Glenn E. Schafe

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.

Modifications in chromatin structure have been widely implicated in memory and cognition, most notably using hippocampal-dependent memory paradigms including object recognition, spatial memory, and contextual fear memory. Relatively little is known, however, about the role of chromatin-modifying enzymes in amygdala-dependent memory formation. Here, we use a combination of biochemical, behavioral, and neurophysiological methods to systematically examine the role of p300/CBP histone acetyltransferase (HAT) activity in the consolidation and reconsolidation of auditory Pavlovian fear memories. We show that local infusions of c646, a selective pharmacological inhibitor of p300/CBP activity, shortly following either fear conditioning or fear memory retrieval impair training and retrieval-related regulation of histone acetylation in the lateral nucleus of the amygdala (LA). Furthermore, we show that intra-LA infusion of c646 significantly impairs fear memory consolidation, reconsolidation, and associated neural plasticity in the LA. Our findings collectively suggest that p300/CBP HAT activity is critical for the consolidation and reconsolidation of amygdala-dependent Pavlovian fear memories.

The Neuronal PAS domain protein 4 (Npas4) is a neuronal activity-dependent immediate early gene that has recently been identified as a transcription factor which regulates the transcription of genes that control inhibitory synapse development and synaptic plasticity. The role Npas4 in learning and memory, however, is currently unknown. Here, we systematically examine the role of Npas4 in auditory Pavlovian fear conditioning, an amygdala-dependent form of emotional learning. In our first series of experiments, we show that Npas4 mRNA and protein are regulated in the rat lateral nucleus of the amygdala (LA) in a learning-dependent manner. Further, knockdown of Npas4 protein in the LA via adeno-associated viral (AAV) mediated gene delivery of RNAi was observed to impair fear memory formation, while innate fear and the expression of fear memory were not affected. In our second series of experiments, we show that Npas4 protein is regulated in the LA by retrieval of an auditory fear memory and that knockdown of Npas4 in the LA impairs retention of a reactivated, but not a non-reactivated, fear memory. Collectively, our findings provide the first comprehensive look at the functional role of Npas4 in learning and memory.

Epigenetic mechanisms have been widely implicated in synaptic plasticity and in memory consolidation, yet little is known about the role of epigenetic mechanisms in memory reconsolidation processes. In the present study, we systematically examine the role of histone acetylation and DNA methylation in the reconsolidation of an amygdala-dependent Pavlovian fear memory. We first show that the acetylation of histone 3 (H3), but not histone 4 (H4), is regulated following auditory fear memory retrieval in the lateral nucleus of the amygdala (LA). We next show that histone deacetylase (HDAC) inhibition in the LA enhances both retrieval-induced histone acetylation and reconsolidation of an auditory fear memory. Conversely, inhibition of DNA methytransferase (DNMT) activity in the LA significantly impairs both retrieval-related H3 acetylation and fear memory reconsolidation. The effects of HDAC and DNMT inhibitors on fear memory reconsolidation were observed to be time-limited and were not evident in the absence of memory reactivation. Further, memories lost following DNMT inhibition were not observed to be vulnerable to spontaneous recovery, reinstatement, or to a shift in testing context, suggesting that memory impairment was not the result of facilitated extinction. Finally, pretreatment with the HDAC inhibitor was observed to rescue the reconsolidation deficit induced by the DNMT inhibitor. These findings collectively suggest that histone acetylation and DNA methylation are critical for reconsolidation of fear memories in the LA.

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.

The activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) is an immediate-early gene that has been widely implicated in synaptic plasticity and in the consolidation of a variety of hippocampal- and amygdala-dependent memory tasks. The functional role of Arc/Arg3.1 in memory reconsolidation processes, however, has not been systematically studied. In the present study, we examined the role of Arc/Arg3.1 in the reconsolidation of an amygdala-dependent auditory pavlovian fear memory. We show that Arc/Arg3.1 protein is regulated in the lateral nucleus of the amygdala (LA) by retrieval of an auditory fear memory. Next, we show that antisense knockdown of Arc/Arg3.1 in the LA impairs fear memory reconsolidation of both a recent (1-d-old) as well as a well-consolidated (2-week-old) fear memory; that is, post-retrieval short-term memory, tested at 3 h after retrieval, is intact, whereas post-retrieval long-term memory, tested ∼24 h after retrieval, is significantly impaired. The effect of Arc/Arg3.1 knockdown was observed to be time limited and specific to an actively reactivated fear memory. Moreover, the reconsolidation deficit induced by Arc/Arg3.1 knockdown was not found to be sensitive to spontaneous recovery, reinstatement, or a shift in the testing context, suggesting that our behavioral effects are not attributable to facilitated extinction. Collectively, our findings provide the first comprehensive look at the functional role of Arc/Arg3.1 in memory reconsolidation processes in the mammalian brain.

The immediate-early gene early growth response gene-1 (EGR-1, zif-268) has been extensively studied in synaptic plasticity and memory formation in a variety of memory systems. However, a convincing role for EGR-1 in amygdala-dependent memory consolidation processes has yet to emerge. In the present study, we have examined the role of EGR-1 in the consolidation and reconsolidation of amygdala-dependent auditory Pavlovian fear conditioning. In our first series of experiments, we show that EGR-1 is regulated following auditory fear conditioning in the lateral nucleus of the amygdala (LA). Next, we use antisense oligodeoxynucleotide (ODN) knockdown of EGR-1 in the LA to show that training-induced expression of EGR-1 is required for memory consolidation of auditory fear conditioning; that is, long-term memory (LTM) is significantly impaired while acquisition and short-term memory (STM) are intact. In a second set of experiments, we show that EGR-1 is regulated in the LA by retrieval of an auditory fear memory. We then show that retrieval-induced expression of EGR-1 in the LA is required for memory reconsolidation of auditory fear conditioning; that is, post-retrieval (PR)-LTM is significantly impaired while memory retrieval and PR-STM are intact. Additional experiments show these effects to be restricted to the LA, to be temporally graded, and unlikely to be due to nonspecific toxicity within the LA. Collectively, our findings strongly implicate a role for EGR-1 in both the initial consolidation and in the reconsolidation of auditory fear memories in the LA.

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”.

We have recently hypothesized that NO-cGMP-PKG signaling in the lateral nucleus of the amygdala (LA) during auditory fear conditioning coordinately regulates ERK-driven transcriptional changes in both auditory thalamic (MGm/PIN) and LA neurons that serve to promote pre- and postsynaptic alterations at thalamo-LA synapses, respectively. In the present series of experiments, we show that N-methyl-d-aspartate receptor (NMDAR)-driven synaptic plasticity and NO-cGMP-PKG signaling in the LA regulate the training-induced expression of ERK and the ERK-driven immediate early genes (IEGs) Arc/Arg3.1, c-Fos, and EGR-1 in the LA and the MGm/PIN. Rats receiving intra-LA infusion of the NR2B selective antagonist Ifenprodil, the NOS inhibitor 7-Ni, or the PKG inhibitor Rp-8-Br-PET-cGMPS exhibited significant decreases in ERK activation and in the training-induced expression of all three IEGs in the LA and MGm/PIN while intra-LA infusion of the PKG activator 8-Br-cGMP had the opposite effect. Remarkably, those rats given intra-LA infusion of the membrane impermeable NO scavenger c-PTIO exhibited significant decreases in ERK activation and ERK-driven IEG expression in the MGm/PIN, but not in the LA. Together with our previous experiments, these results suggest that synaptic plasticity and the NO-cGMP-PKG signaling pathway promote fear memory consolidation, in part, by regulating ERK-driven transcription in both the LA and the MGm/PIN. They further suggest that synaptic plasticity in the LA during fear conditioning promotes ERK-driven transcription in MGm/PIN neurons via NO-driven “retrograde signaling.”

J. Neurochem. (2010) 112, 636–650. Most recent studies aimed at defining the cellular and molecular mechanisms of Pavlovian fear conditioning have focused on protein kinase signaling pathways and the transcription factor cAMP‐response element binding protein (CREB) that promote fear memory consolidation in the lateral nucleus of the amygdala (LA). Despite this progress, there still remains a paucity of information regarding the genes downstream of CREB that are required for long‐term fear memory formation in the LA. We have adopted a strategy of using microarray technology to initially identify genes induced within the dentate gyrus following in vivo long‐term potentiation (LTP) followed by analysis of whether these same genes are also regulated by fear conditioning within the LA. In the present study, we first identified 34 plasticity‐associated genes that are induced within 30 min following LTP induction utilizing a combination of DNA microarray, qRT‐PCR, and in situ hybridization. To determine whether these genes are also induced in the LA following Pavlovian fear conditioning, we next exposed rats to an auditory fear conditioning protocol or to control conditions that do not support fear learning followed by qRT‐PCR on mRNA from microdissected LA samples. Finally, we asked whether identified genes induced by fear learning in the LA are downstream of the extracellular‐regulated kinase/mitogen‐activated protein kinase signaling cascade. Collectively, our findings reveal a comprehensive list of genes that represent the first wave of transcription following both LTP induction and fear conditioning that largely belong to a class of genes referred to as ‘neuronal activity dependent genes’ that are likely calcium, extracellular‐regulated kinase/mitogen‐activated protein kinase, and CREB‐dependent.