Blockade of D-serine signaling and adult hippocampal neurogenesis attenuates remote contextual fear memory following multiple memory retrievals in male mice

Abstract

The retrieval of fear memories induces two opposing processes, reconsolidation, and extinction. The memory reconsolidation is an active process that involves gene expression and updates an existing memory. It is hypothesized that blockade of reconsolidation by manipulating the neurobiological factors, which are mechanistically involved in the process, could weaken or disrupt the original fear memory. The N-methyl-D-aspartate (NMDA) receptor and hippocampal neurogenesis play crucial roles in hippocampus-dependent memory processes, including reconsolidation. Using contextual fear conditioning paradigm with multiple retrievals, we attempted to weaken the original contextual fear memory by repeatedly disrupting retrieval-induced reconsolidation via downregulation of NMDA receptor signaling and inhibition of neurogenesis. In the first experiment, prior to fear conditioning, NMDA receptor signaling was downregulated by the genetic reduction of its co-agonist, D-serine, and the neurogenesis was dampened by focal X-ray irradiation on the hippocampus. We found that simultaneous D-serine reduction and neurogenesis dampening resulted in a progressive decrease in freezing following each retrieval, leading to an attenuation of remote contextual fear memory on day 28. In the second experiment using the same behavioral protocols, after conditioning, pharmacological approaches were conducted to simultaneously block D-serine signaling and neurogenesis, resulting in a similar suppressive effect on the remote fear memory. The present findings provide insights for understanding the role of D-serine-mediated NMDA receptor signaling and neurogenesis in memory retrieval and the maintenance of remote fear memory, and improving the efficacy of exposure-based therapy for the treatment of post-traumatic stress disorder (PTSD).

Keywords: D-serine; hippocampal neurogenesis; multiple retrievals; reconsolidation; remote memory.

FIGURE 1

Hippocampal neurogenesis dampening attenuates remote fear memory following multiple retrievals in SRRKO mice. (A) Experimental schedule for X-ray irradiation, contextual fear conditioning, and multiple retrievals. Two different protocols, one with multiple retrievals (Ret), and another without memory retrieval (No–Ret), were used. (B) No significant difference was observed in freezing between WT (n = 11), SRRKO (n = 12), WT-X-ray (n = 14), and SRRKO-X-ray (n = 13) mice during fear conditioning. (C) During the course of four retrieval sessions, SRRKO-X-ray mice exhibited a significant reduction in freezing levels compared to other three groups in the tests on day 14 and 28. (D) During the remote test on day 28, SRRKO-X-ray mice exhibited significantly lower freezing levels compared to the other three groups. (E) Under the condition without intervention of multiple retrievals, no significant difference was observed in freezing level between four groups (WT, n = 9; SRRKO, n = 12; WT-X-ray, n = 10; SRRKO-X-ray, n = 10) during the remote memory test on day 28. Data are presented as the means ± SEM. ^**p < 0.01.

FIGURE 2

Effects of X-ray irradiation on adult hippocampal neurogenesis. (A) Immediately after the fourth retrieval on day 28, mouse brains of the four groups, WT, WT-X-ray, SRRKO, and SRRKO-X-ray, were removed and stained using anti-doublecortin (DCX) antibody (magenta) and DRAQ5 (blue). Scale bars, 100 μm. (B) Quantification of DCX-positive (DCX⁺) cells in the dentate gyrus (DG) of WT and SRRKO mice with or without X-ray irradiation (WT, n = 7; SRRKO, n = 7; WT-X-ray, n = 4; SRRKO-X-ray, n = 4). Data are presented as the means ± SEM. ^*p < 0.05, ^**p < 0.01.

FIGURE 3

Pharmacological blockade of D-serine signaling and hippocampal neurogenesis attenuates remote fear memory following multiple retrievals. (A) Experimental schedule for contextual fear conditioning, multiple retrievals, and drug treatments. Two different protocols, one with multiple retrievals (Ret) and another without memory retrieval (No–Ret) were used. In the two behavioral tests, mice were divided into four groups: control (saline injection and sham operation), TMZ + HA-966 (TMZ injection and HA-966 administration), HA-966 (saline injection and HA-966 administration), and TMZ (TMZ injection and sham operation) group, respectively. (B) During the fear conditioning, no significant difference could be observed in the freezing level between the four groups (control, n = 10; HA-966 + TMZ, n = 10; HA-966, n = 8; TMZ, n = 10). (C) During the course of four retrieval sessions, mice treated with TMZ and HA-966 exhibited a significant decrease in freezing levels on day 28 than that on day 1. In contrast, multiple retrievals did not affect freezing level in the other three groups. (D) TMZ + HA966 group (n = 10) exhibited significantly lower freezing levels compared to the other three groups during memory test on day 28 following multiple retrievals. (E) Under the condition without intervention of multiple retrievals, no significant difference was observed in freezing level between four groups (control, n = 10; HA-966 + TMZ, n = 10; HA-966, n = 8; TMZ, n = 10) during the remote memory test on day 28. Data are presented as the means ± SEM. ^*p < 0.05.

FIGURE 4

Effects of TMZ and HA-966 treatment on hippocampal neurogenesis. (A) Immediately after the fourth retrieval on day 28, mouse brains were removed and stained using anti-doublecortin (DCX) antibody (magenta) and DRAQ5 (blue). Scale bar, 100 μm. (B) Quantification of DCX-positive (DCX⁺) cells in the dentate gyrus (DG) of control (n = 4), TMZ (n = 4), TMZ + HA966 (n = 4), and HA-966 (n = 3) groups. Data are presented as the means ± SEM. *p < 0.05.

FIGURE 5

Effects of blockade of D-serine signaling and hippocampal neurogenesis on remote fear memory following extended and brief retrievals. (A) Experimental schedule for contextual fear conditioning, multiple retrievals, and TMZ treatment. Two different protocols, extended retrievals with 30 min re-exposure (as an extinction procedure) and brief retrievals with 5 min re-exposure, were used. (B) During the course of four brief retrievals on day 1, 7, 14, and 28, TMZ-treated SRRKO mice exhibited significantly lower freezing levels compared to TMZ-treated WT mice (WT, n = 6; SRRKO, n = 5). (C) During the remote test on day 28, WT mice receiving brief retrievals (WT-5 min, n = 6) exhibited a significantly higher freezing level compared to the other three groups, SRRKO mice receiving brief retrievals (SRRKO-5 min, n = 5), WT mice receiving extended retrievals (WT-30 min, n = 11), and SRRKO mice receiving extended retrievals (SRRKO-30min, n = 8). Data are presented as the means ± SEM. **p < 0.01.