Genetic and Optic Dissection of AMPK Dynamics in Neurotransmission

AMP活化蛋白激酶（AMPK），一种进化上保守的丝氨酸/苏氨酸激酶，由两种激酶刺激， 降低细胞能量状态和增加钙，是一个重要的球员之间的界面作用， 新陈代谢和大脑功能。除了肥胖和糖尿病等代谢性疾病，异常AMPK 活性与具有功能失调的神经传递的多种神经障碍有关。的 然而，负责这些作用的AMPK的神经生物学机制在很大程度上是未知的。最近 研究表明，下丘脑中表达刺豚鼠相关肽（AgRP）的神经元， 进食和能量平衡的控制器，接受强烈的兴奋性突触输入和它们的兴奋性突触 可塑性在调节AgRP神经元放电和相关进食中起重要作用。重要的是我们的前任 研究结果表明，禁食显著诱导树突棘发生，突触能突触发生， 而这种禁食诱导的可塑性需要AgRP上的突触后NMDA受体 神经元并本质上有助于其禁食诱导的激活。神经生物学机制， 然而，禁食诱导的AgRP神经元可塑性的基础尚不清楚。在这方面，AMPK在 下丘脑被禁食激活，并且操纵该区域中的AMPK活性影响进食。在 此外，当在脑片中刺激腺苷酸时，AMPK增加对AgRP的神经递质输入 神经元这些发现表明AMPK可能触发这种禁食诱导的可塑性。但鉴于 AMPK在脑中的广泛表达及其在细胞生物学中的多方面作用，无论AMPK在AgRP中 神经元介导禁食诱导的进食仍在争论中。禁食如何调节AMPK动力学也是 不清楚通过采用一系列神经元特异性方法，包括神经元特异性转基因和 敲除小鼠系，cre依赖性AAV病毒载体，双光子激光扫描显微镜（2 PLSM） 结合全细胞膜片钳电生理学，特别是基于2 PLSM的荧光寿命 成像（FLIM），该提案旨在提供一个独特的，多方面的研究，以了解AMPK信号， 其在AgRP神经元的神经传递中的生理学。根据我们令人信服的初步调查结果，我们 假设AgRP神经元中AMPK参与的突触后通路驱动禁食诱导的兴奋性 突触可塑性和由这一通路引起的可塑性解释了AMPK对能量的影响 平衡（目标1）。我们进一步假设AMPK作为不同输入的关键整合者（如 禁食、生长素释放肽和瘦素），并介导突触和细胞变化（目的2）。我们 突触可塑性和AMPK的新发现将为摄食回路提供创新的知识。给定 AMPK及其底物的广泛分布，AMPK在AgRP神经元中参与的未被覆盖的通路 可能在下丘脑内外都起作用，对许多人来说有重要意义。 突触可塑性在其中起关键调节作用的过程。

AMP-activated protein kinase (AMPK), an evolutionarily conserved serine/threonine kinase stimulated by both decreased cellular energy status and increased calcium, is an important player acting at the interface between metabolism and brain function. In addition to metabolic diseases like obesity and diabetes, abnormal AMPK activities have been implicated in a variety of neurological disorders with dysfunctional neurotransmission. The neurobiological mechanisms of AMPK responsible for these effects, however, are largely unknown. Recent studies have suggested that agouti-related peptide (AgRP)-expressing neurons in the hypothalamus, a master controller of feeding and energy balance, receive intense glutamatergic input and their excitatory synaptic plasticity plays an essential role in regulating AgRP neuron firing and related feeding. Importantly, our prior findings demonstrate that fasting significantly induces dendritic spinogenesis, glutamatergic synaptogenesis, and firing in AgRP neurons, and this fasting-induced plasticity requires postsynaptic NMDA receptors on AgRP neurons and contributes essentially to their fasting-induced activation. The neurobiological mechanism that underlies fasting-induced plasticity in AgRP neurons, however, is left unknown. In this context, AMPK in the hypothalamus is activated by fasting and manipulation of AMPK activity in this region affects feeding. In addition, when stimulated pharmacologically in brain slices, AMPK increases glutamatergic input to AgRP neurons. These findings suggest that AMPK likely trigger this fasting-induced plasticity. However, given the wide expression of AMPK in the brain and its multi-faceted roles in cellular biology, whether AMPK in AgRP neurons mediates fasting-induced feeding is still in debate. How fasting modulates AMPK dynamics is also unclear. By employing a battery of neuron-specific approaches, including neuron-specific transgenic and knockout mouse lines, cre-dependent AAV viral vectors, 2-photon laser scanning microscopy (2PLSM) combined with whole cell patch-clamp electrophysiology, and particularly 2PLSM-based fluorescence lifetime imaging (FLIM), this proposal aims to provide a unique, multi-faceted study to understand AMPK signaling and its physiology in the neurotransmission of AgRP neurons. Based on our compelling preliminary findings, we hypothesize that a postsynaptic pathway engaged by AMPK in AgRP neurons drives fasting induced excitatory synaptic plasticity and the plasticity brought about by this pathway accounts for the effects of AMPK on energy balance (Aim 1). We further hypothesize that AMPK functions as a critical integrator of diverse inputs (such as fasting, ghrelin, and leptin) of AgRP neurons and mediates both synaptic and cellular changes (Aim 2). Our novel findings on synaptic plasticity and AMPK will provide innovative knowledge in the feeding circuits. Given the wide distribution of AMPK and its substrates, the uncovered pathway engaged by AMPK in AgRP neurons will likely operate both within and beyond the hypothalamus, and have important implications for many processes where synaptic plasticity plays a key regulatory role.