Abstract

(R,S)-ketamine elicits rapid-acting and sustained antidepressant actions in treatment-resistant patients with depression. (R)-ketamine produces longer-lasting antidepressant effects than (S)-ketamine in rodents; however, the precise molecular mechanisms underlying antidepressant actions of (R)-ketamine remain unknown. Using isobaric Tag for Relative and Absolute Quantification, we identified nuclear receptor-binding protein 1 (NRBP1) that could contribute to different antidepressant-like effects of the two enantiomers in chronic social defeat stress (CSDS) model. NRBP1 was localized in the microglia and neuron, not astrocyte, of mouse medial prefrontal cortex (mPFC). (R)-ketamine increased the expression of NRBP1, brain-derived neurotrophic factor (BDNF), and phosphorylated cAMP response element binding protein (p-CREB)/CREB ratio in primary microglia cultures thorough the extracellular signal-regulated kinase (ERK) activation. Furthermore, (R)-ketamine could activate BDNF transcription through activation of CREB as well as MeCP2 (methyl-CpG binding protein 2) suppression in microglia. Single intracerebroventricular (i.c.v.) injection of CREB-DNA/RNA heteroduplex oligonucleotides (CREB-HDO) or BDNF exon IV-HDO blocked the antidepressant-like effects of (R)-ketamine in CSDS susceptible mice. Moreover, microglial depletion by colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX3397 blocked the antidepressant-like effects of (R)-ketamine in CSDS susceptible mice. In addition, inhibition of microglia by single i.c.v. injection of mannosylated clodronate liposomes (MCLs) significantly blocked the antidepressant-like effects of (R)-ketamine in CSDS susceptible mice. Finally, single i.c.v. injection of CREB-HDO, BDNF exon IV-HDO or MCLs blocked the beneficial effects of (R)-ketamine on the reduced dendritic spine density in the mPFC of CSDS susceptible mice. These data suggest a novel ERK-NRBP1-CREB-BDNF pathways in microglia underlying antidepressant-like effects of (R)-ketamine.

Conflict of interest statement

KH is the inventor of filed patent applications on “The use of (R)-ketamine in the treatment of psychiatric diseases”, “(S)-norketamine and salt thereof as pharmaceutical”, “(R)-ketamine and derivative thereof as prophylactic or therapeutic agent for neurodegeneration disease or recognition function disorder”, “Preventive or therapeutic agent and pharmaceutical composition for inflammatory diseases or bone diseases”, and “(R)-ketamine and its derivatives as a preventive or therapeutic agent for a neurodevelopmental disorder” by the Chiba University. KH also declares that he has received research support and consultant from Dainippon Sumitomo, Otsuka, Taisho, and Perception Neuroscience. Other authors declare no competing interests.

Methods and materials

Animals

Detailed information of animals was shown in the Supplementary Information.

Chronic social defeat stress (CSDS) model

CSDS was performed according to the previous reports. (for details, see Supplementary Information).

Compounds and cell cultures

Detailed information of the compounds including (R)-ketamine, (S)-ketamine, (2R,6R)-hydroxynorketamine [(2R,6R)-HNK], lipopolysaccharide (LPS), SL327 (ERA inhibitor), PLX3397 [colony-stimulating factor 1 receptor (CSF1R) inhibitor], mannosylated clodronate liposomes (MCLs), the antisense oligonucleotides and cRNA for targeting CREB or BDNF exon IV, and CREB-DNA/RNA heteroduplex oligonucleotides (HDO) or BDNF exon IV-HDO was shown in the Supplementary Information. Detailed information of cells cultures such as HEK293T, BV2 cells and primary microglia was also shown in the Supplementary Information.

iTRAQ analysis

(R)-ketamine (10 mg/kg as HCL salt) or (S)-ketamine (10 mg/kg as HCL salt) was administered intraperitoneally (i.p.) to CSDS susceptible mice. Tissues of mPFC were collected 7 days after a single administration of ketamine enantiomers. iTRAQ analysis of mPFC samples was performed at Aproscience Co., Ltd (now: Integrale Co., Ltd., Naruto, Tokushima, Japan).

Injection and behavioral tests

Detailed method of intracerebroventricular (i.c.v.) injection was shown in Supplementary Information. (R)-ketamine (10 mg/kg as HCL salt) was administered i.p. to CSDS susceptible mice. Behavioral tests, including locomotion test (LMT), forced swimming test (FST), and 1% sucrose preference test (SPT), were performed according to the previous reports. Behavioral experiments were performed in a blind manner.

Immunoprecipitation, quantitative real-time PCR assay, western blotting assay, luciferase assay, chromatin immunoprecipitation (ChIP) assay, immunofluorescence staining, and dendritic spine analysis

We performed immunoprecipitation, quantitative real-time PCR, western blot, luciferase assay, ChIP assay, immunofluorescence staining, and dendritic spine analysis for in vitro and/or in vivo experiments (for details, see Supplementary Information).

Statistical analysis

All data results were expressed as the mean ± standard error of the mean (S.E.M.). All data were analyzed using PASW Statistics 20 software. Differences among the groups were evaluated using one-way analysis of variance, followed by post hoc Fisher least significant difference test. Student’s t test was used to compare the differences between two groups. The P values <0.05 were considered to be significant.

Results

Figure 1: Effects of (R)-ketamine and (S)-ketamine on the BDNF activation.

Figure 2 : Effects of CREB-HDO on beneficial effects of (R)-ketamine on depression-like behaviors, and expression of p-CREB, CREB, BDNF, MeCP2, and CD11b in the mPFC.

Figure 3 : Effects of BDNF exon IV-HDO on antidepressant-like effects of (R)-ketamine.

Figure 4: Effects of PLX3397 and MCLs on the antidepressant-like effects of (R)-ketamine in the CSDS susceptible mice.

Figure 5: Effects of CREB-HDO, BDNF exon IV-HDO, or MCLs on beneficial effects of (R)-ketamine for reduced dendritic spine density in the mPFC of CSDS susceptible mice.