Astrocytes are the most numerous and diverse non-neuronal cells within the central nervous system (CNS). Although astrocytes have been shown to play important roles in normal CNS development, function, and the pathology of the nervous system, the cellular mechanisms which carry out these function are still poorly understood. Recent patch clamp studies have demonstrated that astrocytes can express most voltage- sensitive ionic currents that are found in neurons, including voltage- sensitive Na+, K+, Ca++, and Cl- currents. Of these ionic currents, the presence of voltage-sensitive Na+ currents and their role in astrocytes is particularly interesting because astrocytes have been considered to be electrically non-excitable. Further interest comes from the finding that two distinct Na+ currents (termed neuronal and glial type) with different biophysical and pharmacological characteristics have been observed in astrocytes derived form various regions of the CNS. At present, the molecular basis for the generation of these two distinct astrocytes Na+ currents is unknown. Recently, it has been demonstrated that brain Na+ channel mRNAs (both alpha and beta-1 subtypes) are expressed in rat astrocytes. In the present study, the investigator proposes to test the hypotheses that astrocytes express functional Na+ channels and that the beta-1 subunit plays a key role in expressing neuronal or glial-type Na+ currents. The combination of biochemical, biophysical, and molecular biological approached will be employed to test these hypotheses, including in situ hybridization, immunocytochemistry, immunoprecipitation, and whole-cell patch clamp methods. Specifically, the investigator proposes to : 1) test the hypothesis that brain Na+ channel beta-1 subunit is differentially expressed in freshly isolated and cultured astrocytes from rat spinal cord and hippocampus using combined in situ hybridization and immunocytochemistry; 2) determine whether or not astrocytes co-express brain Na+ channel alpha and beta-1 subunits; 3)test the hypothesis that brain Na+ channel alpha subunits generate voltage-activated Na+ currents in astrocytes by performing the antisense gene knockout experiment; 4)test the hypothesis that the Na+ channel beta-1 subunit is an important factor to generate neuronal-type Na+ currents in astrocytes by performing the antisense suppression and overexpression experiments. The results obtained form this study will offer many new insights as to the nature of astrocyte Na+ channels, their functional roles, and will eventually help to further explain neuron-astrocyte interaction and the involvement of astrocytes in normal and pathophysiological CNS conditions.
