Masashi Nibuya

Effect of paroxetine on a model of posttraumatic stress disorder in rats

To better understand neurochemical and psychopharmacological aspects of post-traumatic stress disorder (PTSD), it is necessary to establish an animal model of PTSD in which behavioral changes persist after the initial traumatization. We administered inescapable electric foot-shock (IS) to male Wistar rats in a shuttle-box with the gate closed. With or without paroxetine (PRX; belonging to selective serotonin reuptake inhibitors) treatment for 2 weeks after IS, we performed an avoidance/escape task session in the shuttle-box using signal lights as non-specific anxiogenic stimulants. Locomotor activity decreased before the task session and avoidance behavior increased during the session. Two weeks of PRX administration reduced hypervigilant behavior during the task session. We considered these behavioral changes as representative of numbing/avoidance and hypervigilance, referred to as bi-directional changes in PTSD.

Stress vulnerabilities in an animal model of post-traumatic stress disorder

We have studied the effects of inescapable electric foot shocks (ISs) on rats by using a subsequent avoidance/escape task performed in a shuttle box as an animal model of post-traumatic stress disorder (PTSD). In this study, the behavioral differences and the effects of chronic stress exposure prior to IS were examined among male rats of the Wistar, Fischer 344, and Lewis strains. In concordance with our previous report on the Wistar rats, we observed the characteristic features of PTSD in all three rat strains tested, that is, the hyperactive and hypoactive bidirectional behavioral changes that are associated with hypervigilant and hyperarousal behavior, and the numbing and avoidant behavior, respectively. The induction of hypoactive behaviors after IS was most exaggerated in the Fischer and Lewis strains. Although the count of hyperactive behaviors was maximal in the Fischer strain both at basal levels without IS and after IS, the increase in the rate of hyperactive behaviors by IS was the most prominent in the Lewis strain. In addition, preloaded chronic variable stress (CVS) enhanced the degree of hyperactive behavioral changes in the Wistar strain. Thus, we consider that the present study further validates the use of shuttle box paradigm as an animal model of PTSD by demonstrating the vulnerability due to genetic background and environmental preloaded stress.

Maternal Separation Enhances Conditioned Fear and Decreases the mRNA Levels of the Neurotensin Receptor 1 Gene with Hypermethylation of This Gene in the Rat Amygdala

Stress during postnatal development is associated with an increased risk for depression, anxiety disorders, and substance abuse later in life, almost as if mental illness is able to be programed by early life stressors. Recent studies suggest that such “programmed” effects can be caused by epigenetic regulation. With respect to conditioned fear, previous studies have indicated that early life stress influences its development in adulthood, whereas no potential role of epigenetic regulation has been reported. Neurotensin (NTS) is an endogenous neuropeptide that has receptors densely located in the amygdala and hippocampus. Recently, NTS systems have constituted an emerging target for the treatment of anxiety. The aim of the present work is to clarify whether the NTS system is involved in the disturbance of conditioned fear in rats stressed by maternal separation (MS). The results showed that MS enhanced freezing behaviors in fear-conditioned stress and reduced the gene expression of NTS receptor (NTSR) 1 but not of NTS or NTSR2 in the amygdalas of adult rats. The microinjection of a NTSR1 antagonist into the amygdala increased the percentage of freezing in conditioned fear, whereas the microinjection of NTSR1 agonist decreased freezing. These results suggest that NTSR1 in the amygdala may play a role in the effects of MS on conditioned fear stress in adult rats. Moreover, MS increased DNA methylation in the promoter region of NTSR1 in the amygdala. Taken together, MS may leave epigenetic marks in the NTSR1 gene in the amygdala, which may enhance conditioned fear in adulthood. The MS-induced alternations of DNA methylation in the promoter region of NTSR1 in the amygdala may be associated with vulnerability to the development of anxiety disorders and depression in adulthood.

Behavioral stress and activated serotonergic neurotransmission induce XBP-1 splicing in the rat brain.

The splicing of 26 nucleotides in the coding region of the X-box binding protein-1 (XBP-1) transcript to generate a mature active transcription factor is a part of the unfolded protein response to intracellular endoplasmic reticulum stress. In this study, we demonstrated that XBP-1 splicing is promptly induced in the rat brain including the hippocampus by both inescapable electric foot shock (IS) and pharmacologically manipulated activation of 5-hydroxytryptamine release in a dose-dependent manner. By administering ketanserin, a 5-hydroxytryptamine 2A antagonist, however, we could only partially block the increased splicing by IS and observed that the splicing was not influenced by lithium carbonate pretreatment. Although it is still unclear whether the enhanced unfolded protein response functions neuroprotectively by modulating the rate of general translation and increasing chaperone proteins or whether it eventually induces cellular damage by triggering apoptosis, the present results indicate the possible existence of a new adaptive intracellular signaling pathway in the brain that responds to environmentally challenged behavioral stress loading.

Electroconvulsive seizures enhance autophagy signaling in rat hippocampus.

The putative antidepressive mechanisms of a series of electroconvulsive seizures (ECS) are the following: 1) downregulation of monoaminergic receptor expression in several brain regions, 2) upregulation of the expression of brain-derived neurotrophic factor (BDNF), and 3) increased neurogenesis in the hippocampus. In this study, we used Western blot techniques to present another mechanism in which ECS enhances the autophagy signaling that is involved in the machinery related to synaptic and neural plasticity. Antibodies for conjugated Atg5-Atg12 (58kD) and cleaved light chain protein 3-II (LC3-II; 14 kD) were used to detect autophagy signals. An antibody for cleaved caspase-3 (17 kD) was used to detect alterations in apoptotic signals. Mature BDNF (14kD) expression in the hippocampus was evaluated in order to qualify the effectiveness of the ECS or stress-loading treatment. While significantly increased autophagy signals and no increases in apoptotic signals were detected in the ECS-treated rat hippocampus, the reverse (increased apoptotic signals and no altered autophagy signals) was observed in stressed rat hippocampus. No neuronal cell loss but new mossy fiber sprouting has been reported to accompany multiple ECS treatments, and recent studies have revealed that autophagy processes regulate the number of specific neurotransmitter receptors and the plasticity of synaptic components. The present study illustrated the neuroplastic and neurotrophic profiles of ECS and the neurotoxic impact of severe stress loading on hippocampal regions. This is the first report to demonstrate increased autophagy signals in ECS-treated rat hippocampus and no alterations in autophagy signals in stress-loaded rat hippocampus.

Relationship between post-traumatic stress disorder-like behavior and reduction of hippocampal 5-bromo-2'-deoxyuridine-positive cells after inescapable shock in rats.

Aim:  Inescapable shocks (IS) have been reported to reduce the number of 5‐bromo‐2′‐deoxyuridine (BrdU)‐positive cells in hippocampus. Antidepressants prevent this reduction, and the role of neurogenesis in depression is now suggested. It has been reported, however, that the number of BrdU‐positive cells was not different between the rats that developed learned helplessness and those that did not. This suggests that reduction of neurogenesis does not constitute a primary etiology of depression. It has been previously shown that IS can cause various post‐traumatic stress disorder (PTSD)‐like behavioral changes in rats. The aim of the present was therefore to examined whether the reduction of BrdU‐positive cells relates to any PTSD‐like behavioral changes in this paradigm.

Environmental enrichment enhances autophagy signaling in the rat hippocampus.

The findings that antidepressive treatments increase hippocampal neurotrophins have led researchers to emphasize the importance of neurogenesis, formation of new dendrites, and survival of neurons in the brain. However, it is difficult to maintain neural plasticity just by enriching the environment to facilitate formation of new networks. Neural plasticity also requires a degradation process that clears off unnecessary and undesirable components. We have recently reported an increase in autophagy signaling (wherein the cell digests components of itself) that has the potential of enhancing neuronal and synaptic plasticity after multiple sessions of electroconvulsive seizure treatment. The present study revealed an increase in autophagy signaling in the rat hippocampus following 2 weeks of environmental enrichment (EE), a procedure known to elicit antidepressive and anxiolytic behavioral changes in various animal paradigms. Western blot analysis showed an increase in hippocampal expression of microtubule-associated protein light chain 3-II (LC3-II), which is lipidated from LC3-I, in rats in the EE group. The effectiveness of the 2-week EE housing condition was validated by anxiolytic effects observed in the elevated plus maze test, enhanced habituation in the open field test, and elevation of hippocampal brain-derived neurotrophic factor expression. In addition, we showed that the EE housing condition ameliorated numbing/avoidance behaviors, but not hypervigilant behaviors, in an animal model of post-traumatic stress disorder (PTSD). This is the first report to show that EE can increase autophagy signaling and improve numbing/avoidance behaviors in an animal model of PTSD.

Decreased Plasma Brain-Derived Neurotrophic Factor and Vascular Endothelial Growth Factor Concentrations during Military Training

Decreased concentrations of plasma brain-derived neurotrophic factor (BDNF) and serum BDNF have been proposed to be a state marker of depression and a biological indicator of loaded psychosocial stress. Stress evaluations of participants in military mission are critically important and appropriate objective biological parameters that evaluate stress are needed. In military circumstances, there are several problems to adopt plasma BDNF concentration as a stress biomarker. First, in addition to psychosocial stress, military missions inevitably involve physical exercise that increases plasma BDNF concentrations. Second, most participants in the mission do not have adequate quality or quantity of sleep, and sleep deprivation has also been reported to increase plasma BDNF concentration. We evaluated plasma BDNF concentrations in 52 participants on a 9-week military mission. The present study revealed that plasma BDNF concentration significantly decreased despite elevated serum enzymes that escaped from muscle and decreased quantity and quality of sleep, as detected by a wearable watch-type sensor. In addition, we observed a significant decrease in plasma vascular endothelial growth factor (VEGF) during the mission. VEGF is also neurotrophic and its expression in the brain has been reported to be up-regulated by antidepressive treatments and down-regulated by stress. This is the first report of decreased plasma VEGF concentrations by stress. We conclude that decreased plasma concentrations of neurotrophins can be candidates for mental stress indicators in actual stressful environments that include physical exercise and limited sleep.

Low-level laser therapy for prevention of noise-induced hearing loss in rats

Noninvasive low-level laser therapy (LLLT) is neuroprotective, but the mechanism of this effect is not fully understood. In this study, the use of LLLT as a novel treatment for noise-induced hearing loss (NIHL) is investigated. Sprague-Dawley rats were exposed to intense noise and their right ears were irradiated with an 808nm diode laser at an output power density of 110 or 165mW/cm(2) for a 30min period for 5 consecutive days. Measurement of the auditory brainstem response revealed an accelerated recovery of auditory function in the groups treated with LLLT compared with the non-treatment group at days 2, 4, 7 and 14 after noise exposure. Morphological observations also revealed a significantly higher outer hair cell survival rate in the LLLT groups. Immunohistochemical analyses for inducible nitric oxide synthase (iNOS) and cleaved caspase-3 were used to examine oxidative stress and apoptosis. Strong immunoreactivities were observed in the inner ear tissues of the non-treatment group, whereas these signals were decreased in the LLLT group at 165mW/cm(2) power density. Our findings suggest that LLLT has cytoprotective effects against NIHL via the inhibition of iNOS expression and apoptosis.

The structural equation analysis of childhood abuse, adult stressful life events, and temperaments in major depressive disorders and their influence on refractoriness

Background Previous studies have shown the interaction between heredity and childhood stress or life events on the pathogenesis of a major depressive disorder (MDD). In this study, we tested our hypothesis that childhood abuse, affective temperaments, and adult stressful life events interact and influence the diagnosis of MDD. Patients and methods A total of 170 healthy controls and 98 MDD patients were studied using the following self-administered questionnaire surveys: the Patient Health Questionnaire-9 (PHQ-9), the Life Experiences Survey, the Temperament Evaluation of the Memphis, Pisa, Paris, and San Diego Autoquestionnaire, and the Child Abuse and Trauma Scale (CATS). The data were analyzed with univariate analysis, multivariable analysis, and structural equation modeling. Results The neglect scores of the CATS indirectly predicted the diagnosis of MDD through cyclothymic and anxious temperament scores of the Temperament Evaluation of the Memphis, Pisa, Paris, and San Diego Autoquestionnaire in the structural equation modeling. Two temperaments – cyclothymic and anxious – directly predicted the diagnosis of MDD. The validity of this result was supported by the results of the stepwise multivariate logistic regression analysis as follows: three factors – neglect, cyclothymic, and anxious temperaments – were significant predictors of MDD. Neglect and the total CATS scores were also predictors of remission vs treatment-resistance in MDD patients independently of depressive symptoms. Limitations The sample size was small for the comparison between the remission and treatment-resistant groups in MDD patients in multivariable analysis. Conclusion This study suggests that childhood abuse, especially neglect, indirectly predicted the diagnosis of MDD through increased affective temperaments. The important role as a mediator of affective temperaments in the effect of childhood abuse on MDD was suggested.