ULTRASTRUCTURAL BASIS FOR SYNAPTIC VESICLE RECYCLING IN CALYX OF HELD

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 听性脑干的HOLD和其他大型神经末梢是声音定位电路的关键元件。我们的目标是揭示结构转换和细胞通讯的特征，这些结构和细胞通讯表征了成杯生长锥与其目标的接触，以及听觉脑干中大型神经末梢的突触组装和稳定的早期阶段。我们的中心假设是，在末端在细胞体上扩张以形成花萼之前，发生花丝的输入之间的竞争。这一假设是基于我们实验室的工作，该工作揭示了小鼠在出生后(P)2天到P4天之间迅速形成花瓣。根据物种的不同，花萼包含数百到2000多个活动区，其中许多位于称为线粒体相关黏附复合体(MACs)的专门细胞器复合体附近。以前已经用标准的透射电子显微镜(STEM)描述了Mac的结构，它显示出连接突触前和突触后膜的细丝将线粒体拴在一个粘附点上。用共聚焦荧光成像和电子断层扫描研究了P0P4期间斜方体内侧核(MNTB)细胞的神经支配。这段时间早于包裹MNTB细胞体的未成熟杯状花萼的形成，并与之重叠。这些实验将精确定位突触前和突触后伙伴之间建立单神经支配的时间段，并突出可能预测输入成败的结构和功能差异。此外，我们还将描述支持高速率神经传递的成熟花萼的结构特征，这是该终末的特征。由于Calyx-MNTB接触的大小和重建所需的高分辨率，需要对细胞结构域进行连续的体积成像。该项目将需要高分辨率、广域、大面积的图像数字记录。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The calyx of Held and other large nerve terminals of the auditory brainstem are key elements of sound localization circuitry. Our goal is to reveal structural transformations and cellular communication that characterize contact of the calycigenic growth cone with its target and the early stages of synapse assembly and stabilization at large nerve terminals in the auditory brainstem. Our central hypothesis is that competition among calycigenic inputs precedes expansion of the terminal over the cell body to form a calyx. This hypothesis is based on work from our laboratory that reveals rapid formation of the calyx in mice between postnatal days (P)2 and P4. The calyx contains hundreds to greater than two thousand active zones, depending upon the species, many of which are located nearby specialized organelle complexes termed mitochondrion-associated adherens complexes (MACs). MAC structure had been described previously using standard transmission electron microscopy (sTEM), which revealed filaments tethering the mitochondrion to a punctum adherens that links the pre- and postsynaptic membranes. Confocal fluorescence imaging and electron tomography are being employed to study medial nucleus of the trapezoid body (MNTB) cell innervation during P0P4. This time period precedes and overlaps the formation of immature, cup-shaped calyces that envelop the MNTB cell body. These experiments will pinpoint time periods during which mono-innervation is established between pre- and postsynaptic partners and highlight structural and functional differences that may predict winning and losing inputs. In addition, we will describe structural features of mature calyces that support high-rate neurotransmission that is characteristic of this terminal. Because of the size of the calyx-MNTB contact and the desired high resolution of the reconstructions, serial volumetric imaging of domains of cells will be required. This project will require high-resolution, wide-field, large-area digital recording of images.