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

BackgroundIn 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 methodsHippocampal neurons were incubated at day 15 in culture with the specific GABAA receptor agonist muscimol (50 μM) 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.ResultsMuscimol, 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.ConclusionNeuronal Ca2+-oscillations mediate neuronal differentiation and are involved in synaptogenesis. By acting via the GABAA receptor, midazolam exerts its toxic effect through the suppression of neuronal Ca2+-oscillations, a reduction in synapsin expression and consecutively reduced synaptic integrity.

► Intravenous anesthetics are potentially neurotoxic to the developing brain. ► Ca2+-oscillations are responsible for neuronal differentiation and synaptogenesis. ► Blocking the GABAA receptor induces apoptosis in hippocampal cell cultures. ► The long-term application of midazolam suppresses neuronal Ca2+-oscillations. ► This was associated with a decrease in synapsin expression and synaptic integrity.