Neurons and Glia in vitro

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DESCRIPTION (provided by applicant): Emerging evidence suggests that gap junction hemichannels (HCs) can act as stand alone channels in astrocytes. The actions of gap junctions and HCs are fundamentally different and the Biology of astrocyte HCs is poorly understood. Unlike gap junctions, which connect the interiors of two cells, open HCs directly connect intracellular and extracellular spaces. The proposed experiments will explore the properties of these channels in astrocytes and begin the process of critically testing how they participate in both normal as well as pathological events in the brain. The following hypotheses will be tested: 1) Astrocytes express functional HCs. 2) HCs are gated and/or modulated by multiple factors including divalent cations, pH, membrane potential, glutamate receptor stimulation, prior HC activity and environment. 3) HCs influence transmembrane gradients of glutamate, ions and other small molecules. HCs open during intense neural activity and may participate in normal brain functions. 4) HCs are activated during ischemia and contribute to the pathophysiology of this condition. These experiments will use primary cultured astrocytes, brain slices and an advantageous CMS white matter preparation, the acutely isolated mouse optic nerve. Tissue will be obtained from wild type and from genetically altered mice that do not express specific proteins that form HCs in astrocytes. The behavior of astrocyte HCs will be studied using ion imaging, dye uptake, HPLC amino acid measurements and immunocytochemistry. The proposed studies will provide useful new information about astrocyte HCs, a neglected channel with unique characteristics. These results will have implications for both normal as well as pathological brain function.