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

Background: In immature neurons anesthetics induce apoptosis and influence neuronal differentiation. Neuronal Ca2+-oscillations regulate differentiation and synaptogenesis. We examined the effects of the long-term blockade of hippocampal Ca2+-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 (50 mu M) or with midazolam (100 and 300 nM), respectively, for 24 h. TUNEL and activated-Caspase-3 staining were used to detect apoptotic neurons. Ca2+-oscillations were detected using the Ca2+-sensitive dye FURA-2 and dual wavelength excitation fluorescence microscopy. Synapsin was identified with confocal anti-synapsin immunofluorescence microscopy. Results: Muscimol, when applied for 24 h, decreased the amplitude and frequency Ca2+-oscillations significantly. Midazolam concentration-dependently suppressed the amplitude and frequency of the Ca2+-oscillations. This was associated by a downregulation of the synapsin expression 24 h after washout. Conclusion: Neuronal Ca2+-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 Ca2+-oscillations, a reduction in synapsin expression and consecutively reduced synaptic integrity. (C) 2011 Elsevier Ireland Ltd. All rights reserved.