Toxic effects of midazolam on differentiating neurons in vitro as a consequence of suppressed neuronal Ca2+-oscillations

Abstract

Background: In immature neurons anesthetics induce apoptosis and influence neuronal differentiation. Neuronal Ca(2+)-oscillations regulate differentiation and synaptogenesis. We examined the effects of the long-term blockade of hippocampal Ca(2+)-oscillations with midazolam on neuronal synapsin expression.

Material and methods: Hippocampal neurons were incubated at day 15 in culture with the specific GABA(A) receptor agonist muscimol (50μM) or with midazolam (100 and 300nM), respectively, for 24h. TUNEL and activated-Caspase-3 staining were used to detect apoptotic neurons. Ca(2+)-oscillations were detected using the Ca(2+)-sensitive dye FURA-2 and dual wavelength excitation fluorescence microscopy. Synapsin was identified with confocal anti-synapsin immunofluorescence microscopy.

Results: Muscimol, when applied for 24h, decreased the amplitude and frequency Ca(2+)-oscillations significantly. Midazolam concentration-dependently suppressed the amplitude and frequency of the Ca(2+)-oscillations. This was associated by a downregulation of the synapsin expression 24h after washout.

Conclusion: Neuronal Ca(2+)-oscillations mediate neuronal differentiation and are involved in synaptogenesis. By acting via the GABA(A) receptor, midazolam exerts its toxic effect through the suppression of neuronal Ca(2+)-oscillations, a reduction in synapsin expression and consecutively reduced synaptic integrity.