Tetramethylenedisulfotetramine alters Ca²⁺ dynamics in cultured hippocampal neurons: Mitigation by NMDA receptor blockade and GABA_A receptor-positive modulation

Tetramethylenedisulfotetramine (TETS) is a potent convulsant that is considered a chemical threat agent. We characterized TETS as an activator of spontaneous Ca²⁺ oscillations and electrical burst discharges in mouse hippocampal neuronal cultures at 13-17 days in vitro using FLIPR Fluo-4 fluorescence measurements and extracellular microelectrode array recording. Acute exposure to TETS (≥ 2μM) reversibly altered the pattern of spontaneous neuronal discharges, producing clustered burst firing and an overall increase in discharge frequency. TETS also dramatically affected Ca²⁺ dynamics causing an immediate but transient elevation of neuronal intracellular Ca²⁺ followed by decreased frequency of Ca²⁺ oscillations but greater peak amplitude. The effect on Ca²⁺ dynamics was similar to that elicited by picrotoxin and bicuculline, supporting the view that TETS acts by inhibiting type A gamma-aminobutyric acid (GABA_A) receptor function. The effect of TETS on Ca²⁺ dynamics requires activation of N-methyl-d-aspartic acid (NMDA) receptors, because the changes induced by TETS were prevented by MK-801 block of NMDA receptors, but not nifedipine block of L-type Ca²⁺ channels. Pretreatment with the GABA_A receptor-positive modulators diazepam and allopregnanolone partially mitigated TETS-induced changes in Ca²⁺ dynamics. Moreover, low, minimally effective concentrations of diazepam (0.1μM) and allopregnanolone (0.1μM), when administered together, were highly effective in suppressing TETS-induced alterations in Ca²⁺ dynamics, suggesting that the combination of positive modulators of synaptic and extrasynaptic GABA_A receptors may have therapeutic potential. These rapid throughput in vitro assays may assist in the identification of single agents or combinations that have utility in the treatment of TETS intoxication.