Department of Anatomy and Cell Biology, University of Heidelberg, 69120 Heidelberg, Germany.
J Comp Neurol. 2010 Apr 1;518(7):1008-29. doi: 10.1002/cne.22260.
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.
巴松管和短笛有助于活跃区（AZ）的细胞基质的形成，而 AZ 是神经末梢中神经递质释放的部位。在这里，我们研究了在出生后第 9 天至第 21 天的大鼠听壶中，Bassoon 和 Piccolo 的 3D 定位，这个时期是听力起始的特征，具有明显的结构和功能变化。通过对含有膜锚定绿色荧光蛋白（mGFP）标记的听壶的脑桥切片进行免疫组织化学（IHC），鉴定了 Bassoon 和 Piccolo。通过使用共聚焦显微镜和 3D 重建，我们研究了 Bassoon 和 Piccolo 在由 mGFP 描绘的听壶中的分布。这使我们能够将听壶的 IHC 信号与位于听壶柄之间的空间中的非听壶信号区分开来，后者可能模拟听壶样分布。我们发现，这两种蛋白质都排列成簇，大小类似于 AZ。这些簇位于面向主细胞的突触前膜附近或与突触小泡（SV）簇重叠。只有大约 60%的 Bassoon 和 Piccolo 簇重叠，而其余簇主要含有 Bassoon 或 Piccolo，表明这些蛋白质在单个神经末梢内的靶向不同，并且潜在地具有不同的 AZ 功能特性。Bassoon 和 Piccolo 簇的总数，可能近似于 AZ 的数量，在 P9 时为 405 +/- 35 个，在 P21 时为 601 +/- 45 个（平均值 +/- SEM，n = 12）。相对于 P9 和 P21 的听壶体积进行归一化，簇的密度相似，表明簇的绝对数量，而不是密度，可能有助于与听力起始相关的功能成熟。
