Voltage-sensitive dye imaging from the nervous system

描述（由申请人提供）：新的或改进的工具来更好地理解神经元如何处理信息和调节活动是至关重要的。由于神经元的主要功能是处理电信号，因此最需要改进的一种工具是使用电压敏感探针的膜电位成像（Vm成像）。该应用程序旨在建立一个高速成像系统，该系统将具有覆盖监测大脑切片中神经元活动所需的全部空间和时间分辨率的灵敏度。目前，这样的系统还不存在。空间分辨率将从记录来自单个树突棘的虚拟机信号到同时监测大型神经元群的活动。在虚拟机成像中，亚细胞、单脊柱分辨率已被尝试过（Nuriya等，2006），但从未取得令人满意的结果。然而，单棘分辨率是必不可少的，因为单个树突棘上的兴奋性突触是短期和长期可塑性机制的基本单位。同时，在宏观层面上，可塑性的细胞机制必须与不同脑区持续突触可塑性的组织和整合方面相结合。监测大的神经元群是必要的，以提供可塑性表达的大区域的脑回路的空间地图。为了准确地监测动作电位（AP）信号，将利用高速CCD相机提供每秒2000 - 10,000帧的高时间分辨率。控制高速，高空间分辨率Vm成像灵敏度的基本原理表明了如何实施所提出的改进。此外，所提出的系统的所有必要组件都是可用的，并且已经经过测试并证明是功能性的（初步实验）。成像系统将使用532 nm的倍频二极管泵浦Nd:YVO4作为宽视场荧光显微镜的激发源，并使用高速CCD相机进行采集。本提案的另一个目的是进一步开发和测试新的电压敏感探针和方案，以最小的细胞内标记选择性标记单个神经元的质膜，以实现最大的信噪比（S/N）。公共卫生相关性：对神经系统中正常突触功能和可塑性的研究寻求对神经回路控制行为的机制的基本见解。通过这些见解，有可能澄清影响数百万人的疾病的根本原因，包括精神分裂症、情绪障碍、退行性脑疾病、癫痫和自闭症等神经发育障碍。

DESCRIPTION (provided by applicant): New or improved tools to gain a better understanding of how neurons process information and modulate activity are critical. Because the principal function of a neuron is to process electrical signals, one tool that is most in need for improvement is membrane potential imaging with voltage-sensitive probes (Vm imaging). This application seeks to build a high speed imaging system that will have the sensitivity to cover the full range of spatial and temporal resolutions required in monitoring neuronal activity in brain slices. Presently, such a system is not in existence. The spatial resolution will range from recording Vm-signals from individual dendritic spines to monitoring simultaneously activity of large ensembles of neurons. Subcellular, single spine resolution in Vm-imaging has been attempted (Nuriya et al, 2006) but never satisfactory achieved. Single spine resolution is, however, essential because excitatory synapses on individual dendritic spines are elementary units underlying mechanisms of short and long term plasticity. At the same time, cellular mechanisms of plasticity must be integrated in the context of organizational and integrative aspects of persistent synaptic plasticity in different brain region at the macroscopic level. Monitoring large neuronal ensembles is needed to provide spatial maps of plasticity expression over large areas of the brain circuitry. In order to accurately monitor action potential (AP) signals, high-temporal resolution of 2,000 - 10,000 frames per second will be provided by utilizing a high-speed CCD camera. The basic principles governing the sensitivity of high-speed, high-spatial resolution Vm- imaging indicate how proposed improvements can be implemented. Also, all necessary components for the proposed system are available and have been tested and demonstrated to be functional (preliminary experiments). The imaging system will use a frequency-doubled diode-pumped Nd:YVO4 at 532 nm as an excitation source in wide-field epi-fluorescence microscopy and a high-speed CCD camera for acquisition. An additional aim of this proposal is to further develop and test new voltage-sensitive probes and protocols for selectively labeling the plasma membrane of individual neurons with minimal intracellular labeling in order to achieve the maximum signal-to-noise (S/N). PUBLIC HEALTH RELEVANCE: The research on normal synaptic function and plasticity in the nervous system seeks fundamental insights into the mechanisms by which neural circuits control behavior. Through these insights, it may become possible to clarify the root causes of diseases that affect millions, including schizophrenia, mood disorders, degenerative brain disorders, epilepsy, and neurodevelopmental disorders such as autism.