Investigations into hydrogen sulfide-induced suppression of neuronal activity in vivo and calcium dysregulation in vitro

Acute exposure to high concentrations of hydrogen sulfide (H(2)S) leads to sudden death and, if survived, lingering neurological disorders. Clinical signs include seizures, loss of consciousness, and dyspnea. The proximate mechanisms underlying H(2)S-induced acute toxicity and death have not been clearly elucidated. We investigated electrocerebral, cardiac, and respiratory activity during H(2)S exposure using electroencephalogram (EEG), electrocardiogram, and plethysmography. H(2)S suppressed electrocerebral activity and disrupted breathing. Cardiac activity was comparatively less affected. To test whether Ca(2+) dysregulation contributes to H(2)S-induced EEG suppression, we developed an in vitro real-time rapid throughput assay measuring patterns of spontaneous synchronized Ca(2+) oscillations in cultured primary cortical neuronal networks loaded with the indicator Fluo-4 using the fluorescent imaging plate reader (FLIPR-Tetra(®)). Sulfide >5 ppm dysregulated synchronous calcium oscillation (SCO) patterns in a dose-dependent manner. Inhibitors of NMDA and AMPA receptors magnified H(2)S-induced SCO suppression. Inhibitors of L-type voltage-gated Ca(2+) channels and transient receptor potential (TRP) channels prevented H(2)S-induced SCO suppression. Inhibitors of T-type voltage-gated Ca(2+) channels, ryanodine receptors, and sodium channels had no measurable influence on H(2)S-induced SCO suppression. Exposures to >5 ppm sulfide also suppressed neuronal electrical activity in primary cortical neurons measured by multielectrode array (MEA), an effect alleviated by pretreatment with the nonselective TRP channel inhibitor, 2-aminoethoxydiphenylborate (2-APB). 2-APB also reduced primary cortical neuronal cell death from sulfide exposure. These results improve our understanding of the role of different Ca(2+) channels in acute H(2)S-induced neurotoxicity and identify TRP channel modulators as novel structures with potential therapeutic benefits.