Probing neural circuits underlying satiety

精确定位参与饱腹感的POMC神经元的下游投射靶点。 假设：下丘脑POMCARC神经元投射调节下丘脑室旁核(PVN)中的MC4R+神经元，以发出饱足感的信号。 基本原理：研究表明，MC4R基因敲除小鼠的下丘脑室旁核和杏仁核神经元亚群中MC4R活性的选择性恢复可以挽救MC4R基因敲除诱导的肥胖(Balthasar 2005)。我们的实验室获得了一个MC4R-T2a-Cre小鼠品系，它在Mc4r基因的内源启动子下表达Cre重组酶，从而可以精确地标记、操作和记录这一特定亚群的活性。我们的实验室还可以获得POMC-CRE小鼠品系，用于探测神经连接和调制。本实验旨在阐明MC4Rs在其中表达以调节饱腹感的解剖结构，以及POMCARC神经元的联系和调节作用。 方法：我们将通过将MC4R-T2a-CRE与GFP/FLOX报告鼠杂交，并结合顺行追踪(通过将表达突触素的Cre依赖的腺相关病毒(AAV)注射到POMC-CRE小鼠的弓状核(ARC))和免疫组织化学来鉴定MC4R表达的神经元，绘制POMCARC在这个复杂网络中的投射图。基于这些示踪研究，我们将选择性地针对光激活的离子通道，将视紫红质2(ChR2)特异性地引导到POMCARC神经元(通过立体定位注射依赖于CRE的AAV-ChR2)，然后选择性地对不同的POMC终端场进行光刺激，并评估由此产生的行为输出。这些体内光遗传学研究将梳理出POMCARC神经元功能相关的下游部位，这些部位减少食物摄入量和体重，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；MC4R-T2a-Cre小鼠，这允许对两个独立的神经种群进行双峰调节，以验证顺序神经网络。例如，我们可以敏锐地激活PVN的POMC终端场，我们假设这将导致摄食减少，同时抑制相关的下游MC4RPVN神经元(通过表达抑制性GPCRs或离子泵的Cre依赖病毒)，我们假设这将扭转食物摄入量的下降。这些闭塞研究非常优雅，使实验者能够无与伦比地控制这些特定的电路。 预测、解释和未来的实验：我们预计这些实验不会有任何问题。已经产生了标记MC4R+神经元的初步数据，我们随后将评估POMCARC在下丘脑内外对这种细胞的投射。此外，这里提出的病毒已经被一些实验室成功使用。我们预测，PVN中POMCARC终端场的激活将减少摄食行为，最终减轻体重。此外，我们有初步数据表明，DREADD介导的MC4RPVN神经元的急性抑制(一种药物遗传学方法，用于通过外源受体的特定表达以及随后通过药理惰性配体的结合和激活来敏锐地操纵神经活动；Krash等人，2011)可以驱动取食行为，并且MC4RPVN神经元从AGRP神经元(利用AgRP-res-Cre小鼠)直接接受单突触输入，这两条强有力的证据都表明，POMCARC神经元在这些下游靶点上扮演了相反的角色。

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.