Fluoroethylnormemantine, a Novel NMDA Receptor Antagonist, for the Prevention and Treatment of Stress-Induced Maladaptive Behavior

Abstract

Background: Major depressive disorder is a common, recurrent illness. Recent studies have implicated the NMDA receptor in the pathophysiology of major depressive disorder. (R,S)-ketamine, an NMDA receptor antagonist, is an effective antidepressant but has numerous side effects. Here, we characterized a novel NMDA receptor antagonist, fluoroethylnormemantine (FENM), to determine its effectiveness as a prophylactic and/or antidepressant against stress-induced maladaptive behavior.

Methods: Saline, memantine (10 mg/kg), (R,S)-ketamine (30 mg/kg), or FENM (10, 20, or 30 mg/kg) was administered before or after contextual fear conditioning in 129S6/SvEv mice. Drug efficacy was assayed using various behavioral tests. Protein expression in the hippocampus was quantified with immunohistochemistry or Western blotting. In vitro radioligand binding was used to assay drug binding affinity. Patch clamp electrophysiology was used to determine the effect of drug administration on glutamatergic activity in ventral hippocampal cornu ammonis 3 (vCA3) 1 week after injection.

Results: Given after stress, FENM decreased behavioral despair and reduced perseverative behavior. When administered after re-exposure, FENM facilitated extinction learning. As a prophylactic, FENM attenuated learned fear and decreased stress-induced behavioral despair. FENM was behaviorally effective in both male and female mice. (R,S)-ketamine, but not FENM, increased expression of c-fos in vCA3. Both (R,S)-ketamine and FENM attenuated large-amplitude AMPA receptor-mediated bursts in vCA3, indicating a common neurobiological mechanism for further study.

Conclusions: Our results indicate that FENM is a novel drug that is efficacious when administered at various times before or after stress. Future work will further characterize FENM's mechanism of action with the goal of clinical development.

Keywords: Antidepressant; Anxiety; Depression; Fear; NMDAR; Prophylactic.

Figure 1.

FENM is a novel antidepressant when administered after stress exposure. (A) Experimental design. (B) Freezing was comparable across all groups during CFC training. (C, D) During CFC re-exposure, freezing was comparable across all groups. (E) On day 1 of the FST, FENM (20 mg/kg), but no other drugs or doses tested, reduced immobility time compared with controls. (F, G) On day 2 of the FST, (R,S)-ketamine and FENM at all doses, but not memantine, significantly reduced immobility time when compared with saline mice. (H–J) In the OF, all groups of mice traveled a comparable distance traveled and spent a comparable amount of time in the center of the arena. (K) In the MB task, FENM (20 mg/kg) decreased the number of marbles buried when compared with saline. (L, M) In the EPM, memantine, (R,S)-ketamine, and FENM did not alter distance traveled or the time spent in the open arms and center of the maze. (N) In the NSF, memantine, (R,S)-ketamine, and FENM did not alter the latency to feed in the open arena. (O, P) Latency to feed and food eaten in the home cage were comparable across all drug groups. n = 6 male mice per group; error bars represent ± SEM; **p < .01, ***p < .001. CFC, contextual fear conditioning; EPM, elevated plus maze; FENM, fluoroethylnormemantine; FST, forced swim test; K, (R,S)-ketamine; M, memantine; MB, marble burying; NSF, novelty-suppressed feeding; OF, open field; Sal, saline.

Figure 2.

FENM is also an effective antidepressant after a shorter intertrial interval between stress and behavioral despair assays. (A) Experimental design. (B–E) During CFC re-exposures 1 and 2, all groups of mice exhibited comparable freezing. (F, G) On day 1 of the FST, all groups exhibited comparable immobility time. (H, I) On day 2 of the FST, FENM, but not (R,S)-ketamine, significantly reduced immobility time when compared with saline mice. (J, K) In the OF, all groups of mice traveled a comparable distance traveled and spent a comparable amount of time in the center of the arena. (L) In the MB task, all groups of mice buried a comparable number of marbles. (M, N) Behavior was comparable across all groups in the EPM. (O, P) In the NSF, (R,S)-ketamine and FENM did not alter the latency to feed in the open arena. (Q, R) Latency to feed and food eaten in the HC were comparable across all groups. n = 6 male mice per group; error bars represent ± SEM; **p < .01. CFC, contextual fear conditioning; EPM, elevated plus maze; FENM, fluoroethylnormemantine; FST, forced swim test; HC, home cage; K, (R,S)-ketamine; MB, marble burying; NSF, novelty-suppressed feeding; OF, open field; Sal, saline.

Figure 3.

FENM attenuates learned fear when administered after an extinction trial. (A) Saline, (R,S)-ketamine (30 mg/kg), or FENM (20 mg/kg) was administered 5 minutes after re-exposure 1 (24 hours before re-exposure 2). (B, C) During re-exposure 1, freezing was comparable across all groups. (D, E) During re-exposure 2, FENM, but not (R,S)-ketamine, significantly decreased fear expression when compared with saline. During day 1 (F, G) and day 2 (H, I) of the FST, all groups exhibited a comparable immobility time. (J–L) In the OF and MB assays, behavior was comparable across all groups. (M, N) In the EPM, the time spent in and entries into the open arms was comparable in all groups. In the NSF, FENM and (R,S)-ketamine did not alter the latency to eat in (O, P) the NSF arena or (Q) the HC. (R) All groups ate a comparable amount of food. n = 7–11 male mice per group; error bars represent ± SEM; *p < .05. CFC, contextual fear conditioning; EPM, elevated plus maze; FENM, fluoroethylnormemantine; FST, forced swim test; HC, home cage; K, (R,S)-ketamine; MB, marble burying; NSF, novelty-suppressed feeding; OF, open field; Sal, saline.

Figure 4.

FENM attenuates learned fear and protects against stress-induced behavioral despair when administered 1 week before CFC. (A) Experimental protocol. (B) Freezing was comparable across all groups during CFC training. (C, D) (R,S)-ketamine (30 mg/kg) and FENM (20 and 30 mg/kg) significantly decreased fear expression. (E) On FST day 1, memantine (10 mg/kg) significantly increased immobility time when compared with saline-administered mice. (F, G) On day 2 of the FST, (R,S)-ketamine and FENM (20 and 30 mg/kg) significantly reduced immobility time when compared with saline controls. (H–J) In the OF, (R,S)-ketamine and FENM did not alter distance traveled or time spent in the center of the arena. (K) In the MB task, all groups buried a comparable number of marbles. (L, M) (R,S)-ketamine and FENM did not alter distance traveled or time spent in the open arms and center of the EPM. (N) Memantine, (R,S)-ketamine and FENM did not alter latency to feed in the NSF. (O) Memantine significantly reduced latency to feed in the HC during the NSF. (P) Food eaten in the NSF was comparable across all drug groups. n = 5–12 male mice per group; error bars represent ± SEM; *p < .05, **p < .01, ***p < .001. CFC, contextual fear conditioning; EPM, elevated plus maze; FENM, fluoroethylnormemantine; FST, forced swim test; HC, home cage; K, (R,S)-ketamine; M, memantine; MB, marble burying; NSF, novelty-suppressed feeding; OF, open field; Sal, saline.

Figure 5.

FENM does not bind to additional receptors nor alter neural activity or protein expression during fear retrieval. (A) Behavioral protocol. Male mice were given a single administration of saline, (R,S)-ketamine (30 mg/kg), or FENM (20 mg/kg) 1 week before three-shock CFC training. Five days later, mice were re-exposed to the training context. One hour after re-exposure, mice were sacrificed, and brain tissue was collected for use in either immunohistochemistry or Western blotting analysis. (B, C) Mice in all groups froze at comparable levels during CFC training. (D, E) Upon re-exposure, (R,S)-ketamine– and FENM-administered mice exhibited significantly less freezing when compared with saline controls. (F) Representative images of c-fos immunohistochemical staining in the (top) dorsal hippocampus and (bottom) ventral hippocampus. (G–J) Levels of c-fos⁺ neurons were comparable across all groups in the dDG, dorsal hilus, dCA3, and dCA1. (K, L) All groups exhibited comparable levels of c-fos⁺ neurons in the vDG and ventral hilus. (M) Mice administered (R,S)-ketamine, but not FENM, had significantly higher numbers of c-fos⁺ neurons in vCA3. (N) c-fos expression was comparable across all groups in the vCA1. (O, R) Western blot analysis revealed comparable expression of NR2A and GluR1 subunits of the NMDA receptor and AMPA receptor, respectively, in the hippocampus. (P, Q) FENM did not significantly bind to any targets tested, indicating that the compound exerts selective affinity for the NMDA receptor. Error bars represent ± SEM; scale bars = 250 or 25 μm (insets); *p < .05, **p < .01. β-tub, beta-tubulin; CA, cornu ammonis; CFC, contextual fear conditioning; dCA, dorsal CA; dDG, dorsal DG; DG, dentate gyrus; FENM, fluoroethylnormemantine; K, (R, S)-ketamine; Sac, sacrifice; Sal, saline; vCA, ventral CA; vDG, ventral DG.

Figure 6.

Similar to (R,S)-ketamine, FENM reduces AMPAR-mediated bursts in hippocampal CA3. (A) Mice were administered saline, (R,S)-ketamine (30 mg/kg), or FENM (20 mg/kg) 1 week before whole-cell voltage clamp electrophysiology. Representative EPSCs in a (B) saline-administered, (C) (R,S)-ketamine–administered, and (D) FENM-administered mouse. (E) Representative expanded view of a large-amplitude AMPAR-mediated burst, smaller-amplitude AMPAR-mediated EPSC, and NMDAR-mediated EPSC. Decay time was measured from 10%−90% of the peak amplitude. (F) Scatter plot indicating that a saline-administered mouse displayed large bursts of AMPAR-mediated EPSCs in addition to small-amplitude NMDAR-mediated currents, which were revealed by washing in NBQX. (G) Large-amplitude AMPAR-mediated bursts and NMDAR-mediated EPSCs were attenuated in (R,S)-ketamine mice. (H) In FENM-administered mice, large-amplitude AMPAR-mediated bursts were also robustly attenuated, but NMDAR-mediated EPSCs were not significantly altered. (I) Large AMPAR-mediated burst amplitude was attenuated in experimental groups compared with saline controls. (J) There was a trending but not significant reduction in the mean amplitude of spontaneous AMPAR-mediated currents in (R,S)-ketamine– and FENM-administered mice. (K) The mean amplitude of NMDAR-mediated currents was significantly reduced in (R,S)-ketamine–, but not in FENM-administered mice. (L) AMPAR-mediated bursts were blocked by both (R,S)-ketamine and FENM drug administration. (M) The mean frequency of all AMPAR-mediated EPSCs within a 20-second recording period was not significantly altered by drug administration. (N) The frequency of NMDAR-mediated EPSCs was comparable across all groups. n = 4 cells per group; error bars represent ± SEM; *p < .05. AMPAR, AMPA receptor; CA3, cornu ammonis area 3; EPSC, excitatory postsynaptic current; FENM, fluoroethylnormemantine; K, (R, S)-ketamine; NMDAR, NMDA receptor; Sal, saline.