This placebo-controlled animal study (n=135) investigated the possibility of synergistic interactions between the antidepressant imipramine (10-20mg/kg) with ketamine (5-10mg/kg). The results indicate that co-administration of imipramine with ketamine may induce a more pronounced antidepressant activity than treatment with each antidepressant alone.
A growing body of evidence has pointed to the N-methyl-d-aspartate (NMDA) receptor antagonists as a potential therapeutic target for the treatment of major depression. The present study investigated the possibility of synergistic interactions between antidepressant imipramine with the uncompetitive NMDA receptor antagonist ketamine. Wistar rats were acutely treated with ketamine (5 and 10 mg/kg) and imipramine (10 and 20 mg/kg) and then subjected to forced swimming tests. The cAMP response element bindig (CREB) and brain-derived neurotrophic factor (BDNF) protein levels and protein kinase C (PKC) and protein kinase A (PKA) phosphorylation were assessed in the prefrontal cortex, hippocampus and amygdala by imunoblot. Imipramine at the dose of 10 mg/kg and ketamine at the dose of 5 mg/kg did not have effect on the immobility time; however, the effect of imipramine (10 and 20 mg/kg) was enhanced by both doses of ketamine. Ketamine and imipramine alone or in combination at all doses tested did not modify locomotor activity. Combined treatment with ketamine and imipramine produced stronger increases of CREB and BDNF protein levels in the prefrontal cortex, hippocampus and amygdala, and PKA phosphorylation in the hippocampus and amygdala and PKC phosphorylation in prefrontal cortex. The results described indicate that co-administration of antidepressant imipramine with ketamine may induce a more pronounced antidepressant activity than treatment with each antidepressant alone. This finding may be of particular importance in the case of drug-resistant patients and could suggest a method of obtaining significant antidepressant actions whilst limiting side effects.
Papers cited by this study that are also in Blossom
Berman, R. M., Cappiello, A., Anand, A. et al. · Biological Psychiatry (2000)
Papers in Blossom that reference this study
Brake, W. G., Gagne, C., Piot, A. · Frontiers in Psychiatry (2022)
Zheng, W., Zhou, Y. L., Wang, C. Y. et al. · PeerJ (2021)
Wilkowska, A., Szałach, L., Cubała, W. J. · Neuropsychiatric Disease And Treatment (2020)
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.
Evidence is emerging for a role for glutamate neurotransmission dysfunctional via N-methyl-d-aspartate (NMDA) receptor in major depression. Research in this area has been made because antidepressant drugs used show therapeutic efficacy in a maximum of 60-70% of depressive patients, therefore there is a strong need for alternative antidepressive treatments. Several studies have shown that NMDA receptor antagonists (such as, memantine and ketamine) have antidepressant effect in animal models, as well as in humans. Furthermore, other studies have showed changes in NMDA receptor subunits. In situ hybridization studies showed mRNA reduced levels of the NR2A and NR2B subunit of the NMDA receptors in the perirhinal cortexand the NR1 subunit of the NMDA receptor in hippocampusin patients with major depression. In addition, reduced levels of NR2A and NR2B subunits of the NMDA receptor and PSD-95 (which interact with the NR2B subunit of NMDA receptor) were found in prefrontal cortex in major depression patients. Ketamine is a NMDA receptor antagonist for glutamate and has been shown to have antidepressant effects in animal models, as well, antidepressant effects in humans. Morphological changes have been reported in the hippocampus, prefrontal cortexand amygdala. One mechanism by which brain impairments may correspond with depression is via neurotrophic factors and related signaling cascades. Neurotrophic factors regulate neural growth and differentiation during development and are regulators of plasticity and survival of adult neurons and glia. Several studies have pointed to the role of brain-derived-neurotrophic factor (BDNF) in major depression. Decreased levels of BDNF have been shown in animal models of depression and humans with depression. Conversely, administration of antidepressant drugs increases BDNF expressionand brain infusion of BDNF produces antidepressant-like actions in rats. In addition, the NMDA receptor antagonist ketamine increases BDNF protein levels in the rat hippocampus. In fact, NMDA receptor trafficking is critical to regulate various forms of synaptic plasticity in the CNS. Cyclic AMP response element binding protein (CREB) is a transcription activator that has critical roles in response to many signal transduction cascades activated by hormones, growth factor, synaptic activity and other cellular stimuli implicated in neuronal plasticity. Moreover CREB is implicated in both stressand antidepressant-induced transcriptional regulation. Phosphorylation, and therefore activation of CREB can be induced by a number of upstream signaling cascades. The regular pathway that leads to CREB phosphorylation is the cAMP protein kinase A (PKA), the catalytic subunits of PKA phosphorylate nuclear substrates, including transcription factors. Recent studies have focused on the role of PKA in various psychiatric disorders, including major depression. In fact, low PKA activity was observed in fibroblasts culture from a group of depressed patients. One study also showed that PKA activity, (but not the protein kinase C (PKC)), was reduced in post mortem brain from people with a history of major depression. In contrast, other studies using both human peripheral and post mortem brain tissue from depressed patient's exhibited reduced PKC activity. PKC plays a major role in the regulation of neuronal excitability, neurotransmitter release, and alterations in gene expression and plasticity. Moreover, Szabo et al.suggest that PKC may play an important role in regulating NMDA receptor functions. Generally, drug-resistant depression is treated with combinations of various antidepressants, however with only moderate therapeutic efficacy. The aim of the present study was to test if a combination of the currently used antidepressant imipramine and NMDA receptor antagonist ketamine would produce synergistic antidepressive-like effects in the forced swim test, BDNF and CREB protein levels and PKA and PKC phosphorylation in rat brain.
Male Adult Wistar rats (60 days old) were obtained from the UNESC (University of Southern Santa Catarina, Criciúma, SC, Brazil) breeding colony. They were housed five per cage with food and water available ad libitum and were maintained on a 12-h light/dark cycle (lights on at 7:00 a.m.). All experimental procedures involving animals were performed in accordance with the NIH Guide for the Care and Usage of Laboratory Animals and the Brazilian Society for Neuroscience and Behavior (SBNeC) recommendations for animal care and with approval by local Ethics Committee under protocol number 95/2009.
Ketamine employed in the present study was purchased from Fort Dodge Animal Health (Fort Dodge, IA, USA) as injectable solution (concentration 0.1 g/ml), and imipramine from Novartis Pharmaceutical Industry (Basel, Switzerland). Different groups of rats, 9 groups in total, (n = 15 each) were administered intraperitoneally 60 min before the test session, i.e. forced swimming or openfield tests, with 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; ketamine 20 mg/kg + imipramine 20 mg/kg. The Imipramine and ketamine were dissolved in saline immediately before the injections. In relation to the co-administration of drugs, they were injected one immediately after another, for example, saline was administered then the ketamine and then imipramine. All treatments were administered in a volume of 1 ml/kg. The range of doses of ketamine and imipramine employed in this work was chosen based on our previous study.
The forced swimming test was conducted according to previous reports. The test involves two individual exposures to a cylindrical tank filled with water in which rats cannot touch the bottom of the tank or escape. The tank is made of transparent Plexiglas, 80 cm tall, 30 cm in diameter, and filled with water (22-23 • C) to a depth of 40 cm. The water in the tank was changed after each rat. For the first exposure, rats without drug treatment were placed in the water for 15 min (pre-test session). Twenty-four hours later, rats were placed in the water again for a 5 min session (test session), and the immobility time of rats were recorded in seconds. Rats were treated with ketamine, imipramine or saline only 60 min before the second exposure to the cylindrical tank of water (test session). In a separate series of experiments, naïve rats were treated with ketamine and imipramine and saline 60 min before the exposure to the open-field apparatus, in order to assess possible effects of drug treatment on spontaneous locomotor activity. Analysis of the rats spontaneous activity was carried out in an open field apparatus, which is an arena 45 × 60 cm surrounded by 50 cm high walls made of brown plywood with a frontal glass wall. The floor of the open field was divided into 9 rectangles (15 × 20 cm each) by black lines. Animals were gently placed on the left rear quadrant, and left to explore the arena for 5 min. The number of horizontal (crossing) and vertical (rearing) activities performed by each rat during the 5-min observation period were counted by an expert observer. After the behavioral tests the animals were killed by decapitation and the skulls were immediately removed and prefrontal cortex, hippocampus and amygdala were quickly isolated by hand dissection using a magnifying glass and a thin brush.
The Prefrontal cortex, hippocampus and amygdala tissues were excised. The tissues were homogenized immediately in extraction buffer (mM) (1% Triton-X 100, 100 Tris, pH 7.4, containing 100 sodium pyrophosphate, 100 sodium fluoride, 10 EDTA, 10 sodium vanadate, 2 PMSF and 0.1 mg of aprotinin/ml) at 4 • C with a Polytron PTA 20S generator (Brinkmann Instruments model PT 10/35) operated at maximum speed for 30 s. The extracts were centrifuged at 11,000 rpm and 4 • C in a Beckman 70.1 Ti rotor (Palo Alto, CA) for 40 min to remove insoluble material, and the supernatants of these tissues were used for protein quantification, using the Bradford method. Proteins were denatured by boiling insample buffer containing 100 mM DTT. After this, 0.2 mg of protein extracts obtained from each tissue were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-BDNF, anti-CREB, anti-PKA and anti-PKC. Antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Chemiluminescent detection was performed with horseradish peroxidase-conjugate secondary antibodies. Visualization of protein bands was performed by exposure of membranes to RX-films. The original membrane was stripped and reblotted with actin loading protein (bands not show). After transfer, the membrane was stained with Ponceau and bands were visualized, photographed and quantified before the primary antibody, to control the transfer. Band intensities were quantitated by optical densitometry (Scion Image software, ScionCorp, Frederick, MD) of the developed autoradiographs.
All data is presented as mean ± S.E.M. Differences among experimental groups in the forced swimming, open field test and in the assessment of BDNF, CREB, PKA and PKC were determined by one-way ANOVA, followed by Tukey post-hoc test when ANOVA was significant; p values less than 0.05 were considered to be statistical significant.
As depicted in Fig., ketamine at the dose of 10 mg/kg (but not 5 mg/kg) and imipramine at the dose of 20 mg/kg (but not 10 mg/kg) induced antidepressant-like activity in the Porsolt's test when given alone, and co-administration of NMDA receptor antagonist ketamine at all doses with antidepressant imipramine produced synergistic effect (Fig.; p < 0.05). In the open-field test, the treatment with ketamine and imipramine alone or in combination at all doses tested did not modify the number of crossing or rearing activities compared to saline treated-rats (Fig.and; p > 0.05). Fig.illustrates the synergistic effects of ketamine (5 and 10 mg/kg) and imipramine (10 and 20 mg/kg) in BDNF and CREB protein levels and PKA and PKC phosphorylation in the prefrontal cortex, hippocampus and amygdala. Ketamine at the dose of 5 mg/kg and imipramine at the dose of 10 mg/kg increased BDNF protein levels in the prefrontal cortex (Fig.; p < 0.05); ketamine at the dose of 10 mg/kg and imipramine at the dose of 20 mg/kg alone and co-administrated with ketamine at the dose of 5 mg/kg and imipramine at the doses of 10 and 20 mg/kg decreased BDNF protein levels in the prefrontal cortex, compared to saline group (Fig.; p < 0.05). In contrast, synergism was observed for the combination of ketamine at the dose of 10 mg/kg and imipramine at the doses of 10 and 20 mg/kg in the prefrontal cortex (Fig.; p < 0.05). In the hippocampus, ketamine at the dose of 5 mg/kg and imipramine at the dose of 10 mg/kg increased BDNF protein levels (Fig.; p < 0.05); however, ketamine at the dose of 10 mg/kg and imipramine at the dose of 20 mg/kg and ketamine at the dose of 5 mg/kg in combination with imipramine at the doses of 10 and 20 mg/kg decreased BDNF protein levels in the hippocampus, compared to saline group (Fig.; p < 0.05). The combination of ketamine at the dose of 10 mg/kg with imipramine at the doses of 10 and 20 mg/kg produced synergism in the hippocampus (Fig.; p < 0.05). In the amygdala, ketamine at the dose of 5 mg/kg and imipramine at the doses of 10 and 20 mg/kg alone increased BDNF protein levels and ketamine at the dose of 5 mg/kg in combination with imipramine at the doses of 10 and 20 mg/kg also increased BDNF protein levels in the amygdala. Synergistic effect was observed with ketamine at the doses of 10 mg/kg and imipramine at the dose of 20 mg/kg (Fig.; p < 0.05). CREB protein levels increased in the prefrontal cortex with ketamine at the doses of 10 and 20 mg/kg and with imipramine at the dose of 20 mg/kg alone. Ketamine at the dose of 5 mg/kg in combination with imipramine at the doses of 10 and 20 mg/kg also increased CREB protein levels in the prefrontal cortex. Synergistic effect was observed with ketamine at doses of 10 mg/kg with imipramine at the doses of 10 and 20 mg/kg (Fig.; p < 0.05). In the hippocampus, ketamine at the dose of 10 mg/kg and imipramine at the dose of 10 mg/kg alone decreased CREB protein levels, ketamine at the dose of 5 mg/kg in combination with imipramine at the dose of 10 mg/kg and ketamine at the dose of 10 mg/kg in combination with imipramine at the dose of 20 mg/kg also decreased CREB protein levels in the hippocampus. In contrast, ketamine at the dose of 5 mg/kg, with imipramine at the dose of 20 mg/kg and ketamine at the dose of 10 mg/kg with imipramine at the dose of 10 mg/kg produced a synergistic effect in the hippocampus, compared to control group (Fig.; p < 0.05). In the amygdala, only ketamine at the dose of 10 mg/kg in combination with imipramine at the doses of 10 mg/kg and 20 mg/kg increased CREB protein levels (Fig.; p < 0.05). The PKC phosphorylation in the prefrontal cortex increased at all doses of ketamine and imipramine alone or in combination, compared to control group. Synergistic effect was observed with ketamine at the dose of 10 mg/kg in combination with imipramine at the dose of 20 mg/kg (Fig.; p < 0.05). In the hippocampus and amygdala, treatment with ketamine and imipramine alone or in combination at all doses tested did not alter the PKC phosphorylation compared to saline treated-rats (Fig.; p < 0.05). The PKA phosphorylation in prefrontal cortex increased with imipramine at the doses of 10 and 20 mg/kg alone, and ketamine at the dose of 10 mg/kg in combination with imipramine at the dose of 20 mg/kg, but no synergistic effect was observed (Fig.; p < 0.05). In the hippocampus and amygdala imipramine at all doses alone or in combination with ketamine increased PKC phosphorylation compared to control group. Synergistic effect was observed with ketamine at the dose of 10 mg/kg in combination with imipramine at the doses of 10 and 20 mg/kg (Fig.; p < 0.05).
Our results indicate that the NMDA receptor antagonist, ketamine, reduced the immobility time in the forced swimming test in rats and showed efficacies compared to tricyclic antidepressant imipramine. This is consistent with previous findings from our group, which showed that there were dose-dependent decreases in the immobility time in rats following administration of ketamine and imipramine. Several studies have supported an antidepressant action for ketamine in basicand clinicalstudies. In 2000, a pilot study showed that a single dose of ketamine produced antidepressant effects in patients suffering from major depression. Moreover, in 2006, this study was extended to a higher number of patients, and they found that the acute administration of ketamine rapidly improved depressive symptoms in patients with major depression. Recently, another pilot study showed that ketamine was well tolerated in patients with treatment-resistant major depression and may have rapid antidepressant properties. In the present study, we showed that a synergistic effect was seen when imipramine was given jointly with ketamine and they did not affect spontaneous locomotor activity in the open-field test. Rogóz et al.have also previously showed the synergistic effect of imipramine given jointly with NMDA receptor antagonists, amantadine, memantine or neramexane, and similar synergism was observed with fluoxetine given jointly with NMDA receptor antagonists. In the present findings, we showed a synergistic effect in CREB and BDNF protein levels and PKA and PKC phosphorylation in the prefrontal cortex, hippocampus and amygdala. The hippocampus is a limbic structure that is important in the control of learning and memory and in the regulation of the HPA axis, moreover, the hippocampus has connections with amygdala and prefrontal cortex and the hippocampus, prefrontal cortex and amygdala have been implicated in major depression. Over the past decades, many studies have suggested that neurotrophins and related signaling cascades might be involved in the pathophysiology of mood disorders. In fact, several neurotrophic factors are associated with depression or antidepressant action. Additionally, NMDA receptor antagonists have demonstrated alter BDNF levels, suggesting that changes are mediated by glutamate action through NMDA receptor. Previous studies from our group have demonstrated that acute treatment with ketamine and memantine, but not imipramine, increased BDNF protein levels in rat hippocampus. However, chronic treatment with both drugs, ketamine, memantine and imipramine, did not modify BDNF protein levels in rat hippocampus, suggesting that these contrasting effects could be due to adaptive mechanisms or induction of tolerance to the effects of ketamine on BDNF protein levels. Repeated treatment with antidepressant fluoxetine and NMDA receptor antagonist amantadine given separately increased BDNF gene expression in rat brain and treatment jointly of fluoxetine and amantadine induced a more potent increase in the BDNF levels and mRNA BDNF gene expression. In addition, maternal deprivation produces a persistent reduction in the expression of the BDNF, as well as of the glutamate NMDA receptor subunits NR-2A and NR-2B in rat hippocampus and, to lesser extent, in cortical areas. Moreover, reduced levels of NR2A and NR2B subunits of NMDA receptor were demonstrated in the prefrontal cortex in major depression. There are relevant literature studies showering alterations of CREB in response to antidepressant treatment. Qi et al.identified in their studies disrupted activities of the CREB in the hippocampus and prefrontal cortex in rats that displayed depressive-like behaviors after receiving chronic forced swim stress. Additionally, the reduction in the activity of the CREB in both regions and the depressive-like behaviors exhibited in stressed rats were reversed by chronic fluoxetine treatment. Many other studies have demonstrated CREB changes and antidepressant effects in animal models. In our present data, we showed synergistic effect in the CREB protein levels in the hippocampus, prefrontal cortex and amygdala. Very recently, the roles of the proteins, PKA and PKC, have been studied in depression. Low PKA activity has been demonstrated in fibroblasts cultureand in post mortem brainfrom depressed patients. Reduced PKC activity was showed in human peripheral and post mortem brain of depressed patients. Nevertheless, in other studies, PKC activity did not alter in post mortem brain. We showed in this study the synergistic effect of ketamine and imipramine in the PKC phosphorylation only in the prefrontal cortex and in the PKA phosphorylation in the hippocampus and amygdala. PKC phosphorylation of the NMDA receptor site GluN1S896 was also enhanced in the prefrontal cortex of animals treated with the antidepressant imipramine, suggesting that PKC may play an important role in regulating NMDA receptor functions. In conclusion, this present study indicates that coadministration of antidepressant imipramine and NMDA receptor antagonist, ketamine, may induce a more pronounced antidepressive activity than treatment with imipramine alone. Our study may be important in the case of drug-resistant patients, and to produce a rapid onset antidepressant action. In addition, we suggested that synergistic effect by ketamine plus imipramine in BDNF and CREB protein levels and PKA and PKC phosphorylation may be involved in regulation of NMDA receptor.
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Cameron, L. P., Benson, C. J., Dunlap, L. E. · ACS Chemical Neuroscience (2018)