大脑皮层GABA能投射神经元的分型和环路连接

There are two major classes of neurons in the neocortex: glutamatergic excitatory neurons and GABAergic inhibitory neurons. Glutamatergic pyramidal cells constitute the major cortical output channels, whereas GABAergic neurons project by and large locally, which are often referred to as ‘interneurons’. Even though it has been long known that some neocortical GABAergic cells can project at long distance and connect functional distinct brain regions, these cortical-cortical and cortical-fugal GABAergic projection neurons were often neglected when studying neocortical GABAergic neurons. In this proposal, we plan to implement multiple combinatorial strategies and a suite of genetic and viral tools that enable genetic targeting of GABAergic projection neuron subtypes. Strategically designed Flp and Cre pairs could be used to combine multiple features, such as expression of two marker genes, or the expression of one marker gene and the axonal projection pattern defined by retrograde transportation of viral particles. For example, cortical-cortical projecting Type I nNOS neurons co-expressing nNOS and SST could be labelled by intersection of nNOS-CreER and SST-ires-Flp. PV+ and VIP+ GABAergic projection neurons could be labelled in transgenic Cre or Flp mouse line injected with Flp or Cre expressing retrograde-transported virus. Besides, the development of “TripleTrigger” system and its extension allowed retrograde tracing of monosynaptic inputs onto GABAergic projection neurons. Utilizing these tools, we plan to analyze and compare the connectivity and electrophysiological properties of neocortical GABAergic projection neurons in a subtype-specific way. This study will not only be an important step towards understanding the logic of inhibitory circuit assembly, but also will facilitate our future research about the function of neocortical GABAergic projection neurons.

大脑皮层的GABA能神经元主要在本地建立抑制性突触，常被称为中间神经元。但是，部分GABA能神经元可对其它皮层分区或皮层外脑区进行远距离轴突投射，即GABA能投射神经元。这类神经元在大脑皮层中广泛存在，可能参与调控多种重要脑功能，但迄今为止对其发育起源、亚型分类、环路连接和功能的研究匮乏、认识有限。申请人新近发展了多系统组合遗传靶标神经元亚型的策略和工具，为深入研究前述科学问题提供了基础和契机。在本项目中，申请人计划组合运用转基因小鼠和病毒技术，对大脑皮层中表达不同标记基因的GABA能投射神经元亚型进行特异靶标和环路示踪，解析其环路连接模式，追踪其环路构筑的发育过程，检测其电生理和形态特征，并通过亚型之间的相互比较，揭示亚型特异性环路连接规律。这项研究将为探索GABA能投射神经元的功能提供必需的技术手段和有指导价值的研究思路，对于全面理解大脑皮层抑制性神经环路的构成和运行机制具有重要意义。

大脑皮层中的GABA能神经元主要在本地建立抑制性突触，因此常被称为中间神经元。但是，部分GABA能神经元也可进行远距离投射，为皮层间的信息交换与对外的信息输出提供了独立于兴奋性投射之外的重要渠道。但是，受阻于研究技术的局限，迄今为止对皮层GABA能投射神经元（GABAergic projection neuron, GPN）亚型分类与环路联接模式的认知仍十分有限。本项目发展了多系统组合标记皮层GABA能投射神经元亚型并进行环路示踪的遗传策略，着重研究了I型nNOS神经元的输入和输出，分别在群体和个体水平分析了该类神经元的轴突投射模式。此外，我们也利用顺行和逆行病毒对其他亚型的皮层GABA能投射神经元群体水平上的投射模式进行了探索。这项研究为探索皮层GPN的功能研究提供了必要工具和参考信息，对于全面深入了解皮层抑制性神经环路的构筑规律和运行机制具有重要意义。