Interactions Among Hippocampal Interneuron Circuits

描述（申请人提供）：本项目的主要假设：内源性阿片类药物、大麻素和CCK系统通过海马中间神经元相互作用，从而共同调节锥体细胞的兴奋性。低频神经元群体振荡（尤其是 θ“？”，4-12 Hz 和伽马“？”，30-80 Hz，节律）代表对高阶认知处理至关重要的大脑状态。 GABA能篮状细胞微电路的活性是多种形式的节律产生所必需的，但有关潜在细胞机制的许多问题尚未解决，很大程度上是因为现有工具不足以选择性地研究稀疏且广泛分散的细胞电路。称为低频振荡 (LFO) 的模型现象捕获了更为复杂的体内大脑节律的一些特性。本申请将使用新的光遗传学方法来解决有关微电路级别 LFO 机制的问题。目标是阐明可能有助于阐明体内现象的基本细胞电路特性。分子生物学工具将用于通过病毒载体将光敏分子（“视蛋白”）引入目标细胞群。该载体含有双flox反向基因，用于融合蛋白，该融合蛋白由视蛋白和荧光标记蛋白组成。将载体引入在细胞特异性启动子控制下表达 Cre 重组酶的转基因小鼠的大脑中。我们将视蛋白靶向海马中表达小白蛋白 (PV) 或表达胆囊收缩素 (CCK) 的中间神经元，或内侧隔膜中表达乙酰胆碱 (ACh) 的细胞。根据其分子特性，特定视蛋白的光激活将兴奋或抑制表达它的细胞。我们将使用兴奋剂 拟议实验中的视蛋白、Channelrhodopsin2 和抑制性视蛋白、Halorhodopsin。即使细胞分散在组织中，适当波长的闪光也会激发或抑制特定的细胞网络。光遗传学方法将补充单个神经元的高分辨率电生理分析。在海马体中，μ-阿片受体 ?OR 或大麻素受体 CB1R 分别以高密度分离在 PV 或 CCK 细胞上。我们将询问受 ?OR 和 CB1R 调节的各个微电路如何相互作用以促进抑制性 LFO。具体目标是： 目标#1：检验 CCK 和 PV 电池相互影响的假设。目标#2：测试 CCK 和 PV 电池 IPSP 共同生成 LFO-LFP 的假设。目标#3：测试 LFO-IPSC 和 LFO-LFP 可以由内源性 ACh 触发的假设。 公共健康相关性： 健康相关性：抑制性微电路功能紊乱与精神分裂症、自闭症和阿尔茨海默病等精神疾病有关。此外，阿片类药物和大麻素药物具有相互作用影响行为的作用，但它们相互作用的方式尚不清楚。详细了解 PV 和 CCK 细胞微电路之间的串扰将对重要的神经系统问题产生影响。

DESCRIPTION (provided by applicant): Major hypothesis of this project: The endogenous opioid, cannabinoid, and CCK systems mutually interact via hippocampal interneurons and thereby jointly regulate the excitability of pyramidal cells. Low frequency neuronal population oscillations (especially theta "?", 4-12 Hz, and gamma "?", 30-80 Hz, rhythms) represent brain states that are crucial for high order cognitive processing. Activity in GABAergic basket cell microcircuits is required for several forms of rhythm generation, yet many questions regarding the underlying cellular mechanisms are unresolved, largely because available tools are inadequate for selectively investigating the sparse and widely dispersed cellular circuits. Model phenomena, called low frequency oscillations (LFOs), capture some of the properties of the much more complex in vivo brain rhythms. The present application will use new optogenetic methods to address questions about mechanisms of LFOs at the microcircuit level. The goal is to elucidate fundamental cell circuit properties that may help illuminate the in vivo phenomena. Molecular biological tools will be used to introduce light-sensitive molecules ("opsins") into targeted cell groups via viral vectors. The vectors contain a double-floxed, inverted gene for a fusion protein consisting of an opsin plus a fluorescent marker protein. Vectors are introduced into the brains of transgenic mice expressing Cre-recombinase under the control of cell-specific promoters. We will target the opsins to either the parvalbumin (PV) - expressing, or cholescystokinin (CCK) - expressing interneurons in hippocampus, or acetylcholine (ACh) - expressing cells in the medial septum. Depending on its molecular properties, light-activation of a particular opsin will either excite or inhibit the cell expressing it. We will use the excitatory opsin, Channelrhodopsin2, and the inhibitory opsin, Halorhodopsin in the proposed experiments. Flashes of light of an appropriate wavelength will either excite or inhibit defined networks of cells, even though the cells are scattered in the tissue. Optogenetic methods will complement high resolution electrophysiological analysis of individual neurons. In the hippocampus, the mu-opioid receptor, ?OR, or the cannabinoid receptor, CB1R, are segregated at high density on the PV or CCK cells, respectively. We will ask how the individual microcircuits regulated by ?ORs and CB1Rs interact to foster inhibitory LFOs. The Specific Aims are to: Aim #1: Test the hypothesis that CCK and PV cells mutually influence each other. Aim #2: Test the hypothesis that CCK and PV cell IPSPs collectively generate LFO-LFPs. Aim #3: Test the hypothesis that LFO-IPSCs and LFO-LFPs can be triggered by endogenous ACh. PUBLIC HEALTH RELEVANCE: Health Relevance: Disordered function of inhibitory microcircuits has been implicated in psychiatric disorders such as schizophrenia, autism, and Alzheimer's Disease, among others. In addition, opiate and cannabinoid drugs have actions that interact in affecting behavior, and yet the ways in which they interact is not known. A detailed understanding of the cross-talk between PV and CCK cell microcircuits will impact on important neurological problems.