Chromatin remodelling associated with transcriptional activation of silent genes involves phosphorylation at Serine-10 and acetylation at Lysine-14 in the N-terminal tails of the nucleosomal protein histone H3. We have identified neurons predominantly in the dentate gyrus showing a speckled nuclear immunoreactivity pattern for phosphorylated histone H3 [i.e. P(Ser10)-H3] and phospho-acetylated histone H3 [i.e. P(Ser10)-Ac(Lys14)-H3]. Forced swimming increased the number of P(Ser10)-H3-positive [P(Ser10)-H3+] neurons in the rat and mouse dentate gyrus. Exposure of mice to a predator had a similar effect, but exposing rats to ether vapour or a cold environment evoked no change in the number of P(Ser10)-H3+ dentate neurons, indicating that the effect of stress on histone H3 phosphorylation is stressor-specific. The forced swimming-induced increase in dentate P(Ser10)-H3+ neurons peaked at 8-24 h, was restricted to NeuN+ (i.e. mature) neurons, and occurred mainly in the middle and superficial aspects of the granular cell layer. Moreover, this increase showed stimulus strength dependency (i.e. swimming at 19 degrees C produced a larger increase than swimming at 25 degrees C) and could be blocked by the glucocorticoid receptor (GR) antagonists RU 38486 and ORG 34517. Under these experimental conditions, when the forced swimming-induced behavioural immobility response was determined in a re-test 24 h after the initial forced swim test, striking correlations were observed between the phosphorylation of histone H3 in dentate gyrus granule neurons and the acquired immobility response. Our data indicate that stressful events with a strong psychological component such as forced swimming evoke distinct GR-dependent histone modifications in mature dentate gyrus granule neurons that may participate in the behavioural adaptation of the organism to this event.
The transcription factor cAMP response element-binding protein (CREB) plays a critical role in plasticity processes underlying learning and memory. We investigated the phosphorylation of CREB in rat brain after forced swimming, a stressor known to impact on higher limbic and neocortical brain areas. As shown by immunohistochemistry, forced swimming increased phosphorylated CREB (P-CREB) levels in the dentate gyrus, all neocortical areas, the medial, lateral and basolateral nuclei of the amygdala, cerebellum but not in the hypothalamic paraventricular nucleus. Distinct differences in the P-CREB pattern were observed in the deeper vs. superficial layers of the neocortex. The response in P-CREB was stressor type-specific because exposure to either ether or a cold environment was ineffective. The forced swimming-induced changes in P-CREB levels showed a biphasic time-course: an early peak detected at 15 min was followed by a marked drop at 60 min; a second rise starting after 1-2 h, reached maximal values between 6 and 8 h, and remained elevated for at least 48 h. Examination of the neuroanatomical induction pattern of the CRE-inducible immediate early gene product c-fos revealed that it was only partly overlapping with that of P-CREB. Western analyses showed that only the 43-kDa CREB protein (an enhancer of CRE-containing promotors) was phosphorylated after forced swimming, while other members of the CREB/ATF family (CREM, ATF-1 and ATF-2) remained unaffected. The NF-kappaB pathway was not activated, indicating that forced swimming does not unspecifically evoke transcription factor activation. Thus, in contrast to physical stressors, such as ether or cold exposure, forced swimming, a stressor with a strong psychological component, elicits the recruitment of the CREB pathway in a widespread manner in the limbic system and neocortex; brain regions known to be implicated in various forms of (stress-related) learning and memory.
BackgroundModified Vaccinia Ankara (MVA) is a live, viral vaccine under advanced development as a non-replicating smallpox vaccine. A randomised, double-blind, placebo-controlled phase III clinical trial was conducted to demonstrate the humoral immunogenic equivalence of three consecutively manufactured MVA production lots, and to confirm the safety and tolerability of MVA focusing on cardiac readouts.MethodsThe trial was conducted at 34 sites in the US. Vaccinia-naïve adults aged 18-40 years were randomly allocated to one of four groups using a 1:1:1:1 randomization scheme. Subjects received either two MVA injections from three consecutive lots (Groups 1-3), or two placebo injections (Group 4), four weeks apart. Everyone except personnel involved in vaccine handling and administration was blinded to treatment. Safety assessment focused on cardiac monitoring throughout the trial. Vaccinia-specific antibody titers were measured using a Plaque Reduction Neutralization Test (PRNT) and an Enzyme-Linked Immunosorbent Assay (ELISA). The primary immunogenicity endpoint was Geometric Mean Titers (GMTs) after two MVA vaccinations measured by PRNT at trial visit 4. This trial is registered with ClinicalTrials.gov, number NCT01144637.ResultsBetween March 2013 and May 2014, 4005 subjects were enrolled and received at least one injection of MVA (n = 3003) or placebo (n = 1002). The three MVA lots induced equivalent antibody titers two weeks after the second vaccination, with seroconversion rates of 99·8% (PRNT) and 99·7% (ELISA). Overall, 180 (6·0%) subjects receiving MVA and 29 (2·9%) subjects in the placebo group reported at least one unsolicited Adverse Event (AE) that was considered trial-related. Vaccination was well tolerated without significant safety concerns, particularly regarding cardiac assessment.ConclusionsThe neutralizing and total antibody titers induced by each of the three lots were equivalent. No significant safety concerns emerged in this healthy trial population, especially regarding cardiac safety, thus confirming the excellent safety and tolerability profile of MVA.Trial registrationClinicalTrials.gov NCT01144637
Glucocorticoid receptor (GR) antagonists can block the retention of the immobility response in the forced swimming test. Recently, we showed that forced swimming evokes a distinct spatiotemporal pattern of cAMP-responsive element-binding protein (CREB) phosphorylation in the dentate gyrus (DG) and neocortex. In the present study, we found that chronic treatment of rats with the selective GR antagonist ORG 34116 decreased the immobility time in the forced swim test, increased baseline levels of phosphorylated CREB (P-CREB) in the DG and neocortex and affected the forced swimming-induced changes in P-CREB levels in a time- and site-specific manner. Overall, we observed that, in control rats, forced swimming evoked increases in P-CREB levels in the DG and neocortex, whereas in ORG 34116-treated animals a major dephosphorylation of P-CREB was observed. These observations underscore an important role of GRs in the control of the phosphorylation state of CREB which seems to be of significance for the immobility response in the forced swim test and extend the molecular mechanism of action of GRs in the brain.
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