理功能的调节，本研究对未定带脑区的神经元进行了神经元亚类的分型并对其功能进行了探究。运用单细胞核RNA测序技术检测了成年小鼠未定带脑区的神经元分子亚类，本研究发现了在未定带脑区区域特异性高表达的神经元标志物，并鉴定了未定带脑区不同区域神经元亚类特异的基因标志物。在脑组织水平通过荧光组织原位杂交（RNAscope）检测了神经元基因标志物的区域分布特征：发现在未定带脑区前端神经元高表达Ecel1及Cdh23，在未定带脑区的中段神经元高表达Pde11a、Ptprk、Nos1及Pax6。我们进一步针对未定带脑区区域特异性表达基因，构建了新的未定带脑区前端和中段神经元特异标记的转基因小鼠、结合在体光遗传学研究未定带脑区不同GABA能神经元亚类的行为学功能。研究结果揭示了未定带脑区GABA能神经元具有复杂的基因表达和功能抑制性，且基因表达和神经元的脑区投射特征共同决定了神经元亚类的功能。研究成果阐明了未定带脑区前端SST神经元至丘脑连接核（nucleus reuniens,Re）投射调控先天恐惧防御行为，未定带脑区前端Ecel1神经元至导水管周围灰质（periaqueductal gray，PAG）GABA能投射调控理毛行为，及未定带脑区中段Pde11a神经元至脑桥网状结构区（pontine reticular formation，PnO）GABA能投射调控睡眠觉醒的神经环路机制，拓展了未定带脑区神经元亚型的功能图谱。