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Abstract:
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A major and irreversible effect seen in the Fetal Alcohol Syndrome is microencephaly. The mechanism(s) underlying this effect remains unknown. Data from our laboratory demonstrate that ethanol inhibits muscarinic-induced proliferation of astroglia. An important component of the intracellular signaling cascade following muscarinic stimulation is an increase in intracellular calcium. This dissertation research tests the hypothesis that ethanol exposure during development causes central nervous system dysfunction through effects on astrocytes, specifically on their calcium response to muscarinic stimulation, thereby interfering with the normal mitogenic response to muscarinic agonists. Calcium imaging techniques were used to quantitate the carbachol-induced calcium responses in primary rat cortical astrocytes and in human 132 1N1 astrocytoma cells. The effects of ethanol on these responses were also investigated. Carbachol. induced a concentration-dependent increase in intracellular calcium, via the M3 receptor, which consisted of an initial spike from IP3-sensitive stores, followed by a sustained elevation and oscillations, which were dependent upon extracellular calcium. Protein kinase C was also found to modulate these effects. Ethanol selectively inhibited these calcium responses in a concentration- and duration-dependent manner. Acute (5 min) ethanol exposure had no effect, while short-term exposure (30 min) to high, concentrations (100--250 mM) partially inhibited these responses, and long-term exposure (24 h) inhibited these responses to a greater extent and at a lower concentration (10 mM). The inhibition was variable, but was more consistent at higher concentrations of ethanol. The effects persisted after ethanol removal, and responses returned to control levels after 24 h. A similar time-course of ethanol effects was seen on carbachol-induced 3H-thymidine incorporation. Further experiments determined the calcium-dependency of muscarinic-induced proliferation. Directly increasing calcium with ionomycin induced proliferation in these cells. Removal of extracellular calcium, or pre-incubation with nickel or cobalt, but not verapamil, nifedipine or SKF-96365, inhibited this thymidine incorporation. These data suggest that the calcium responses induced by muscarinic stimulation in astroglial cells are indirectly inhibited by ethanol and that this inhibition may mediate ethanol's inhibition of glial cell proliferation. This mechanism may be involved in the microencephaly seen in the Fetal Alcohol Syndrome. |