We propose to use a combination of electrophysiological, confocal imaging and molecular approaches to investigate the role of astrocytes in synaptic transmission in situ.
Specific Aim 1 will investigate the morphological contacts between astrocytes and between astrocytes and neurons in CA1 s. radiatum. It remains unclear whether neuronal dendrites are embedded within an astrocytic syncytium or make discrete contacts with astrocytic processes within the synaptic field of CA1 s. radiatum. Distinguishing between these two possibilities is essential to understand how astrocytes interact with neurons in this region.
Specific Aim 2 will examine the hypotheses that calcium waves move through astrocytic processes in situ and that the level of stimulation affects the distance or volume filled by a calcium wave. Our current view is that there are microdomains within astrocytic syncytium and/or along astrocytic processes that interact with local neuronal elements. We propose to test these hypotheses by releasing caged IP3 to trigger calcium increases within the astrocytic syncytium or along astrocytic processes.
Specific Aim 3 will test the hypothesis that increasing calcium at discrete points within astrocytic processes/syncytium leads to localized, glutamate-dependent calcium increases within nearby dendrites. The results of these experiments will be critical in determining the impact of astrocytic signaling on neuronal activity. We hypothesize that localized increases in astrocytic calcium will affect a small cluster of dendritic spines.
Specific Aim 4 will use molecular strategies to disrupt receptor-mediated increases in astrocytic calcium in situ. We propose to make a mouse line expressing a dominant-negative mutation of Gq driven by the transcriptional control unit of GFAP for selective expression in astrocytes. Gq is the G-protein that generally couples receptors to phospholipase C and calcium mobilization. The dominant-negative mutation that will be used, Galphaq305-359, blocks receptor-mediated increases in calcium in vitro and in vivo without affecting receptor signaling through either Gs or Gi.
Specific Aim 5 will use mice expressing Galphaq305-359 in astrocytes to investigate the role of astrocytic receptors at the Schaffer collateral-CA1 synapse. It is our premise that while astroglia in vitro exhibit properties that would enable them to modulate neuronal activity in vivo, it is essential to develop in vivo model systems whereby the role of astrocytic signaling in brain function and dysfunction can be examined. A major goal of this proposal is to develop such a model system and to test the hypothesis that astrocytic receptors coupled to calcium mobilization regulate neuronal activity.
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