Figure 6.

Participation of CaMKII, melatonin receptors, and calmodulin (CaM) on cluster formation. Olfactory neuronal precursors were cultured for 4 days and then preincubated in serum-free medium with the VEH, 10 µM KN-62, an inhibitor of CaM Kinase II, 10 µM luzindole, an MT1 and MT2 melatonin receptor antagonist, or 10 µM trifluoperazine (TFP), a CaM antagonist, during 15 min. Afterwards, the combination of 10⁻⁷ M ketamine with 10⁻⁷ M melatonin (KET/MEL) was added for 6 h. Cells were fixed and stained with an anti-nestin an anti-doublecortin antibodies, followed by secondary antibodies as described in Materials and Methods. Panel A shows representative images of clusters formed in the presence of the VEH or the KET/MEL combination. Bar = 10 µm. Panel B shows a table and a bar graph with the results of frequency formation of clusters. Results are the mean of 7000 cells/coverslip analyzed by duplicate per treatment (n = 4). Each coverslip has an area of 113 mm² which nearly corresponds to 340 fields observed with the 40X objective. Coverslips were scanned by confocal microscopy with the Image J (NIH) and Zenblue (Zeiss) software. The experiment was repeated two times.