Ultrastructural Imaging of Activity-Dependent Synaptic Membrane-Trafficking Events in Cultured Brain Slices.
Ultrastructural Imaging of Activity-Dependent Synaptic Membrane-Trafficking Events in Cultured Brain Slices.
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培养脑切片中活动依赖性突触膜运输事件的超微结构成像。
DOI：
10.1016/j.neuron.2020.09.004
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发表时间：
2020-12-09
期刊：
影响因子：
16.2
通讯作者：
Cooper BH
中科院分区：
文献类型：
--
作者：
Imig C;López-Murcia FJ;Maus L;García-Plaza IH;Mortensen LS;Schwark M;Schwarze V;Angibaud J;Nägerl UV;Taschenberger H;Brose N;Cooper BH
电子显微镜可以以纳米级精度解析突触超微结构，但捕获时间分辨的、活动依赖的突触膜运输事件仍然具有挑战性，特别是在组织环境中功能不同的突触。我们提出了一种结合光遗传刺激耦合冷冻固定（“闪冻”）和电子显微镜的方法，以高时空分辨率在培养的小鼠脑组织突触中观察突触前囊泡组织的膜运输事件和突触状态特异性变化。根据我们的实验流程，电生理和“快速冷冻”电子显微镜实验可以在相同的条件下在人工脑脊液中单独进行，而不需要添加外部冷冻保护剂，否则需要在冷冻时保存足够的组织。使用这种方法，我们揭示了停靠囊泡的耗竭，并解决了持续刺激下海马苔藓纤维突触单个突触前活跃区的代偿性膜循环事件。复杂神经回路中已识别突触的快速冷冻功能EM活动诱导功能状态与超微结构的直接关联已识别突触内吞作用的时空组织解剖Imig等人提出了电生理和光刺激耦合高压冷冻和电镜下进行的实验流程脑组织培养中几乎相同的条件。为了证明这一原理，他们捕捉了囊泡池的变化，并在确定的活动状态下以高时空分辨率绘制了海马苔藓纤维钮扣的内吞事件。
Electron microscopy can resolve synapse ultrastructure with nanometer precision, but the capture of time-resolved, activity-dependent synaptic membrane-trafficking events has remained challenging, particularly in functionally distinct synapses in a tissue context. We present a method that combines optogenetic stimulation-coupled cryofixation (“flash-and-freeze”) and electron microscopy to visualize membrane trafficking events and synapse-state-specific changes in presynaptic vesicle organization with high spatiotemporal resolution in synapses of cultured mouse brain tissue. With our experimental workflow, electrophysiological and “flash-and-freeze” electron microscopy experiments can be performed under identical conditions in artificial cerebrospinal fluid alone, without the addition of external cryoprotectants, which are otherwise needed to allow adequate tissue preservation upon freezing. Using this approach, we reveal depletion of docked vesicles and resolve compensatory membrane recycling events at individual presynaptic active zones at hippocampal mossy fiber synapses upon sustained stimulation. High-pressure freezing of cultured brain tissue without cryoprotectants for EM Flash-and-freeze functional EM of identified synapses in complex neural circuits Direct correlation of activity-induced functional states and ultrastructure Dissection of the spatiotemporal organization of endocytosis in identified synapses Imig et al. present an experimental workflow for electrophysiological and light-stimulation-coupled high-pressure freezing and electron microscopy to be performed under near-identical conditions in brain tissue cultures. Demonstrating proof of principle, they capture vesicle pool changes and map endocytic events in hippocampal mossy fiber boutons with high spatiotemporal resolution during defined activity states.
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影响因子：
7.8
作者：
Heuser, J E;Reese, T S
通讯作者：
Reese, T S
DOI：
10.1073/pnas.1720648115
发表时间：
2018-05-22
影响因子：
11.1
作者：
Helassa N;Dürst CD;Coates C;Kerruth S;Arif U;Schulze C;Wiegert JS;Geeves M;Oertner TG;Török K
通讯作者：
Török K
影响因子：
16.6
作者：
He E;Wierda K;van Westen R;Broeke JH;Toonen RF;Cornelisse LN;Verhage M
通讯作者：
Verhage M
影响因子：
2.5
作者：
Fiala, JC;Kirov, SA;Harris, KM
通讯作者：
Harris, KM
影响因子：
2.3
作者：
Frotscher, Michael;Zhao, Shanting;Studer, Daniel
通讯作者：
Studer, Daniel