Defining the role of a prefrontal-midbrain circuit in exploratory behavior

摘要 研究神经回路是如何调节自然行为的，对我们理解大脑至关重要。 探索行为是啮齿动物在自然界生存所必需的，在自由活动中无处不在。 啮齿动物实验。一种可能调节自然探索行为的神经回路是来自 前额叶皮质(PL，啮齿动物前额叶皮质的一部分)至腹侧被盖区(VTA)。对PL-VTA的刺激 投射导致速度加快，但并不是有益的，尽管直接投射具有众所周知的有益效果。 刺激VTA-多巴胺神经元。再加上VTA被牵连到 探索，我的中心假设是PL-VTA神经元通过 下游对非多巴胺能VTA神经元亚群的影响。 为了研究这一假设，我将首先开发一种基于机器学习的无偏见方法来量化 未经训练的、自由移动的老鼠行为(实验1.1)。我的方法将利用多视角、高 分辨率视频、有监督的身体部位跟踪和基于无监督的机器学习的姿势聚类 方法：研究方法。我将使用这种方法来确定哪些特定的行为(养育、行走、梳理、嗅探等) 在存在和不存在奖赏的情况下，受到PL-VTA细胞的光遗传刺激或抑制的影响 (实验1.2)。初步数据证明了我的行为量化方法的可行性和 提示刺激PL-VTA可增加探索行为。 接下来，为了识别基因和靶向定义的VTA细胞，这些细胞优先与PL突触 投射方面，我将结合使用光遗传学、体外电生理学、转基因小鼠和逆行 追踪。首先，我将使用转基因小鼠来测量专门针对VTA的PL输入的功能强度 多巴胺能、GABA能或谷氨酸能细胞(实验2.1)。在另一组小鼠中，我将使用 逆行追踪以确定PL突触是否优先投射到伏隔核或VTA细胞 杏仁核(实验2.2)。在了解了哪些遗传和目标定义的VTA亚群收到PL后 输入，我将使用逆行追踪和转基因标记相结合的方法来具体识别 遗传和预测定义的VTA亚群接受PL输入(实验2.3)。最后，我将使用 逆行依赖病毒选择性地用ChR2感染VTA亚群，并使用我的行为 量化管道，以调查体内对这一亚群的刺激是否概括了 刺激PL-VTA的探索效应(实验2.4)。 因此，提出的项目将开发一种新的方法来量化自然主义行为，并将其 具有多种实验方法的计算方法，包括体内和体外光遗传学，自然 行为和突触生理学。这些实验将提供关于PL-VTA如何 回路调节自然的运动输出，独立于VTA-多巴胺活动的强化效应。

ABSTRACT Investigating how neural circuits mediate natural behavior is a critical to our understanding of the brain. Exploratory behaviors are necessary for rodent survival in the natural world and ubiquitous in freely-moving rodent experiments. One neural circuit that may mediate natural exploratory behaviors are the projections from prelimbic cortex (PL, part of rodent prefrontal cortex) to ventral tegmental area (VTA). Stimulation of PL-VTA projections causes increased velocity but is not rewarding, despite the well-known rewarding effect of direct stimulation of VTA-dopamine neurons. Taken together with the fact that VTA has been implicated in exploration, my central hypothesis is that PL-VTA neurons mediate natural exploratory behaviors through downstream effects on a subpopulation of non-dopaminergic VTA neurons. To investigate this hypothesis, I will first develop an unbiased, machine-learning-based method to quantify untrained, freely-moving mouse behaviors (Experiment 1.1). My method will leverage multi-view, high resolution video, supervised body part tracking, and unsupervised machine learning-based postural clustering methods. I will use this method to identify which specific behaviors (rearing, walking, grooming, sniffing, etc.) are affected by optogenetic stimulation or inhibition of PL-VTA cells in the presence and absence of rewards (Experiment 1.2). Preliminary data demonstrates the feasibility of my behavioral quantification method and suggests that PL-VTA stimulation increases exploratory behaviors. Next, to identify the genetically- and target-defined VTA cells that are preferentially synapsed onto by PL projections, I will use a combination of optogenetics, in vitro electrophysiology, transgenic mice, and retrograde tracing. First, I will use transgenic mice to measure the functional strength of PL input specifically onto VTA cells that are dopaminergic, GABAergic, or glutamatergic (Experiment 2.1). In a different set of mice, I will use retrograde tracing to identify if PL synapses preferentially onto VTA cells that project to nucleus accumbens or amygdala (Experiment 2.2). After learning which genetic and target-defined VTA subpopulations receive PL input, I will use retrograde tracing in combination with transgenic labeling to identify specifically which genetically and projection-defined VTA subpopulation receives PL input (Experiment 2.3). Finally, I will use a retrograde cre-dependent virus to selectively infect this VTA subpopulation with ChR2 and use my behavioral quantification pipeline to investigate whether in vivo stimulation of this subpopulation recapitulates the exploratory effects of PL-VTA stimulation (Experiment 2.4). Thus, the project proposed will develop a novel method for quantifying naturalistic behavior and integrate this computational method with diverse experimental methods, including in vivo and in vitro optogenetics, natural behavior, and synaptic physiology. These experiments will contribute novel information on how the PL-VTA circuit mediates naturalistic motor output, independent of the reinforcement effects of VTA-dopamine activity.