Shigeto Yamamoto

Effects of single prolonged stress and D-cycloserine on contextual fear extinction and hippocampal NMDA receptor expression in a rat model of PTSD

Although the impaired extinction of traumatic memory is one of the hallmark symptoms of posttraumatic stress disorder (PTSD), the underlying mechanisms of impaired extinction are unclear and effective pharmacological interventions have not yet been developed. Single prolonged stress (SPS) has been proposed as an animal model of PTSD, since rats subjected to SPS (SPS rats) show enhanced negative feedback of the HPA axis and increased contextual fear, which are characteristics similar to those observed in patients with PTSD. In this study, using SPS rats, we examined (a) the ability of SPS to impair fear extinction, (b) whether D-cycloserine (DCS) can alleviate impaired fear extinction in SPS rats, and (c) the effect of SPS and/or DCS on the levels of N-methyl-D-aspartate (NMDA) receptor subunit mRNAs in the rat hippocampus during extinction training. SPS rats exhibited impaired fear extinction in the contextual fear test, which was alleviated by the repeated administration of DCS. The effect of enhanced extinction, induced by the administration of DCS to SPS rats, was maintained for one week following extinction training. SPS induced significant upregulation of the levels of NMDA receptor subunit mRNAs before and during the period of extinction training, while repeated administration of DCS eliminated the enhanced mRNA levels of NMDARs. Behavioral analyses indicated that SPS is an appropriate animal model of PTSD and that DCS may be effective in the treatment of PTSD. These findings suggest that DCS, irrespective of its mechanistic involvement in the enhancement of fear extinction, may help to reverse hippocampal plasticity, and thus reverse the NMDA compensatory alterations.

Single immobilization stress differentially alters the expression profile of transcripts of the brain-derived neurotrophic factor (BDNF) gene and histone acetylation at its promoters in the rat hippocampus.

Decreased levels of brain-derived neurotrophic factor (BDNF) in the hippocampus are implicated in the pathophysiology of major depression, although the mechanism has yet to be characterized. Epigenetic studies revealed that DNA methylation and histone modifications at the promoter of exons of the BDNF gene are the pivotal factors in the regulation of BDNF transcription. Histone acetylation regulates gene transcription through chromatin remodelling. We examined the influence of a single immobilization stress (SIS) at 2 h and 24 h afterwards on the levels of total BDNF mRNA with each exon mRNA by quantitative real-time PCR, acetylated histone at the promoters of the BDNF gene by chromatin immunoprecipitation followed by real-time PCR, and BDNF protein by ELISA in the rat hippocampus. SIS significantly decreased the levels of total BDNF mRNA with significantly reduced levels of exons I and IV mRNA followed by a significant reduction in BDNF protein 4 h after SIS. Significant decreases in the levels of acetylated histone H3, but not H4, were found at the promoters of exons I, IV, and VI. In contrast, no marked changes in the levels of either acetylated histone or BDNF mRNA and protein were found 24 h after SIS. This study demonstrated the involvement of histone acetylation in the regulation of BDNF transcription by SIS, and the plastic change in histone acetylation after SIS. These findings suggest that stress affects BDNF gene transcription via epigenetic regulation, and glucocorticoid may be involved in this regulation.

Enhanced hippocampal BDNF/TrkB signaling in response to fear conditioning in an animal model of posttraumatic stress disorder

Because the majority of patients with posttraumatic stress disorder (PTSD) exhibit long-lasting traumatic fear memory, we hypothesize that enhanced fear memory consolidation is closely involved in the pathophysiology of PTSD. Brain-derived neurotrophic factor (BDNF) and its receptor, tyrosine kinase receptor B (TrkB), are crucial for hippocampal-dependent learning and memory. In particular, differential induction of BDNF gene transcripts mediated by histone acetylation plays a role in the consolidation of fear memory. In the present study, total and exon-specific mRNA and protein levels of BDNF and TrkB in the hippocampus after contextual fear conditioning (FC) were compared between rats subjected to single prolonged stress (SPS) and sham treatment. In addition, we examined the degree of histone acetylation at the promoter of each exon of the BDNF gene by chromatin immunoprecipitation (ChIP). We previously demonstrated a significant increase in contextual freezing in SPS rats. In the present study, SPS rats also showed increased total BDNF mRNA (including exons I, IV) and BDNF protein levels in the hippocampus after FC, accompanied by increased acetylation of histone H3 and H4 at the promoter of exon I and IV relative to sham-treated rats. Furthermore, the TrkB protein levels in the hippocampus of SPS rats were significantly higher than those in sham rats. These findings suggest that the enhanced levels of BDNF as well as TrkB along with epigenetic regulation of the BDNF gene during fear memory consolidation is, at least in part, associated with long-lasting fear memory in patients with PTSD.

Epigenetic Regulation of BDNF Gene in Response to Stress

Neuronal plasticity induced by changes in synaptic morphology and function is well known to play a pivotal role in leaning and memory as well as adaptation to stress. It is suggested that these plastic changes are due to orchestration of alterations in gene expression in the brain. Recent advances in molecular biology have provided evidence that epigenetic mechanisms, such as DNA methylation and histone modification, are crucial to gene transcription in the mammalian brain. Our research group has recently investigated the involvement of histone actylation at the promoter of the brain-derived neurotrophic factor (BDNF) gene in stress-induced reduction in BDNF, as well as in fear conditioning-induced enhancement of BDNF, in the rat hippocampus. The results of the stress study demonstrated that single-immobilization stress significantly reduced the levels of total, exon I, and exon IV BDNF mRNA, and also significantly reduced acetylation levels of histone H3, but not H4, at the promoter of exons I, IV, and VI. The results of the fear conditioning study showed that footshock stress significantly increased the levels of total, exon I, and exon IV BDNF mRNA, with significantly increased acetylation levels of both histone H3 and H4, at the promoter of exons I and IV, followed by enhanced freezing to fear-context exposure. These findings suggest that changes in BDNF transcription in the rat hippocampus in response to stressful stimuli are, at least in part, regulated by histone acetylation status.

Neonatal tactile stimulation reverses the effect of neonatal isolation on open-field and anxiety-like behavior, and pain sensitivity in male and female adult Sprague–Dawley rats

It is well known that early life events induce long-lasting psychophysiological and psychobiological influences in later life. In rodent studies, environmental enrichment after weaning prevents the adulthood behavioral and emotional disturbances in response to early adversities. We compared the behavioral effect of neonatal isolation (NI) with the effect of NI accompanied by tactile stimulation (NTS) to determine whether NTS could reverse or prevent the effects of NI on the adulthood behavioral and emotional responses to environmental stimuli. In addition, we also examined the sex difference of the NTS effect. Measurements of body weights, an open-field locomotor test, an elevated plus maze test, a hot-plate test, and a contextual fear-conditioning test were performed on postnatal day 60. As compared with rats subjected to NI, rats subjected to NTS showed significantly higher activity and exploration in the open-field locomotor test, lower anxiety-like behavior in the elevated plus maze test, and significantly prolonged latencies in the hot-plate test, and this effect was equal among males and females. In the contextual fear-conditioning test, whereas NTS significantly reduced the enhanced freezing time due to NI in females, no significant difference in the freezing time between NI and NTS was found in males. These findings indicate that adequate tactile stimulation in early life plays an important role in the prevention of disturbances in the behavioral and emotional responses to environmental stimuli in adulthood induced by early adverse experiences.

Vorinostat, a histone deacetylase inhibitor, facilitates fear extinction and enhances expression of the hippocampal NR2B-containing NMDA receptor gene

Histone acetylation, which alters the compact chromatin structure and changes the accessibility of DNA to regulatory proteins, is emerging as a fundamental mechanism for regulating gene expression. Histone deacetylase (HDAC) inhibitors increase histone acetylation and enhance fear extinction. In this study, we examined whether vorinostat, an HDAC inhibitor, facilitates fear extinction, using a contextual fear conditioning (FC) paradigm, in Sprague-Dawley rats. We found that vorinostat facilitated fear extinction. Next, the levels of global acetylated histone H3 and H4 were measured by Western blotting. We also assessed the effect of vorinostat on the hippocampal levels of NMDA receptor mRNA by real-time quantitative PCR (RT-PCR) and protein by Western blotting. 2 h after vorinostat administration, the levels acetylated histones and NR2B mRNA, but not NR1 or NR2A mRNA, were elevated in the hippocampus. The NR2B protein level was elevated 4 h after vorinostat administration. Last, we investigated the levels of acetylated histones and phospho-CREB (p-CREB) binding at the promoter of the NR2B gene using the chromatin immunoprecipitation (ChIP) assay followed by RT-PCR. The ChIP assay revealed increases in the levels of acetylated histones and they were accompanied by enhanced binding of p-CREB to its binding site at the promoter of the NR2B gene 2 h after vorinostat administration. These findings suggest that vorinostat increases the expression of NR2B in the hippocampus by enhancing histone acetylation, and this process may be implicated in fear extinction.

The potential use of histone deacetylase inhibitors in the treatment of depression.

Numerous preclinical studies demonstrate that changes in gene expression in the brain occur in animal models of depression using exposure to stress, such as social defeat and leaned helplessness, and that repeated administration of antidepressants ameliorates these stress-induced changes in gene expression. These findings suggest that alteration in gene transcription in the central nervous system in response to stress plays an important role in the pathophysiology of depression. Recent advances in epigenetics have led to the realization that chromatin remodeling mediated by histone deacetylase (HDAC) is closely involved in the regulation of gene transcription. In this context, we first review several preclinical studies demonstrating the antidepressant-like efficacy of HDAC inhibitors. We then suggest the efficacy of HDAC inhibitors in treatment-resistant depression based on the mechanism of action of HDAC. Finally, we discuss the possibility of using HDAC inhibitors in patients with treatment-resistant depression.

Alterations in the hippocampal glycinergic system in an animal model of posttraumatic stress disorder

Previous studies have demonstrated that rats subjected to single prolonged stress (SPS) exhibit posttraumatic stress disorder (PTSD)-like symptoms, such as enhanced contextual fear in response to trauma-related and trauma-unrelated events. Furthermore, we previously reported that upregulation of hippocampal glycine transporter 1 (GlyT-1) mRNA after context exposure could be the initial mechanism underlying impaired fear extinction in SPS rats. To clarify the involvement of the hippocampal glycinergic system in impaired fear extinction in SPS rats, we measured the time course of changes in the duration of freezing and the hippocampal levels of Gly-T1 mRNA using contextual fear conditioning (FC) and extinction training. We also used in vivo microdialysis to measure the concentration of extracellular glycine in the hippocampus during the time interval between FC and the first context exposure. SPS rats exhibited increased and sustained contextual fear responses. The enhanced contextual fear response in SPS rats was associated with a sustained increase in hippocampal levels of Gly-T1 mRNA after FC relative to sham rats, and by a decrease in the extracellular glycine concentration. GlyT-1 mRNA levels in rats that underwent repeated extinction training were significantly lower than in rats that did not undergo extinction training. These findings indicate that reduced activity of the hippocampal glycinergic system could be closely involved in impaired fear extinction in SPS rats, suggesting that activation of the glycinergic system by d-cycloserine or GlyT-1 inhibitors may ameliorate the impairment of fear extinction.

Maternal postpartum learned helplessness (LH) affects maternal care by dams and responses to the LH test in adolescent offspring

It is known that the early environment affects the mental development of rodent and human offspring. However, it is not known specifically whether a postpartum depressive state influences the depressive state in offspring. Using learned helplessness (LH) in rats as an animal model of depression, we examined the influence of maternal postpartum LH on responses to the LH test of offspring. Dam rats were judged as LH or non-helpless (nLH) on postnatal days (PN) 2-3, and maternal behavior was recorded during PN2-14. On PN 45-46, offspring were subjected to the LH test. Plasma corticosterone (CORT) levels, hippocampal levels of glucocorticoid receptor (GR) and brain-derived neurotrophic factor (BDNF) mRNA were measured before and after the LH test in offspring. Active nursing in LH dams was significantly lower than that in nLH dams. Susceptibility to LH in the offspring of LH dams was significantly higher than in those of nLH dams, and was negatively correlated with active nursing by LH dams. The GR mRNA levels before and after the LH test were lower in the offspring of LH dams than in those of nLH dams, and the reduced basal GR mRNA and protein might have resulted in the higher CORT response after the LH test. There was no significant difference in BDNF mRNA in the offspring of LH and nLH dams. These findings suggest that early postpartum LH decreased active nursing and increased depression-like behavior in the adolescent offspring via dysfunction of the hypothalamic-pituitary-adrenal axis.