Class I histone deacetylases inhibition reverses memory impairment induced by acute stress in mice

Abstract

While chronic stress induces learning and memory impairments, acute stress may facilitate or prevent memory consolidation depending on whether it occurs during the learning event or before it, respectively. On the other hand, it has been shown that histone acetylation regulates long-term memory formation. This study aimed to evaluate the effect of two inhibitors of class I histone deacetylases (HDACs), 4-phenylbutyrate (PB) and IN14 (100 mg/kg/day, ip for 2 days), on memory performance in mice exposed to a single 15-min forced swimming stress session. Plasma corticosterone levels were determined 30 minutes after acute swim stress in one group of mice. In another experimental series, independent groups of mice were trained in one of three different memory tasks: Object recognition test, Elevated T maze, and Buried food location test. Subsequently, the hippocampi were removed to perform ELISA assays for histone deacetylase 2 (HDAC2) expression. Acute stress induced an increase in plasma corticosterone levels, as well as hippocampal HDAC2 content, along with an impaired performance in memory tests. Moreover, PB and IN14 treatment prevented memory loss in stressed mice. These findings suggest that HDAC2 is involved in acute stress-induced cognitive impairment. None of the drugs improved memory in non-stressed animals, indicating that HDACs inhibitors are not cognitive boosters, but rather potentially useful drugs for mitigating memory deficits.

Fig 1. Experimental design.

All mice were habituated to the experimental room for 5 minutes one day before training on Day 1. On Day 2, spontaneous motor activity (SMA) was assessed in all groups. Ten min later, one group was stressed through forced swimming for 15 min and the other remained in their home cage. Fifteen minutes later stressed and non-stressed animals were sacrificed and blood samples were collected for plasma corticosterone levels analysis. In another experimental series, independent groups of mice were trained in the Object recognition test (ORT), Elevated T maze (ETM), and Buried food location test (BFLT). Long-term memory (LTM)-tests were carried out 24 h later (Day 3) for ORT groups and 48 h later (Day 4) for ETM and BFLT groups. Sixty minutes later, hippocampal tissue samples were collected for ELISA assays to determine HDAC2 content in the ETM groups only.

Fig 2. Diagram of the buried food location test.

Top view of the arena, where the buried food reward (fruit loop) location in quadrant I (black dot) is indicated. (A) Acquisition session consisting of training 6 trials, where the mice were placed randomly inside the box. (B) Long-term spatial memory retention testing without food reward. Plus symbol, rectangle with diagonal stripes and circle correspond to the real spatial clues that were placed on the walls of the experimental room.

Fig 3. Effect of IN14 and PB on acute swim stress-induced increase in plasma corticosterone level.

All values are expressed as mean ± SEM (n  =  4) * P < 0.05; ** P < 0.01; *** P < 0.001; ^&& P < 0.01, Tukey test. VEH, PB, and IN14 mice were not subjected to any behavioral manipulations. VEH: vehicle; PB: 4-phenylbutyrate.

Fig 4. Motor activity after PB and IN14 injection in mice exposed to acute swim stress.

The results are expressed as the mean ± SEM of spontaneous motor activity: total, ambulatory, and vertical, (n = 8 per group). Vertical activity (right scale): ** p<0.01, Tukey test. VEH: vehicle; PB: 4-phenylbutyrate.

Fig 5. Effect of PB and IN14 on swim stress-induced short- and long-term memory impairment in the NORT.

A) Swim stress impaired short- and long-term memory retrieval. B) Lack of effects of PB and IN14 (both 100 mg/kg) in non-stressed mice performance. C) Effects of PB and IN14 on swim stress-induced short- and long-term memory impairment. ** p< 0.01; *** p< 0.001 vs. is respective vehicle group. ^&p <0.05, PB vs. IN14. Data are expressed as mean ± SEM, n = 8 per group. VEH: vehicle; PB: 4-phenylbutyrate.

Fig 6. Effect of PB and IN14 on swim stress-induced long-term memory deficit in the ETM.

A) Experimental protocol of the ETM task (see 2.4, Methods section). B) ETM learning curves during the training session. AL1 vs. AL2, AL3, and AL4, ***p <0.001, Sidák test for all groups, except AL2 of the VEH-S group; VEH-S vs. VEH, PB-S, IN14-S, ^&&&p<0.001 for AL2, Tukey test. Retention latencies are shown on the right axis. VEH vs. VEH-S; VEH-S vs. PB-S and VEH-S vs. IN14-S, ^&&&p< 0.001, Tukey test. C) Escape latencies (EL), no significant differences were observed among the groups, p = 0.0963, one-way ANOVA. Data expressed as mean ± SEM, n = 8 per group. AL: acquisition latency; VEH: vehicle; PB: 4-phenylbutyrate.

Fig 7. Effect of PB and IN14 on swim stress-induced memory impairment in the BFLT.

A) Learning curves of mice during the training session, *** p < 0.001 for AL1 vs. AL3, AL4, AL5, AL6, Sidák test; ### p < 0.001, VEH vs. VEH-S, Tukey test. B) Retention latencies (LR) in the long-term retrieval test. C) Time in the target quadrant during long-term retrieval testing. D) and E) Time spent in the target (T) and opposite O) quadrants of the arena during long-term retrieval testing. The VEH group and the groups treated with PB (-/+ stress) and IN14 (-/+ stress) spend more time in the target quadrant than in the opposite one, unlike the VEH-S. Data are presented as mean + S.E.M. n = 10. AL: acquisition latency; VEH: vehicle; PB: 4-phenylbutyrate. * p < 0.05, **p < 0.01, ***p < 0.001, post hoc Tukey test.

Fig 8. Increased HDAC2 expression in the hippocampus after swim stress.

IN14 and PB, prevented acute stress-increased expression of HDAC2 in the mouse hippocampus. n = 4. * p < 0.05, *** p< 0.001, Tukey test. Bla: blank; HDAC2: Histone deacetylase-2; VEH: vehicle; PB: 4-phenylbutyrate.