Optical detection of neuron connectivity by random access two-photon microscopy
Optical detection of neuron connectivity by random access two-photon microscopy
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DOI：
10.1016/j.jneumeth.2016.01.023
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发表时间：
2016-04-01
影响因子：
3
通讯作者：
Inoue，Takafumi
中科院分区：
文献类型：
--
作者：
Shafeghat，Nasrin;Heidarinejad，Morteza;Inoue，Takafumi
背景有关神经元网络内突触连接的分布、强度和方向的知识对于理解大脑功能至关重要。使用多个电极的电生理学提供了非常高的时间分辨率，但不能产生足够的空间信息来解析神经元连接拓扑。使用单细胞分辨率的光记录技术为提供空间信息提供了希望。尽管来自数百个神经元的钙成像提供了网络内神经连接的新视图，但钙反应的动力学不够快，无法以高保真度解析每个动作电位事件。因此，不可能检测神经元连接的方向。新方法我们利用电压敏感染料的 DiO/DPA 组合的快速动力学和大动态范围以及定制的随机访问双光子显微镜的快速扫描速度来解析培养物中多个神经元的每个动作电位事件。结果从培养的海马神经元进行长达 100 分钟的长时间记录，产生了足够数量的尖峰事件，用于分析突触连接。神经元对的互相关分析清楚地区分了突触连接的神经元对与连接方向。与现有方法比较本研究中使用的电压敏感染料对动作电位的长时间记录比以前的研究要长得多。同时进行光学电压和钙测量表明，电压敏感染料能够比钙指示剂更可靠地检测放电事件。结论这种新颖的方法揭示了神经元网络功能结构的新观点。
BackgroundKnowledge about the distribution, strength, and direction of synaptic connections within neuronal networks are crucial for understanding brain function. Electrophysiology using multiple electrodes provides a very high temporal resolution, but does not yield sufficient spatial information for resolving neuronal connection topology. Optical recording techniques using single-cell resolution have provided promise for providing spatial information. Although calcium imaging from hundreds of neurons has provided a novel view of the neural connections within the network, the kinetics of calcium responses are not fast enough to resolve each action potential event with high fidelity. Therefore, it is not possible to detect the direction of neuronal connections.New methodWe took advantage of the fast kinetics and large dynamic range of the DiO/DPA combination of voltage sensitive dye and the fast scan speed of a custom-made random-access two-photon microscope to resolve each action potential event from multiple neurons in culture.ResultsLong-duration recording up to 100 min from cultured hippocampal neurons yielded sufficient numbers of spike events for analyzing synaptic connections. Cross-correlation analysis of neuron pairs clearly distinguished synaptically connected neuron pairs with the connection direction.Comparison with existing methodThe long duration recording of action potentials with voltage-sensitive dye utilized in the present study is much longer than in previous studies. Simultaneous optical voltage and calcium measurements revealed that voltage-sensitive dye is able to detect firing events more reliably than calcium indicators.ConclusionsThis novel method reveals a new view of the functional structure of neuronal networks.