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.     

Failure of Long-Term Memory Formation in Juvenile Snails Is Determined by Acetylation Status of Histone H3 and Can Be Improved by NaB Treatment

Background

Animals’ capacities for different forms of learning do not mature simultaneously during ontogenesis but the molecular mechanisms behind the delayed development of specific types of memory are not fully understood. Mollusks are considered to be among the best models to study memory formation at the molecular level. Chromatin remodeling in developmental processes, as well as in long-term memory formation, was recently shown to play a major role. Histone acetylation is a key process in the chromatin remodeling and is regulated through the signaling cascades, for example MAPK/ERK. Previously, we found that MAPK/ERK is a key pathway in the formation of the food aversion reflex in Helix. Pretreatment with upstream ERK kinase inhibitor PD98059 prevented food avoidance learning in adult Helix. In contrast to adult snails, juveniles possess immature plasticity mechanisms of the avoidance reflex until the age of 2–3 months while the MAPK/ERK cascade is not activated after aversive learning. In the present study, we focused on the potential MAPK/ERK target - histone H3.

Methodology/Principal Findings

Here we found that a significant increase in histone H3 acetylation occurs in adult animals after learning, whereas no corresponding increase was observed in juveniles. The acetylation of histone H3 is regulated by ERK kinase, since the upstream ERK kinase inhibitor PD98059 prevented the increase of histone H3 acetylation upon learning. We found that the injection of histone deacetylase inhibitor sodium butyrate (NaB) prior to training led to induction in histone H3 acetylation and significantly ameliorated long-term memory formation in juvenile snails.

Conclusions/Significance

Thus, MAPK/ERK-dependent histone H3 acetylation plays an essential role in the formation of food aversion in Helix. Dysfunction of the MAPK/ERK dependent histone H3 acetylation might determine the deficiency of avoidance behavior and long-term plasticity in juvenile animals. Stimulation of histone H3 acetylation in juvenile animals by NaB promoted avoidance plasticity.     

Regulation of histone acetylation during memory formation in the hippocampus

Formation of long term memory begins with the activation of many disparate signaling pathways that ultimately impinge on the cellular mechanisms regulating gene expression. We investigated whether mechanisms regulating chromatin structure were activated during the early stages of long term memory formation in the hippocampus. Specifically, we investigated hippocampal histone acetylation during the initial stages of consolidation of long term association memories in a contextual fear conditioning paradigm. Acetylation of histone H3 in area CA1 of the hippocampus was regulated in contextual fear conditioning, an effect dependent on activation of N-methyl-D-aspartic acid (NMDA) receptors and ERK, and blocked using a behavioral latent inhibition paradigm. Activation of NMDA receptors in area CA1 in vitro increased acetylation of histone H3, and this effect was blocked by inhibition of ERK signaling. Moreover, activation of ERK in area CA1 in vitro through either the protein kinase C or protein kinase A pathways, biochemical events known to be involved in long term memory formation, also increased histone H3 acetylation. Furthermore, we observed that elevating levels of histone acetylation through the use of the histone deacetylase inhibitors trichostatin A or sodium butyrate enhanced induction of long term potentiation at Schaffer-collateral synapses in area CA1 of the hippocampus, a candidate mechanism contributing to long term memory formation in vivo. In concert with our findings in vitro, injection of animals with sodium butyrate prior to contextual fear conditioning enhanced formation of long term memory. These results indicate that histone-associated heterochromatin undergoes changes in structure during the formation of long term memory. Mimicking memory-associated changes in heterochromatin enhances a cellular process thought to underlie long term memory formation, hippocampal long term potentiation, and memory formation itself.     

Changes in histone modifications during in vitro maturation of porcine oocytes

Nuclear core histone modifications influence chromosome structures and functions. Recently, the involvement of histone acetylations in the cell memory of gene expression has been suggested in mouse oocyte maturation. At present, there is little available data on histone modifications in mammalian oocyte maturation. In the present study, we examined changes in the acetylation of histone H3 lysines 9 (H3K9) and 14 (H3K14), and histone H4 lysines 5 (H4K5), 8 (H4K8) and 12 (H4K12), and trimethylation of H3K9 during in vitro maturation of porcine oocytes. Immunocytochemical analyses revealed that the all of the lysines examined were highly acetylated in the germinal vesicle stage, and this level of acetylation was maintained until the first prometaphase. In the first metaphase, the lysines near the N-terminal end, H3K9 and H4K5, were completely deacetylated. The acetylation of the lysines far from the N-terminal end, H3K14, H4K8, and H4K12, was markedly decreased but still present. The acetylations were increased transiently at the first anaphase and telophase, and then decreased again at the second metaphase to the same level as the first metaphase. Since effective concentrations of trichostatin A (TSA) to inhibit the deacetylation were different in various lysine residues, multiple histone deacetylases (HDACs) were suggested to function during meiotic maturation. The trimethylation of H3K9 was maintained in a high level throughout maturation. These results suggest that the histone acetylation during porcine oocyte maturation is precisely controlled by the cell cycle.     

Roles of NO Signaling in Long-Term Memory Formation in Visual Learning in an Insect

Many insects exhibit excellent capability of visual learning, but the molecular and neural mechanisms are poorly understood. This is in contrast to accumulation of information on molecular and neural mechanisms of olfactory learning in insects. In olfactory learning in insects, it has been shown that cyclic AMP (cAMP) signaling critically participates in the formation of protein synthesis-dependent long-term memory (LTM) and, in some insects, nitric oxide (NO)-cyclic GMP (cGMP) signaling also plays roles in LTM formation. In this study, we examined the possible contribution of NO-cGMP signaling and cAMP signaling to LTM formation in visual pattern learning in crickets. Crickets that had been subjected to 8-trial conditioning to associate a visual pattern with water reward exhibited memory retention 1 day after conditioning, whereas those subjected to 4-trial conditioning exhibited 30-min memory retention but not 1-day retention. Injection of cycloheximide, a protein synthesis inhibitor, into the hemolymph prior to 8-trial conditioning blocked formation of 1-day memory, whereas it had no effect on 30-min memory formation, indicating that 1-day memory can be characterized as protein synthesis-dependent long-term memory (LTM). Injection of an inhibitor of the enzyme producing an NO or cAMP prior to 8-trial visual conditioning blocked LTM formation, whereas it had no effect on 30-min memory formation. Moreover, injection of an NO donor, cGMP analogue or cAMP analogue prior to 4-trial conditioning induced LTM. Induction of LTM by an NO donor was blocked by DDA, an inhibitor of adenylyl cyclase, an enzyme producing cAMP, but LTM induction by a cAMP analogue was not impaired by L-NAME, an inhibitor of NO synthase. The results indicate that cAMP signaling is downstream of NO signaling for visual LTM formation. We conclude that visual learning and olfactory learning share common biochemical cascades for LTM formation.     

BDNF Activates mTOR to Regulate GluR1 Expression Required for Memory Formation

Background

The mammalian target of Rapamycin (mTOR) kinase plays a key role in translational control of a subset of mRNAs through regulation of its initiation step. In neurons, mTOR is present at the synaptic region, where it modulates the activity-dependent expression of locally-translated proteins independently of mRNA synthesis. Indeed, mTOR is necessary for different forms of synaptic plasticity and long-term memory (LTM) formation. However, little is known about the time course of mTOR activation and the extracellular signals governing this process or the identity of the proteins whose translation is regulated by this kinase, during mnemonic processing.

Methodology/Principal Findings

Here we show that consolidation of inhibitory avoidance (IA) LTM entails mTOR activation in the dorsal hippocampus at the moment of and 3 h after training and is associated with a rapid and rapamycin-sensitive increase in AMPA receptor GluR1 subunit expression, which was also blocked by intra-hippocampal delivery of GluR1 antisense oligonucleotides (ASO). In addition, we found that pre- or post-training administration of function-blocking anti-BDNF antibodies into dorsal CA1 hampered IA LTM retention, abolished the learning-induced biphasic activation of mTOR and its readout, p70S6K and blocked GluR1 expression, indicating that BDNF is an upstream factor controlling mTOR signaling during fear-memory consolidation. Interestingly, BDNF ASO hindered LTM retention only when given into dorsal CA1 1 h after but not 2 h before training, suggesting that BDNF controls the biphasic requirement of mTOR during LTM consolidation through different mechanisms: an early one involving BDNF already available at the moment of training, and a late one, happening around 3 h post-training that needs de novo synthesis of this neurotrophin.

Conclusions/Significance

In conclusion, our findings demonstrate that: 1) mTOR-mediated mRNA translation is required for memory consolidation during at least two restricted time windows; 2) this kinase acts downstream BDNF in the hippocampus and; 3) it controls the increase of synaptic GluR1 necessary for memory consolidation.     

Critical Period of Memory Enhancement during Taste Avoidance Conditioning in Lymnaea stagnalis

The present study investigated the optimal training procedure leading to long-lasting taste avoidance behavior in Lymnaea. A training procedure comprising 5 repeated pairings of a conditional stimulus (CS, sucrose), with an unconditional stimulus (US, a tactile stimulation to the animal’s head), over a 4-day period resulted in an enhanced memory formation than 10 CS-US repeated pairings over a 2-day period or 20 CS-US repeated pairings on a single day. Backward conditioning (US-CS) pairings did not result in conditioning. Thus, this taste avoidance conditioning was CS-US pairing specific. Food avoidance behavior was not observed following training, however, if snails were immediately subjected to a cold-block (4°C for 10 min). It was critical that the cold-block be applied within 10 min to block long-term memory (LTM) formation. Further, exposure to the cold-block 180 min after training also blocked both STM and LTM formation. The effects of the cold-block on subsequent learning and memory formation were also examined. We found no long lasting effects of the cold-block on subsequent memory formation. If protein kinase C was activated before the conditioning paradigm, snails could still acquire STM despite exposure to the cold-block.     

Roles of Calcium/Calmodulin-Dependent Kinase II in Long-Term Memory Formation in Crickets

Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key molecule in many systems of learning and memory in vertebrates, but roles of CaMKII in invertebrates have not been characterized in detail. We have suggested that serial activation of NO/cGMP signaling, cyclic nucleotide-gated channel, Ca²⁺/CaM and cAMP signaling participates in long-term memory (LTM) formation in olfactory conditioning in crickets, and here we show participation of CaMKII in LTM formation and propose its site of action in the biochemical cascades. Crickets subjected to 3-trial conditioning to associate an odor with reward exhibited memory that lasts for a few days, which is characterized as protein synthesis-dependent LTM. In contrast, animals subjected to 1-trial conditioning exhibited memory that lasts for only several hours (mid-term memory, MTM). Injection of a CaMKII inhibitor prior to 3-trial conditioning impaired 1-day memory retention but not 1-hour memory retention, suggesting that CaMKII participates in LTM formation but not in MTM formation. Animals injected with a cGMP analogue, calcium ionophore or cAMP analogue prior to 1-trial conditioning exhibited 1-day retention, and co-injection of a CaMKII inhibitor impaired induction of LTM by the cGMP analogue or that by the calcium ionophore but not that by the cAMP analogue, suggesting that CaMKII is downstream of cGMP production and Ca²⁺ influx and upstream of cAMP production in biochemical cascades for LTM formation. Animals injected with an adenylyl cyclase (AC) activator prior to 1-trial conditioning exhibited 1-day retention. Interestingly, a CaMKII inhibitor impaired LTM induction by the AC activator, although AC is expected to be a downstream target of CaMKII. The results suggest that CaMKII interacts with AC to facilitate cAMP production for LTM formation. We propose that CaMKII serves as a key molecule for interplay between Ca²⁺ signaling and cAMP signaling for LTM formation, a new role of CaMKII in learning and memory.     

大鼠基底外侧杏仁核组蛋白乙酰化对吗啡成瘾记忆的调控作用

为了探索组蛋白乙酰化对吗啡成瘾记忆相关分子表达调控机制,文章选取健康成年雄性SD大鼠34只,随机分为正常对照组(n = 6)及基底外侧杏仁核(Basolateral amygdala, BLA)颅内定位手术组(n =28)。在条件性位置偏爱(Conditioned place preference, CPP)训练阶段,大鼠BLA内给予组蛋白去乙酰化酶抑制剂曲古抑菌素A(Trichostafin A, TSA)并且腹腔注射吗啡溶液(10.0 mg/kg),对照组给予相同体积的10%二甲基亚砜(Dimethyl sulfoxide,DMSO)或盐水。应用蛋白质印记方法,检测吗啡诱导大鼠CPP建立后BLA内组蛋白H3K14乙酰化和脑源性神经营养因子(Brain-derived neurotrophic factor, BDNF)蛋白表达水平。结果显示,腹腔注射10 mg/kg吗啡能成功建立CPP。吗啡、TSA联合给药组大鼠比单纯吗啡给药组大鼠表现出更强烈的CPP(P<0.0001)。吗啡和TSA都能使BLA内的组蛋白H3乙酰化水平和BDNF的表达显著增高(P < 0.0001),同时二者之间具有协同作用。结果表明,大鼠BLA内组蛋白乙酰化水平与吗啡成瘾记忆形成有关,抑制BLA内组蛋白去乙酰化酶(Histone deacetylases, HDACs)的活性可强化吗啡诱导的线索记忆的形成;大鼠BLA内BDNF参与了吗啡诱导的线索记忆的形成并可能受到组蛋白乙酰化的调控。     

Dynamic Acetylation of All Lysine 4–Methylated Histone H3 in the Mouse Nucleus: Analysis at c-fos and c-jun

A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4)–methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9–methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9–methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.     

Combining Stressors That Individually Impede Long-Term Memory Blocks All Memory Processes

The effects of stress on memory are typically assessed individually; however, in reality different stressors are often experienced simultaneously. Here we determined the effect that two environmentally relevant stressors, crowding and low calcium availability, have on memory and neural activity following operant conditioning of aerial respiration in the pond snail, Lymnaea stagnalis. We measured aerial breathing behaviour and activity of a neuron necessary for memory formation, right pedal dorsal 1 (RPeD1), in the central pattern generator (CPG) that drives aerial respiration in untrained animals, and assessed how these traits changed following training. In naïve animals both crowding and combined stressors significantly depressed burst activity in RPeD1 which correlated with a depression in aerial breathing behaviour, whereas low calcium availability had no effect on RPeD1 activity. Following training, changes in burst activity in RPeD1 correlated with behavioural changes, decreasing relative to their naïve state at 3 h and 24 h in control conditions when both intermediate-term memory (ITM: 3 h) and long-term memory (LTM: 24 h) are formed, at 3 h but not 24 h when exposed to individual stressors when only ITM is formed, and did not change in combined stressors (i.e. when no memory is formed). Additionally, we also found that Lymnaea formed short-term memory (STM: 10 min) in the presence of individual stressors or under control conditions, but failed to do so in the presence of combined stressors. Our data demonstrate that by combining stressors that individually block LTM only we can block all memory processes. Therefore the effects of two stressors with similar individual affects on memory phenotype may be additive when experienced in combination.     

Dynamic acetylation of all lysine 4-methylated histone H3 in the mouse nucleus: analysis at c-fos and c-jun

A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4)–methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9–methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9–methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.     

Exposure to bisphenol-A affects fear memory and histone acetylation of the hippocampus in adult mice

Bisphenol-A (BPA), an environmental endocrine disruptor, has been reported to possess weak estrogenic, anti-estrogenic, and anti-androgen properties. Previous evidence indicates that perinatal exposure to low levels of BPA affects anxiety-like and cognitive behaviors in adult rodents. The present study aims to investigate the effect of BPA on emotional memory using the contextual fear conditioning of male mice in adulthood exposed to BPA for 90 days. The results indicated that exposure to BPA increased the freezing time 1 h and 24 h after fear conditioning training. Furthermore, western blot analyses showed that BPA exposure decreased the level of N-methyl-d-aspartic acid (NMDA) receptor subunit NR1 and increased the expression of histone deacetylase 2 (HDAC2) before fear conditioning training in the hippocampus of male mice. One and twenty-four hours after fear conditioning training, BPA enhanced the changes of the expressions of NR1, phosphorylated extracellular regulated protein kinases (ERK1/2), and histone acetylation induced by contextual fear conditioning in the hippocampus. These results suggest that long term exposure to BPA enhanced fear memory by the concomitant increased level of NMDA receptor and/or the enhanced histone acetylation in the hippocampus, which may be associated with activation of ERK1/2 signaling pathway.