[The delayed sensitization of CRH response developed after chronic variable stress on the acoustic startle reflex].

Substantial evidence indicates that brain neurons containing and secreting norepinephrine (NE) and corticotrophin-releasing hormone (CRH) are activated during stress. The acoustic startle reflex (ASR) can be enhanced by CRH neuronal activity in the central nucleus of the amygdala. Our previous study demonstrates an augmentation of the footshock-induced ASR (f-ASR) 1 day after chronic variable stress (CVS) for 13 days. In this study, to evaluate a long-term neural plasticity in NE-CRH systems after CVS, we examined f-ASR 1, 8 or 15 days after CVS. The augmented magnitude of the f-ASR 15 day after CVS was potentiated and delayed compared with that 1 day after CVS. The delayed augmentation of f-ASR was inhibited by repeated treatment with desipramine, maprotiline or paroxetine for 14 days after CVS. A single treatment with any antidepressant agent had no influence the f-ASR while a marked inhibition by a single dose of alprazolam, CRH1-receptor antagonist, prazosin and propranolol was observed. The decreased tyrosine hydroxylase activity in the locus coeruleus and the beta-adrenoceptor down-regulation in the amygdaloid complex might be involved in the inhibiton of the delayed augmentation of f-ASR by repeated antidepressant treatment, leading to the possibility that the delayed sensitization of CRH response to stress after CVS might contribute to the biological mechanism underlying the formation of pathological states such as anxiety and depressive disorders.

Fluvoxamine and sigma-1 receptor agonists dehydroepiandrosterone (DHEA)-sulfate induces the Ser473-phosphorylation of Akt-1 in PC12 cells.

AIMS: The expression of brain-derived neurotrophic factor (BDNF) may be a downstream target of a variety of antidepressant treatments, and selective serotonin reuptake inhibitors (SSRIs) are used clinically for the treatment of depression. BDNF binds to and activates tyrosine kinases receptor (TrkB) to exert its effects. TrkB, after activation by ligands, stimulates phosphoinositide 3-kinase (PI3K). The downstream target of PI3K is Akt-1, a serine-threonine kinase. BDNF has signaling through the PLC-IP(3)/Ca(2+) pathway. Furthermore, the PLC-gamma/IP(3)/Ca(2+) pathway is regulated by the sigma-1 receptors. Here, we examined whether fluvoxamine (FLV) activated Akt-1 and increased phosphorylation of Akt-1 via sigma-1 receptor in PC12 cells. MAIN METHODS: We examined the effect of the SSRI, FLV and BDNF on the phosphorylation levels of serine-threonine kinase Akt-1 in PC12 cells using immunoblotting techniques. KEY FINDINGS: Treatment with 10 microM and 100 microM FLV of PC12 cells stimulated a 2.4- and 3.8-fold maximal increase in Ser(473)-phosphorylated Akt-1 levels at 40 min, respectively. Treatment with 50 ng/ml BDNF also stimulated Ser(473) -phosphorylated Akt-1 by 2.6-fold with a maximal increase at 5 min. In addition, the phosphorylation induced by FLV and BDNF was blocked by LY294002, a selective inhibitor of PI3K. The sigma-1 receptor agonists dehydroepiandrosterone (DHEA)-sulfate also stimulated a 2.1-fold increase in the level of Ser473-phosphorylated Akt-1. SIGNIFICANCE: This study demonstrates that fluvoxamine treatment rapidly increased phosphorylation of Akt-1. And BDNF activated Akt-1 phosphorylation by the TrkB/PI3K/Akt-1 pathway. We conclude that the phosphorylation of Akt-1, downstream of PI3K, was the key to their antidepressant effects.

[Long-lasting sensitization to footshock after chronic variable stress on the acoustic startle reflex].

Corticotropin releasing hormone (CRH)--and norepinephrine (NE) -containing neurons in the brain are activated during stress. We previously reported a decrease in the basal level of CRH immunoreactivity in the central nucleus of the amygdala and the tyrosine hydroxylase immunoreactivity in the locus coeruleus after chronic variable stress (CVS), whereas both responses were augmented by a novel stress (footshock). Since the acoustic startle reflex (ASR) can be enhanced by the CRH neuronal activity in the central nucleus of the amygdala, we examined the influence of footshock on ASR in rats exposed to CVS. The footshock after CVS caused a significant augmentation of ASR compared with the acute footshock. Moreover, the enhanced startle to acute footshock was maximally increased at 6 min and was absent after 40 min, whereas the maximal change of the enhanced startle to footshock after CVS was delayed to 14 min and the significant enhanced startle was found until 180 min. The footshock-enhanced startle after CVS may be related to the augmentation of CRH-NE activity, leading to the possibility that a prolonged CRH hyperactivity to stress might generate a pathophysiology of major depression with a vulnerability to stress.

[Regulation of TH activity by GAD immunoreactivity in rat peri-LC after chronic variable stress].

Locus coeruleus (LC) is the major component of noradrenergic neurons in the brain. The corticotropin-releasing hormone (CRH) and norepinephrine (NE) are suggested to play a role in modulating the central stress response. In a previous study we observed a decrease of the basal level of tyrosine hydroxylase (TH) immunoreactivity (-ir) in the LC of rats treated with chronic variable stress (CVS) for 14 days. Furthermore a novel stressor produced an enhanced response of the TH-ir after CVS. In the present study we examined the effect of CVS on the glutamic acid decarboxylase (GAD)-ir activity of periaqueductal gray (PAG), prepositus hypoglossi (PrH) and peri-LC. The GAD-ir was significantly increased in PrH and peri-LC after CVS. The footshock-induced reactivity in the GAD-ir was decreased in both regions after CVS. Moreover, we investigated the influence of the CRH receptor antagonist, alphah-CRH (i.c.v.) on the CVS-induced activation of the TH-ir in the LC. The alphah-CRH i.c.v. diminished the enhanced-TH reactivity by novel stressor after CVS. Our results suggest that the GABA activity in peri-LC and PrH might regulate the LC-TH response, and also the CRH input from central nucleus of amygdala (CeA) and/or the bed nucleus of stria terminalis (BNST) might regulate the TH reactivity.

[Effect of chronic variable stress on limbic corticotrapin-releasing hormone receptor].

The Corticotropin-releasing hormone (CRH) is a key mediator in the stress response. Two CRH-receptor subtypes have been identified in the brain, CRH-receptor 1 (CRH-R1) and CRH-receptor 2, and stress responses are mediated by the CRH-R1. In this study we have examined the effect of chronic variable stress (CVS) on the CRH-R1 immunoreactivity in the hypothalamic and limbic brain regions of the rat. For CVS we selected the six stressors described in previous reports. Male Wistar rats were randomly exposed to one stressor per a day during 13 days and finally exposed to a novel stressor (footshock). There was a significant increase of the CRH-R1 immunoreactivity 24 hours after CVS in the bed nucleus of the stria terminalis (BNST) and basolateral amygdala (BLA), whereas a single footshock had no influence on the immunoreactivity in any of the regions. A novel stressor after CVS suppressed within 24 hours the CVS-induced increase in the CRH-R1 immuoreactivity in the BNST and BLA. Our results suggest that an up-regulation of the limbic CRH-R1 may contribute to stress sensitization (vulnerability) such as an anxiety, arousal and hypersympathetic autonomic symptoms under stressful situations.

[Effect of concurrent treatment of SSRI on the tyrosine hydroxylase immunoreactivity in the rat locus coeruleus treated with chronic variable stress].

Locus coeruleus (LC) is the major component of noradrenergic neurons in the brain. corticotropine-releasing hormone (CRH) and norepinephrine (NE) are suggested to play significant roles in the pathophysiology of depression, although the involvement of the serotonergic system in the CRH-NE systems is not elucidated. Chronic inescapable and unpredictable stress can result in a sustained dysregulation of both of CRH and NE systems. In the present study we have investigated the TH immunoreactivity in the LC by immunohistochemical staining in rats treated with chronic variable stress (CVS) and concurrent administration with clomipramine or fluvoxamine. There was a significant decrease in TH levels 24 h after the last stressor of CVS, followed by a further decrease in that of 72 h later, whereas a marked increase was observed in TH levels immediately after the last stress of CVS 13 d. Concurrent clomipramine and fluvoxamine treatment prevented the sensitization of TH reactivity and the delayed decrease until 72 h later. These data suggest that an increase in serotonin availability would contribute to the normalization of both hypoactivity and sensitization of LC-NE neurons modified under "chronically stressful" situations.

Chronic carbamazepine treatment increases myristoylated alanine-rich C kinase substrate phosphorylation in the rat cerebral cortex via down-regulation of calcineurin A alpha.

Carbamazepine (CBZ) is generally used as a mood-stabilizing drug for the treatment of bipolar disorders. However, little is known about the molecular mechanisms of CBZ actions in the brain, which account for this therapeutic profile. In the present study, we examined the effects of chronic CBZ treatment on the protein kinase C (PKC) pathway. Male Wistar rats received injections of CBZ once daily for 3-5 weeks. The protein levels of PKC isozymes, calcineurin Aalpha subunit (CaN-Aalpha) and myristoylated alanine-rich C kinase substrate (MARCKS), and phosphorylation of MARCKS in the rat cerebral cortex were determined by immunoblot analysis. The content of CaN-Aalpha mRNA was determined by Northern blot analysis. Nomicr; significant changes were observed in PKC alpha, beta, gamma, delta and epsilon in the cytosol and membrane fractions after 5 weeks of CBZ treatment. There were no significant changes in the actin-binding PKCepsilon. Interestingly, phosphorylation of MARCKS was increased more than twofold, while no significant changes were observed in MARCKS protein level in the cytosol fraction. Furthermore, CaN-Aalpha was significantly decreased at both the protein and mRNA levels. The level of MARCKS phosphorylation is reportedly regulated by the balance between PKC-mediated phosphorylation and CaN-mediated dephosphorylation. Our results indicate that chronic CBZ treatment increases MARCKS phosphorylation via decreasing the content of CaN-Aalpha. Phosphorylation of MARCKS has been reported to play an important role in the release of neurotransmitters, such as noradrenaline and serotonin. Therefore, the increase in phosphorylation of MARCKS observed only after chronic CBZ treatment may be related to the mood-stabilizing effects of CBZ.

[Reduction and sensitization of CRH neuron in rat hypothalamic and extrahypothalamic regions after chronic variable stress].

The hyperactivity of corticotropin-releasing hormone (CRH) neurons of the hypothalamic and/or extrahypothalamic regions is believed to contribute to the pathophysiology of major depression in an experimental animal chronically exposed to stress. In the present study, we examined the effects of chronic variable stress (CVS) and novel stress (footshock) on the CRH immunoreactivity in the hypothalamic paraventricular nucleus (PVN) and subdivision of PVN, and the extrahypothalamic bed nucleus of the stria terminalis (BNST) and the central nucleus of the amygdala (CeA). We observed a significant reduction in CRH levels in the whole PVN, lateral parvocellular part of PVN, BNST and CeA 24 hours after the last stressor of CVS for 13 days. A novel stressor after CVS caused a marked increase in CRH levels in these four regions, followed a further reduction observed 24 hours later. Since the CVS-induced modulation of CRH levels are consistent with an alteration of tyrosine hydroxylase levels in the locus coeruleus, CRH-norepinephrine (NE) interaction in the terminal projection of forebrain NE systems, PVN, BNST and CeA where NE stimulates CRII release, might contribute to the bi-directional change in CRH. The CVS-induced bi-directional changes in CRH of PVN, BNST and CeA might play a role in the formation of the pathophysiology of major depressive disorders.