Calmodulin Dependent Protein Kinase II: An Effector in the Signaling Pathway of Antidepressant-like Behavior Elicited by Ketamine in Combination with Melatonin

Abstract

<b>Abstract ID 21584</b> <b>Poster Board 473</b> Melatonin (MEL; <i>N</i>-acetyl-5-hydroxytryptamine) and ketamine (KET; 2-(2-chlorophenyl)-2-(methylamino) cyclohexan-1-one) elicit antidepressant-like effects in mice and increases neurogenesis in the hippocampus (HP) through stimulation of MEL receptors or by blocking the NMDA receptors, respectively. Non-effective doses KET administered with non-effective MEL doses, increases both processes. The alpha isoform of Calmodulin-dependent kinase II (CaMKII) is a downstream effector in both KET and MEL signaling pathways. MEL activates CaMKII and stimulates dendrite formation and complexity, as well as neurogenesis in the hippocampus (HP). On the other hand, KET through NMDA receptor antagonism increases pospho-CaMKII (p-CaMKII) levels, and glutamate receptor (GluR1) expression and phosphorylation in the prefrontal cortex (PC). The goal of this study was to determine whether MEL in combination with KET at doses that elicit the anti-depressant-like behavior in mice activates CaMKII and GLUR1 signaling pathways in the HP and PC. Male Swiss Webster mice (25–35 g) were managed under the laboratory animal care rules (NIH-85-23, 1985). Vehicle (VEH), or either MEL (16 mg/kg), or KET (1.5 mg/kg) alone or KET in combination with MEL (1.5/16 mg/kg; KET/MEL) were intraperitoneally administered in 10.0 mL/kg body weight, and 30 min later were submitted to the forced swimming test (FST). Mice were decapitated, and the HP and the PC dissected, homogenized, separated by two- or one-dimensional electrophoresis, and assayed by Western blot. We detected αCaMKII, anti-pCaMKII, GluR1 with specific primary and secondary antibodies coupled to either FITC or RITC. Another group of mice, treated as described above were intracardially perfused with 4% paraformaldehyde after the FST. We sectioned brains with a cryostat at -20°C in slices of 30-35 μm. Slices were stained with the antibodies mentioned above and nuclei with DAPI. Data were analyzed with a Student́s t-test or a one-way analysis of variance (ANOVA). p-values ≤ 0.05 was considered as significant. We separated and identified CaMKII and GluR1 by molecular weights and isoelectric point by two-dimensional electrophoresis. The relative amount of CaMKII were increased in mice treated with MEL or KET/MEL in the HP. In the PF, the relative amount of this protein was solely increased in mice treated with KET/MEL. Also, the acidic isoforms of GluR1 were significantly increased in the HP of mice treated with MEL or KET/MEL and in the PC of mice treated with KET or KET/MEL. We showed by Western blot that the relative amount of αCaMKII and pCaMKII were increased in HP of mice administered with KET or the combination of KET/MEL. This result was confirmed by immunostaining of pCaMKII in HP. No differences were observed in the PC. These results indicate that CaMKII expression and activation is selectively caused in the HP by MEL, KET and KET/MEL after an acute administration. Also, we showed that in PF, the amount of the acidic isoforms of GluR1 were increased by KET/MEL. Importantly, KET at the dosage tested <i>per se</i> does not produce the antidepressant-like behavior in mice. Data suggest that the antidepressant-like behavior observed after an acute administration of KET/MEL could be mediated by increasing phosphorylation of both CaMKII and the glutamate receptor GluR1. More experiments are necessary to go deeper into the mechanism of KET/MEL combination. Supported by CONACyT Grant 290526 (GBK) and Grant 252935 (CT).