This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The calyx of Held, perhaps the largest nerve terminal in the central nervous system, is a hallmark structure of brainstem circuits that mediate the early stages of sound localization. Each calyx contains many active zones, ranging from hundreds in rodents to over two thousand in larger animals such as cats. Given this large number of vesicle release sites, the use of glutamate as neurotransmitter and the one-to-one relationship between calyces and their postsynaptic target, principal cells of the medial nucleus of the trapezoid body (MNTB), each calyx is the primary determinant of spike patterns in MNTB neurons. Axons leading into the calyx transmit action potentials at high rates, exceeding 600 Hz, with high temporal precision required by brainstem circuits that encode sound location based on interaural temporal delays and intensity differences. Therefore, calyx structure must be specialized to support such high rates of activity with great temporal precision.The calyx and other large terminals of the auditory brainstem contain a specialized organelle complex that we have termed the mitochondrion-associated adherens complex (MAC). The essential structure of the MAC is a mitochondrion tethered about 200 nm from a punctum adherens, with intervening membranous structures and location typically adjacent to active zones. Based on this proximity to synaptic machinery, the MAC has been implicated in synaptic function, hypothesized to effect vesicle membrane recycling, buffer and shunt Ca2+, manufacture glutamate for vesicle refilling and supply ATP to support all steps in the synaptic vesicle life cycle. Evaluation of MAC structure in the context of these functional hypotheses requires spatial resolution through the depth of the tissue exceeding that available using standard transmission electron microscopy, which is limited to the section thickness of typically 50-100 nm. Currently the best method to obtain high resolution 3-D structural information is electron tomography (ET). In order to support high rates of activity, nerve terminals (and the calyx in particular) must recycle vesicle membrane and promote vesicles from a reserve to active status. The actin cytoskeleton has been implicated in this process, and is thought to direct movement of synaptic vesicles to the active zone by surrounding the reserve pool of vesicles or to provide direct tracks for moving vesicles to the active zone. Filaments have been described linking synaptic vesicles and may anchor onto synapsin protein in the vesicle membrane. These structures may also play roles in vesicle mobilization.
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