Ca2+ entry pathways in mouse spinal motor neurons in culture following in vitro exposure to methylmercury

Abstract

Methylmercury (MeHg) is a widespread environmental toxicant with major actions on the central nervous system. Among the neurons reportedly affected in cases of Hg poisoning are motor neurons; however, the direct cellular effects of MeHg on motor neurons have not been reported. Ratiometric fluorescence imaging, using the Ca2+-sensitive fluorophore fura-2, was used to examine the effect of MeHg on Ca2+ homeostasis in primary cultures of mouse spinal motor neurons. In vitro MeHg exposure at concentrations (0.1–2μM) known to affect other neurons in culture differentially, induced a biphasic rise in fura-2 fluorescence ratio indicating an increase in [Ca2+]i. The time-to-onset of these fura-2 fluorescence ratio changes was inversely correlated with MeHg concentration. TPEN (20μM), a non-Ca2+, divalent cation chelator, reduced the amplitude of the increase in fura-2 fluorescence induced by MeHg in the first phase, indicating that both Ca2+ and non-Ca2+ divalent cations contribute to the MeHg-induced effect. When examining various Ca2+ entry pathways as possible targets contributing to Ca2+ influx, we found that excitatory amino acid receptor blockers MK-801 (15μM), and AP-5 (100μM)—both NMDA receptor-operated ion channel blockers, CNQX (20μM), a non-NMDA receptor blocker, and the voltage-dependent Ca2+ channel blockers nifedipine (1μM) and ω-conotoxin-GVIA (1μM) all significantly delayed the development of increased Ca2+ caused by MeHg. The voltage-dependent Na+ channel blocker tetrodotoxin (TTX, 1μM) did not alter the MeHg-induced increases in fura-2 fluorescence ratio. Thus, MeHg alters Ca2+ homeostasis in mouse spinal motor neurons through excitatory amino acid receptor-mediated pathways, and nifedipine and ω-conotoxin-GVIA-sensitive pathways. Spinal motor neurons are highly sensitive to this effect of acute exposure to MeHg.