Targeted Three-Dimensional Immunohistochemistry Reveals Localization of Presynaptic Proteins Bassoon and Piccolo in the Rat Calyx of Held Before and After the Onset of Hearing
Targeted Three-Dimensional Immunohistochemistry Reveals Localization of Presynaptic Proteins Bassoon and Piccolo in the Rat Calyx of Held Before and After the Onset of Hearing
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DOI：
10.1002/cne.22260
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发表时间：
2010-04-01
影响因子：
2.5
通讯作者：
Kuner, Thomas
中科院分区：
文献类型：
--
作者：
Dondzillo, Anna;Saetzler, Kurt;Kuner, Thomas
巴松管和短笛有助于活跃区（AZ）的细胞基质，神经末梢中神经递质释放的位点。在这里，我们研究了3D定位巴松管和短笛在大鼠肾盏举行出生后第9和21天之间，听力发作期间的特点是明显的结构和功能的变化。通过免疫组织化学（IHC）在用膜锚定绿色荧光蛋白（mGFP）标记的脑干容纳盏切片上鉴定巴松管和短笛。通过使用共聚焦显微镜和三维重建，我们研究了大管和短笛在肾盏描绘的mGFP的分布。这使我们能够区分位于花萼柄之间的空间中的非花萼IHC信号和花萼信号，这可以模拟花萼样分布。我们发现，这两种蛋白质都排列在类似AZ大小的簇中。这些簇位于沿着突触前膜面向主细胞，接近或重叠突触囊泡（SV）集群。只有大约60%的巴松管和短笛集群重叠，而其余的集群主要包含巴松管或短笛，这表明这些蛋白质在单个神经末梢和潜在的异质性AZ功能特性的差异靶向。巴松管和短笛簇的总数（可能接近AZ的数量）在P9时为405 +/- 35，在P21时为601 +/- 45（平均值+/- SEM，n = 12）。归一化为P9和P21时的萼体积，簇的密度相似，这表明簇的绝对数量，而不是密度，可能有助于与听力开始相关的功能成熟。神经学比较杂志518：1008-1029，2010。(C)2009 Wiley-Liss，Inc.
Bassoon and Piccolo contribute to the cytomatrix of active zones (AZ), the sites of neurotransmitter release in nerve terminals. Here, we examined the 3D localization of Bassoon and Piccolo in the rat calyx of Held between postnatal days 9 and 21, the period of hearing onset characterized by pronounced structural and functional changes. Bassoon and Piccolo were identified by immunohistochemistry (IHC) on slices of the brainstem harboring calyces labeled with membrane-anchored green fluorescent protein (mGFP). By using confocal microscopy and 3D reconstructions, we examined the distribution of Bassoon and Piccolo in calyces delineated by mGFP. This allowed us to discriminate calyceal IHC signals from noncalyceal signals located in the spaces between the calyceal stalks, which could mimic a calyx-like distribution. We found that both proteins were arranged in clusters resembling the size of AZs. These clusters were located along the presynaptic membrane facing the principal cell, close to or overlapping with synaptic vesicle (SV) clusters. Only about 60% of Bassoon and Piccolo clusters overlapped, whereas the remaining clusters contained predominantly Bassoon or Piccolo, suggesting differential targeting of these proteins within a single nerve terminal and potentially heterogeneous AZs functional properties. The total number of Bassoon and Piccolo clusters, which may approximate the number of AZs, was 405 +/- 35 at P9 and 601 +/- 45 at P21 (mean +/- SEM, n = 12). Normalized to calyx volume at P9 and P21, the density of clusters was similar, suggesting that the absolute number of clusters, not density, may contribute to the functional maturation associated with hearing onset. J. Comp. Neurol. 518: 1008-1029, 2010. (C) 2009 Wiley-Liss, Inc.