Probing neural circuits underlying satiety

探究饱腹感背后的神经回路

• 批准号: 9549950
• 负责人: Michael J. Krashes
• 金额: $ 33.12万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9549950
• 关键词: Acute; Agonist; Amygdaloid structure; Anatomy; Animals; Behavioral; Binding; Biological Neural Networks; Body Weight; Chronic; Complex; Data; Dependovirus; Eating; Enterobacteria phage P1 Cre recombinase; Feeding behaviors; Feeling; Future; Grant; Hormones; Hypothalamic structure; Immunohistochemistry; Injectable; Injection of therapeutic agent; Ion Channel; Ion Pumps; Knock-out; Knockout Mice; Label; Laboratories; Lateral; Lead; Ligands; Light; Maps; Medial; Mediating; Melanocortin 4 Receptor; Mus; Neurons; Nucleus Accumbens; Nucleus solitarius; Obesity; Output; Pharmacogenetics; Pharmacology; Physiological; Population; Pro-Opiomelanocortin; Protocols documentation; Receptor Gene; Regulation; Reporter; Role; Satiation; Self Stimulation; Signal Transduction; Site; Structure of nucleus infundibularis hypothalami; Structure of terminal stria nuclei of preoptic region; Synaptophysin; Thalamic structure; Tracer; Transgenic Organisms; Virus; base; cell type; design; experimental study; falls; feeding; in vivo; melanocyte; midbrain central gray substance; neural circuit; neuronal cell body; optical imaging; optogenetics; parabrachial nucleus; paraventricular nucleus; preference; promoter; receptor; relating to nervous system; restoration

To pinpoint the downstream projection targets of POMC neurons that mediate satiety. Hypothesis: Hypothalamic POMCARC neuron projections modulate MC4R+ neurons in the paraventricular nucleus of the hypothalamus (PVN) to signal satiety. Rationale: Studies have demonstrated that the selective restoration of MC4R activity in the PVN and a subpopulation of amygdaloid neurons of MC4R-null knockout mice rescued the MC4R knockout-induced obesity (Balthasar 2005). Our lab has access to a MC4R-T2a-Cre mouse line, which expresses Cre recombinase under the endogenous promoter of the Mc4r gene, allowing precise access to mark, manipulate and record activity from this specific subset. Our lab also has access to the POMC-Cre mouse line for the purpose of probing neural connections and modulation. This experiment is designed to elucidate the anatomical structures in which MC4Rs are expressed to mediate satiety, as well as the connections and modulatory roles of POMCARC neurons. Approach: We will identify MC4R-expressing neurons by crossing MC4R-T2a-Cre with a GFPflox/flox reporter mouse and in combination with anterograde tracing (via a Cre-dependent adeno-associated virus (AAV) expressing synaptophysin injected into the arcuate (ARC) nucleus of POMC-Cre mice) and immunohistochemistry, map POMCARC projections in this complex network. Based on these tracing studies, we will selectively target the light-activated ion channel, channelrhodopsin2 (ChR2) specifically to POMCARC neurons (via stereotaxic injection of a Cre-dependent AAV-ChR2), followed by selective photo-stimulation of distinct POMC terminal fields and assess the resulting behavioral output. These in vivo optogenetic studies will tease apart the functionally relevant downstream sites of POMCARC neurons that decrease food intake and body weight, and ultimately lead to a clearer idea of the neural circuits controlling satiety. We will also generate double transgenic POMC-Cre; MC4R-T2a-Cre mice, which allows for bimodal regulation of two separate neural populations, to verify sequential neural networks. For instance, we can acutely activate the POMC terminal field to the PVN, which we hypothesize will result in reduced feeding, and simultaneously inhibit the relevant downstream MC4RPVN neurons (via Cre-dependent viruses expressing inhibitory GPCRs or ion pumps), which we hypothesize will reverse this fall in food intake. These occlusion studies are extremely elegant and grant the experimenter unparalleled control of these specific circuits. Predictions, interpretations & Future Experiments: We do not anticipate any problems with these experiments. Preliminary data has already been generated labeling MC4R+ neurons and we will subsequently assess POMCARC projections to this cell type both within and outside the hypothalamus. In addition, the viruses proposed here have been used successfully by a number of laboratories. We predict that activation of the POMCARC terminal field in the PVN will reduce feeding behavior and ultimately reduce body weight. Furthermore, we have preliminary data showing that DREADD-mediated acute inhibition (a pharmaco-genetic approach used to acutely manipulate neural activity via specific expression of an exogenous receptor and its subsequent binding and activation through a pharmacologically inert ligand; Krashes et al., 2011) of MC4RPVN neurons can drive feeding behavior, and that MC4RPVN neurons receive direct monosynaptic inputs from AGRP neurons (utilizing AgRP-ires-Cre mice), both strong lines of evidence that suggest an opposing role for POMCARC neurons on these downstream targets.

确定POMC神经元下游投射靶点，介导饱腹感。 假设：下丘脑POMCARC神经元投射调节下丘脑室旁核（PVN）中的MC 4 R+神经元以发出饱腹感信号。 基本原理：研究表明，MC 4 R无效敲除小鼠的PVN和杏仁核神经元亚群中MC 4 R活性的选择性恢复挽救了MC 4 R敲除诱导的肥胖症（Balthasar 2005）。我们的实验室已经获得了MC 4 R-T2 a-Cre小鼠系，该小鼠系在Mc 4 r基因的内源启动子下表达Cre重组酶，从而可以精确地标记、操作和记录该特定子集的活性。我们的实验室还可以使用POMC-Cre小鼠系，以探测神经连接和调制。本实验旨在阐明MC 4 R表达介导饱腹感的解剖结构，以及POMCARC神经元的连接和调节作用。 方法：我们将通过将MC 4 R-T2 a-Cre与GFPflox/flox报告小鼠杂交并结合顺行追踪（通过将表达突触体素的Cre依赖性腺相关病毒（AAV）注射到POMC-Cre小鼠的弓状（ARC）核中）和免疫组织化学来鉴定MC 4 R表达神经元，绘制POMCARC在该复杂网络中的投射。基于这些追踪研究，我们将选择性地将光激活离子通道，通道视紫红质2（ChR 2）特异性地靶向POMCARC神经元（通过Cre依赖性AAV-ChR 2的立体定位注射），然后选择性地光刺激不同的POMC终末场并评估所得的行为输出。这些体内光遗传学研究将梳理POMCARC神经元的功能相关下游位点，这些位点减少食物摄入量和体重，并最终导致对控制饱腹感的神经回路有更清晰的认识。我们还将产生双转基因POMC-Cre; MC 4 R-T2 a-Cre小鼠，其允许两个单独的神经群体的双峰调节，以验证顺序神经网络。例如，我们可以急性激活POMC终末场到PVN，我们假设这将导致进食减少，同时抑制相关的下游MC 4 RPVN神经元（通过表达抑制性GPCR或离子泵的Cre依赖性病毒），我们假设这将逆转食物摄入量的下降。这些闭塞研究是非常优雅的，并给予实验者无与伦比的控制这些特定的电路。 预测、解释和未来实验：我们预计这些实验不会有任何问题。标记MC 4 R+神经元的初步数据已经产生，我们随后将评估POMCARC投射到下丘脑内外的这种细胞类型。此外，这里提出的病毒已被许多实验室成功使用。我们预测PVN中POMCARC终末场的激活将减少摄食行为并最终减轻体重。此外，我们有初步数据显示DREADD介导的急性抑制（一种用于通过外源受体的特异性表达及其随后通过非生物活性配体的结合和活化来急性操纵神经活性的药物遗传学方法; Krashes等人，2011）可以驱动进食行为，并且MC 4 RPVN神经元接收来自AGRP神经元的直接单突触输入（利用AgRP-ires-Cre小鼠），这两个强有力的证据表明POMCARC神经元在这些下游靶标上的相反作用。