AGRP NEURONS. NMDARs, Spines, Source of Excitatory Input and Downstream Effectors

描述(申请人提供)：大脑调节进食行为和新陈代谢，然而，负责这一点的神经回路在很大程度上是未知的。大脑的复杂性，以及在清醒、行为正常的动物身上操纵大脑的需要，都是重大的技术挑战。新的、创新的方法的应用可能会大大加快进展。最近，我们和其他人利用药物遗传学和光遗传学技术发现，AgRP神经元驱动强烈的摄食行为。考虑到这一点，现在至关重要的是确定控制AgRP神经元活动的传入信号(激素和神经输入)以及导致AgRP神经元驱动摄食的效应器(GABA、NPY、AgRP及其下游神经回路)。值得注意的是，很少有人注意到传入(上游)神经输入对AgRP神经元的调节。这是不幸的，因为a)对agrp神经元的神经控制缺陷可能对复杂的饮食障碍(从厌食到肥胖)起重要作用，以及b)agrp神经元的激素调节，例如ghrelin，可能通过调节这些输入来发挥作用。最近，我们在初步研究中发现，兴奋性突触可塑性的关键调节者NMDAR在调节AgRP神经元的活动和摄食方面起着关键作用；另一方面，POMC神经元上的NMDAR几乎或根本不起作用。与此一致的是，AgRP神经元有丰富的树突(突触后特化，大多数兴奋性突触驻留在其中，NMDAR在其中操作以控制可塑性)；POMC神经元则相反，没有脊刺。最后，我们发现，AgRP神经元的禁食激活需要NMDAR，并涉及树突的棘突发生，并且很可能增加兴奋性突触发生。因此，NMDAR介导的兴奋性传入AgRP神经元的可塑性在调节AgRP神经元的活动从而调节摄食行为中起着关键的、以前未知的作用。本申请将探讨上述调查结果的主要影响。在目标1中，使用先进的双光子显微镜，我们将机械地研究兴奋性输入通过NMDAR/脊柱对AgRP神经元的“调节”可塑性。在目标2中，我们将利用CRE依赖的单突触狂犬病图谱和通道视紫红质辅助的电路图谱，识别和评估向AgRP神经元发送谷氨酸能输入的传入神经元的功能。在目标3中，利用对不能通过GABA、NPY和/或AgRP发出信号的AgRP神经元的药物发生激活以及对AgRP神经末梢的解剖选择性、光发生激活，我们将识别AgRP神经元的下游效应器(递质和回路)。

DESCRIPTION (provided by applicant): The brain regulates feeding behavior and metabolism however the neurocircuitry responsible for this is largely unknown. The complexity of the brain and the need to manipulate it in awake, behaving animals, represent significant technical challenges. The application of new, innovative approaches is likely to greatly accelerate progress. Recently, we and others, using pharmacogenetic and optogenetic technology, have discovered that AgRP neurons drive intense feeding behavior. With this in mind, it is now critical to determine the afferent signals (hormones and neural inputs) controlling AgRP neuron activity as well as the effectors (GABA, NPY, AgRP, and downstream neural circuits) that bring about AgRP neuron-driven feeding. Remarkably, little attention has been paid to afferent (upstream) neural inputs regulating AgRP neurons. This is unfortunate because a) defective neural control of AgRP neurons could contribute importantly to complex eating disorders (ranging from anorexia to obesity), and b) the hormonal regulation of AgRP neurons, one example being ghrelin, likely works by modulating these inputs. Recently, in Preliminary Studies, we discovered that NMDA receptors (NMDARs), key regulators of excitatory synaptic plasticity, play a critical role in regulating AgRP neuron activity and feeding; NMDARs on POMC neurons, on the other hand, play little or no role. Consistent with this, AgRP neurons have abundant dendritic spines (postsynaptic specializations where most excitatory synapses reside and within which NMDARs operate to control plasticity); POMC neurons, in contrast, lack spines. Finally, we have found that fasting-activation of AgRP neurons require NMDARs and involves dendritic spinogenesis, and very likely increased excitatory synaptogenesis. Thus, NMDAR-mediated plasticity of excitatory input to AgRP neurons plays a key, previously unknown role in regulating AgRP neuron activity and consequently feeding behavior. The present application will pursue key implications of the above-mentioned findings. In Aim 1, using advanced 2-photon microscopy, we will mechanistically investigate "regulatory" plasticity of excitatory inputs, via NMDARs/spines, to AgRP neurons. In Aim 2, using Cre-dependent, Monosynaptic Rabies Mapping and also Channelrhodopsin-Assisted Circuit Mapping, we will identify and assess function of afferent neurons sending glutamatergic input to AgRP neurons. In Aim 3, using pharmacogenetic activation of AgRP neurons unable to signal via GABA, NPY and/or AgRP and also anatomic-selective, optogenetic activation of AgRP nerve terminals, we will identify the downstream effectors (transmitters and circuits) of AgRP neurons.