Neural depolarization triggers Mg²⁺ influx in rat hippocampal neurons

Homeostasis of magnesium ion (Mg²⁺) plays key roles in healthy neuronal functions, and deficiency of Mg²⁺ is involved in various neuronal diseases. In neurons, we have reported that excitotoxicity induced by excitatory neurotransmitter glutamate increases intracellular Mg²⁺ concentration ([Mg²⁺]_i). However, it has not been revealed whether neuronal activity under physiological condition modulates [Mg²⁺]_i. The aim of this study is to explore the direct relationship between neural activity and [Mg²⁺]_i dynamics. In rat primary-dissociated hippocampal neurons, the [Mg²⁺]_i and [Ca²⁺]_i dynamics were simultaneously visualized with a highly selective fluorescent Mg²⁺ probe, KMG-104, and a fluorescent Ca²⁺ probe, Fura Red, respectively. [Mg²⁺]_i increase concomitant with neural activity by direct current stimulation was observed in neurons plated on an indium-tin oxide (ITO) glass electrode, which enables fluorescent imaging during neural stimulation. The neural activity-dependent [Mg²⁺]_i increase was also detected in neurons whose excitability was enhanced by the treatment of a voltage-gated K⁺ channel blocker, tetraethylammonium (TEA) at the timings of spontaneous Ca²⁺ increase. Furthermore, the [Mg²⁺]_i increase was abolished in Mg²⁺-free extracellular medium, indicating [Mg²⁺]_i increase is due to Mg²⁺ influx induced by neural activity. The direct neuronal depolarization by veratridine, a Na⁺ channel opener, induced [Mg²⁺]_i increase, and this [Mg²⁺]_i increase was suppressed by the pretreatment of a non-specific Mg²⁺ channel inhibitor, 2-aminoethoxydiphenyl borate (2-APB). Overall, activity-dependent [Mg²⁺]_i increase results from Mg²⁺ influx through 2-APB-sensitive channels in rat hippocampal neurons.