Ketamine Rescues Hippocampal Reelin Expression and Synaptic Markers in the Repeated-Corticosterone Chronic Stress Paradigm

Abstract

Depression is the leading cause of disability worldwide, which necessitates novel therapeutics and biomarkers to approach treatment of this neuropsychiatric disorder. To assess potential mechanisms underlying the fast-acting antidepressant actions of ketamine we used a repeated corticosterone paradigm in adult male rats to assess the effects of ketamine on reelin-positive cells, a protein largely implicated in the pathophysiology of depression. We also assessed the effects of reelin and ketamine on hippocampal and cerebellar synpatosomes, and on serotonin transporter clustering in peripheral lymphocytes to determine reelin and ketamine's impact at the synaptic and peripheral levels. Reelin and ketamine similarly rescue synaptic expression of mTOR and p-mTOR that were decreased by corticosterone. Reelin, but not ketamine, was able to rescue patterns of serotonin transporter clustering in the periphery. These findings display ketamine as a powerful modulator of reelin expression and lend strength to further evaluation of the putative fast antidepressant-like actions of reelin.

Keywords: antidepressant; hippocampus; ketamine; lymphocytes; mammalian target of rapamycin; reelin; synaptosomes.

Figure 1

Experimental Timeline. The methodological timeline of the study. Rats were on a 12-h light-dark cycle that changed at 7am/pm and were injected between the h of 9am–10am to control for natural fluctuations in corticosterone (CORT) throughout the day. Sacrifices were conducted between the h of 9am–12pm. SNPs, synaptosomes; IHC, immunohistochemistry.

Figure 2

The effect of corticosterone (CORT) and ketamine on reelin-IR cells in the gyrus subgranular zone (SGZ). (A) Representative photomicrographs of reelin-ir cells in the dorsal SGZ. Stereological analyses were undertaken at 40× magnification. (B) Effects of treatment on reelin-ir cells in the dorsal SGZ. CORT significantly decreased the number of positive cells, which was rescued by acute ketamine administration. There were no effects of CORT or reelin in the ventral hippocampus. All data are expressed as mean±SD. Veh/Veh, vehicle/vehicle; Veh/Ket, vehicle/ketamine; CORT/Veh, CORT/vehicle; CORT/Ket, CORT/ketamine. *p < 0.05 vs. Veh/Veh, ^#p < 0.05, **p < 0.01 vs. CORT/Veh.

Figure 3

The effect of corticosterone (CORT) and ketamine on doublecortin (DCX)-IR cells in the gyrus subgranular zone (SGZ). (A) Representative photomicrographs of DCX-ir cells in the SGZ. (B) Effects of treatment on DCX-ir cells. CORT significantly decreased the number of positive cells, which was not rescued with acute ketamine administration. All data are expressed as mean±SD. Veh/Veh, vehicle/vehicle; Veh/Ket, vehicle/ketamine; CORT/Veh, CORT/vehicle; CORT/Ket, CORT/ketamine. **p < 0.01 vs. Veh/Veh, ^##p < 0.01 vs. CORT/Veh.

Figure 4

The effect of ketamine on synaptic-level proteins in hippocampal synaptosomes. (A) Representative Western Blot images of synaptic-level protein expression in the hippocampus after ketamine incubations on synaptosomes derived from corticosterone (CORT)-treated animals. Ladder presented is Spectra Multicolor High Range Protein Ladder (Thermofisher, #26625). (B) Effects of ketamine incubations on postsynaptic density-95 (PSD-95). CORT decreased this expression while all ketamine incubations increased these levels. (C) Effects of ketamine incubations on mammalian target of rapamycin (mTOR). All concentrations of ketamine increased mTOR levels above CORT-treated animals. (D) Effects of ketamine incubations compared to those of reelin on p-mTOR. Only 10nм of ketamine increased p-mTOR levels above CORT-treated animals. (E) Effects of ketamine incubations on the active ratio of mTOR. All data are expressed as mean±SD. K50, 50nм ketamine; K100, 100nм ketamine; K500, 500nм ketamine. ^#p < 0.05, ^##p < 0.01 vs. vehicle, *p < 0.05, **p < 0.01, ***p < 0.001 vs. CORT.

Figure 5

The effect of ketamine on synaptic-level proteins in cerebellar synaptosomes. (A) Representative Western Blot images of synaptic-level protein expression in the cerebellum after ketamine incubations on synaptosomes from corticosterone (CORT)-treated animals. Ladder presented is Spectra Multicolor High Range Protein Ladder (Thermofisher, #26625). (B) Effects of ketamine incubations on levels of postsynaptic density-95 (PSD-95). All levels stayed the same. (C) Effects of ketamine incubations on levels of mammalian target of rapamycin (mTOR). All concentrations of ketamine increased the levels above vehicle and CORT. (D) Effects of ketamine on levels of p-mTOR. All concentrations were significantly different from both vehicle and CORT. (E) Effects of ketamine on the active ratio of mTOR. All data are expressed as mean±SD. K5, 5nм ketamine; K10, 10nм ketamine; K50, 50nм ketamine. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 vs. vehicle, *p < 0.05, ***p < 0.001, ****p < 0.0001 vs. CORT.

Figure 6

The effect of reelin on synaptic-level proteins in cerebellar synaptosomes. (A) Representative Western Blot images of synaptic-level protein expression in the cerebellum after reelin incubations on synaptosomes from corticosterone (CORT)-treated animals. Ladder presented is Spectra Multicolor High Range Protein Ladder (Thermofisher, #26625). (B) Effects of reelin incubations on levels of postsynaptic density-95 (PSD-95). All levels stayed the same. (C) Effects of reelin incubations on levels of mammalian target of rapamycin (mTOR). Lower concentrations increased the levels above vehicle and CORT, but higher concentrations decreased mTOR levels. (D) Effects of reelin on levels of p-mTOR. All concentrations were significantly different from both vehicle and CORT, paralleling the findings with mTOR. (E) Effects of ketamine and reelin on the active ratio of mTOR. All data are expressed as mean±SD. R5, 5nм reelin; R10, 10nм reelin; R50, 50nм reelin. ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 vs. vehicle, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. CORT.

Figure 7

The effect of corticosterone (CORT), reelin, and ketamine on serotonin transporter (SERT) clusters in peripheral lymphocytes. (A) Representative confocal microscopy images for each condition. (B) Effect of treatment on clusters by number and size. (C) Effect of treatment on size of SERT clusters. CORT increased the size of SERT clusters, which was rescued by reelin but not ketamine. (D) Effect of treatment on number of SERT clusters. No changes were found between any groups. All data expressed as mean±SD. ^#p < 0.05 vs. vehicle, *p < 0.05, ^##p < 0.01 vs. CORT.