The Effect of mGluR5 Antagonism During Binge Drinkingon Subsequent Ethanol Intake in C57BL/6J Mice: Sex- and Age-Induced Differences.

BACKGROUND: Binge ethanol (EtOH) intake during adolescence leads to an array of behavioral and cognitive consequences including elevated intake of EtOH during adulthood, with female mice showing greater susceptibility than males. Administration of the metabotropic glutamate receptor 5 (mGluR5) antagonist 3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP) has been shown to reduce EtOH self-administration in adult male mice, but little is known about its effect on female and adolescent mice. METHODS: MTEP (0, 10, 20 mg/kg, i.p.) was repeatedly administered to female and male, adult and adolescent C57BL/6J mice during binge sessions using the scheduled high alcohol consumption paradigm. Next, we assessed whether MTEP administration during binge altered the subsequent 24-hour EtOH intake following a period of abstinence. Finally, we investigated whether MTEP administration during binge followed by an abstinence period altered mRNA of glutamatergic genes within the nucleus accumbens of female mice. RESULTS: MTEP significantly decreased binge EtOH intake in all mice, but only female mice exhibited altered subsequent 24-hour EtOH intake. Interestingly, the alteration in subsequent EtOH intake in female animals was age dependent, with adolescent exposure to MTEP during binge decreasing 24-hour intake and adult exposure to MTEP during binge increasing 24-hour intake. Finally, while there were no effects of MTEP pretreatment on the genes examined, there was a robust age effect found during analysis of mGluR1 (Grm1), mGluR5 (Grm5), the NR2A subunit of the NMDA receptor (Grin2a), phosphatidylinositol 3-kinase (Pik3r1), mammalian target of rapamycin (Mtor), and extracellular signal-regulated kinase (Mapk1) mRNA, with adolescent female animals having lower expression than their adult counterparts. CONCLUSIONS: Collectively, the present findings add to existing evidence implicating the contribution of long-term effects of adolescent binge drinking to enhance alcohol abuse in adulthood, while suggesting that mGluR5 antagonism may not be the best pharmacotherapy to treat binge alcohol consumption in female and adolescent animals.

Body composition changes and inhibition of fat development in vivo implicates androgen in regulation of stem cell lineage allocation.

Androgens regulate body composition in youth and declining testosterone that occurs with aging is associated with muscle wasting, increased fat mass and osteopenia. Transgenic mice with targeted androgen receptor (AR) over-expression in mesenchymal stem cells (MSC) were generated to explore the role of androgen signaling in the regulation of body composition. Transgenic males, but not females, were shorter and have reduced body weight and visceral fat accumulation. Dual-energy X-ray absorptiometry (DXA) revealed significant reductions in fat mass with a reciprocal increase in lean mass, yet no difference in food consumption or locomotor activity was observed. Adipose tissue weight was normal in brown fat but reduced in both gonadal and perirenal depots, and reduced hyperplasia was observed with smaller adipocyte size in visceral and subcutaneous white adipose tissue. Although serum leptin, adiponectin, triglyceride, and insulin levels were no different between the genotypes, intraperitoneal glucose tolerance testing (IPGTT) showed improved glucose clearance in transgenic males. High levels of the AR transgene are detected in MSCs but not in mature fat tissue. Reduced fibroblast colony forming units indicate fewer progenitor cells resident in the marrow in vivo. Precocious expression of glucose transporter 4 (GLUT4), peroxisome proliferator-activated receptor γ (PPARγ), and CCAAT enhancer-binding protein α (C/EBPα) was observed in proliferating precursor cultures from transgenic mice compared to controls. In more mature cultures, there was little difference between the genotypes. We propose a mechanism where enhanced androgen sensitivity can alter lineage commitment in vivo to reduce progenitor number and fat development, while increasing the expression of key factors to promote smaller adipocytes with improved glucose clearance.

Stem cell activation in adults can reverse detrimental changes in body composition to reduce fat and increase lean mass in both sexes.

Detrimental changes in body composition are often associated with declining levels of testosterone. Here, we evaluated the notion that multipotent mesenchymal stem cells, that give rise to both fat and muscle tissue, can play a significant role to alter existing body composition in the adult. Transgenic mice with targeted androgen receptor (AR) overexpression in stem cells were employed. Wild-type littermate and AR-transgenic male and female mice were gonadectomized and left untreated for 2 months. After the hypogonadal period, mice were then treated with 5α-dihydrotestosterone (DHT) for 6 weeks. After orchidectomy (ORX), wild-type males have reduced lean mass and increased fat mass compared to shams. DHT treatment was beneficial to partially restore body composition. In wild-type females, ovariectomy (OVX) produced a similar change but there was no improvement with DHT. In targeted AR transgenic mice, DHT treatment increased lean and reduced fat mass to sham levels. In contrast to wild-type females, DHT treatment in female transgenic mice significantly ameliorated the increased fat and decreased lean mass changes that result after OVX. Our results show that DHT administration reduces fat mass and increases lean mass in wild-type males but not females, indicating that wild-type females are not as sensitive to androgen treatment. Because both male and female transgenic mice are more responsive than wild-type, results suggest that body composition remains linked to stem cell fate in the adult and that targeted androgen signaling in stem cells can play a significant role to reverse detrimental changes in body composition in both sexes.

Individual differences in hyperlipidemia and vitamin E status in response to chronic alcohol self-administration in cynomolgus monkeys.

BACKGROUND: Chronic ethanol self-administration induces oxidative stress and exacerbates lipid peroxidation. α-Tocopherol is a potent lipid antioxidant and vitamin that is dependent upon lipoprotein transport for tissue delivery. METHODS: To evaluate the extent to which vitamin E status is deranged by excessive alcohol consumption, monkeys voluntarily drinking ethanol (1.36 to 3.98 g/kg/d for 19 months, n = 11) were compared with nondrinkers (n = 5, control). RESULTS: Three alcohol-drinking animals developed hyperlipidemia with plasma triglyceride levels (1.8 ± 0.9 mM) double those of normolipidemic (NL) drinkers (0.6 ± 0.2) and controls (0.6 ± 0.3, p < 0.05); elevated plasma cholesterol (3.6 ± 0.5 mM) compared with NL drinkers (2.3 ± 0.2, p < 0.05) and controls (2.9 ± 0.3); and lower plasma α-tocopherol per triglycerides (14 ± 6 mmol/mol) than controls (27 ± 8) and NL drinkers (23 ± 6, p < 0.05). Hyperlipidemic monkey liver α-tocopherol (47 ± 15 nmol/g) was lower than NL drinkers (65 ± 13) and controls (70 ± 15, p = 0.080), as was adipose α-tocopherol (84 ± 37 nmol/g) compared with controls (224 ± 118) and NL drinkers (285 ± 234, p < 0.05). Plasma apolipoprotein (apo) CIII increased compared to baseline at both 12 and 19 months in the normolipidemic (p = 0.0016 and p = 0.0028, respectively) and in the hyperlipidemic drinkers (p < 0.05 and p < 0.05, respectively). Plasma apo H concentrations at 19 months were elevated hyperlipidemics (p < 0.05) relative to concentrations in control animals. C-reactive protein (CRP), a marker of inflammation, was increased compared to baseline at both the 12- and 19-month time points in the normolipidemic (p = 0.005 and p = 0.0153, respectively) and hyperlipidemic drinkers (p = 0.016 and p = 0.0201, respectively). CONCLUSION: A subset of alcohol-drinking monkeys showed a predisposition to alcohol-induced hyperlipidemia. The defect in lipid metabolism resulted in lower plasma α-tocopherol per triglycerides and depleted adipose tissue α-tocopherol, and thus decreased vitamin E status.

Selected line difference in the effects of ethanol dependence and withdrawal on allopregnanolone levels and 5alpha-reductase enzyme activity and expression.

BACKGROUND: Allopregnanolone (ALLO) is a progesterone derivative that rapidly potentiates gamma-aminobutyric acid(A) (GABA(A)) receptor-mediated inhibition and modulates symptoms of ethanol withdrawal. Because clinical and preclinical data indicate that ALLO levels are inversely related to symptoms of withdrawal, the present studies determined whether ethanol dependence and withdrawal differentially altered plasma and cortical ALLO levels in mice selectively bred for differences in ethanol withdrawal severity and determined whether the alterations in ALLO levels corresponded to a concomitant change in activity and expression of the biosynthetic enzyme 5alpha-reductase. METHODS: Male Withdrawal Seizure-Prone (WSP) and -Resistant (WSR) mice were exposed to 72 hours ethanol vapor or air and euthanized at select times following removal from the inhalation chambers. Blood was collected for analysis of ALLO and corticosterone levels by radioimmunoassay. Dissected amygdala, hippocampus, midbrain, and cortex as well as adrenals were examined for 5alpha-reductase enzyme activity and expression levels. RESULTS: Plasma ALLO was decreased significantly only in WSP mice, and this corresponded to a decrease in adrenal 5alpha-reductase expression. Cortical ALLO was decreased up to 54% in WSP mice and up to 46% in WSR mice, with a similar decrease in cortical 5alpha-reductase activity during withdrawal in the lines. While cortical gene expression was significantly decreased during withdrawal in WSP mice, there was a 4-fold increase in expression in the WSR line during withdrawal. Hippocampal 5alpha-reductase activity and gene expression was decreased only in dependent WSP mice. CONCLUSIONS: These results suggest that there are line and brain regional differences in the regulation of the neurosteroid biosynthetic enzyme 5alpha-reductase during ethanol dependence and withdrawal. In conjunction with the finding that WSP mice exhibit reduced sensitivity to ALLO during withdrawal, the present results are consistent with the hypothesis that genetic differences in ethanol withdrawal severity are due, in part, to modulatory effects of GABAergic neurosteroids such as ALLO.

Targeting of androgen receptor in bone reveals a lack of androgen anabolic action and inhibition of osteogenesis: a model for compartment-specific androgen action in the skeleton.

Androgens are anabolic hormones that affect many tissues, including bone. However, an anabolic effect of androgen treatment on bone in eugonadal subjects has not been observed and clinical trials have been disappointing. The androgen receptor (AR) mediates biological responses to androgens. In bone tissue, both AR and the estrogen receptor (ER) are expressed. Since androgens can be converted into estrogen, the specific role of the AR in maintenance of skeletal homoeostasis remains controversial. The goal of this study was to use skeletally targeted overexpression of AR in differentiated osteoblasts as a means of elucidating the specific role(s) for AR transactivation in the mature bone compartment. Transgenic mice overexpressing AR under the control of the 2.3-kb alpha1(I)-collagen promoter fragment showed no difference in body composition, testosterone, or 17ss-estradiol levels. However, transgenic males have reduced serum osteocalcin, CTx and TRAPC5b levels, and a bone phenotype was observed. In cortical bone, high-resolution micro-computed tomography revealed no difference in periosteal perimeter but a significant reduction in cortical bone area due to an enlarged marrow cavity. Endocortical bone formation rate was also significantly inhibited. Biomechanical analyses showed decreased whole bone strength and quality, with significant reductions in all parameters tested. Trabecular morphology was altered, with increased bone volume comprised of more trabeculae that were closer together but not thicker. Expression of genes involved in bone formation and bone resorption was significantly reduced. The consequences of androgen action are compartment-specific; anabolic effects are exhibited exclusively at periosteal surfaces, but in mature osteoblasts androgens inhibited osteogenesis with detrimental effects on matrix quality, bone fragility and whole bone strength. Thus, the present data demonstrate that enhanced androgen signaling targeted to bone results in low bone turnover and inhibition of bone formation by differentiated osteoblasts. These results indicate that direct androgen action in mature osteoblasts is not anabolic, and raise concerns regarding anabolic steroid abuse in the developing skeleton or high-dose treatment in eugonadal adults.

Neurotoxic consequences of chronic alcohol withdrawal: expression profiling reveals importance of gender over withdrawal severity.

While women are more vulnerable than men to many of the medical consequences of alcohol abuse, the role of sex in the response to ethanol is controversial. Neuroadaptive responses that result in the hyperexcitability associated with withdrawal from chronic ethanol likely reflect gene expression changes. We have examined both genders for the effects of withdrawal on brain gene expression using mice with divergent withdrawal severity that have been selectively bred from a genetically heterogeneous population. A total of 295 genes were identified as ethanol regulated from each gender of each selected line by microarray analyses. Hierarchical cluster analysis of the arrays revealed that the transcriptional response correlated with sex rather than with the selected withdrawal phenotype. Consistent with this, gene ontology category over-representation analysis identified cell death and DNA/RNA binding as targeted classes of genes in females, while in males, protein degradation, and calcium ion binding pathways were more altered by alcohol. Examination of ethanol-regulated genes and these distinct signaling pathways suggested enhanced neurotoxicity in females. Histopathological analysis of brain damage following ethanol withdrawal confirmed elevated cell death in female but not male mice. The sexually dimorphic response was observed irrespective of withdrawal phenotype. Combined, these results indicate a fundamentally distinct neuroadaptive response in females compared to males during chronic ethanol withdrawal and are consistent with observations that female alcoholics may be more vulnerable than males to ethanol-induced brain damage associated with alcohol abuse.

Binge Ethanol Drinking Produces Sexually Divergent and Distinct Changes in Nucleus Accumbens Signaling Cascades and Pathways in Adult C57BL/6J Mice.

We previously determined that repeated binge ethanol drinking produced sex differences in the regulation of signaling downstream of Group 1 metabotropic glutamate receptors in the nucleus accumbens (NAc) of adult C57BL/6J mice. The purpose of the present study was to characterize RNA expression differences in the NAc of adult male and female C57BL/6J mice following 7 binge ethanol drinking sessions, when compared with controls consuming water. This binge drinking procedure produced high intakes (average >2.2 g/kg/30 min) and blood ethanol concentrations (average >1.3 mg/ml). Mice were euthanized at 24 h after the 7th binge session, and focused qPCR array analysis was employed on NAc tissue to quantify expression levels of 384 genes in a customized Mouse Mood Disorder array, with a focus on glutamatergic signaling (3 arrays/group). We identified significant regulation of 50 genes in male mice and 70 genes in female mice after 7 ethanol binges. Notably, 14 genes were regulated in both males and females, representing common targets to binge ethanol drinking. However, expression of 10 of these 14 genes was strongly dimorphic (e.g., opposite regulation for genes such as Crhr2, Fos, Nos1, and Star), and only 4 of the 14 genes were regulated in the same direction (Drd5, Grm4, Ranbp9, and Reln). Interestingly, the top 30 regulated genes by binge ethanol drinking for each sex differed markedly in the male and female mice, and this divergent neuroadaptive response in the NAc could result in dysregulation of distinct biological pathways between the sexes. Characterization of the expression differences with Ingenuity Pathway Analysis was used to identify Canonical Pathways, Upstream Regulators, and significant Biological Functions. Expression differences suggested that hormone signaling and immune function were altered by binge drinking in female mice, whereas neurotransmitter metabolism was a central target of binge ethanol drinking in male mice. Thus, these results indicate that the transcriptional response to repeated binge ethanol drinking was strongly influenced by sex, and they emphasize the importance of considering sex in the development of potential pharmacotherapeutic targets for the treatment of alcohol use disorder.

The androgen receptor in bone marrow progenitor cells negatively regulates fat mass.

It is well established that testosterone negatively regulates fat mass in humans and mice; however, the mechanism by which testosterone exerts these effects is poorly understood. We and others have shown that deletion of the androgen receptor (AR) in male mice results in a phenotype that mimics the three key clinical aspects of hypogonadism in human males; increased fat mass and decreased bone and muscle mass. We now show that replacement of the Ar gene specifically in mesenchymal progenitor cells (PCs) residing in the bone marrow of Global-ARKO mice, in the absence of the AR in all other tissues (PC-AR Gene Replacements), completely attenuates their increased fat accumulation. Inguinal subcutaneous white adipose tissue and intra-abdominal retroperitoneal visceral adipose tissue depots in PC-AR Gene Replacement mice were 50-80% lower than wild-type (WT) and 75-90% lower than Global-ARKO controls at 12 weeks of age. The marked decrease in subcutaneous and visceral fat mass in PC-AR Gene Replacements was associated with an increase in the number of small adipocytes and a healthier metabolic profile compared to WT controls, characterised by normal serum leptin and elevated serum adiponectin levels. Euglycaemic/hyperinsulinaemic clamp studies reveal that the PC-AR Gene Replacement mice have improved whole-body insulin sensitivity with higher glucose infusion rates compared to WT mice and increased glucose uptake into subcutaneous and intra-abdominal fat. In conclusion, these data provide the first evidence for an action of androgens via the AR in mesenchymal bone marrow PCs to negatively regulate fat mass and improve metabolic function.

Prefrontal cortex expression of chromatin modifier genes in male WSP and WSR mice changes across ethanol dependence, withdrawal, and abstinence.

Alcohol-use disorder (AUD) is a relapsing disorder associated with excessive ethanol consumption. Recent studies support the involvement of epigenetic mechanisms in the development of AUD. Studies carried out so far have focused on a few specific epigenetic modifications. The goal of this project was to investigate gene expression changes of epigenetic regulators that mediate a broad array of chromatin modifications after chronic alcohol exposure, chronic alcohol exposure followed by 8 h withdrawal, and chronic alcohol exposure followed by 21 days of abstinence in Withdrawal-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) selected mouse lines. We found that chronic vapor exposure to highly intoxicating levels of ethanol alters the expression of several chromatin remodeling genes measured by quantitative PCR array analyses. The identified effects were independent of selected lines, which, however, displayed baseline differences in epigenetic gene expression. We reported dysregulation in the expression of genes involved in histone acetylation, deacetylation, lysine and arginine methylation and ubiquitinationhylation during chronic ethanol exposure and withdrawal, but not after 21 days of abstinence. Ethanol-induced changes are consistent with decreased histone acetylation and with decreased deposition of the permissive ubiquitination mark H2BK120ub, associated with reduced transcription. On the other hand, ethanol-induced changes in the expression of genes involved in histone lysine methylation are consistent with increased transcription. The net result of these modifications on gene expression is likely to depend on the combination of the specific histone tail modifications present at a given time on a given promoter. Since alcohol does not modulate gene expression unidirectionally, it is not surprising that alcohol does not unidirectionally alter chromatin structure toward a closed or open state, as suggested by the results of this study.

Osteoblast and osteocyte apoptosis associated with androgen action in bone: requirement of increased Bax/Bcl-2 ratio.

Both the number and the activity of osteoblasts are critical for normal bone growth and maintenance. Although a potential role for estrogen in protection of bone mass through inhibition of osteoblast apoptosis has been proposed, a function for androgen is much less clear. The aim of this study was to establish a direct role for androgen to influence osteoblast apoptosis both in vitro and in vivo. AR-MC3T3-E1 cells, with androgen receptor (AR) overexpression controlled by the type I collagen promoter, were treated with the non-aromatizable androgen 5alpha-dihydrotestosterone (DHT). Apoptosis was assessed by three different techniques including DNA fragmentation, caspase-3 activation, and changes in mitochondrial membrane potential. Transactivation of AR by DHT enhanced apoptosis while 17beta-estradiol (E(2)) treatment reduced apoptosis in both proliferating preosteoblasts and mature osteocyte-like cells. To explore mechanism, the apoptosis regulators Bcl-2 (antiapoptotic) and Bax (proapoptotic) were evaluated. Western analysis revealed that DHT decreased Bcl-2 resulting in a significantly increased Bax/Bcl-2 ratio. Regulation of Bcl-2 was post-transcriptional since bcl-2 mRNA levels were unaffected by DHT treatment. Furthermore, ubiquitination of Bcl-2 was increased and serine phosphorylation was reduced, consistent with inhibition of MAP kinase signaling by DHT. Increased Bax/Bcl-2 ratio was essential since either Bcl-2 overexpression or Bax downregulation by RNA interference (RNAi) partially abrogated or reversed DHT-enhanced osteoblastic apoptosis. In order to establish physiologic significance in vivo, AR-transgenic mice with AR overexpression in the osteoblast lineage and thus enhanced androgen sensitivity were characterized. In male AR-transgenic mice, increased osteoblast apoptosis was observed in vivo even in association with new bone formation. Thus, although estrogen can be antiapoptotic, androgen stimulates osteoblast and osteocyte apoptosis through an increased Bax/Bcl-2 ratio even in anabolic settings. These results identify a new mechanism for androgen regulation of osteoblast activity distinct from estrogen, and suggest that enhanced apoptosis can be associated with anabolic stimulation of new bone growth. Androgens thus play a distinct role in skeletal homeostasis.

Androgens and bone growth: it's location, location, location.

Androgens increase bone mass in specific skeletal compartments through effects on bone cells, enhancing osteoblast activity but inhibiting that of osteoclasts. The mechanism of action of androgens might involve both classic androgen receptor transcriptional activation and rapid non-genomic effects, and could also be dependent upon low levels of estrogen.

A neurotoxic alcohol exposure paradigm does not induce hepatic encephalopathy.

Alcohol abuse is associated with neurological dysfunction, brain morphological deficits and frank neurotoxicity. Although these disruptions may be a secondary effect due to hepatic encephalopathy, no clear evidence of causality is available. This study examined whether a 72h period of alcohol intoxication known to induce physical dependence, followed by a single withdrawal, was sufficient to induce signs of hepatic encephalopathy in male and female mice. Animals were continuously intoxicated via alcohol vapor inhalation, a procedure previously shown to induce significant neurotoxicity in female mice. At peak synchronized withdrawal (8h following the end of alcohol exposure), blood samples were taken and levels of several liver-regulated markers and brain swelling were characterized. Glutathione levels were also determined in the medial frontal cortex (mFC) and hippocampus. Results revealed elevated levels of cholesterol, albumin, alkaline phosphatase (ALP), alanine aminotransferase (ALT) and decreased levels of blood urea nitrogen and total bilirubin in alcohol-exposed male and female groups compared to controls. Brain water weight was not affected by alcohol exposure, though males tended to have slightly more water weight overall. Alcohol exposure led to reductions in tissue levels of glutathione in both the hippocampus and mFC which may indicate increased oxidative stress. Combined, these results suggest that hepatic encephalopathy does not appear to play a significant role in the neurotoxicity observed following alcohol exposure in this model.

Functional regulation of PI3K-associated signaling in the accumbens by binge alcohol drinking in male but not female mice.

It is well established that binge alcohol consumption produces alterations in Group 1 metabotropic glutamate receptors (mGlus) and related signaling cascades in the nucleus accumbens (NAC) of adult male mice, but female and adolescent mice have not been examined. Thus, the first set of studies determined whether repeated binge alcohol consumption produced similar alterations in protein and mRNA levels of Group 1 mGlu-associated signaling molecules in the NAC of male and female adult and adolescent mice. The adult (9 weeks) and adolescent (4 weeks) C57BL/6J mice were exposed to 7 binge alcohol sessions every 3rd day while controls drank water. Repeated binge alcohol consumption produced sexually divergent changes in protein levels and mRNA expression for Group 1 mGlus and downstream signaling molecules in the NAC, but there was no effect of age. Binge alcohol intake decreased mGlu5 levels in females, whereas it decreased indices of phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), 4E-binding protein 1, and p70 ribosomal protein S6 kinase in males. Expression of genes encoding mGlu1, mGlu5, the NR2A subunit of the NMDA receptor, and Homer2 were all decreased by binge alcohol consumption in males, while females were relatively resistant (only phosphoinositide-dependent protein kinase 1 was decreased). The functional implication of these differences was investigated in a separate study by inhibiting mTOR in the NAC (via infusions of rapamycin) before binge drinking sessions. Rapamycin (50 and 100 ng/side) significantly decreased binge alcohol consumption in males, while consumption in females was unaffected. Altogether these results highlight that mTOR signaling in the NAC was necessary to maintain binge alcohol consumption only in male mice and that binge drinking recruits sexually divergent signaling cascades downstream of PI3K and presumably, Group 1 mGlus. Importantly, these findings emphasize that sex should be considered in the development of potential pharmacotherapeutic targets.

Astrocyte Dysfunction Induced by Alcohol in Females but Not Males.

Chronic alcohol abuse is associated with brain damage in a sex-specific fashion, but the mechanisms involved are poorly described and remain controversial. Previous results have suggested that astrocyte gene expression is influenced by ethanol intoxication and during abstinence in vivo. Here, bioinformatic analysis of astrocyte-enriched ethanol-regulated genes in vivo revealed ubiquitin pathways as an ethanol target, but with sexually dimorphic cytokine signaling and changes associated with brain aging in females and not males. Consistent with this result, astrocyte activation was observed after exposure in female but not male animals, with reduced S100ß levels in the anterior cingulate cortex and increased GFAP(+) cells in the hippocampus. In primary culture, the direct effects of chronic ethanol exposure followed by recovery on sex-specific astrocyte function were examined. Male astrocyte responses were consistent with astrocyte deactivation with reduced GFAP expression during ethanol exposure. In contrast, female astrocytes exhibited increased expression of Tnf, reduced expression of the neuroprotective cytokine Tgfb1, disrupted bioenergetics and reduced excitatory amino acid uptake following exposure or recovery. These results indicate widespread astrocyte dysfunction in ethanol-exposed females and suggest a mechanism that may underlie increased vulnerability to ethanol-induced neurotoxicity in females.

The role of pregnane neurosteroids in ethanol withdrawal: behavioral genetic approaches.

Within the last 20 years, rapid nongenomic actions of steroid hormones have been demonstrated to occur via an interaction with ligand-gated ion channels. For example, the pregnane neurosteroid allopregnanolone (ALLOP) is a potent positive modulator of gamma-aminobutyric acid(A) (GABA(A)) receptors. The physiological significance of fluctuations in endogenous ALLOP levels has been investigated with regard to disease states and the effect of therapeutic agents on ALLOP levels. Because the pharmacological profile of ALLOP is similar to that of ethanol (EtOH), the modulatory effect of pregnane neurosteroids on EtOH dependence and withdrawal will be the focus of this review. Data on the effects of chronic EtOH exposure and withdrawal on pregnane neurosteroid levels, biosynthetic enzymes, and changes in neurosteroid sensitivity will be summarized. Results from genetic animal models indicate that seizure-prone animals have a persistent decrease in endogenous ALLOP levels during EtOH withdrawal in conjunction with tolerance to ALLOP's anticonvulsant effect. Manipulation of endogenous ALLOP levels with finasteride also markedly reduced the severity of chronic EtOH withdrawal. Gene mapping studies provide a hint for an interaction between genes for GABA(A) receptor subunits and the biosynthetic enzyme 5alpha-reductase. Overall, the results are suggestive of a relationship between endogenous pregnane neurosteroid levels and behavioral changes in excitability during EtOH withdrawal, consistent with recent findings in humans. While the findings with ALLOP emphasize the therapeutic potential of neurosteroid treatment during EtOH withdrawal, the gene mapping studies suggest that pregnane neurosteroid biosynthesis may represent a target for therapeutic intervention in the treatment of alcohol dependence.

Neural regulation of bone and the skeletal effects of serotonin (5-hydroxytryptamine).

There is increasing evidence for a contribution of the neural system to the regulation of bone metabolism. The skeleton is richly innervated by both sympathetic and sensory neurons. While these nerves serve sensory and vascular functions, they are also being found to influence bone cell activities. The most convincing evidence for this has been provided by studies into the skeletal effects of the hormone leptin, which has been shown to centrally regulate bone mass, and through studies into the skeletal effects of hypothalamic neuropeptide Y2 and Y4 receptors. This paper discusses recent evidence for the neural regulation of bone metabolism and, in particular, the potential role of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT). Recent studies have demonstrated the presence of functional pathways in bone for both responding to and regulating the uptake of 5-HT. This is of high clinical relevance given the role of the serotonergic system in affective disorders, and the wide use of pharmacological agents that target the 5-HT system to manage these disorders. Initial data suggest that exposure to these agents at different stages during the lifespan may have significant effects on the skeleton.

Alteration of kappa-opioid receptor system expression in distinct brain regions of a genetic model of enhanced ethanol withdrawal severity.

Abrupt withdrawal from chronic alcohol exposure can produce convulsions that are likely due to ethanol (EtOH) neuroadaptations. While significant efforts have focused on elucidating dependence mechanisms, the alterations contributing to EtOH withdrawal severity are less well characterized. The present studies examined the kappa-opioid receptor (KOP-R) system in Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice, selected lines that display severe and mild convulsions upon removal from chronic EtOH exposure. Previous data demonstrated significant increases in whole brain prodynorphin (Pdyn) mRNA in WSP mice only during EtOH withdrawal. No significant effects of EtOH exposure or withdrawal were observed in WSR mice. The present study characterized Pdyn mRNA and the KOP-R in WSP and WSR mice during EtOH withdrawal using in situ hybridization (ISH) and KOP-R autoradiography. Analyses were performed in brain regions that express Pdyn mRNA and/or KOP-R and that might participate in seizure circuitry: the piriform cortex, olfactory tubercle, nucleus accumbens, caudate-putamen, claustrum, dorsal endopiriform nucleus, and cingulate cortex. ISH analyses confirmed previous findings; EtOH withdrawal increased Pdyn mRNA in multiple brain regions of WSP mice, but not WSR. Basal KOP-R binding was higher in WSR mice than in WSP mice, suggesting an anti-convulsant role for receptor activation. Finally, increased KOP-R density was present during EtOH withdrawal in WSP mice. These data suggest that differences in the KOP-R system among the lines might contribute to their selected difference in EtOH withdrawal severity.

Androgen receptor action in osteoblasts in male mice is dependent on their stage of maturation.

Androgen action via the androgen receptor (AR) is essential for normal skeletal growth and bone maintenance post-puberty in males; however, the molecular and cellular mechanisms by which androgens exert their actions in osteoblasts remains relatively unexplored in vivo. To identify autonomous AR actions in osteoblasts independent of AR signaling in other tissues, we compared the extent to which the bone phenotype of the Global-ARKO mouse was restored by replacing the AR in osteoblasts commencing at either the (1) proliferative or (2) mineralization stage of their maturation. In trabecular bone, androgens stimulated trabecular bone accrual during growth via the AR in proliferating osteoblasts and maintained trabecular bone post-puberty via the AR in mineralizing osteoblasts, with its predominant action being to inhibit bone resorption by decreasing the ratio of receptor activator of NF-κB ligand (RANKL) to osteoprotegerin (OPG) gene expression. During growth, replacement of the AR in proliferating but not mineralizing osteoblasts of Global-ARKOs was able to partially restore periosteal circumference, supporting the concept that androgen action in cortical bone to increase bone size during growth is mediated via the AR in proliferating osteoblasts. This study provides further significant insight into the mechanism of androgen action via the AR in osteoblasts, demonstrating that it is dependent on the stage of osteoblast maturation.

Females uniquely vulnerable to alcohol-induced neurotoxicity show altered glucocorticoid signaling.

Women are more sensitive to the harmful effects of alcohol (EtOH) abuse than men, yet the underlying mechanisms remain poorly understood. Previous gene expression analysis of the medial prefrontal cortex (mPFC) following a chronic intoxication paradigm using continuous 72 h vapor inhalation found that females, but not males, exhibit an inflammatory response at peak withdrawal that is associated with cell damage. Given that glucocorticoids can function as anti-inflammatories, are known to increase with EtOH exposure, and influence neurotoxicity, we hypothesized that males and females may exhibit an altered corticosterone (CORT) response following chronic intoxication. Analysis of serum CORT levels revealed the expected increase during withdrawal with no difference between males and females, while control males but not females exhibited higher CORT concentrations than naive animals. Glucocorticoid signaling characterized using focused qPCR arrays identified a sexually dimorphic response in the mPFC during withdrawal, particularly among astrocyte-enriched genes. These genes include aquaporin-1 (Aqp1), sphingosine kinase 1 (Sphk1) and connective tissue growth factor (Ctgf); genes associated with inflammatory signaling, and tissue damage and repair. Bioinformatic analysis also revealed activation of inflammatory signaling and cell death pathways in females. Confirmation studies showed that female mice exhibited significant neuronal degeneration within the anterior cingulate cortex (ACC). By contrast, EtOH exposure lead to a significant reduction in cell death in males. Thus, distinct glucocorticoid signaling pathways are associated with sexually dimorphic neurotoxicity, suggesting one mechanism by which EtOH-exposed females are particularly vulnerable to the damaging effects of alcohol in the CNS.