Neurocircuitry Underlying Leptin Regulation of Energy Balance

描述(申请人提供)：对调节摄食和代谢的神经回路和神经生物学基础知之甚少，这极大地限制了对肥胖和摄食障碍的理解和治疗的进展。缺乏这方面的知识在很大程度上是由于控制这些过程的大脑区域的复杂性--即那些位于下丘脑内并与之相连的区域。每个解剖分区包含许多不同类型的神经元，每种神经元控制着无关、相反或未知的功能。虽然存在关于亚区之间连接的一般信息，但这提供的机制洞察力很少，因为每个亚区内不同神经元的功能是复杂的和/或未知的，连接特定上游神经元和特定下游神经元的标记线也是未知的。从本质上说，我们缺乏下丘脑控制行为和生理的“接线图”。随着最近的技术进步，在表达神经元特异性Cre的小鼠的支持下，现在有可能以细胞特有的方式建立连接和功能。本提案利用这些方法来描绘瘦素调节能量平衡的神经回路。这些研究建立在我们最近的发现基础上，即瘦素的大部分减肥作用是由瘦素介导的 GABA能神经元上的受体。在目标1中，我们开始确定瘦素反应性GABA能传入POMC和AgRP神经元的来源。在初步研究中，利用CRE依赖的单突触狂犬病标测和通道视紫质辅助回路标测，我们已经确定，对于POMC神经元，瘦素反应性GABA能输入完全来自局部神经元(全部在弓状体内)，而对于AgRP神经元，非常强的瘦素反应性GABA能输入来自下丘脑背内侧(DMH)。鉴于这些DMH传入AgRP神经元在控制饥饿方面的关键功能重要性(如目标2所示)，我们正在使用单神经元RNA-Seq来确定它们的身份，检测可能暗示功能的基因(如那些参与神经递质合成和运输的基因、神经肽和受体)，并开始寻找可能的药物靶点。在目标2中，我们使用光遗传学技术在清醒的行为小鼠中确定DMH Leptin受体表达的GABA能传入AgRP神经元在调节饥饿中的作用。在初步研究中，我们发现，这些传入的光基因激活完全阻止了饥饿，甚至是禁食引起的饥饿。因此，这些神经元以前是未知的饥饿的有力调节者。最后，在目标3中，我们在清醒的小鼠身上进行光电极谱记录，以实时确定AgRP神经元及其DMH-瘦素反应性GABA能传入的放电率。通过使用光/ChR2诱发的尖峰信号来识别神经元，这些研究解决了以前难以解决的关于行为和生理扰动对体内放电频率的影响的问题。总而言之，这些研究应该会显著促进我们对瘦素及其调控的回路如何控制进食和能量平衡的理解。

DESCRIPTION (provided by applicant): Little is known about the neurocircuit and neurobiological bases for regulation of feeding and metabolism, and this has greatly limited progress in understanding and treating obesity and feeding disorders. Lack of knowledge in this area is due, in large part, to complexity within brain regions controlling these processes - namely those that lie within and are connected to the hypothalamus. Each anatomic subregion contains many different types of neurons, each controlling unrelated, opposite or unknown functions. While general information exists regarding connectivity between subregions, this provides little mechanistic insight because the functions of the different neurons within each subregion are complex and/or unknown, and the labeled lines connecting specific upstream neurons to specific downstream neurons are also not known. In essence, we lack a "wiring diagram" for hypothalamic control of behavior and physiology. With recent technological advances, enabled by neuron-specific Cre-expressing mice, it is now possible in a cell-specific fashion to establish connectivity and function. The present proposal utilizes such approaches to delineate the neurocircuitry underlying leptin regulation of energy balance. These studies build upon our recent discovery that the majority of leptin's anti-obesity effects are mediated by leptin receptors on GABAergic neurons. In Aim 1, we set out to identify the source of leptin-responsive GABAergic input to POMC and AgRP neurons. In preliminary studies, using Cre-dependent monosynaptic rabies mapping and channelrhodopsin-assisted circuit mapping, we have determined that, for POMC neurons, leptin-responsive GABAergic input is entirely from local neurons (all within the arcuate), while for AgRP neurons, very strong leptin-responsive GABAergic input comes from the dorsomedial hypothalamus (DMH). Given the key functional importance of these DMH afferents to AgRP neurons in controlling hunger (as shown in Aim 2), we are using Single-Neuron RNA-Seq to determine their identity, to detect genes likely to suggest function (such as those involved in neurotransmitter synthesis and transport, neuropeptides and receptors), and to begin a search for possible drug targets. In Aim 2, we are using optogenetic techniques in awake, behaving mice to determine the role of DMH leptin receptor-expressing GABAergic inputs to AgRP neurons in regulating hunger. In preliminary studies we have discovered that optogenetic activation of these afferents completely blocks hunger, even that caused by fasting. Thus, these neurons are previously unknown potent regulators of hunger. Finally, in Aim 3, we are performing optetrode recordings in awake, behaving mice to determine, in real time, the firing rate of AgRP neurons and their DMH leptin-responsive GABAergic afferents. By using light/ChR2- evoked spiking to identify neurons, these studies address previously inaccessible questions regarding effects of behavioral and physiologic perturbations on in vivo firing rates. In total, these studies should significantly advance our understanding of how leptin, and the circuits it regulates, control eating and energy balance.