Adrenal-dependent and -independent stress-induced Per1 mRNA in hypothalamic paraventricular nucleus and prefrontal cortex of male and female rats.

Oscillating clock gene expression gives rise to a molecular clock that is present not only in the body's master circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), but also in extra-SCN brain regions. These extra-SCN molecular clocks depend on the SCN for entrainment to a light:dark cycle. The SCN has limited neural efferents, so it may entrain extra-SCN molecular clocks through its well-established circadian control of glucocorticoid hormone secretion. Glucocorticoids can regulate the normal rhythmic expression of clock genes in some extra-SCN tissues. Untimely stress-induced glucocorticoid secretion may compromise extra-SCN molecular clock function. We examined whether acute restraint stress during the rat's inactive phase can rapidly (within 30 min) alter clock gene (Per1, Per2, Bmal1) and cFos mRNA (in situ hybridization) in the SCN, hypothalamic paraventricular nucleus (PVN), and prefrontal cortex (PFC) of male and female rats (6 rats per treatment group). Restraint stress increased Per1 and cFos mRNA in the PVN and PFC of both sexes. Stress also increased cFos mRNA in the SCN of male rats, but not when subsequently tested during their active phase. We also examined in male rats whether endogenous glucocorticoids are necessary for stress-induced Per1 mRNA (6-7 rats per treatment group). Adrenalectomy attenuated stress-induced Per1 mRNA in the PVN and ventral orbital cortex, but not in the medial PFC. These data indicate that increased Per1 mRNA may be a means by which extra-SCN molecular clocks adapt to environmental stimuli (e.g. stress), and in the PFC this effect is largely independent of glucocorticoids.

Effect of Infant Formula on Streptococcus Mutans Biofilm Formation.

OBJECTIVE: This study investigated the effect that infant formula had on biofilm growth of Streptococcus mutans. Specifically, it compared biofilm growth in media containing lactose-based and sucrose-based formulas. It also analyzed biofilm formation with formulas of varying iron content. Biofilm growth was tested with the specific infant formula components sucrose, lactose, and ferric chloride. The study was designed to determine if these types of infant formulas and components affected S. mutans biofilm formation differently. STUDY DESIGN: A 24-hour culture of S. mutans was treated with various concentrations of infant formula diluted in bacteriological media. To test for biofilm formation, S. mutans was cultured with and without the infant formula and formula components. The biofilms were washed, fixed, and stained with crystal violet. The absorbance was measured to evaluate biofilm growth and total absorbance. RESULTS: Sucrose-based formulas provided significant increases in biofilm growth when compared to lactose-based formulas at two dilutions (1:5, 1:20). Similac Sensitive RS (sucrose-based) at most dilutions provided the most significant increase in biofilm growth when compared to the control. Sucrose tested as an individual component provided more of a significant increase on biofilm growth than lactose or iron when compared to the control. A low iron formula provided a significant increase in biofilm growth at one dilution (1:5) when compared to formula containing a normal iron content. There was no significant difference in biofilm growth when comparing high iron formula to normal iron formula or low iron formula. There was no significant difference when comparing Similac PM 60/40 (low iron formula) to Similac PM 60/40 with additional ferric chloride. CONCLUSION: The results of this study demonstrated that sucrose-based formula provided more of a significant increase in biofilm growth compared to lactose-based formula. Sucrose alone provided a significant increase of biofilm growth at more dilutions when compared to the control than lactose and iron. The amount of iron in formula had a significant effect on biofilm formation only when comparing low iron formula to normal iron formula at the highest concentration (1:5). There was no significant difference in biofilm growth when iron was added to the low iron formula. The information obtained expands current knowledge regarding the influence of infant formula on the primary dentition and reinforces the importance of oral hygiene habits once the first tooth erupts.

CRTC2 activation in the suprachiasmatic nucleus, but not paraventricular nucleus, varies in a diurnal fashion and increases with nighttime light exposure.

Entrainment of the intrinsic suprachiasmatic nucleus (SCN) molecular clock to the light-dark cycle depends on photic-driven intracellular signal transduction responses of SCN neurons that converge on cAMP response element-binding protein (CREB)-mediated regulation of gene transcription. Characterization of the CREB coactivator proteins CREB-regulated transcriptional coactivators (CRTCs) has revealed a greater degree of differential activity-dependent modulation of CREB transactivational function than previously appreciated. In confirmation of recent reports, we found an enrichment of crtc2 mRNA and prominent CRTC2 protein expression within the SCN of adult male rats. With use of a hypothalamic organotypic culture preparation for initial CRTC2-reactive antibody characterization, we found that CRTC2 immunoreactivity in hypothalamic neurons shifted from a predominantly cytoplasmic profile under basal culture conditions to a primarily nuclear localization (CRTC2 activation) 30 min after adenylate cyclase stimulation. In adult rat SCN, we found a diurnal variation in CRTC2 activation (peak at zeitgeber time of 4 h and trough at zeitgeber time of 16-20 h) but no variation in the total number of CRTC2-immunoreactive cells. There was no diurnal variation of CRTC2 activation in the hypothalamic paraventricular nucleus, another site of enriched CRTC2 expression. Exposure of rats to light (50 lux) for 30 min during the second half of their dark (nighttime) phase produced CRTC2 activation. We observed in the SCN a parallel change in the expression of a CREB-regulated gene (FOS). In contrast, nighttime light exposure had no effect on CRTC2 activation or FOS expression in the paraventricular nucleus, nor did it affect corticosterone hormone levels. These results suggest that CRTC2 participates in CREB-dependent photic entrainment of SCN function.

Atrazine inhibits pulsatile gonadotropin-releasing hormone (GnRH) release without altering GnRH messenger RNA or protein levels in the female rat.

Atrazine (ATR) is a commonly used pre-emergence/early postemergence herbicide. Previous work has shown that exposure to high doses of ATR in rats results in blunting of the hormone-induced luteinizing hormone (LH) surge and inhibition of pulsatile LH release without significantly reducing pituitary sensitivity to a gonadotropin-releasing hormone (GnRH) agonist. Accompanying the reduction in the LH surge was an attenuation of GnRH neuronal activation. These findings suggest that ATR exposure may be acting to inhibit GnRH release. In this study, we examined GnRH directly to determine the effect of high doses of ATR on GnRH pulsatile release, gene expression, and peptide levels in the female rat. Ovariectomized adult female Wistar rats were treated with ATR (200 mg/kg) or vehicle for 4 days via gavage. Following the final treatment, GnRH release was measured from ex vivo hypothalamic explants for 3 h. In another experiment, animals were administered either vehicle or ATR (50, 100, or 200 mg/kg) daily for 4 days. Following treatment, in situ hybridization was performed to examine total GnRH mRNA and the primary GnRH heterogeneous nuclear RNA transcript. Finally, GnRH immunoreactivity and total peptide levels were measured in hypothalamic tissue of treated animals. ATR treatment resulted in no changes to GnRH gene expression, peptide levels, or immunoreactivity but a reduction in GnRH pulse frequency and an increased pulse amplitude. These findings suggest that ATR acts to inhibit the secretory dynamics of GnRH pulses without interfering with GnRH mRNA and protein synthesis.

The differential effect of atrazine on luteinizing hormone release in adrenalectomized adult female Wistar rats.

High doses of atrazine (ATR), administered for 4 days, suppress luteinizing hormone (LH) release and increase adrenal hormones levels. Considering the known inhibitory effects of adrenal hormones on the hypothalamo-pituitary-gonadal axis, we investigated the possible role the adrenal gland has in mediating ATR inhibition of LH release. To determine the extant and duration of adrenal activation, ovariectomized Wistar rats were given a single dose of ATR (0, 50, or 200 mg/kg), and corticosterone (CORT) levels were assayed at multiple time points posttreatment. CORT levels were increased within 20 min and remained elevated over 12 h postgavage in 200-mg/kg animals. To determine the effects of adrenalectomy on ATR inhibition of the LH surge and pulsatile LH release, adrenalectomized (ADX) or sham-operated ovariectomized rats were treated for 4 days with ATR (0, 10, 100, or 200 mg/kg), and an LH surge was induced with hormone priming. In the afternoon following the last dose of ATR, blood was sampled hourly for 9 h. Another cohort of ovariectomized rats was examined for pulsatile patterns of LH secretion after ATR (0, 50, or 200 mg/kg) and sampled every 5 min for 3 h. ADX had no effect on ATR inhibition of the LH surge but prevented the ATR disruption of pulsatile LH release. These data indicate that ATR selectively affects the LH pulse generator through alterations in adrenal hormone secretion. Adrenal activation does not play a role in ATR's suppression of the LH surge, and therefore ATR may work centrally to alter the preovulatory LH surge in female rats.

Applications of ultrasound to enhance fluidized bed drying of Ascophyllum Nodosum: Drying kinetics and product quality assessment.

In this study, ultrasound either as a pretreatment technique or as an integrated technique was employed to enhance fluidized bed drying of Ascophyllum nodosum, and drying kinetics and dried product quality were assessed. In order to compare technology efficiency and dried product qualities, oven drying and fluidized bed drying (FBD) were employed. The novel drying methods included airborne ultrasound-assisted fluidized bed drying (AUA), ultrasound pre-treatment followed by FBD (USP), and hot water blanching pre-treatment followed byFBD (HWB). Six drying kinetics models were used to describe the drying curves, among which the Page model was the best in fitting USP and AUA. Model by Millidi et al. was employed to describe HWB. Airborne ultrasound in AUA did not reduce energy consumption or drying time, but retained total phenolic content (TPC) as well as colour, and exhibited the highest yield among the novel drying methods. USP and HWB showed lower energy consumption and drying time considerably, but the TPC was the lowest among the studied methods. At the same time, USP dried product exhibited the lowest aw, followed by HWB and then AUA. This studyalso demonstrated that FBD could be a very practical drying method on Irish brown seaweed, and ultrasound-assisted drying methods may have potential developments in Irish brown seaweed drying process.

Antimicrobial effects of airborne acoustic ultrasound and plasma activated water from cold and thermal plasma systems on biofilms.

Bacterial biofilms are difficult to inactivate due to their high antimicrobial resistance. Therefore, new approaches are required for more effective bacterial biofilm inactivation. Airborne acoustic ultrasound improves bactericidal or bacteriostatic activity which is safe and environmentally friendly. While, plasma activated water (PAW) is attracting increasing attention due to its strong antimicrobial properties. This study determined efficacy of combined airborne acoustic ultrasound and plasma activated water from both cold and thermal plasma systems in inactivating Escherichia coli K12 biofilms. The application of airborne acoustic ultrasound (15 min) alone was significantly more effective in reducing E. coli counts in 48 and 72 h biofilms compared to 30 min treatment with PAW. The effect of airborne acoustic ultrasound was more pronounced when used in combination with PAW. Airborne acoustic ultrasound treatment for 15 min of the E. coli biofilm followed by treatment with PAW significantly reduced the bacterial count by 2.2-2.62 Log10 CFU/mL when compared to control biofilm treated with distilled water. This study demonstrates that the synergistic effects of airborne acoustic ultrasound and PAW for enhanced antimicrobial effects. These technologies have the potential to prevent and control biofilm formation in food and bio-medical applications.

Effects of atrazine and its withdrawal on gonadotropin-releasing hormone neuroendocrine function in the adult female Wistar rat.

High doses of the commonly used herbicide atrazine have been shown to suppress luteinizing hormone (LH) release. To determine whether atrazine alters the function of gonadotropin-releasing hormone (GnRH) neurons, we examined the effects of atrazine on GnRH neuronal activation and the subsequent release of LH normally associated with ovulation. Ovariectomized adult Wistar rats were administered atrazine (50, 100, or 200 mg/kg of body weight daily by gavage) or vehicle for 4 days. Animals were primed with estrogen and progesterone to induce an evening LH surge. Blood samples were obtained over the afternoon and evening using an indwelling right atrial cannula, and plasma was assayed for LH and FSH. Another cohort of animals was transcardially perfused in the afternoon to examine GnRH activation using FOS immunoreactivity. Results of these studies show that 4-day treatment with atrazine resulted in a significant reduction in the magnitude of the LH and FSH surges, and this corresponds to a decrease in GnRH neurons expressing FOS immunoreactivity. To determine if the effects of atrazine were long lasting, additional studies were performed examining LH levels and GnRH activation 2 days and 4 days after atrazine withdrawal. Within 4 days (but not 2 days) after cessation of atrazine treatment, measures of hypothalamic-pituitary-gonadal (HPG) activation returned to normal. These data indicate that atrazine affects neuroendocrine function in the female rat by actions at the level of the GnRH neuron and that the acute effects of high doses of atrazine can be reversed within 4 days after withdrawal of treatment.

Coordination between Prefrontal Cortex Clock Gene Expression and Corticosterone Contributes to Enhanced Conditioned Fear Extinction Recall.

Post-traumatic stress disorder (PTSD) is associated with impaired conditioned fear extinction learning, a ventromedial prefrontal cortex (vmPFC)-dependent process. PTSD is also associated with dysregulation of vmPFC, circadian, and glucocorticoid hormone function. Rats have rhythmic clock gene expression in the vmPFC that requires appropriate diurnal circulatory patterns of corticosterone (CORT), suggesting the presence of CORT-entrained intrinsic circadian clock function within the PFC. We examined the role of vmPFC clock gene expression and its interaction with CORT profiles in regulation of auditory conditioned fear extinction learning. Extinction learning and recall were examined in male rats trained and tested either in the night (active phase) or in the day (inactive phase). Using a viral vector strategy, Per1 and Per2 clock gene expression were selectively knocked down within the vmPFC. Circulating CORT profiles were manipulated via adrenalectomy (ADX) ± diurnal and acute CORT replacement. Rats trained and tested during the night exhibited superior conditioned fear extinction recall that was absent in rats that had knock-down of vmPFC clock gene expression. Similarly, the superior nighttime extinction recall was absent in ADX rats, but restored in ADX rats given a combination of a diurnal pattern of CORT and acute elevation of CORT during the postextinction training consolidation period. Thus, conditioned fear extinction learning is regulated in a diurnal fashion that requires normal vmPFC clock gene expression and a combination of circadian and training-associated CORT. Strategic manipulation of these factors may enhance the therapeutic outcome of conditioned fear extinction related treatments in the clinical setting.

Characterization of Activation of the Hypothalamic-Pituitary-Adrenal Axis by the Herbicide Atrazine in the Female Rat.

Atrazine (ATR) is a commonly used pre-emergence and early postemergence herbicide. Rats gavaged with ATR and its chlorometabolites desethylatrazine (DEA) and deisopropylatrazine (DIA) respond with a rapid and dose-dependent rise in plasma corticosterone, whereas the major chlorometabolite, diaminochlorotriazine (DACT), has little or no effect on corticosterone levels. In this study, we investigated the possible sites of ATR activation of the hypothalamic-pituitary-adrenal (HPA) axis. ATR treatment had no effect on adrenal weights but altered adrenal morphology. Hypophysectomized rats or rats under dexamethasone suppression did not respond to ATR treatment, suggesting that ATR does not directly stimulate the adrenal gland to induce corticosterone synthesis. Immortalized mouse corticotrophs (AtT-20) and primary rat pituitary cultures were treated with ATR, DEA, DIA, or DACT. None of the compounds induced an increase in ACTH secretion or potentiated ACTH release in conjunction with CRH on ACTH release. In female rats gavaged with ATR, pretreatment with the CRH receptor antagonist astressin completely blocked the ATR-induced rise in corticosterone concentrations, implicating CRH release in ATR-induced HPA activation. Intracerebroventricular infusion of ATR, DEA, and DIA but not DACT at concentrations equivalent to peak plasma concentrations after gavage dosing resulted in an elevation of plasma corticosterone concentrations. However, ATR did not induce c-Fos immunoreactivity in the paraventricular nucleus of the hypothalamus. These results indicate that ATR activates the HPA axis centrally and requires CRH receptor activation, but it does not stimulate cellular pathways associated with CRH neuronal excitation.

The androgen 5alpha-dihydrotestosterone and its metabolite 5alpha-androstan-3beta, 17beta-diol inhibit the hypothalamo-pituitary-adrenal response to stress by acting through estrogen receptor beta-expressing neurons in the hypothalamus.

Estrogen receptor beta (ERbeta) and androgen receptor (AR) are found in high levels within populations of neurons in the hypothalamus. To determine whether AR or ERbeta plays a role in regulating hypothalamo-pituitary-adrenal (HPA) axis function by direct action on these neurons, we examined the effects of central implants of 17beta-estradiol (E2), 5alpha-dihydrotestosterone (DHT), the DHT metabolite 5alpha-androstan-3beta, 17beta-diol (3beta-diol), and several ER subtype-selective agonists on the corticosterone and adrenocorticotropin (ACTH) response to immobilization stress. In addition, activation of neurons in the paraventricular nucleus (PVN) was monitored by examining c-fos mRNA expression. Pellets containing these compounds were stereotaxically implanted near the PVN of gonadectomized male rats. Seven days later, animals were killed directly from their home cage (nonstressed) or were restrained for 30 min (stressed) before they were killed. Compared with controls, E2 and the ERalpha-selective agonists moxestrol and propyl-pyrazole-triol significantly increased the stress induced release of corticosterone and ACTH. In contrast, central administration of DHT, 3beta-diol, and the ERbeta-selective compound diarylpropionitrile significantly decreased the corticosterone and ACTH response to immobilization. Cotreatment with the ER antagonist tamoxifen completely blocked the effects of 3beta-diol and partially blocked the effect of DHT, whereas the AR antagonist flutamide had no effect. Moreover, DHT, 3beta-diol, and diarylpropionitrile treatment significantly decreased restraint-induced c-fos mRNA expression in the PVN. Together, these studies indicate that the inhibitory effects of DHT on HPA axis activity may be in part mediated via its conversion to 3beta-diol and subsequent binding to ERbeta.

Estrogen receptor-beta mediates dihydrotestosterone-induced stimulation of the arginine vasopressin promoter in neuronal cells.

Arginine vasopressin (AVP) is a neuropeptide involved in the regulation of fluid balance, stress, circadian rhythms, and social behaviors. In the brain, AVP is tightly regulated by gonadal steroid hormones in discrete regions with gonadectomy abolishing and testosterone replacement restoring normal AVP expression in adult males. Previous studies demonstrated that 17beta-estradiol, a primary metabolite of testosterone, is responsible for restoring most of the AVP expression in the brain after castration. However, 5alpha-dihydrotestosterone (DHT) has also been shown to play a role in the regulation of AVP expression, thus implicating the involvement of both androgen and estrogen receptors (ER). Furthermore, DHT, through its conversion to 5alpha-androstane-3beta,17beta-diol, has been shown to modulate estrogen response element-mediated promoter activity through an ER pathway. The present study addressed two central hypotheses: 1) that androgens directly modulate AVP promoter activity and 2) the effect is mediated by an estrogen or androgen receptor pathway. To that end, we overexpressed androgen receptor, ERbeta, and ERbeta splice variants in a neuronal cell line and measured AVP promoter activity using a firefly luciferase reporter assay. Our results demonstrate that DHT and its metabolite 5alpha-androstane-3beta,17beta-diol stimulate AVP promoter activity through ERbeta in a neuronal cell line.

Dynamic glucocorticoid-dependent regulation of Sgk1 expression in oligodendrocytes of adult male rat brain by acute stress and time of day.

Recent studies support plasticity in adult brain white matter structure and myelination in response to various experiential factors. One possible contributor to this plasticity may be activity-dependent modulation of serum- and glucocorticoid-inducible kinase 1 (Sgk1) expression in oligodendrocytes. We examined whether Sgk1 expression in adult rat brain white matter is increased by acute stress-induced elevations in endogenous corticosterone and whether it fluctuates with diurnal variations in corticosterone. We observed rapid increases (within 30 min) in Sgk1 mRNA in the corpus callosum in response to acute stress, as well as large increases at the beginning of the rat's active period (the time of peak corticosterone secretion). These increases were absent in adrenalectomized rats. Corticosterone treatment of adrenalectomized rats also rapidly increased corpus callosum Sgk1 mRNA. The majority of Sgk1 mRNA in corpus callosum was co-localized with myelin basic protein mRNA, suggesting that mature oligodendrocytes respond dynamically to acute stress and circadian rhythms. The regulation of Sgk1 expression by acute stress and time of day was selective for white matter, with limited alteration of Sgk1 expression by these factors in hippocampus and somatosensory cortex. These results indicate a unique sensitivity of oligodendrocyte Sgk1 expression to activity-dependent fluctuations in corticosterone hormone secretion, and raises the prospect that hypothalamic-pituitary-adrenal axis dysregulation or glucocorticoid pharmacotherapy may compromise the normal activity-dependent interactions between oligodendrocytes and neurons.

Progestin receptor expression in the developing rat brain depends upon activation of estrogen receptor alpha and not estrogen receptor beta.

Perinatal 17beta-estradiol (E2) rapidly and markedly affects the morphological and neurochemical organization of the vertebrate brain. For instance, the sex difference in perinatal progestin receptor (PR) immunoreactivity in the medial preoptic nucleus (MPN) of the rat brain is due to the intracellular conversion of testosterone into E2 in males. Neonatal alpha-fetoprotein prevents circulating estrogens from accessing the brain, therefore, to overcome alpha-fetoprotein sequestration of E2, estrogen replacement studies during development have used natural and synthetic estrogen dosages in the milligram to microgram range. These levels could be considered as supraphysiological. Moreover, it is not clear through which ER subtype E2 acts to induce PR expression in the neonatal rat MPN because E2 binds similarly to estrogen receptor (ER)alpha and ERbeta. Consequently, we investigated whether nanogram levels of E2 affected PR protein and mRNA levels in the neonatal MPN. Furthermore, propylpyrazole-triol (PPT), a highly selective agonist for ERalpha, and diarylpropionitrile (DPN), a highly selective agonist for ERbeta, were used to determine if E2-dependent PR expression in the neonatal rat is mediated through ERalpha and/or ERbeta. Immunocytochemistry and quantitative real-time RT-PCR determined that as little as 100 ng E2 significantly induced PR protein and mRNA in the female and neonatally castrated male MPN on PN 4, indicating that the neonatal rat brain is highly sensitive to circulating estrogens. PPT, but not DPN, induced PR expression in the neonatal MPN and arcuate nucleus (Arc), demonstrating that PR expression in the neonatal rat brain depends solely on E2 activated ERalpha. In the lateral bed nucleus of the stria terminalis (BSTL), neither PPT nor DPN affected PR expression, suggesting the presence of a gonadal hormone-independent PR regulatory mechanism.

Diurnal Corticosterone Presence and Phase Modulate Clock Gene Expression in the Male Rat Prefrontal Cortex.

Mood disorders are associated with dysregulation of prefrontal cortex (PFC) function, circadian rhythms, and diurnal glucocorticoid (corticosterone [CORT]) circulation. Entrainment of clock gene expression in some peripheral tissues depends on CORT. In this study, we characterized over the course of the day the mRNA expression pattern of the core clock genes Per1, Per2, and Bmal1 in the male rat PFC and suprachiasmatic nucleus (SCN) under different diurnal CORT conditions. In experiment 1, rats were left adrenal-intact (sham) or were adrenalectomized (ADX) followed by 10 daily antiphasic (opposite time of day of the endogenous CORT peak) ip injections of either vehicle or 2.5 mg/kg CORT. In experiment 2, all rats received ADX surgery followed by 13 daily injections of vehicle or CORT either antiphasic or in-phase with the endogenous CORT peak. In sham rats clock gene mRNA levels displayed a diurnal pattern of expression in the PFC and the SCN, but the phase differed between the 2 structures. ADX substantially altered clock gene expression patterns in the PFC. This alteration was normalized by in-phase CORT treatment, whereas antiphasic CORT treatment appears to have eliminated a diurnal pattern (Per1 and Bmal1) or dampened/inverted its phase (Per2). There was very little effect of CORT condition on clock gene expression in the SCN. These experiments suggest that an important component of glucocorticoid circadian physiology entails CORT regulation of the molecular clock in the PFC. Consequently, they also point to a possible mechanism that contributes to PFC disrupted function in disorders associated with abnormal CORT circulation.

The androgen metabolite, 5alpha-androstane-3beta, 17beta-diol, is a potent modulator of estrogen receptor-beta1-mediated gene transcription in neuronal cells.

5alpha-Androstane-3beta, 17beta-diol (3betaAdiol) is a metabolite of the potent androgen, 5alpha-dihydrotestosterone. Recent studies showed that 3betaAdiol binds to estrogen receptor (ER)-beta and regulates growth of the prostate gland through an estrogen, and not androgen, receptor-mediated pathway. These data raise the possibility that 3betaAdiol could regulate important physiological processes in other tissues that produce 3betaAdiol, such as the brain. Although it is widely accepted that the brain is a target for 5alpha-dihydrotestosterone action, there is no evidence that 3betaAdiol has a direct action in neurons. To explore the molecular mechanisms by which 3betaAdiol might act to modulate gene transcription in neuronal cells, we examined whether 3betaAdiol activates ER-mediated promoter activity and whether ER transactivation is facilitated by a classical estrogen response element (ERE) or an AP-1 complex. The HT-22 neuronal cell line was cotransfected with an expression vector containing ERalpha, ER-beta1, or the ERbeta splice variant, ER-beta2 and one of two luciferase-reporter constructs containing either a consensus ERE or an AP-1 enhancer site. Cells were treated with 100 nM 17beta-estradiol, 100 nM 3betaAdiol, or vehicle for 15 h. We show that 3betaAdiol activated ER-beta1-induced transcription mediated by an ERE equivalent to that of 17beta-estradiol. By contrast, 3betaAdiol had no effect on ERalpha- or ER-beta2-mediated promoter activity. Moreover, ER-beta1 stimulated transcription mediated by an ERE and inhibited transcription by an AP-1 site in the absence of ligand binding. These data provide evidence for activation of ER signaling pathways by an androgen metabolite in neuronal cells.

Variations in Phase and Amplitude of Rhythmic Clock Gene Expression across Prefrontal Cortex, Hippocampus, Amygdala, and Hypothalamic Paraventricular and Suprachiasmatic Nuclei of Male and Female Rats.

The molecular circadian clock is a self-regulating transcription/translation cycle of positive (Bmal1, Clock/Npas2) and negative (Per1,2,3, Cry1,2) regulatory components. While the molecular clock has been well characterized in the body's master circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), only a few studies have examined both the positive and negative clock components in extra-SCN brain tissue. Furthermore, there has yet to be a direct comparison of male and female clock gene expression in the brain. This comparison is warranted, as there are sex differences in circadian functioning and disorders associated with disrupted clock gene expression. This study examined basal clock gene expression (Per1, Per2, Bmal1 mRNA) in the SCN, prefrontal cortex (PFC), rostral agranular insula, hypothalamic paraventricular nucleus (PVN), amygdala, and hippocampus of male and female rats at 4-h intervals throughout a 12:12 h light:dark cycle. There was a significant rhythm of Per1, Per2, and Bmal1 in the SCN, PFC, insula, PVN, subregions of the hippocampus, and amygdala with a 24-h period, suggesting the importance of an oscillating molecular clock in extra-SCN brain regions. There were 3 distinct clock gene expression profiles across the brain regions, indicative of diversity among brain clocks. Although, generally, the clock gene expression profiles were similar between male and female rats, there were some sex differences in the robustness of clock gene expression (e.g., females had fewer robust rhythms in the medial PFC, more robust rhythms in the hippocampus, and a greater mesor in the medial amygdala). Furthermore, females with a regular estrous cycle had attenuated aggregate rhythms in clock gene expression in the PFC compared with noncycling females. This suggests that gonadal hormones may modulate the expression of the molecular clock.

Adrenal-dependent diurnal modulation of conditioned fear extinction learning.

Post traumatic stress disorder (PTSD) is associated with altered conditioned fear extinction expression and impaired circadian function including dysregulation of glucocorticoid hormone secretion. We examined in adult male rats the relationship between conditioned fear extinction learning, circadian phase, and endogenous glucocorticoids (CORT). Rats maintained on a 12h light:dark cycle were trained and tested across 3 separate daily sessions (conditioned fear acquisition and 2 extinction sessions) that were administered during either the rats' active or inactive circadian phase. In an initial experiment we found that rats at both circadian phases acquired and extinguished auditory cue conditioned fear to a similar degree in the first extinction session. However, rats trained and tested at zeitgeber time-16 (ZT16) (active phase) showed enhanced extinction memory expression during the second extinction session compared to rats trained and tested at ZT4 (inactive phase). In a follow-up experiment, adrenalectomized (ADX) or sham surgery rats were similarly trained and tested across 3 separate daily sessions at either ZT4 or ZT16. ADX had no effect on conditioned fear acquisition or conditioned fear memory. Sham ADX rats trained and tested at ZT16 exhibited better extinction learning across the two extinction sessions compared to all other groups of rats. These results indicate that conditioned fear extinction learning is modulated by time of day, and this diurnal modulation requires the presence of adrenal hormones. These results support an important role of CORT-dependent circadian processes in regulating conditioned fear extinction learning, which may be capitalized upon to optimize effective treatment of PTSD.

Absence of glucocorticoids augments stress-induced Mkp1 mRNA expression within the hypothalamic-pituitary-adrenal axis.

Stress-induced activation of hypothalamic paraventricular nucleus (PVN) corticotropin-releasing hormone (CRH) neurons trigger CRH release and synthesis. Recent findings have suggested that this process depends on the intracellular activation (phosphorylation) of ERK1/2 within CRH neurons. We have recently shown that the presence of glucocorticoids constrains stress-stimulated phosphorylation of PVN ERK1/2. In some peripheral cell types, dephosphorylation of ERK has been shown to be promoted by direct glucocorticoid upregulation of the MAP kinase phosphatase 1 (Mkp1) gene. In this study, we tested the hypothesis that glucocorticoids regulate Mkp1 mRNA expression in the neural forebrain (medial prefrontal cortex, mPFC, and PVN) and endocrine tissue (anterior pituitary) by subjecting young adult male Sprague-Dawley rats to various glucocorticoid manipulations with or without acute psychological stress (restraint). Restraint led to a rapid increase in Mkp1 mRNA within the mPFC, PVN, and anterior pituitary, and this increase did not require glucocorticoid activity. In contrast to glucocorticoid upregulation of Mkp1 gene expression in the peripheral tissues, we found that the absence of glucocorticoids (as a result of adrenalectomy) augmented basal mPFC and stress-induced PVN and anterior pituitary Mkp1 gene expression. Taken together, this study indicates that the presence of glucocorticoids may constrain Mkp1 gene expression in the neural forebrain and endocrine tissues. This possible constraint may be an indirect consequence of the inhibitory influence of glucocorticoids on stress-induced activation of ERK1/2, a known upstream positive regulator of Mkp1 gene transcription.

Estrogen receptor ß expression in the mouse forebrain: age and sex differences.

Estrogen receptors regulate multiple brain functions, including stress, sexual, and memory-associated behaviors as well as controlling neuroendocrine and autonomic function. During development, estrogen signaling is involved in programming adult sex differences in physiology and behavior. Expression of estrogen receptor α changes across development in a region-specific fashion. By contrast, estrogen receptor ß (ERß) is expressed in many brain regions, yet few studies have explored sex and developmental differences in its expression, largely because of the absence of selective reagents for anatomical localization of the protein. This study utilized bacterial artificial chromosome transgenic mice expressing ERß identified by enhanced green fluorescent protein (EGFP) to compare expression levels and distribution of ERß in the male and female mouse forebrain on the day of birth (P0), on postnatal day 4 (P4), and on P21. By using qualitative analysis, we mapped the distribution of ERß-EGFP and found developmental alterations in ERß expression within the cortex, hippocampus, and hypothalamic regions including the arcuate, ventromedial, and paraventricular nuclei. We also report a sex difference in ERß in the bed nucleus of the stria terminalis, with males showing greater expression at P4 and P21. Another sex difference was found in the anteroventral periventricular nucleus of P21, but not P0 or P4, mice, in which ERß-EGFP-immunoreactive cells were densely clustered near the third ventricle in females but not males. These developmental changes and sex differences in ERß indicate a mechanism through which estrogens might differentially affect brain functions or program adult physiology at select times during development.