Genetic and Optic Dissection of AMPK Dynamics in Neurotransmission

AMP激活的蛋白激酶(AMPK)，一种进化上保守的丝氨酸/苏氨酸激酶 细胞能量状态降低和钙离子增加是起作用的重要因素 新陈代谢和大脑功能。除了肥胖和糖尿病等代谢性疾病外，AMPK异常 活动与多种神经功能障碍和神经传递功能障碍有关。这个 然而，AMPK导致这些效应的神经生物学机制在很大程度上还不清楚。近期 研究表明，刺鼠相关肽(AgRP)在下丘脑表达的神经元是 摄食和能量平衡的控制器，接受强烈的谷氨酸能输入和兴奋性突触 可塑性在调节AgRP神经元的放电和相关的摄食方面起着至关重要的作用。重要的是，我们的副院长 研究结果表明，禁食显著诱导树突棘发生，谷氨酸能突触发生， 这种禁食诱导的可塑性需要AgRP上的突触后NMDA受体 并对其禁食诱导的激活起主要作用。神经生物学机制 然而，禁食诱导AgRP神经元可塑性的机制尚不清楚。在这方面，AMPK在 下丘脑通过禁食而激活，该区域AMPK活性的调节会影响摄食。在……里面 此外，当药物刺激脑片时，AMPK增加谷氨酸能对AgRP的输入 神经元。这些发现表明，AMPK可能触发了这种禁食诱导的可塑性。然而，鉴于 AMPK在脑内的广泛表达及其在细胞生物学中的多方面作用 神经元在禁食诱导的进食中起中介作用仍存在争议。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 基因敲除小鼠系、依赖cre的AAV病毒载体、双光子激光扫描显微镜(2PLSM) 结合全细胞膜片钳电生理学，特别是基于2PLSM的荧光寿命 成像(FLIM)，这项建议旨在提供一个独特的，多方面的研究，以了解AMPK信号和 其在AgRP神经元神经传递中的生理学作用。基于我们令人信服的初步调查结果，我们 AMPK参与AgRP神经元突触后通路驱动禁食引起兴奋性反应的假说 突触可塑性及其所带来的可塑性解释了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集成器(例如 空腹、Ghrelin和Leptin)和调节突触和细胞的变化(目标2)。我们的 有关突触可塑性和AMPK的新发现将为馈入回路提供创新知识。vt.给出 AMPK及其底物在AgRP神经元中的广泛分布--AMPK参与的未知途径 可能会在下丘脑内部和外部运行，并对许多 突触可塑性发挥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.