Cano-Astorga Nicolás, Plaza-Alonso Sergio, Turegano-Lopez Marta, Rodrigo-Rodríguez José, Merchan-Perez Angel, DeFelipe Javier
Laboratorio Cajal de Circuitos Corticales, Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, Madrid, Spain.
Instituto Cajal, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
Front Neuroanat. 2024 Apr 5;18:1348032. doi: 10.3389/fnana.2024.1348032. eCollection 2024.
The brain contains thousands of millions of synapses, exhibiting diverse structural, molecular, and functional characteristics. However, synapses can be classified into two primary morphological types: Gray's type I and type II, corresponding to Colonnier's asymmetric (AS) and symmetric (SS) synapses, respectively. AS and SS have a thick and thin postsynaptic density, respectively. In the cerebral cortex, since most AS are excitatory (glutamatergic), and SS are inhibitory (GABAergic), determining the distribution, size, density, and proportion of the two major cortical types of synapses is critical, not only to better understand synaptic organization in terms of connectivity, but also from a functional perspective. However, several technical challenges complicate the study of synapses. Potassium ferrocyanide has been utilized in recent volume electron microscope studies to enhance electron density in cellular membranes. However, identifying synaptic junctions, especially SS, becomes more challenging as the postsynaptic densities become thinner with increasing concentrations of potassium ferrocyanide. Here we describe a protocol employing Focused Ion Beam Milling and Scanning Electron Microscopy for studying brain tissue. The focus is on the unequivocal identification of AS and SS types. To validate SS observed using this protocol as GABAergic, experiments with immunocytochemistry for the vesicular GABA transporter were conducted on fixed mouse brain tissue sections. This material was processed with different concentrations of potassium ferrocyanide, aiming to determine its optimal concentration. We demonstrate that using a low concentration of potassium ferrocyanide (0.1%) improves membrane visualization while allowing unequivocal identification of synapses as AS or SS.
大脑包含数以十亿计的突触，呈现出多样的结构、分子和功能特征。然而，突触可分为两种主要的形态类型：格雷I型和II型，分别对应科隆尼尔的不对称（AS）和对称（SS）突触。AS和SS的突触后致密部分别较厚和较薄。在大脑皮层中，由于大多数AS突触是兴奋性的（谷氨酸能），而SS突触是抑制性的（γ-氨基丁酸能），确定这两种主要皮层突触类型的分布、大小、密度和比例至关重要，这不仅有助于从连接性方面更好地理解突触组织，而且从功能角度来看也是如此。然而，一些技术挑战使突触研究变得复杂。亚铁氰化钾已被用于最近的体电子显微镜研究中，以增强细胞膜中的电子密度。然而，随着亚铁氰化钾浓度的增加，突触后致密部变薄，识别突触连接，尤其是SS突触，变得更具挑战性。在这里，我们描述了一种采用聚焦离子束铣削和扫描电子显微镜研究脑组织的方法。重点是明确识别AS和SS类型。为了验证使用该方法观察到的SS突触是γ-氨基丁酸能的，对固定的小鼠脑组织切片进行了囊泡γ-氨基丁酸转运体免疫细胞化学实验。该材料用不同浓度的亚铁氰化钾处理，旨在确定其最佳浓度。我们证明，使用低浓度的亚铁氰化钾（0.1%）可改善膜的可视化，同时能明确识别突触为AS或SS类型。
