Involvement of the GABAa receptor in calcium-dependent toxicity of methylmercury in developing and aged cerebellum
Methylmercury (MeHg) is an environmental neurotoxicant of concern because of its bioaccumulation in marine ecosystems and the susceptibility of developing cerebellum to toxicity. MeHg accumulates in the central nervous system with a long latency to toxicity and excretion. It induces permanent motor dysfunction through selective toxicity in cerebellar granule cells (CGCs), with developing CGCs being a particular target. Though the exact pathways by which MeHg induces neuronal death and dysfunction are not fully characterized, one of the common, central pathways by which it is hypothesized to act is through disruption of intracellular calcium concentration ([Ca2+]i). [Ca2+]i is kept low in neurons and small changes are critical for neuronal signaling. Disruption of these concentrations occurs in several cell models, characterized as an irreversible increase in [Ca2+]i over time. We sought to characterize disruption of [Ca2+]i in cerebellar slices, which retain many characteristics of whole tissue. One pathway by which methylmercury may act on intracellular calcium is through the γ-aminobutyric acid type A receptor (GABAAR), a normally inhibitory receptor that is excitatory in developing CGCs and possibly in axons and presynaptic terminals. This excitation is coupled to influx of [Ca2+]i and is critical to developmental migration of neurons and to neurotransmitter release. The interaction of GABAAR s with MeHg is partially characterized by electrophysiology and there are several experiments that suggest certain subunits of the receptors are associated with cell and developmental susceptibility. We hypothesized that both MeHg and GABAARs could have effects on [Ca2+]i in CGCs in acutely-isolated slices of cerebellum and that these effects would be dependent on age and GABAAR subunit expression. To monitor [Ca2+]i we used concentration-dependent fluorophores and imaging on confocal and epifluorescent microscopes. We first tested for [Ca2+]i changes in neonatal rat, a model frequently used previously for MeHg toxicity studies. In neonatal rat CGCs, MeHg caused a concentration-dependent increase in [Ca2+]i-dependent fluorescence within minutes. These fluorescence increases included up to about 2-fold in mature CGCs compared to starting fluorescence and up to about 3-fold increase in susceptible developing CGCs. Muscimol, a GABAAR agonist, and bicuculline, a GABAAR antagonist, are both able to reduce the effects of MeHg on [Ca2+]i. MeHg is able to increase [Ca2+]i in cerebellar slices from mice as well, and a knockout of the GABAAR α6 subunit is able to increase the susceptibility of CGCs, particularly developing CGCs, with similar 2-3 foldincreases in fluorescence. Muscimol and bicuculline reduce MeHg effects in these mice as well. Compared to developmental toxicity, aging and lifelong exposures to MeHg have not been characterized nearly as fully, and as part of a comprehensive study, the effects of MeHg and several other [Ca2+]i-dependent mechanisms were tested with these fluorescence microscopy approaches. Acute application of MeHg appears to cause 2-fold increases in fluorescence that are not dependent on age or chronic treatment. However, chronic treatment with MeHg appears to be able to increase both spontaneous and induced Ca2+ activity in slices, and chronic treatment with isradipine appears to reduce this activity, indicating chronic, non-lethal concentrations can havesubtle effects on Ca2+-dependent mechanisms. Finally, this dissertation provides some characterization of spontaneous [Ca2+]i transients in slices, indicating age and genotype have effects on spontaneous CGC activity. Altogether, the results of this dissertation indicate that GABAAR s have a partial role in the toxicity of MeHg as a pathway by which MeHg appears to be able to disrupt [Ca2+]i as interference by GABAAR modulators and alterations to subunit expression also alter MeHg effects on [Ca2+]i. MeHg exposure may in turn alter GABAAR signaling as indicated by changes to muscimol responses in chronically-exposed mice.
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- In Collections
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Electronic Theses & Dissertations
- Copyright Status
- In Copyright
- Material Type
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Theses
- Authors
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Bradford, Aaron Blackwood
- Thesis Advisors
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Atchison, William D.
- Committee Members
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LaPres, John
Chan, Christina
Kuo, Min Hao
DeWitt, David
- Date
- 2013
- Subjects
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GABA--Receptors
Methylmercury
Neurotoxicology
- Degree Level
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Doctoral
- Language
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English
- Pages
- xxiii, 225 pages
- ISBN
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9781303445941
1303445948