title: The toxic effects of s(+)-ketamine on differentiating neurons in vitro as a consequence of suppressed neuronal Ca2+ oscillations.
abstract: BACKGROUND
  In the immature brain, neuronal Ca2+ oscillations are present during a time period of high plasticity and regulate neuronal differentiation and synaptogenesis. In this study we examined the long-term blockade of hippocampal Ca2+ oscillations, the role of the N-methyl-D-aspartate (NMDA) receptors and the effects of S(+)-ketamine on neuronal synapsin expression.
  
  
  METHODS
  Hippocampal neurons were incubated at day 15 in culture with the specific NMDA receptor antagonists dizocilpine (MK 801, 100 μM) or S(+)-ketamine (3 μM to 25 μM) for 24 hours. Terminal-deoxynucleotidyl-transferase (TUNEL) and activated caspase3 were used to detect apoptotic neurons. Ca2+ oscillations were detected after loading the neurons with the Ca2+-sensitive dye fura-2AM, and dual wavelength excitation fluorescence microscopy was performed. Ca2+/calmodulin kinase II (CaMKII) was measured using Western blots. Synapsin was identified with confocal antisynapsin immunofluorescence.
  
  
  RESULTS
  Blocking the NMDA receptor with MK 801 or 25 μM S(+)-ketamine resulted in a significant increase in apoptotic neurons. MK 801 led to a significant increase in cytosolic Ca2+ concentration and reduction of the amplitude and frequency of the Ca2+ oscillations. Similar to MK 801, the long-term application of S(+)-ketamine resulted in a significant increase in cytosolic Ca2+ concentration 24 hours after washout. This was associated with a down-regulation of the CaMKII and a reduction of the synapsin 24 hours after washout.
  
  
  CONCLUSIONS
  Neuronal Ca2+ oscillations mediate neuronal differentiation and synaptogenesis via activating CaMKII. By acting via the NMDA receptor, S(+)-ketamine exerts its toxic effect through the suppression of neuronal Ca2+ oscillations, down-regulation of the CaMKII, and consecutively reduced synaptic integrity.
authors.affiliation: Department of Anesthesiology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 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: Wolfgang
authors.initials: W
authors.lastname: Zink
authors.forename: York
authors.initials: Y
authors.lastname: Zausig
authors.forename: Bernhard M
authors.initials: BM
authors.lastname: Graf
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D020013:Calcium Signaling / Q000187:drug effects*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000096:biosynthesis / Q000235:genetics; D053148:Caspase 3 / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D018691:Excitatory Amino Acid Antagonists / Q000633:toxicity*; D005260:Female; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects; D020287:In Situ Nick-End Labeling; D007649:Ketamine / Q000633:toxicity*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000187:drug effects*; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects; D013237:Stereoisomerism; D016704:Synapsins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D018691:Excitatory Amino Acid Antagonists; D016194:Receptors, N-Methyl-D-Aspartate; D016704:Synapsins; D007649:Ketamine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D053148:Caspase 3