DNA methyltransferase activity is required for memory-related neural plasticity in the lateral amygdala

Abstract

We have previously shown that auditory Pavlovian fear conditioning is associated with an increase in DNA methyltransferase (DNMT) expression in the lateral amygdala (LA) and that intra-LA infusion or bath application of an inhibitor of DNMT activity impairs the consolidation of an auditory fear memory and long-term potentiation (LTP) at thalamic and cortical inputs to the LA, in vitro. In the present study, we use awake behaving neurophysiological techniques to examine the role of DNMT activity in memory-related neurophysiological changes accompanying fear memory consolidation and reconsolidation in the LA, in vivo. We show that auditory fear conditioning results in a training-related enhancement in the amplitude of short-latency auditory-evoked field potentials (AEFPs) in the LA. Intra-LA infusion of a DNMT inhibitor impairs both fear memory consolidation and, in parallel, the consolidation of training-related neural plasticity in the LA; that is, short-term memory (STM) and short-term training-related increases in AEFP amplitude in the LA are intact, while long-term memory (LTM) and long-term retention of training-related increases in AEFP amplitudes are impaired. In separate experiments, we show that intra-LA infusion of a DNMT inhibitor following retrieval of an auditory fear memory has no effect on post-retrieval STM or short-term retention of training-related changes in AEFP amplitude in the LA, but significantly impairs both post-retrieval LTM and long-term retention of AEFP amplitude changes in the LA. These findings are the first to demonstrate the necessity of DNMT activity in the consolidation and reconsolidation of memory-associated neural plasticity, in vivo.

Keywords: DNA methytransferase; Epigenetics; Fear conditioning; Neural plasticity; Reconsolidation.

Figure 1. Intra-LA infusion of an inhibitor of DNMT activity impairs fear memory consolidation and training-related neural plasticity in the LA

(a) Rats were habituated to handling and given two days of baseline recording sessions in which AEFPs were recorded from the LA. On the 4^th day, rats were fear conditioned with 3 tone pip series (CS)-shock (US) pairings followed 1 hr later by intra-LA infusion of either vehicle (n = 6), RG108 (500 ng/side; n = 6) or 5-AZA (500 ng/side; n = 6). Rats in each group were then tested for STM and LTM 3 and 21 hrs later, respectively, while AEFPs were recorded from the LA. (b) Mean (± SEM) percent freezing during the STM and LTM tests in vehicle, RG108 and 5-AZA infused groups. *p<0.05 relative to vehicle-infused controls. (c) Mean (± SEM) percent of change in AEFP amplitude during the STM and LTM tests in vehicle, RG108 and 5-AZA-infused rats, relative to baseline. *p<0.05 relative to vehicle-infused controls. (d) Correlation between freezing scores and AEFP amplitudes in RG108- and 5-AZA-treated rats during the LTM test, each expressed as a percentage of freezing and AEFP amplitude change during the STM test. (e) Representative AEFPs recorded from the LA for each group during baseline (light gray trace), STM and LTM sessions (darker traces). Scale bar =10 μV, 5ms.

Figure 2. Intra-LA infusion of an inhibitor of DNMT activity impairs fear memory reconsolidation and retention of memory-associated neural plasticity in the LA

(a) Rats were habituated and given two days of baseline AEFP recording sessions, followed by fear conditioning with 3 tone pip series-(CS) shock (US) pairings. Twenty four hrs following training rats were given a memory reactivation session consisting of a single tone pip series CS presentation followed 1 hr later by intra-LA infusion of vehicle (n = 6), RG108 (500 ng/side; n = 6) or 5-AZA (500 ng/side; n = 7). Rats in each group were then tested for PR-STM and PR-LTM 3 and 21 hrs later, respectively, while AEFPs were recorded from the LA. (b) Memory retrieval data during the reactivation session for the vehicle, RG108 and 5-AZA-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, RG108 and 5-AZA-infused groups. *p<0.05 relative to vehicle-infused controls. (d) Mean (± SEM) percent change in AEFP amplitude during the PR-STM and PR-LTM tests in vehicle, RG108 and 5-AZA-infused rats, relative to baseline. *p<0.05 relative to vehicle-infused controls. (e) Correlation between freezing scores and AEFP amplitudes in RG108- and 5-AZA-treated rats during the PR-LTM test, each expressed as a percentage of freezing and AEFP amplitude change during the PR-STM.test. (f) 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, 5ms.

Figure 3. Intra-LA infusion of an inhibitor of DNMT activity in the absence of fear memory retrieval has no effect on fear memory reconsolidation or the retention of memory-associated neural plasticity in the LA

(a) Rats were given two days of baseline AEFP recording sessions, followed by fear conditioning with three tone pip series (CS)-shock (US) pairings. Twenty four hrs following training rats were given a ‘no reactivation’ session in which they were placed in the testing context but not presented with a tone pip series. One hour following the ‘no reactivation’ session, rats received infusion of vehicle (n = 5), RG108 (500 ng/side; n = 5) or 5-AZA (500 ng/side; n = 4). Rats in each group were then tested for ‘PR’-STM and ‘PR’-LTM 3 and 21 hrs later, respectively, while AEFPs were recorded from the LA. (b) Memory retrieval data for the vehicle, RG108 and 5-AZA-infused groups during the ‘no reactivation’ session. (c) Mean (± SEM) percent freezing during the ‘PR’-STM and ‘PR’-LTM tests in vehicle, RG108 and 5-AZA-infused groups. (d) Mean (± SEM) percent change in AEFP amplitude during the ‘PR’-STM and ‘PR’-LTM tests in vehicle, RG108 and 5-AZA-infused rats, relative to baseline. (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, 5ms.

Figure 4. Electrode placements in each experiment

(a) Histological location of recording electrode placements for rats infused with vehicle (black circles), 5-AZA (gray circles) or RG108 (white circles) in the consolidation experiment (Figure 1). (b) Histological location of recording electrode placements for rats infused with vehicle (black circles), 5-AZA (gray circles) or RG108 (white circles) in the reconsolidation experiment (Figure 2). (c) Histological location of recording electrode placements for rats infused with vehicle (black circles), 5-AZA (gray circles) or RG108 (white circles) in the non-reactivated experiment (Figure 3). All panels adapted from Paxinos and Watson (1998). LA = lateral amygdala; CE = central nucleus of amygdala; B = basal nucleus of the amygdala