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Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses
Nature Protocols ( IF 14.8 ) Pub Date : 2021-05-14 , DOI: 10.1038/s41596-021-00526-0
David Vandael 1 , Yuji Okamoto 1 , Carolina Borges-Merjane 1 , Victor Vargas-Barroso 1 , Benjamin A Suter 1 , Peter Jonas 1
Affiliation  

Rigorous investigation of synaptic transmission requires analysis of unitary synaptic events by simultaneous recording from presynaptic terminals and postsynaptic target neurons. However, this has been achieved at only a limited number of model synapses, including the squid giant synapse and the mammalian calyx of Held. Cortical presynaptic terminals have been largely inaccessible to direct presynaptic recording, due to their small size. Here, we describe a protocol for improved subcellular patch-clamp recording in rat and mouse brain slices, with the synapse in a largely intact environment. Slice preparation takes ~2 h, recording ~3 h and post hoc morphological analysis 2 d. Single presynaptic hippocampal mossy fiber terminals are stimulated minimally invasively in the bouton-attached configuration, in which the cytoplasmic content remains unperturbed, or in the whole-bouton configuration, in which the cytoplasmic composition can be precisely controlled. Paired pre–postsynaptic recordings can be integrated with biocytin labeling and morphological analysis, allowing correlative investigation of synapse structure and function. Paired recordings can be obtained from mossy fiber terminals in slices from both rats and mice, implying applicability to genetically modified synapses. Paired recordings can also be performed together with axon tract stimulation or optogenetic activation, allowing comparison of unitary and compound synaptic events in the same target cell. Finally, paired recordings can be combined with spontaneous event analysis, permitting collection of miniature events generated at a single identified synapse. In conclusion, the subcellular patch-clamp techniques detailed here should facilitate analysis of biophysics, plasticity and circuit function of cortical synapses in the mammalian central nervous system.



中文翻译:

用于皮质突触的单按钮刺激和同时突触前和突触后记录的亚细胞膜片钳技术

对突触传递的严格研究需要通过同时记录突触前末端和突触后目标神经元来分析单一突触事件。然而,这仅在有限数量的模型突触中实现,包括鱿鱼巨型突触和 Held 的哺乳动物花萼。由于其体积小,皮质突触前终端在很大程度上无法直接进行突触前记录。在这里,我们描述了一种在大鼠和小鼠脑切片中改进亚细胞膜片钳记录的协议,突触在基本完整的环境中。切片准备需要约 2 小时,记录约 3 小时,事后形态分析需要 2 天。单突触前海马苔藓状纤维末端在连接结构中被微创刺激,其中细胞质含量保持不受干扰,或在整体结构中,其中细胞质成分可以精确控制。成对的突触后记录可以与生物胞素标记和形态分析相结合,从而可以对突触结构和功能进行相关研究。成对的录音可以从大鼠和小鼠切片中的苔藓纤维末端获得,这意味着它适用于转基因突触。配对记录也可以与轴突束刺激或光遗传学激活一起进行,从而可以比较同一靶细胞中的单一和复合突触事件。最后,配对记录可以与自发事件分析相结合,允许收集在单个识别突触处生成的微型事件。综上所述,

更新日期:2021-05-14
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