Rapastinel (GLYX-13) has therapeutic potential for the treatment of post-traumatic stress disorder: Characterization of a NMDA receptor-mediated metaplasticity process in the medial prefrontal cortex of rats

Abstract

Rapastinel (GLYX-13) is a NMDA receptor modulator with glycine-site partial agonist properties. It is a robust cognitive enhancer and shows rapid and long-lasting antidepressant properties in both animal models and in humans. Contextual fear extinction (CFE) in rodents has been well characterized and used extensively as a model to study the neurobiological mechanisms of post-traumatic stress disorder (PTSD). Since CFE is NMDA receptor modulated and neural circuitry in the medial prefrontal cortex (MPFC) regulates both depression and PTSD, studies were undertaken to examine the effects of rapastinel for its therapeutic potential in PTSD and to use rapastinel as a tool to study its underlying glutamatergic mechanisms. A 21-day chronic mild unpredictable stress (CUS) rat model was used to model depression and PTSD. The effects of CUS alone compared to No CUS controls, and the effects of rapastinel (3 mg/kg IV) on CUS-treated animals were examined. The effect of rapastinel was first assessed using CUS-treated rats in three depression models, Porsolt, sucrose preference, and novelty-induced hypophagia tests, and found to produce a complete reversal of the depressive-like state in each model. Rapastinel was then assessed in a MPFC-dependent positive emotional learning paradigm and in CFE and again a reversal of the impairments induced by CUS treatment was observed. Both synaptic plasticity and metaplasticity, as measured by the induction of long-term potentiation in rat MPFC slice preparations, was found to be markedly impaired in CUS-treated animals. This impairment was reversed when CUS-treated rats were administered rapastinel and tested 24 h later. Transcriptomic analysis of MPFC mRNA expression in CUS-treated rats corroborated the link between rapastinel's behavioral effects and synaptic plasticity. A marked enrichment in both the LTP and LTD connectomes in rapastinel-treated CUS rats was observed compared to CUS-treated controls. The effects of rapastinel on depression models, PEL, and most importantly on CFE demonstrate the therapeutic potential of rapastinel for the treatment of PTSD. Moreover, rapastinel appears to elicit its therapeutic effects through a NMDA receptor-mediated, LTP-like, metaplasticity process in the MPFC.

Keywords: Depression; GLYX-13; LTP; Medial Prefrontal Cortex; NMDA Receptor.

Figure 1. Rapastinel (3 mg/kg IV) produces a rapid-acting and long-lasting antidepressant-like effect in a CUS paradigm

(A) Schematic demonstrating the timeline for CUS exposure, drug administration, and behavioral testing. Numbers in parentheses represent days after drug administration. Rats were exposed to CUS and administered a single dose of rapastinel (3 mg/kg IV) or 0.9% sterile saline vehicle (1 ml/kg IV) on day 21. Mean ± SEM floating time in the (B) Porsolt forced swim test (FS) in CUS treated rats treated with a single dose of Rapastinel (3 mg/kg IV; n = 10), saline vehicle (n = 10), or No CUS exposed control rats (n = 9) tested 1 hr, 1 day, 1 week, and 2 weeks post-dosing. Mean ± SEM (C) latency to feed in the novelty induced hypophagia (NIH) test (2 days post-dosing), (D) sucrose preference (SP) in the sucrose preference test, 3 days post-dosing, and (E) change in body weight (2 weeks post-dosing compared to CUS day 0). * P < .05 Fisher’s PLSD post hoc test vs. No CUS group or CUS vehicle group.

Figure 2. Rapastinel (3 mg/kg IV) facilitates MPFC-dependent positive emotional learning and fear extinction in CUS-treated rats

(A) Mean ± SEM hedonic USVs in response to a conditioned stimuli that predicts heterospecific play 3 hrs post-dosing with Rapastinel (3 mg/kg IV) or 0.9% sterile saline vehicle (1 ml/kg IV) in 2–3 month old adult male SD rats exposed to a depressogenic regimen of chronic unpredictable stress (CUS; 2 stressors / day for 21 consecutive days) or non-stressed control rats (No CUS). A similar increase in PEL was seen in animals tested 2 weeks post-dosing (results section). (B–C) Mean ± SEM % time freezing in CUS treated animals dosed with rapastinel (3 mg/kg IV) or sterile saline vehicle (1 ml/kg IV) 1 hr before extinction day 1. Three shocks (0.5 mA, 1 s) were administered 90, 210, and 330 sec after animals were placed in the conditioning chamber on D0. During extinction, rats were submitted to a 5 min non-reinforced test trial every 24 hrs for 6 consecutive days (D1-6), and on day 14 post-conditioning (consolidation trial). Freezing was quantified via FreezeView software and was measured at baseline (30–60 sec) on D0, and during the last 3 min of each extinction trial. N = 9 – 14 rats per group. * P < .05 Fisher’s PLSD post hoc test vs. No CUS group (A–B), * P < .05 within subjects t-test, 2 tailed (C).

Figure 3. In vitro application of Rapastinel (100 nM) enhances the magnitude of LTP in the MPFC

(A) Time course, and (B) Mean ± SEM 45 min post-theta burst stimulation, of MPFC layer II/III evoked normalized field EPSP slopes in rat slices recorded from MPFC layer IV. Three submaximal bouts of high-frequency layer II/III stimulation (10 × 100 Hz / 5 pulse bursts each, applied at an inter-burst interval of 200 ms) were applied at 3 minute intervals, and LTP was measured 45–47 min post-tetanus (shaded). Rapastinel (20 – 1,000 nM) was bath applied starting 10 min before tetanus, and remained in the bath throughout the experiment. N = 5–11 slices per group. * P < .05 Fisher’s PLSD post hoc test vs. aCSF control group.

Figure 4. Rapastinel (3 mg/kg IV) rescues CUS-induced deficits in LTP in the MPFC 24 hrs post-dosing

(A) Time course, and (B) Mean ± SEM 45 min post-theta burst stimulation of normalized field EPSP slopes evoked in layer II/III and recorded in layer IV of rat MPFC slices from 2–3 month old adult male SD rats exposed to a depressogenic regimen of 21 days of Chronic Unpredictable stress (CUS; 2 stressors / day for 21 consecutive days; red circles and bar), versus control animals (No CUS; black circles and bar). CUS-treated animals were dosed with rapastinel (3 mg/kg IV; blue circles and bar) or 0.9% sterile saline vehicle (1 ml/kg IV) 24 hrs before ex-vivo LTP testing. LTP was measured 45–47 min post-tetanus (shaded). N = 8–10 slices per group. * P < .05 Fisher’s PLSD post hoc test.