Ketamine plus imipramine treatment induces antidepressant-like behavior and increases CREB and BDNF protein levels and PKA and PKC phosphorylation in rat brain

Evidence reviewed by Réus and colleagues frames NMDA receptor dysfunction in glutamate neurotransmission as a contributing factor in major depression, and highlights ketamine—an uncompetitive NMDA receptor antagonist—as producing rapid antidepressant effects in both animal models and clinical studies. The introduction summarises prior findings linking depression to reductions in neurotrophic support (notably brain-derived neurotrophic factor, BDNF), alterations in NMDA receptor subunit expression, and changes in intracellular signalling pathways involving cyclic AMP response element binding protein (CREB), protein kinase A (PKA) and protein kinase C (PKC). Existing antidepressants achieve therapeutic benefit in only around 60–70% of patients, motivating investigation of alternative or adjunctive treatments and of molecular mechanisms that might underlie rapid antidepressant action.

Adult male Wistar rats (60 days old) were used. Animals were group-housed with ad libitum food and water under a 12-h light/dark cycle. All procedures complied with institutional and national animal-care guidelines and an ethics protocol was reported. Drug treatments were administered intraperitoneally 60 min before behavioural testing. Nine experimental groups (n = 15 per group) received the following treatments (all volumes 1 ml/kg): saline + saline; saline + ketamine 5 mg/kg; saline + ketamine 10 mg/kg; saline + imipramine 10 mg/kg; saline + imipramine 20 mg/kg; ketamine 5 mg/kg + imipramine 10 mg/kg; ketamine 5 mg/kg + imipramine 20 mg/kg; ketamine 10 mg/kg + imipramine 10 mg/kg; and ketamine 20 mg/kg + imipramine 20 mg/kg. Co‑administrations were injected sequentially (one immediately after another). Ketamine was used as an injectable solution and imipramine was dissolved in saline immediately before injection; dose ranges were chosen based on previous work by the group. Behavioural assessment comprised the forced swimming test and an open-field locomotor test. The forced swimming test followed the standard two-session protocol: a 15 min pre-test without drug, followed 24 h later by a 5 min test session in which immobility time was recorded; treatments were given 60 min before the second (test) session. Naïve rats were used in a separate series to assess spontaneous locomotor activity in an open-field arena during a 5 min observation (horizontal crossings and vertical rearing counted by an observer). After behavioural testing rats were killed by decapitation and the prefrontal cortex, hippocampus and amygdala were dissected. Tissue homogenates were prepared in detergent-containing extraction buffer and centrifuged; supernatants were quantified by Bradford assay. Protein samples (0.2 mg per lane) were denatured, separated by SDS-PAGE, transferred to nitrocellulose membranes and probed with antibodies against BDNF, CREB, PKA and PKC (antibodies from Santa Cruz Biotechnology). Membranes were detected by chemiluminescence; membranes were stripped and re‑probed with actin as a loading control and Ponceau staining was used to check transfer. Band intensities were measured by optical densitometry (Scion Image). Statistical comparisons used one-way ANOVA followed by Tukey post-hoc tests where appropriate. Data are presented as mean ± S.E.M., and p < 0.05 was considered statistically significant.

In the forced swimming test ketamine at 10 mg/kg and imipramine at 20 mg/kg produced antidepressant-like decreases in immobility when administered alone; ketamine 5 mg/kg and imipramine 10 mg/kg did not alter immobility time by themselves. Co-administration of ketamine (both 5 and 10 mg/kg) with imipramine (10 and 20 mg/kg) produced a synergistic reduction in immobility time (reported as p < 0.05). Open-field testing showed no significant changes in horizontal crossings or rearing for any treatment, alone or combined (p > 0.05), indicating that the behavioural effects were not attributable to altered locomotor activity. Biochemical analyses revealed dose- and region-dependent effects on BDNF and CREB protein levels and on PKA and PKC phosphorylation across prefrontal cortex, hippocampus and amygdala. The authors report that certain low-dose combinations (for example ketamine 5 mg/kg with imipramine 10 mg/kg) increased BDNF in prefrontal cortex and hippocampus (p < 0.05), whereas higher single doses (ketamine 10 mg/kg or imipramine 20 mg/kg) and some other combinations were associated with decreased BDNF relative to saline in those regions. Nevertheless, combinations of ketamine 10 mg/kg with imipramine 10 or 20 mg/kg are described as producing synergistic increases in BDNF in both prefrontal cortex and hippocampus (p < 0.05). In the amygdala, several treatments including ketamine 5 mg/kg and imipramine 10 or 20 mg/kg alone, and those doses combined, increased BDNF, with a reported synergistic effect for ketamine 10 mg/kg plus imipramine 20 mg/kg (p < 0.05). CREB protein levels showed region- and dose-specific changes. In prefrontal cortex CREB increased after ketamine 10 and 20 mg/kg and imipramine 20 mg/kg, and also after ketamine 5 mg/kg combined with imipramine 10 or 20 mg/kg; synergistic increases were reported for ketamine 10 mg/kg combined with imipramine 10 or 20 mg/kg (p < 0.05). In the hippocampus some single doses (ketamine 10 mg/kg, imipramine 10 mg/kg) and certain combinations decreased CREB, while other combinations (ketamine 5 mg/kg + imipramine 20 mg/kg; ketamine 10 mg/kg + imipramine 10 mg/kg) produced synergistic increases compared with control (p < 0.05). In the amygdala, increases in CREB were observed only for ketamine 10 mg/kg combined with imipramine 10 or 20 mg/kg (p < 0.05). PKC phosphorylation in prefrontal cortex was increased by ketamine and imipramine at all tested doses, alone or combined, with a synergistic increase for ketamine 10 mg/kg plus imipramine 20 mg/kg (p < 0.05). No changes in PKC phosphorylation were reported for hippocampus and amygdala at the doses tested. Reported PKA phosphorylation changes were region- and dose-dependent: prefrontal cortex PKA phosphorylation increased with imipramine 10 and 20 mg/kg alone and with the ketamine 10 mg/kg plus imipramine 20 mg/kg combination (no synergism reported in PFC). In hippocampus and amygdala the text indicates increased phosphorylation with imipramine alone or combined, and a synergistic effect with ketamine 10 mg/kg combined with imipramine 10 or 20 mg/kg (p < 0.05). The extracted results contain some dose- and region-specific contrasts and apparent inconsistencies in direction for single versus combined treatments; where reported, significance is stated as p < 0.05.

Réus and colleagues interpret their findings as showing that ketamine produces antidepressant-like effects in the forced swimming test and that co-administration of ketamine with the tricyclic antidepressant imipramine yields synergistic behavioural effects without altering spontaneous locomotion. They place these results in the context of previous animal and clinical studies reporting rapid antidepressant actions of ketamine and prior reports of synergism when conventional antidepressants are paired with NMDA receptor antagonists. At the molecular level the authors argue that the combined treatment produced more pronounced changes in neurotrophic signalling and intracellular kinases—specifically BDNF and CREB protein levels and phosphorylation of PKA and PKC—across brain regions implicated in mood regulation (prefrontal cortex, hippocampus and amygdala). They suggest these changes could mediate the enhanced behavioural response seen with combination treatment and note that prior work has reported acute increases in hippocampal BDNF after ketamine but not after imipramine, with possible tolerance or adaptive effects after repeated exposure. The discussion acknowledges variable findings in the literature regarding PKA and PKC activity in depression and points to region- and treatment-specific differences observed here; for example, PKC phosphorylation changes were most evident in prefrontal cortex, whereas PKA phosphorylation increases were reported in hippocampus and amygdala for certain treatments. The authors mention that differing results across studies may reflect adaptive mechanisms, treatment duration, or methodological factors. Finally, they propose that coadministration of ketamine and imipramine could be of interest for drug-resistant patients and for achieving a more rapid onset of antidepressant action, and they suggest that modulation of BDNF, CREB and kinase signalling may be involved in regulation of NMDA-receptor-related pathways. The extracted text does not present an explicit list of study limitations beyond noting contrasting effects that might reflect adaptive mechanisms or tolerance, nor does it report long-term or repeated‑dose data in this paper.