A Role for Nitric Oxide-Driven Retrograde Signaling in the Consolidation of a Fear Memory

In both invertebrate and vertebrate models of synaptic plasticity, signaling via the putative “retrograde messenger” nitric oxide (NO) has been hypothesized to serve as a critical link between functional and structural alterations at pre- and postsynaptic sites. However, while in vitro models of synaptic plasticity have consistently implicated NO signaling in linking postsynaptic induction mechanisms with accompanying presynaptic changes, a convincing role of such “retrograde signaling” in mammalian memory formation has remained elusive. Using auditory Pavlovian fear conditioning, we show that synaptic plasticity and NO signaling in the lateral nucleus of the amygdala (LA) regulate the expression of the ERK-driven immediate early gene early growth response gene I (EGR-1) in regions of the auditory thalamus that are presynaptic to the LA. Further, antisense knockdown of EGR-1 in the auditory thalamus impairs both fear memory consolidation and the training-induced elevation of two presynaptically localized proteins in the LA. These findings indicate that synaptic plasticity and NO signaling in the LA during auditory fear conditioning promote alterations in ERK-driven gene expression in auditory thalamic neurons that are required for both fear memory consolidation as well as presynaptic correlates of fear memory formation in the LA, and provide general support for a role of NO as a “retrograde signal” in mammalian memory formation

Synaptic Plasticity and NO-cGMP-PKG Signaling Regulate Pre- and Postsynaptic Alterations at Rat Lateral Amygdala Synapses Following Fear Conditioning

In vertebrate models of synaptic plasticity, signaling via the putative “retrograde messenger” nitric oxide (NO) has been hypothesized to serve as a critical link between functional and structural alterations at pre- and postsynaptic sites. In the present study, we show that auditory Pavlovian fear conditioning is associated with significant and long-lasting increases in the expression of the postsynaptically-localized protein GluR1 and the presynaptically-localized proteins synaptophysin and synapsin in the lateral amygdala (LA) within 24 hrs following training. Further, we show that rats given intra-LA infusion of either the NR2B-selective antagonist Ifenprodil, the NOS inhibitor 7-Ni, or the PKG inhibitor Rp-8-Br-PET-cGMPS exhibit significant decreases in training-induced expression of GluR1, synaptophysin, and synapsin immunoreactivity in the LA, while those rats infused with the PKG activator 8-Br-cGMP exhibit a significant increase in these proteins in the LA. In contrast, rats given intra-LA infusion of the NO scavenger c-PTIO exhibit a significant decrease in synapsin and synaptophysin expression in the LA, but no significant impairment in the expression of GluR1. Finally, we show that intra-LA infusions of the ROCK inhibitor Y-27632 or the CaMKII inhibitor KN-93 impair training-induced expression of GluR1, synapsin, and synaptophysin in the LA. These findings suggest that the NO-cGMP-PKG, Rho/ROCK, and CaMKII signaling pathways regulate fear memory consolidation, in part, by promoting both pre- and post-synaptic alterations at LA synapses. They further suggest that synaptic plasticity in the LA during auditory fear conditioning promotes alterations at presynaptic sites via NO-driven “retrograde signaling”

Epigenetic Alterations Are Critical for Fear Memory Consolidation and Synaptic Plasticity in the Lateral Amygdala

Epigenetic mechanisms, including histone acetylation and DNA methylation, have been widely implicated in hippocampal-dependent learning paradigms. Here, we have examined the role of epigenetic alterations in amygdala-dependent auditory Pavlovian fear conditioning and associated synaptic plasticity in the lateral nucleus of the amygdala (LA) in the rat. Using Western blotting, we first show that auditory fear conditioning is associated with an increase in histone H3 acetylation and DNMT3A expression in the LA, and that training-related alterations in histone acetylation and DNMT3A expression in the LA are downstream of ERK/MAPK signaling. Next, we show that intra-LA infusion of the histone deacetylase (HDAC) inhibitor TSA increases H3 acetylation and enhances fear memory consolidation; that is, long-term memory (LTM) is enhanced, while short-term memory (STM) is unaffected. Conversely, intra-LA infusion of the DNA methyltransferase (DNMT) inhibitor 5-AZA impairs fear memory consolidation. Further, intra-LA infusion of 5-AZA was observed to impair training-related increases in H3 acetylation, and pre-treatment with TSA was observed to rescue the memory consolidation deficit induced by 5-AZA. In our final series of experiments, we show that bath application of either 5-AZA or TSA to amygdala slices results in significant impairment or enhancement, respectively, of long-term potentiation (LTP) at both thalamic and cortical inputs to the LA. Further, the deficit in LTP following treatment with 5-AZA was observed to be rescued at both inputs by co-application of TSA. Collectively, these findings provide strong support that histone acetylation and DNA methylation work in concert to regulate memory consolidation of auditory fear conditioning and associated synaptic plasticity in the LA

Auditory fear conditioning regulates histone acetylation and DNMT expression in the LA.

<p>(A) Schematic of the behavioral protocol. Rats were habituated to handling, trained with 3 tone-shock pairings, and sacrificed at 30, 60, or 90 min later. (<i>B</i>) Representative Western blots for acetylated histone (top), total histone (middle), and DNMT expression (bottom) at each time point. (<i>C</i>) Mean (±SEM) acetyl-H3 and acetyl-H4 immunoreactivity from LA punches taken from Naïve (n = 7) and trained rats sacrificed at 30 min (n = 8), 60 min (n = 8), or 90 min (n = 7). Here, acetyl-H3 and acetyl-H4 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the Naïve group. (*) <i>p</i><0.05 relative to all other time points. (<i>D</i>) Mean (±SEM) total-H3 and total-H4 immunoreactivity from LA punches taken from the samples in (B). Here, total-H3 and total-H4 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the Naïve group. (E) Mean (±SEM) DNMT3A/3B immunoreactivity from LA punches taken from Naïve (n = 7) and trained rats sacrificed at 30 min (n = 8), 60 min (n = 7), or 90 min (n = 8). (*) <i>p</i><0.05 relative to all other time points. Here, DNMT3A/3B protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the Naïve group.</p

Training-related regulation of histone H3 acetylation and DNMT3A expression in the LA is specific to tone-shock pairing.

<p>(A) Schematic of the behavioral protocol. In two separate experiments, rats were either given no stimulation (“Naïve”), 3 tone-alone presentations (“Tone Alone”), 3 immediate shocks (“Imm. Shock”), or 3 tone-shock pairings (“Paired”) and sacrificed 90 min later. (<i>B</i>) Representative Western blots for acetylated and total histone H3 (top) and DNMT3A/B expression (bottom) in each group. (<i>C</i>) Mean (±SEM) acetyl-H3 and total H3 immunoreactivity from LA punches taken from Naïve (n = 8), Tone Alone (n = 7), Imm. Shock (n = 8), and Paired (n = 6) rats. Here, acetyl-H3 and total H3 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the Naïve group. (*) <i>p</i><0.05 relative to all other groups. (<i>D</i>) Mean (±SEM) DNMT3A/B immunoreactivity from LA punches taken from Naïve (n = 8), Tone Alone (n = 8), Imm. Shock (n = 8), and Paired (n = 8) rats. Here, DNMT3A/B protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the Naïve group. (*) <i>p</i><0.05 relative to all other groups.</p

Regulation of Npas4 mRNA and protein in the LA following fear conditioning.

<p>(A) Schematic of the behavioral protocol for qRT-PCR and Western experiments. (B) Time course analysis of Npas4 mRNA expression in the LA following fear conditioning using qRT-PCR (n = 8/group). *p<0.05 relative to the other groups. (C) Regulation of Npas4 mRNA in the LA using qRT-PCR. Rats were sacrificed 30 min following exposure to fear conditioning (Paired; n = 8), tone alone (Tone Alone; n = 6) or the training environment alone (Box; n = 7). *p<0.05 relative to Box and Tone Alone groups. (D) Regulation of Npas4 mRNA in the LA using qRT-PCR. Rats were sacrificed 30 min following exposure to fear conditioning (Paired; n = 7), immediate shock (Imm. Shock; n = 8) or no stimulation (Naïve; n = 7). *p<0.05 relative to Naïve and Imm. Shock groups. (E) Western blot analysis of Npas4 protein in the LA. Rats were sacrificed 90 min following exposure to fear conditioning (Paired; n = 7), tone alone (Tone Alone; n = 7), immediate shock (Imm. Shock; n = 8) or no stimulation (Naïve; n = 8). *p<0.05 relative to the other groups. Representative Western blots are shown in the inset.</p

Histone acetylation and DNA methylation interact to regulate memory consolidation in the LA.

<p>(A) Schematic of the behavioral protocol. Rats were trained with 3 tone-shock pairings followed 1 hr later by intra-LA infusion of either vehicle (n = 7) or 5-AZA (n = 7) and sacrificed 30 min after infusion. <i>(B)</i> Mean (±SEM) acetyl-H3 and acetyl-H4 immunoreactivity from punches taken from the LA. Here, acetyl-H3 and acetyl-H4 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the vehicle group. (*) <i>p</i><0.05 relative to vehicle group. (<i>C</i>) Mean (±SEM) total-H3 and total-H4 immunoreactivity from the samples in (B). Here, total-H3 and total-H4 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the vehicle-infused group. (<i>D</i>) Representative blots for acetyl-H3/H4 and total-H3/H4, respectively. (<i>E</i>) Schematic of the behavioral protocol. Rats were trained and immediately after given intra-LA infusion of either (1 µg in 0.5 µl/side) TSA or Vehicle (0.5 µl/side) followed 60 min later by intra-LA infusion of (1 µg in 0.5 µl/side) 5-AZA or Vehicle (0.5 µl/side), creating the following groups: Veh-Veh (<i>n</i> = 6), Veh-5-AZA (<i>n</i> = 5), and TSA-5-AZA (<i>n</i> = 6). LTM was examined 24 hrs later. (<i>F</i>) Post-shock freezing scores in each group immediately after the conditioning trials. (<i>G</i>) Mean (±SEM) LTM retention test scores across each trial. (<i>H</i>) Histological verification of cannula placements for rats infused with Vehicle-Vehicle (black circles), or Vehicle-5-AZA (gray circles), or TSA-5-AZA (white circles). Panels adapted from Paxinos and Watson <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019958#pone.0019958-Paxinos1" target="_blank">[40]</a>.</p

Intra-LA infusion of a DNMT inhibitor impairs auditory fear memory consolidation.

<p>(A) Schematic of behavioral protocol. Rats were conditioned with 3 tone-shock pairings, followed by intra-LA infusion of vehicle or 5-AZA 1 hr later (n = 5, each group). LTM was assessed ∼24 hr after training in a distinct context. Rats were re-conditioned drug free and re-tested for LTM ∼1 week later. (<i>B</i>) Mean (±SEM) post-shock freezing scores in each group following each conditioning trial. (<i>C</i>) Mean (±SEM) LTM retention test scores across each trial. (<i>D</i>) Mean (±SEM) LTM retention re-test scores across each trial following re-conditioning one week later. (<i>E</i>) Schematic of behavioral protocol. Rats were conditioned with 3 tone-shock pairings, followed 1 hr later by intra-LA infusion of vehicle (n = 4) or 5-AZA (n = 4). STM was assessed 1 hr after training in a distinct context. (<i>F</i>) Mean (±SEM) post-shock freezing scores in each group following each conditioning trial. (<i>G</i>) Mean (±SEM) STM retention test scores across each trial. (<i>H</i>) Histological verification of cannula placements for rats in LTM (left) and STM (right) experiments infused with vehicle (black circles) or 5-AZA (white circles). Panels adapted from Paxinos and Watson <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019958#pone.0019958-Paxinos1" target="_blank">[40]</a>.</p

Regulation of histone H3 acetylation and DNMT3A expression in the LA following fear conditioning is ERK-dependent.

<p>(A) Schematic of the behavioral protocol. Rats received intra-LA infusion of either vehicle (n = 7) or U0126 (n = 8) followed 30 min later by fear conditioning consisting of 3 tone-shock pairings and were sacrificed 90 min after training. (<i>B</i>) Representative Western blots for acetylated histone (top), total histone (middle), and DNMT3A expression (bottom) at each time point. (<i>C</i>) Mean (±SEM) acetyl-H3 and acetyl-H4 immunoreactivity from punches taken from the LA. Here, acetyl-H3 and acetyl-H4 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the vehicle-infused group. (*) <i>p</i><0.05 relative to vehicle group. (<i>D</i>) Mean (±SEM) total-H3 and total-H4 immunoreactivity from the samples in (C). Here, total-H3 and total-H4 protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the vehicle-infused group. (<i>E</i>) Mean (±SEM) DNMT3A immunoreactivity from punches taken from the LA from vehicle (n = 7) and U0126-treated rats (n = 7). Here, DNMT3A protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the vehicle-infused group. (*) <i>p</i><0.05 relative to vehicle group.</p

Knockdown of Npas4 <i>in vitro</i>.

<p>(A) HEK293 cells were co-transfected with plasmids expressing Npas4-RFP and plasmids expressing shRNAs designed to deplete Npas4 mRNA [shNpas4(1) (ii, v) and shNpas4(5) (iii, vi)]. A control shRNA (shSCRM) (i, iv) that does not target Npas4 was used as a control. Ninety-six hrs following transfection, cells were visualized by fluorescence microcopy (20X) for the presence of the shRNA GFP plasmids, (panels i, ii, iii) and Npas4-RFP (panels iv, v, and vi). Cells transfected with shNPAS4(1) and shNPAS4(5) exhibited significantly less Npas4-RFP compared to cells transfected with shSCRM, suggesting that the shRNAs against Npas4 are targeting the Npas4-RFP mRNA for degradation resulting in less Npas4-RFP protein (panels v. and vi. versus iv.). Exposure times were the same for visualization of GFP images and RFP images, respectively. (B) qRT-PCR analysis of Npas4 mRNA from samples prepared as described above. Both shNpas4(1) and shNpas4(5) significantly depleted Npas4 mRNA relative to the shSCRM control (n = 3, each group). p<0.05 relative to the shSCRM group.</p