A naturally-occurring histone acetyltransferase inhibitor derived from Garcinia indica impairs newly acquired and reactivated fear memories

Abstract

The study of the cellular and molecular mechanisms underlying the consolidation and reconsolidation of traumatic fear memories has progressed rapidly in recent years, yet few compounds have emerged that are readily useful in a clinical setting for the treatment of anxiety disorders such as post-traumatic stress disorder (PTSD). Here, we use a combination of biochemical, behavioral, and neurophysiological methods to systematically investigate the ability of garcinol, a naturally-occurring histone acetyltransferase (HAT) inhibitor derived from the rind of the fruit of the Kokum tree (Garcina indica), to disrupt the consolidation and reconsolidation of Pavlovian fear conditioning, a widely studied rodent model of PTSD. We show that local infusion of garcinol into the rat lateral amygdala (LA) impairs the training and retrieval-related acetylation of histone H3 in the LA. Further, we show that either intra-LA or systemic administration of garcinol within a narrow window after either fear conditioning or fear memory retrieval significantly impairs the consolidation and reconsolidation of a Pavlovian fear memory and associated neural plasticity in the LA. Our findings suggest that a naturally-occurring compound derived from the diet that regulates chromatin function may be useful in the treatment of newly acquired or recently reactivated traumatic memories.

Figure 1. Intra-LA infusions of garcinol impair training-related acetylation of histone H3 and fear memory consolidation.

(a) Schematic of the behavioral protocol. Rats were fear conditioned with three tone-shock pairings followed 1 hr later by intra-LA infusion of either vehicle (n = 7) or garcinol (500 ng/side; n = 7) and were sacrificed 30 min later. A third group did not receive conditioning and was infused with vehicle prior to sacrifice (n = 7). Separate groups of rats were fear conditioned with three tone-shock pairings followed 1 hr later by intra-LA infusion of either vehicle (n = 9) or garcinol (500 ng/side; n = 8) and tested for STM and LTM 3 and 21 hrs later, respectively. (b) Western blot analysis of acetylated and total (non-acetylated) histone H3 from LA homogenates from naïve (N)-Vehicle, fear conditioned (FC)-Vehicle and FC-Garcinol groups. * p<0.05 relative to FC-vehicle and N-Vehicle groups. Representative Western blots are depicted in the inset. (c) Mean (± SEM) percent freezing during the STM and LTM tests in vehicle and garcinol-infused groups. A third group is depicted that received infusion of either vehicle (n = 8) or garcinol (n = 6) 6 hrs following fear conditioning (‘delayed infusion’) followed by a LTM test 21 hrs later. (d) Cannula placements for rats infused with either vehicle (black circles) or garcinol (gray circles). *p<0.05 relative to vehicle-infused controls.

Figure 2. Garcinol impairs retrieval-related acetylation of histone H3 in the LA and fear memory reconsolidation.

(a) Schematic of the behavioral protocol. Rats were fear conditioned with three tone-shock pairings. Twenty four hrs following training rats were given a memory reactivation session consisting of a single tone CS presentation followed 1 hr later by intra-LA infusions of vehicle (n = 8) or garcinol (500 ng/side; n = 7). All rats were sacrificed 30 min following infusion. A third group did not receive conditioning or retrieval testing and was infused with vehicle prior to sacrifice (n = 7). Separate groups of rats were fear conditioned followed 24 hr later by a memory reactivation session consisting of a single tone CS presentation followed 1 hr later by intra-LA infusion of vehicle (n = 9) or garcinol (500 ng/side; n = 8). Two additional groups of rats were given a ‘no-reactivation’ session followed by infusion of vehicle (n = 7) or garcinol (500 ng/side; n = 5). All rats were then tested for PR-STM and PR-LTM 3 and 21 hrs later, respectively. (b) Cannula placements for rats infused with either vehicle (black circles) or garcinol (gray circles). (c) Memory retrieval data for the Reactivated (R)-Garcinol and R-Vehicle groups used in the Western blotting experiments. *p<0.05 relative to the pre-CS period. (d) Western blot analysis of acetylated and total histone H3 from LA homogenates taken from Naïve (N)-vehicle, R-Vehicle and R-Garcinol groups. * p<0.05 relative to R-Vehicle and N-Vehicle groups. Representative Western blots are depicted in the inset. (e) Memory retrieval data for the Reactivated (R)-Garcinol and R-Vehicle groups in the behavioral experiments. *p<0.05 relative to the pre-CS period. (f) Mean (± SEM) percent freezing during the PR-STM and PR-LTM tests in R-Vehicle and R-Garcinol groups. A third group is depicted that received infusion of either vehicle (n = 9) or garcinol (n = 6) 6 hrs following retrieval (‘delayed infusion’) followed by a PR-LTM test 21 hrs later. *p<0.05 relative to vehicle-infused controls. (g) Memory retrieval data for the Non-reactivated (NR)-Garcinol and NR-Vehicle groups. (h) Mean (± SEM) percent freezing during the ‘PR’-STM and ‘PR’-LTM tests in NR-Vehicle and NR-Garcinol groups.

Figure 3. The effect of garcinol on fear memory reconsolidation is not sensitive to spontaneous recovery, reinstatement, or to a shift in testing context.

(a) Schematic of the behavioral protocol (see text for details). (b) Memory retrieval data for rats given intra-LA infusion of vehicle (n = 6) or garcinol (n = 6). *p<0.05 relative to the pre-CS period. (c) Mean (± SEM) percent freezing during the PR-STM and PR-LTM tests in vehicle and garcinol-infused rats. *p<0.05 relative to vehicle-infused controls. (d) Mean (± SEM) percent freezing during the spontaneous recovery, reinstatement, and context shift tests. (e) Cannula placements for rats infused with either vehicle (black circles) or garcinol (gray circles). *p<0.05 relative to vehicle-infused controls.

Figure 4. Intra-LA infusion of garcinol impairs the reconsolidation of a ‘well-consolidated’ fear memory.

(a) Rats were fear conditioned with three tone-shock pairings. Two weeks following training rats were given a memory reactivation session consisting of a single tone CS presentation followed 1 hr later by intra-LA infusion of vehicle (n = 5) or garcinol (500 ng/side; n = 6). (b) Memory retrieval data for the vehicle and garcinol-infused groups. *p<0.05 relative to the pre-CS period. (c) Mean (± SEM) percent freezing during the PR-STM and PR-LTM tests in vehicle and garcinol-infused rats. (d) Cannula placements for rats infused with either vehicle (black circles) or garcinol (gray circles). *p<0.05 relative to vehicle-infused controls.

Figure 5. Intra-LA infusion of garcinol impairs fear memory consolidation and the consolidation of training-related neural plasticity in the LA.

(a) Rats were given two baseline AEFP recording sessions on separate days followed by fear conditioning with three tone-pip-shock pairings followed 1 hr later by intra-LA infusion of either vehicle (n = 8) or garcinol (500 ng/side; n = 7). Rats in each group were then tested for STM and LTM 3 and 21 hrs later while AEFPs were recorded from the LA. (b) Mean (± SEM) percent freezing during the STM and LTM tests in vehicle and garcinol-infused groups. (c) Mean (± SEM) percent of change in AEFP amplitude during the STM and LTM tests in vehicle and garcinol-infused rats, relative to baseline. *p<0.05 relative to vehicle-infused controls. (d) Representative AEFPs recorded from the LA for each group during baseline (light gray trace), STM and LTM sessions (darker traces). Scale bar  = 10 µV, 5 ms. (e) Electrode placements for rats infused with either vehicle (black circles) or garcinol (gray circles).

Figure 6. Intra-LA infusion of garcinol impairs fear memory reconsolidation and memory-related neural plasticity in the LA.

(a) Rats were given two baseline AEFP recording sessions on separate days followed by fear conditioning with three tone-pip-shock pairings. Twenty four hrs following training rats were given a memory reactivation session consisting of a single tone-pip CS presentation followed 1 hr later by intra-LA infusions of vehicle (n = 5) or garcinol (500 ng/side; n = 7). Rats in each group were then tested for PR-STM and PR-LTM 3 and 21 hrs later while AEFPs were recorded from the LA. (b) Memory retrieval data for the vehicle and garcinol-infused groups. *p<0.05 relative to the pre-CS period. (c) Mean (± SEM) percent freezing during the PR-STM and PR-LTM tests in vehicle and garcinol-infused groups. (d) Mean (± SEM) percent of change in AEFP amplitude during the PR-STM and PR-LTM tests in vehicle and garcinol-infused rats, relative to baseline. *p<0.05 relative to vehicle-infused controls. (e) Representative AEFPs recorded from the LA for each group during baseline (light gray trace), PR-STM and PR-LTM sessions (darker traces). Scale bar  = 10 µV, 5 ms. (f) Electrode placements for rats infused with either vehicle (black circles) or garcinol (gray circles).

Figure 7. Intra-LA infusion of garcinol in the absence of fear memory retrieval has no effect on fear memory reconsolidation or memory-related neural plasticity in the LA.

(a) Rats were given two baseline AEFP recording sessions on separate days followed by fear conditioning with three tone-pip-shock pairings. Twenty four hrs following training rats were given a ‘no-reactivation’ session followed by infusion of vehicle (n = 7) or garcinol (500 ng/side; n = 6). Rats in each group were then tested for ‘PR’-STM and ‘PR’-LTM 3 and 21 hrs later while AEFPs were recorded from the LA. (b) Memory retrieval data for the vehicle and garcinol-infused groups. (c) Mean (± SEM) percent freezing during the ‘PR’-STM and ‘PR’-LTM tests in vehicle and garcinol-infused groups. (d) Mean (± SEM) percent of change in AEFP amplitude during the ‘PR’-STM and ‘PR’-LTM tests in vehicle and garcinol-infused rats, relative to baseline. *p<0.05 relative to vehicle-infused controls. (e) Representative AEFPs recorded from the LA for each group during baseline (light gray trace), ‘PR’-STM and ‘PR’-LTM sessions (darker traces). Scale bar  = 10 µV, 5 ms. (f) Electrode placements for rats infused with either vehicle (black circles) or garcinol (gray circles).

Figure 8. Systemic injection of garcinol impairs the consolidation and reconsolidation of a fear memory.

(a) Schematic of the behavioral protocol. In the consolidation experiment, rats were fear conditioned with two tone-shock pairings followed 30 min later by i.p. injection of either garcinol (10 mg/kg; n = 8) or vehicle (n = 8). STM was examined 3 hrs later and LTM 21 hrs following injections. In the reconsolidation experiment, rats were fear conditioned with two tone-shock pairings followed 24 hrs later by fear memory reactivation session and i.p. injection of either garcinol (R-Garcinol; n = 9) or vehicle (R-Vehicle; n = 9). A third group received garcinol following a no-reactivation control session (NR-Garcinol; n = 8). All rats received tests of PR-STM and PR-LTM 3 hrs and 21 hrs after injections, respectively. (b) Mean (± SEM) percent freezing during the STM and LTM tests in vehicle and garcinol-infused groups in the consolidation experiment. * p<0.05 relative to vehicle group. (c) Memory retrieval data for the R-Vehicle, R-Garcinol, and NR-Garcinol groups in the reconsolidation experiment. *p<0.05 relative to the pre-CS period. (d) Mean (± SEM) percent freezing during the PR-STM and PR-LTM tests in R-Vehicle, R-Garcinol, and NR-Garcinol groups. *p<0.05 relative to vehicle-infused controls.