Vollum Institute, Oregon Health and Science University, Portland, Oregon, United States of America.
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, United States of America.
PLoS One. 2022 Aug 12;17(8):e0271799. doi: 10.1371/journal.pone.0271799. eCollection 2022.
Ionotropic glutamate receptors (iGluRs) at postsynaptic terminals mediate the majority of fast excitatory neurotransmission in response to release of glutamate from the presynaptic terminal. Obtaining structural information on the molecular organization of iGluRs in their native environment, along with other signaling and scaffolding proteins in the postsynaptic density (PSD), and associated proteins on the presynaptic terminal, would enhance understanding of the molecular basis for excitatory synaptic transmission in normal and in disease states. Cryo-electron tomography (ET) studies of synaptosomes is one attractive vehicle by which to study iGluR-containing excitatory synapses. Here we describe a workflow for the preparation of glutamatergic synaptosomes for cryo-ET studies. We describe the utilization of fluorescent markers for the facile detection of the pre and postsynaptic terminals of glutamatergic synaptosomes using cryo-laser scanning confocal microscope (cryo-LSM). We further provide the details for preparation of lamellae, between ~100 to 200 nm thick, of glutamatergic synaptosomes using cryo-focused ion-beam (FIB) milling. We monitor the lamella preparation using a scanning electron microscope (SEM) and following lamella production, we identify regions for subsequent cryo-ET studies by confocal fluorescent imaging, exploiting the pre and postsynaptic fluorophores.
离子型谷氨酸受体（iGluRs）位于突触后终端，介导来自突触前终端的谷氨酸释放后大多数快速兴奋性神经递质传递。获得 iGluRs 在其天然环境中的分子组织、突触后密度（PSD）中的其他信号转导和支架蛋白以及突触前终端上的相关蛋白的结构信息，将增强对正常和疾病状态下兴奋性突触传递的分子基础的理解。通过冷冻电子断层扫描（ET）研究突触体是研究包含 iGluR 的兴奋性突触的一种有吸引力的方法。在这里，我们描述了一种用于冷冻 ET 研究的谷氨酸能突触体的制备工作流程。我们描述了使用荧光标记物来方便地检测谷氨酸能突触体的前突触和后突触末端，使用冷冻激光扫描共聚焦显微镜（cryo-LSM）。我们进一步提供了使用冷冻聚焦离子束（FIB）铣削制备厚度约为 100 至 200nm 的谷氨酸能突触体薄片的详细信息。我们使用扫描电子显微镜（SEM）监测薄片的制备情况，并且在薄片生产后，我们通过共聚焦荧光成像来识别后续冷冻 ET 研究的区域，利用前突触和后突触荧光团。
