title: 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Anesthesiology, University of Regensburg, Regensburg, Germany. barbara.sinner@klinik.uni-regensburg.de
authors.forename: Barbara
authors.initials: B
authors.lastname: Sinner
authors.forename: Oliver
authors.initials: O
authors.lastname: Friedrich
authors.forename: York
authors.initials: Y
authors.lastname: Zausig
authors.forename: Thomas
authors.initials: T
authors.lastname: Bein
authors.forename: Bernhard M
authors.initials: BM
authors.lastname: Graf
mesh_terms: D000818:Animals; D002118:Calcium / Q000037:antagonists & inhibitors* / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D008874:Midazolam / Q000633:toxicity*; D009433:Neural Inhibition / Q000187:drug effects / Q000502:physiology*; D055495:Neurogenesis / Q000187:drug effects* / Q000502:physiology; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar
chemical_list: D008874:Midazolam; D002118:Calcium