Methylmercury (MeHg) is a prominent environmental neurotoxicant which is responsible for several episodes of mass poisoning, and remains an environmental concern today- especially among populations with high dietary intake of fish. Cerebellar-based ataxia is a hallmark sign of MeHg poisoning. The granule cell layers of the cerebellum are particularly sensitive to MeHg - especially during development. The primary objective of this project is to understand the basis for this unique sensitivity of cerebellar granule neurons to MeHg. Recent data from our lab suggest that GABAA receptor function is highly sensitive to inhibition by MeHg-particularly in cerebellar granule cells. Inasmuch as these cells express a unique phenotype of GABAA receptor, we propose to investigate the hypothesis that disruption of inhibitory GABAergic transmission at granule neuron synapses is uniquely sensitive to MeHg due to the presence of a unique phenotype of GABAA receptor expressed in these cells. GABAA receptors are heteropentameric membrane proteins consisting of combinations of several distinct subunits. They are responsible for mediating fast inhibitory synaptic transmission in the mammalian CNS by gating entry of Cl-. Typically, GABAA receptors consist of combinations of alpha, beta, gamma, or delta, subunits; multiple isoforms of many of the subunits exist. Subunit composition has been shown to affect pharmacological sensitive of GABAA receptors. Only granule cells contain the a6 subunit which lacks benzodiazapine agonist sensitivity; they frequently also contain the delta subunit. These two subunits also confer sensitive to Zn2+, which is released in cerebellar granule neurons by MeHg, and which inhibits GABAA receptor function. The approach will involve application of whole cell voltage clamp techniques to native cerebellar neurons in culture or in isolated slice to compare the sensitivity of the GABAA receptors to MeHg in granule cells and purkinje cells, which lack the alpha6 and delta subunit. Additionally, heterologous expression of cloned GABAA receptors in HEK 293 cells will be used to examine specifically the role that the alpha6 and delta subunit play in sensitivity to MeHg.
Specific aims are to determine 1) Does MeHg differentially affect the functional properties of GABAA receptors in granule cells and Purkinje cells? If so, by what mechanism(s)? 2) Are recombinant GABAA receptors containing alpha6 subunits more sensitive to MeHg than those containing alpha1, subunits, and what if any role do other subunits, specifically gamma or delta subunits, play in MeHg-induced effects on GABAA receptors? 3) Are the effects of MeHg on cerebellar synaptic transmission, particularly inhibitory synaptic transmission, altered by knockout or mutation of genes for subunits such as the alpha6 or delta subunit which are unique to cerebellar granule cells?
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