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

描述（由申请人提供）：大脑调节摄食行为和新陈代谢，但对此负责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、有行为的动物中操纵它的需要，代表着重大的技术挑战。新的创新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神经元驱动进食的效应器（GABA， NPY， AgRP和下游神经回路）是至关重要的。值得注意的是，很少有人关注调节AgRP神经元的传入（上游）神经输入。这是不幸的，因为a) AgRP神经元的神经控制缺陷可能对复杂的饮食失调（从厌食症到肥胖）起重要作用，b) AgRP神经元的激素调节，例如胃饥饿素，可能通过调节这些输入起作用。最近，在前期研究中，我们发现NMDA受体（NMDARs）作为兴奋性突触可塑性的关键调节因子，在调节AgRP神经元的活动和摄食中起着关键作用；另一方面，POMC神经元上的NMDARs作用很小或没有作用。与此一致的是，AgRP神经元具有丰富的树突棘（突触后特化，大多数兴奋性突触存在，NMDARs在其中运作以控制可塑性）；相比之下，POMC神经元缺乏棘。最后，我们发现AgRP神经元的禁食激活需要NMDARs，并涉及树突脊髓发生，并且很可能增加兴奋性突触发生。因此，nmdar介导的AgRP神经元兴奋性输入的可塑性在调节AgRP神经元活动和摄食行为中起着关键的、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，我们将机械地研究通过NMDARs/棘对AgRP神经元的兴奋性输入的“调节”可塑性。在Aim 2中，我们将使用cre依赖的单突触狂犬病映射和通道视紫红质辅助电路映射，识别和评估传入神经元向AgRP神经元发送谷氨酸能输入的功能。在Aim 3中，通过药物遗传学激活无法通过GABA、NPY和/或AgRP发出信号的AgRP神经元，以及解剖选择性的AgRP神经末梢的光遗传学激活，我们将确定AgRP神经元的下游效应器（递质和电路）。

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.