Defining the cellular metabolic responses to brain activity using fluorescent biosensors

项目摘要： 大脑新陈代谢必须适应动态范围广泛的能量需求 单元格类型到单元格类型。虽然对大脑活动增加的代谢反应是众所周知的 对于功能性核磁共振信号，这种代谢反应的性质仍存在很大争议。一直以来 新陈代谢在包括癫痫在内的几种神经疾病中起着关键的调节作用 和神经退行性疾病。然而，人们对神经元兴奋性的耦合机制知之甚少。 代谢，虽然主要的代谢途径已经很好地建立了几十年。 脑代谢的实时动力学研究由于空间和空间分布的不充分而受到阻碍 这些方法的时间分辨率，但这样的限制现在可以通过使用遗传- 编码的荧光生物传感器。我将使用传感器的组合来确定葡萄糖的消耗量 分别为NADH/NAD+和ATP/ADP比值。这些传感器可以给出校准后的定量读数 在完整的脑组织中，通过使用具有比率或荧光寿命的双光子显微镜 成像(电影)。钙离子指示剂RCaMP1h和电生理记录将为我提供读数 神经活动的特征。海马CA3区神经元可通过突触兴奋(这也与星形胶质细胞接触)或 反方向的。 这项拟议的研究结合了葡萄糖、ATP、NADH和钙离子生物传感器的使用 先进的电生理和光学双光子成像技术研究人体的能量代谢 脑片中的神经元和星形胶质细胞。目标1是描述葡萄糖、NADH和ATP的动态变化。 静息状态下海马片星形胶质细胞和神经元(谷氨酸能和GABA能)的水平 和活动状态。这项工作还将探索糖酵解对神经元活动诱导的贡献 能量代谢加快。目标2是确定将神经元活动与一种 新陈代谢增强，无论是触发刺激(Na+，K+和Ca~(2+)流量)还是随后的信号(如 亲离子或G蛋白偶联受体和蛋白激酶)。

Project Abstract: Brain metabolism must accommodate a wide dynamic range of energy demands from time to time and from cell type to cell type. Although the metabolic response to increased brain activity is the basis of well-known functional MRI signals, the nature of this metabolic response is still very controversial. It has been demonstrated that metabolism plays a key regulatory role in several neurological conditions, including epilepsy and neurodegenerative diseases. However, little is known about the mechanisms coupling neuronal excitability to metabolism, though the main metabolic pathways have been well established for decades now. The study of real-time dynamics of brain metabolism has been hampered by the insufficient spatial and temporal resolution of the methods, but such limitations can now be overcome by the use of genetically- encoded fluorescent biosensors. I will use a combination of sensors to determine glucose consumption as well as the NADH/NAD+ and ATP/ADP ratios, respectively. These sensors can give calibrated quantitative readouts within intact brain tissue, by using two-photon microscopy with either ratiometric or fluorescence lifetime imaging (FLIM). The Ca2+ indicator RCaMP1h and electrophysiologic recordings will provide me with a readout of neuronal activity. Hippocampal CA3 neurons can be excited synaptically (which also engages astrocytes) or antidromically. The proposed research combines the use of fluorescent glucose, ATP, NADH and Ca2+ biosensors with advanced electrophysiological and optical 2-photon imaging techniques to study the energy metabolism of neurons and astrocytes in brain slices. Aim 1 is to characterize the dynamics of glucose and NADH and ATP levels in the astrocytes and neurons (both glutamatergic and GABAergic) in hippocampal slices during resting and active states. This work will also explore the glycolytic contribution to the neuronal activity-induced acceleration of energy metabolism. Aim 2 is to determine the mechanisms linking the neuronal activity to an increased metabolism, both the triggering stimuli (Na+, K+ and Ca2+ fluxes) and subsequent signaling (such as ionotropic or G-protein coupled receptors and protein kinases).