Developmental Neuroscience, Munroe-Meyer Institute, University of Nebraska Medical Center Omaha, NE, USA.
Materials Science and Engineering Program, University of California San Diego, La Jolla, CA, USA.
Front Cell Neurosci. 2014 May 21;8:142. doi: 10.3389/fncel.2014.00142. eCollection 2014.
Synaptic activity is intimately linked to neuronal structure and function. Stimulation of live cultured primary neurons, coupled with fluorescent indicator imaging, is a powerful technique to assess the impact of synaptic activity on neuronal protein trafficking and function. Current technology for neuronal stimulation in culture include chemical techniques or microelectrode or optogenetic based techniques. While technically powerful, chemical stimulation has limited spatial resolution and microelectrode and optogenetic techniques require specialized equipment and expertise. We report an optimized and improved technique for laser based photoconductive stimulation of live neurons using an inverted confocal microscope that overcomes these limitations. The advantages of this approach include its non-invasive nature and adaptability to temporal and spatial manipulation. We demonstrate that the technique can be manipulated to achieve spatially selective stimulation of live neurons. Coupled with live imaging of fluorescent indicators, this simple and efficient technique should allow for significant advances in neuronal cell biology.
突触活动与神经元的结构和功能密切相关。刺激活培养的原代神经元，并结合荧光指示剂成像，是一种评估突触活动对神经元蛋白运输和功能影响的强大技术。目前用于培养中的神经元刺激的技术包括化学技术或基于微电极或光遗传学的技术。虽然在技术上很强大，但化学刺激的空间分辨率有限，而微电极和光遗传学技术需要专门的设备和专业知识。我们报告了一种优化和改进的技术，用于使用倒置共聚焦显微镜对活神经元进行基于激光的光导刺激，该技术克服了这些限制。这种方法的优点包括其非侵入性和对时间和空间操作的适应性。我们证明可以操纵该技术以实现对活神经元的空间选择性刺激。与荧光指示剂的实时成像相结合，这种简单有效的技术应该可以在神经元细胞生物学方面取得重大进展。
