Circuit mapping by ultraviolet uncaging of glutamate.
Circuit mapping by ultraviolet uncaging of glutamate.
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DOI：
10.1101/pdb.prot070664
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发表时间：
2012-09-01
影响因子：
--
通讯作者：
Shepherd, Gordon M G
中科院分区：
文献类型：
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作者：
Shepherd, Gordon M G
在激光光刺激中，脑切片中的小簇神经元通过局灶性谷氨酸释放而被诱导激发动作电位，并且通过突触事件的细胞内记录来评估光激发的突触前神经元和单个突触后神经元之间的突触连接。通过扫描仪，这个过程可以在刺激位置的模式阵列上顺序重复，生成神经元突触输入的局部来源的地图。激光扫描光刺激（LSPS）的基础上图案谷氨酸uncaging提供了一个有效的，定量的，光电生理的方式来映射突触电路在脑切片。用于电路映射的基于谷氨酸的光刺激的功效（与电刺激相反）源自以高精度和速度刺激神经元的能力，并且不刺激通道轴突。该协议描述了组件，组装和操作的激光扫描显微镜紫外线（UV）撑开，沿着与实验方法的电路映射在脑切片。它提出了一个通用的方法和一套使用“标准”的LSPS方法的基础上，使用紫外激光，一对扫描镜检流计，膜片钳设置，和开源数据采集软件的单光子谷氨酸uncaging定量电路映射的指导方针。
In laser photostimulation, small clusters of neurons in brain slices are induced to fire action potentials by focal glutamate uncaging, and synaptic connectivity between photoexcited presynaptic neurons and individual postsynaptic neurons is assessed by intracellular recording of synaptic events. With a scanner, this process can be repeated sequentially across a patterned array of stimulus locations, generating maps of neurons' local sources of synaptic inputs. Laser scanning photostimulation (LSPS) based on patterned glutamate uncaging offers an efficient, quantitative, optical-electrophysiological way to map synaptic circuits in brain slices. The efficacy of glutamate-based photostimulation for circuit mapping (in contrast to electrical stimulation) derives from the ability to stimulate neurons with high precision and speed, and without stimulating axons of passage. This protocol describes the components, assembly, and operation of a laser scanning microscope for ultraviolet (UV) uncaging, along with experimental methods for circuit mapping in brain slices. It presents a general approach and a set of guidelines for quantitative circuit mapping using "standard" LSPS methods based on single-photon glutamate uncaging using a UV laser, a pair of scanning mirror galvanometers, a patch-clamp setup, and open-source data acquisition software.