Flavoprotein autofluorescence imaging of neuronal activation in the cerebellar cortex in vivo
Flavoprotein autofluorescence imaging of neuronal activation in the cerebellar cortex in vivo
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DOI：
10.1152/jn.01275.2003
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发表时间：
2004-07-01
影响因子：
2.5
通讯作者：
Ebner, TJ
中科院分区：
文献类型：
--
作者：
Reinert, KC;Dunbar, RL;Ebner, TJ
在分离的细胞培养和脑片中，自体荧光已被用作神经元活性的间接测量，但在体内仅有有限的程度。内源荧光信号反映了神经元活动和线粒体代谢之间的耦合，由黄素蛋白或烟酰胺腺嘌呤二核苷酸(NADH)的氧化/还原引起。本研究评价了氯胺酮/赛拉津麻醉小鼠小脑皮质中这些自体荧光信号的存在和性质。表面刺激未染色的小脑皮质引起一束窄而横向的光活动，包括大幅度、短潜伏期的荧光增加和较长持续时间的减少。该自发荧光信号的最佳激发波长为420-490 nm，发射波长为515-570 nm，与黄素蛋白的来源一致。光信号的幅度与刺激的幅度和频率呈线性关系，其持续时间与刺激的持续时间呈线性关系。阻断突触传递表明，大部分自体荧光信号归因于激活平行纤维的突触后靶点。假设是黄素蛋白氧化和随后还原的结果，用氰化钠阻断线粒体呼吸或用二苯碘使黄素蛋白失活，大大减少了光学信号。这种自发荧光信号的减少是在不改变电生理反应的突触前和突触后成分的情况下完成的。反射成像和阻断一氧化氮合酶的结果表明，荧光信号不是血红蛋白氧合或血流变化的结果。因此，这种黄素蛋白的自发荧光信号为监测体内神经元的活动及其与线粒体代谢的关系提供了强有力的工具。
Autofluorescence has been used as an indirect measure of neuronal activity in isolated cell cultures and brain slices, but only to a limited extent in vivo. Intrinsic fluorescence signals reflect the coupling between neuronal activity and mitochondrial metabolism, and are caused by the oxidation/reduction of flavoproteins or nicotinamide adenine dinucleotide ( NADH). The present study evaluated the existence and properties of these autofluorescence signals in the cerebellar cortex of the ketamine/xylazine anesthetized mouse in vivo. Surface stimulation of the unstained cerebellar cortex evoked a narrow, transverse beam of optical activity consisting of a large amplitude, short latency increase in fluorescence followed by a longer duration decrease. The optimal wavelengths for this autofluorescence signal were 420 - 490 nm for excitation and 515 - 570 nm for emission, consistent with a flavoprotein origin. The amplitude of the optical signal was linearly related to stimulation amplitude and frequency, and its duration was linearly related to the duration of stimulation. Blocking synaptic transmission demonstrated that a majority of the autofluorescence signal is attributed to activating the postsynaptic targets of the parallel fibers. Hypothesized to be the result of oxidation and subsequent reduction of flavoproteins, blocking mitochondrial respiration with sodium cyanide or inactivation of flavoproteins with diphenyleneiodonium substantially reduced the optical signal. This reduction in the autofluorescence signal was accomplished without altering the presynaptic and postsynaptic components of the electrophysiological response. Results from reflectance imaging and blocking nitric oxide synthase demonstrated that the epifluorescence signal is not the result of changes in hemoglobin oxygenation or blood flow. This flavoprotein autofluorescence signal thus provides a powerful tool to monitor neuronal activity in vivo and its relationship to mitochondrial metabolism.