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. Studies of the past decade have significantly changed the view of the roles of glial cells in the central nervous system (CNS). Like other glial subtypes, astrocytes were once thought to be support cells, serving the metabolic needs of the neuron, however, it is now known that they also take up external K+, and remove glutamate from the synaptic cleft. There is evidence, however, that the role of astrocytes in the CNS extends beyond that of neuronal support, as they can also release neurotransmitters in response to elevated internal calcium, and this released neurotransmitter can modulate neurons (Haydon, 2001). The ability of astrocytes to integrate neuronal inputs and to respond with calcium signals that can cause the release of chemical transmitters indicates that astrocytes are likely to serve key roles in information processing. While recent work, including that of this group, has shown that calcium signaling in astrocytes can modulate synaptic transmission (Araque et al. 1998a, 1999a, 1999b, 2000; Haydon 2001; Kang et al. 1998; Robitaille, 1998), the roles of these signaling elements in nervous system function still remain to be elucidated. This project involves the use of male Swiss Webster mice, ranging from from three weeks of age to adults. Our goal is to determine whether the processes of astrocytes contain chemical transmitter filled vesicles, and/or proteins associated with vesicles, such as vesicular glutamate transporter at EM level. The long-term goal of our study is to elucidate the integrative properties of astrocytes and to determine how these non-neuronal cells regulate synaptic plasticity. A crucial first step towards this goal is to determine of the spatial juxtaposition of astrocytic glutamate release sites relative to neuronal synapses. Until we can quantitatively determine the location of these astrocytic release sites and their association with neuronal metabotropic glutamate receptors (mGluRs) and NMDA receptors we are unable to develop accurate models of neuro-glial interactions. Dr. Haydon and colleagues are collaborating with NCMIR researchers using advanced correlative techniques in light and electron microscopy to: (1) map the locations of astrocytic vesicles, which mediate the exocytic release of glutamate from these glial cells, and (2) study the role of calcium-dependent chemical transmitter release from astrocytes in controlling the synapse. We are currently working through the logistics for these collaborative studies, including institutional animal use protocol amendments, specimen preparation details and procurement of necessary reagents. Electron tomography experiments will begin soon and will include immunolabeling brain sections with antibodies against various vesicle proteins (anti-vesicular glutamate transporters, anti-synaptobrevin and anti-synaptotagmin. The immunolabeling will be examined with regard to proximity to astrocytes and neurons by members of Dr. Haydon s group via telemicroscopy to increase the data throughput while reducing travel time. The use of thick section electron tomography will allow us to investigate the presence of few vesicles within the entire process of an astrocyte or neuron in one section. We will localize these vesicles and use the information to build an accurate model of the astrocyte-neuronal interface. Using the Monte Carlo simulation program MCELL, this model will be used to conduct computer simulations of the role of calcium-dependent chemical transmitter release from astrocytes in controlling the synapse.
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