Glutamate N-methyl-D-aspartate receptor antagonists rapidly reverse behavioral and synaptic deficits caused by chronic stress exposure

Abstract

Background: Despite widely reported clinical and preclinical studies of rapid antidepressant actions of glutamate N-methyl-D-aspartate (NMDA) receptor antagonists, there has been very little work examining the effects of these drugs in stress models of depression that require chronic administration of antidepressants or the molecular mechanisms that could account for the rapid responses.

Methods: We used a rat 21-day chronic unpredictable stress (CUS) model to test the rapid actions of NMDA receptor antagonists on depressant-like behavior, neurochemistry, and spine density and synaptic function of prefrontal cortex neurons.

Results: The results demonstrate that acute treatment with the noncompetitive NMDA channel blocker ketamine or the selective NMDA receptor 2B antagonist Ro 25-6981 rapidly ameliorates CUS-induced anhedonic and anxiogenic behaviors. We also found that CUS exposure decreases the expression levels of synaptic proteins and spine number and the frequency/amplitude of synaptic currents (excitatory postsynaptic currents) in layer V pyramidal neurons in the prefrontal cortex and that these deficits are rapidly reversed by ketamine. Blockade of the mammalian target of rapamycin protein synthesis cascade abolishes both the behavioral and biochemical effects of ketamine.

Conclusions: The results indicate that the structural and functional deficits resulting from long-term stress exposure, which could contribute to the pathophysiology of depression, are rapidly reversed by NMDA receptor antagonists in a mammalian target of rapamycin dependent manner.

Figure 1

NMDA receptor antagonists produce rapid antidepressant responses in a CUS paradigm. (A) Schematic demonstrating the time line for CUS exposure, drug administration, and behavioral testing. Numbers in parentheses represents days after drug administration. Rats were exposed to CUS and administered ketamine or Ro 25-6981 (both at 10 mg/kg, i.p) on day 21. The SPT was conducted 1 day later (B, D) and NSFT 2 day after drug treatment (C, E). Ketamine and Ro 25-6981 administration in CUS rats reversed the decreased sucrose preference and increased latency to feed to the level of non-stressed control rats. The SPT was also conducted at 3, 5, and 7 days after ketamine or Ro 25-6981 (F,G). Baseline was measured on day 21 before drug injections. Values represent mean ± SEM [n = 6 per group. **P < 0.01, analysis of variance (ANOVA)].

Figure 2

Rapid behavioral actions of ketamine in the CUS model require mTOR signaling. On day 21, rapamycin (0.2 nmol, ICV) or DMSO was infused 30 min before ketamine (10 mg/kg, i.p.) or vehicle treatment. The SPT was conducted 1 day (A) and the NSFT 2 days after drug administration. Pre-treatment with rapamycin completely abolished the behavioral actions of ketamine in both SPT (A) and NSFT (B). Values represent mean ± SEM (n = 6 per group; **P < 0.01, ANOVA).

Figure 3

CUS exposure decreases synaptic proteins: rapid reversal by ketamine. Rats were exposed to CUS and on day 21 were infused with vehicle or rapamycin (0.2 nmol, ICV) 30 minutes before ketamine (10 mg/kg, i.p.) or vehicle treatment. Levels of synaptic proteins in PFC synaptoneurosome were determined by western blot 2 days later. (A) Representative western blot images of synapsin I, GluR1, and PSD95 are shown, and (B) Results were quantified and are the mean ± SEM, percent of control (n = 6 animals; **P < 0.01, ANOVA). Levels of GAPDH were determined to control for differences in amounts of protein loading. CUS decreased expression of synapsin I, PSD95 and GluR1, and this deficit was completely reversed by a single dose of ketamine. Pre-treatment with rapamycin completely abolished the effects of ketamine, but rapamycin infusion alone did not affect synaptic protein levels.

Figure 4

CUS exposure decreases spine density in PFC layer V pyramidal cells: rapid reversal by ketamine. Animals were exposed to CUS for 21 d and then received ketamine injections (10 mg/kg, i.p.). Twenty-four hours later slices of PFC were prepared for whole-cell recordings followed by neurobiotin labeling, and post hoc two-photon microscopy image of the neurobiotin-labeled layer V pyramidal cells. (A) Representative images are shown of high magnification Z-stack projections of distal and proximal segments of the layer V pyramidal cell apical tuft dendrites (Scale: 5 μm). The density of spines was analyzed using Neurolucida Explorer (version 9) and the results are the mean ± SEM (~12 cells from 4 rats in each group; *P < 0.05; **P < 0.01, ANOVA). CUS decreased spine density of both distal and proximal segments of the apical tuft, and this deficit was completely reversed by ketamine treatment (bar graphs to right of images). (B) Quantification of distal and proximal spine head diameter and spine length. (C) Cumulative fraction curves for distal and proximal tuft spine diameter; note large decrease in population of large diameter, mushroom spines (>0.8 μm; vertical dashed line) in the CUS group as compared to other groups in the distal tuft; stress-induced changes are less pronounced in the proximal tuft, consistent with previous studies (13).

Figure 5

CUS exposure decreases EPSC responses in PFC layer V pyramidal cells: rapid reversal by ketamine. (A) Sample whole cell voltage-clamp traces of 5-HT and hypocretin-induced EPSCs in slices from non-stressed control, CUS, or CUS + ketamine rats are shown (24 hr post drug treatment). (B) Frequency of 5-HT- and hypocretin-induced EPSCs and (C) Cumulative probability distributions showing that CUS exposure causes decreases in amplitude (P < 0.0001 for 5-HT, KS-z value = 3.89; p < 0.05 for Hcrt, KS-z value = 1.92) (n = 12 neurons/group; *P < 0.05, ANOVA). These deficits were completely reversed by a single dose of ketamine (B, C).