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、大麻和CCK系统通过海马神经元相互作用，从而共同调节锥体细胞的兴奋性。低频神经元群体振荡(特别是4-12赫兹的theta“？”和30-80 hz的伽马“？”节律)代表着对高级认知处理至关重要的大脑状态。GABA能篮子细胞微电路中的活动是几种形式的节律产生所必需的，然而关于潜在的细胞机制的许多问题仍然没有得到解决，主要是因为现有的工具不足以选择性地研究稀疏和广泛分布的细胞电路。被称为低频振荡(LFO)的模型现象捕捉到了体内复杂得多的大脑节律的一些特性。本申请将使用新的光遗传学方法在微电路水平上解决LFO机制的问题。我们的目标是阐明基本的细胞电路属性，这可能有助于阐明体内现象。分子生物学工具将被用来通过病毒载体将光敏分子(“Opsins”)引入靶细胞群。这些载体包含一个双花倒置基因，用于融合蛋白，该融合蛋白由视蛋白和荧光标记蛋白组成。在细胞特异性启动子的控制下，将载体导入表达Cre重组酶的转基因小鼠的大脑。我们将针对表达小白蛋白(PV)或胆囊素(CCK)的海马区中间神经元，或表达乙酰胆碱(ACh)的内侧隔区的细胞作为视蛋白。根据其分子特性，特定视蛋白的光激活将刺激或抑制表达它的细胞。我们将使用激动剂 视黄素、视紫红质2，以及抑制视黄素、卤视紫红质的实验建议。适当波长的闪光会刺激或抑制特定的细胞网络，即使这些细胞分散在组织中。光遗传学方法将补充对单个神经元的高分辨率电生理分析。在海马区，mU阿片受体或大麻素受体CB1R分别高密度地分布在PV或CCK细胞上。我们将询问受？ORS和CB1Rs调控的单个微电路如何相互作用来促进抑制性LFO。具体目的是：目的1：检验CCK和PV细胞相互影响的假说。目的#2：验证CCK和PV细胞IPSP共同产生LFO-LFP的假设。目的#3：验证内源性ACh可触发LFO-IPSCs和LFO-LFP的假设。 公共卫生相关性：健康相关性：抑制微电路功能紊乱与精神疾病有关，如精神分裂症、自闭症和阿尔茨海默病等。此外，鸦片类药物和大麻类药物在影响行为方面有相互作用，但它们相互作用的方式尚不清楚。对PV和CCK细胞微电路之间的串扰的详细了解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.