BRCA1 protein dose-dependent risk for embryonic oxidative DNA damage, embryopathies and neurodevelopmental disorders with and without ethanol exposure.

Although widely known as a tumor suppressor, the breast cancer 1 susceptibility protein (BRCA1) is also important in development, where it regulates fetal DNA repair pathways that protect against DNA damage caused by physiological and drug-enhanced levels of reactive oxygen species (ROS). We previously showed that conditional heterozygous (+/-) knockout (cKO) mouse embryos with a minor 28% BRCA1 deficiency developed normally in culture, but when exposed to the ROS-initiating drug, alcohol (ethanol, EtOH), exhibited embryopathies not evident in wild-type (+/+) littermates. Herein, we characterized a directBrca1 +/- knockout (KO) model with a 2-fold greater (58%) reduction in BRCA1 protein vs. the cKO model. We also characterized and compared learning & memory deficits in both the cKO and KO models. Even saline-exposed Brca1 +/- vs. +/+ KO progeny exhibited enhanced oxidative DNA damage and embryopathies in embryo culture and learning & memory deficits in females in vivo, which were not observed in the cKO model, revealing the potential pathogenicity of physiological ROS levels. The embryopathic EtOH concentration for cultured direct KO embryos was half that for cKO embryos, and EtOH affected Brca1 +/+ embryos only in the direct KO model. The spectrum and severity of EtOH embryopathies in culture were greater in both Brca1 +/- vs. +/+ embryos, and direct KO vs. cKO +/- embryos. Motor coordination deficits were evident in both male and female Brca1 +/- KO progeny exposed in utero to EtOH. The results in our direct KO model with a greater BRCA1 deficiency vs. cKO mice provide the first evidence for BRCA1 protein dose-dependent susceptibility to developmental disorders caused by physiological and drug-enhanced oxidative stress.

Altered Epigenetic Marks and Gene Expression in Fetal Brain, and Postnatal Behavioural Disorders, Following Prenatal Exposure of Ogg1 Knockout Mice to Saline or Ethanol.

Oxoguanine glycosylase 1 (OGG1) is widely known to repair the reactive oxygen species (ROS)-initiated DNA lesion 8-oxoguanine (8-oxoG), and more recently was shown to act as an epigenetic modifier. We have previously shown that saline-exposed Ogg1 -/- knockout progeny exhibited learning and memory deficits, which were enhanced by in utero exposure to a single low dose of ethanol (EtOH) in both Ogg1 +/+ and -/- progeny, but more so in Ogg1 -/- progeny. Herein, OGG1-deficient progeny exposed in utero to a single low dose of EtOH or its saline vehicle exhibited OGG1- and/or EtOH-dependent alterations in global histone methylation and acetylation, DNA methylation and gene expression (Tet1 (Tet Methylcytosine Dioxygenase 1), Nlgn3 (Neuroligin 3), Hdac2 (Histone Deacetylase 2), Reln (Reelin) and Esr1 (Estrogen Receptor 1)) in fetal brains, and behavioural changes in open field activity, social interaction and ultrasonic vocalization, but not prepulse inhibition. OGG1- and EtOH-dependent changes in Esr1 and Esr2 mRNA and protein levels were sex-dependent, as was the association of Esr1 gene expression with gene activation mark histone H3 lysine 4 trimethylation (H3K4me3) and gene repression mark histone H3 lysine 27 trimethylation (H3K27me3) measured via ChIP-qPCR. The OGG1-dependent changes in global epigenetic marks and gene/protein expression in fetal brains, and postnatal behavioural changes, observed in both saline- and EtOH-exposed progeny, suggest the involvement of epigenetic mechanisms in developmental disorders mediated by 8-oxoG and/or OGG1. Epigenetic effects of OGG1 may be involved in ESR1-mediated gene regulation, which may be altered by physiological and EtOH-enhanced levels of ROS formation, possibly contributing to sex-dependent developmental disorders observed in Ogg1 knockout mice. The OGG1- and EtOH-dependent associations provide a basis for more comprehensive mechanistic studies to determine the causal involvement of oxidative DNA damage and epigenetic changes in ROS-mediated neurodevelopmental disorders.

The performance of saliva test strips for determining ethanol levels, as compared to gas chromatography and breathalyser methods.

We set out to determine the performance of the Testi Technologies enzymatic assay saliva ethanol test strips of three different detection levels: 0 g/L, 0.2 g/L and 0.5 g/L, using as the reference method a gas chromatography analyser (GC). Alcohol levels were measured in 104 volunteers at up to three points in time, using up to three test strips per measurement, while gathering blood samples and breathalyser readings in parallel. The plasma alcohol concentrations (PAC) were determined from the plasma samples using GC. The qualitative results of the test strips were compared to the quantitative results from the reference method, as well as the breathalyser readings, and the amount of true and false positive and true and false negative results were classified using predetermined cut-off levels. The best performing test strips were the 0 g/L and the 0.2 g/L strips. The 0 g/L strips had a sensitivity and specificity of 1.00, as false negatives and false positives were not detected. The 0.2 g/L strips had a sensitivity and specificity [95% confidence interval (CI)] of 0.98 (0.96 - 1.00) and 0.83 (0.62 - 1.00) respectively, an accuracy of 0.97 (0.95 - 0.99), and a diagnostic odds ratio of 205.00 (35.33 - 1189.66). The test strips perform their intended purpose of screening for alcohol consumption well, with their great sensitivity as a defining property compared to other testing methods. For them to be able to be implemented in a clinical setting however, further refinement of the tests' characteristics would be required.

Breast cancer 1 (BRCA1) protection in altered gene expression and neurodevelopmental disorders due to physiological and ethanol-enhanced reactive oxygen species formation.

The breast cancer 1 (Brca1) susceptibility gene regulates the repair of reactive oxygen species (ROS)-mediated DNA damage, which is implicated in neurodevelopmental disorders. Alcohol (ethanol, EtOH) exposure during pregnancy causes fetal alcohol spectrum disorders (FASD), including abnormal brain function, associated with enhanced ROS-initiated DNA damage. Herein, oxidative DNA damage in fetal brains and neurodevelopmental disorders were enhanced in saline-exposed +/- vs. +/+ Brca1 littermates. A single EtOH exposure during gestation further enhanced oxidative DNA damage, altered the expression of developmental/DNA damage response genes in fetal brains, and resulted in neurodevelopmental disorders, all of which were BRCA1-dependent. Pretreatment with the ROS inhibitor phenylbutylnitrone (PBN) blocked DNA damage and some neurodevelopmental disorders in both saline- and EtOH-exposed progeny, corroborating a ROS-dependent mechanism. Fetal BRCA1 protects against altered gene expression and neurodevelopmental disorders caused by both physiological and EtOH-enhanced levels of ROS formation. BRCA1 deficiencies may enhance the risk for FASD.

Effect of moderate prenatal ethanol exposure on the differential expression of two histamine H3 receptor isoforms in different brain regions of adult rat offspring.

We have reported that prenatal alcohol exposure (PAE) elevates histamine H3 receptor (H3R) agonist-mediated inhibition of glutamatergic neurotransmission in the dentate gyrus. Here, we hypothesized that PAE alters the expression of two prominent H3R isoforms namely, the rH3A and rH3C isoforms, which have differing intrinsic activities for H3R agonists, in a manner that may contribute to heightened H3R function in PAE rats. In contrast to our predictions, we found different effects of sex and PAE in various brain regions with significant interactions between sex and PAE in dentate gyrus and entorhinal cortex for both isoforms. Subsequently, to confirm the PAE-and sex-induced differences on H3R isoform mRNA expression, we developed a polyclonal antibody selective for the rH3A inform. Western blots of rH3A mRNA-transfected HEK-293 cells identified a ~ 48 kDa band of binding consistent with the molecular weight of rH3A, thus confirming antibody sensitivity for rH3A protein. In parallel, we also established a pan-H3R knockout mice line to confirm antibody specificity in rodent brain membranes. Both qRT-PCR and H3R agonist-stimulated [35S]-GTPγS binding confirmed the absence of mH3A mRNA and H3 receptor-effector coupling in H3R knockout (KO) mice. Subsequent western blotting studies in both rat and mouse brain membranes were unable to detect rH3A antibody binding at ~48 kDa. Rather, the H3RA antibody bound to a ~ 55 kDa band in both rat and mouse membranes, including H3R KO mice, suggesting H3RA binding was not specific for H3Rs in rodent membranes. Subsequent LC/MS analysis of the ~55 kDa band in frontal cortical membranes identified the highly abundant beta subunit of ATPase in both WT and KO mice. Finally, LC/MS analysis of the ~48 kDa band from rH3A mRNA-transfected HEK-293 cell membranes was able to detect rH3A protein, but its presence was below the limits of quantitative reliability. We conclude that PAE alters rH3A and rH3C mRNA expression in some of the same brain regions where we have previously reported PAE-induced alterations in H3R-effector coupling. However, interpreting the functional consequences of altered H3R isoform expression was limited given the technical challenges of measuring the relatively low abundance of rH3A protein in native membrane preparations.

Factors predicting general health concerns and atypical behaviours in children with prenatal alcohol exposure and other adverse exposures.

Background: Prenatal alcohol exposure (PAE) can have significant negative consequences on the health outcomes of children. Children with PAE often experience other prenatal and postnatal adverse exposures. Increased rates of general health concerns and atypical behaviours are seen in both children with PAE as well as with other patterns of adverse exposures, although these have not been systematically described. The association between multiple adverse exposures and adverse health concerns and atypical behaviours in children with PAE is unknown. Methods: Demographic information, medical history, adverse exposures, health concerns, and atypical behaviours were collected from children with confirmed PAE (n = 22; 14 males, age range = 7.9-15.9 years) and their caregivers. Support vector machine learning classification models were used to predict the presence of health concerns and atypical behaviours based on adverse exposures. Associations between the sums of adverse exposures, health concerns, and atypical behaviours were examined using correlation analysis. Results: All children experienced health concerns, the most common being sensitivity to sensory inputs (64%; 14/22). Similarly, all children engaged in atypical behaviours, with atypical sensory behaviour (50%; 11/22) being the most common. Prenatal alcohol exposure was most important factor for predicting some health concerns and atypical behaviours, and alone and in combination with other factors. Simple associations between adverse exposures could not be identified for many health concerns and atypical behaviours. Conclusion: Children with PAE and other adverse exposures experience high rates of health concerns and atypical behaviours. This study demonstrates the complex effects of multiple adverse exposures on health and behaviour in children.

Prenatal alcohol exposure results in brain region- and sex-specific changes in circHomer1 expression in adult mouse brain.

Circular RNAs (circRNAs) are a novel category of covalently-closed non-coding RNAs mainly derived from the back-splicing of exons or introns of protein-coding genes. In addition to their inherent high overall stability, circRNAs, have been shown to have strong functional effects on gene expression via a multitude of transcriptional and post-transcriptional mechanisms. Furthermore, circRNAs, appear to be particularly enriched in the brain and able to influence both prenatal development and postnatal brain function. However, little is known about the potential involvement of circRNAs in the long term influence of prenatal alcohol exposure (PAE) in the brain and their relevance for Fetal Alcohol Spectrum Disorders (FASD). Using circRNA-specific quantification, we have found that circHomer1, an activity-dependent circRNA derived from Homer protein homolog 1 (Homer1) and enriched in postnatal brain, is significantly down-regulated in the male frontal cortex and hippocampus of mice subjected to modest PAE. Our data further suggest that the expression of H19, an imprinted embryonic brain-enriched long non-coding RNA (lncRNA), is significantly up-regulated in the frontal cortex of male PAE mice. Furthermore, we show opposing changes in the developmental- and brain region specific- expression of circHomer1 and H19. Lastly, we show that knockdown of H19 results in robust increases in circHomer1 but not linear HOMER1 mRNA expression in human glioblastoma cell lines. Taken together, our work uncovers notable sex- and brain region-specific alterations in circRNA and lncRNA expression following PAE and introduces novel mechanistic insights with potential relevance to FASD.

Cumulative Coffee Consumption as a Protective Factor for Head and Neck Cancer in Brazil.

There is still no consensus in the literature regarding the role of coffee in head and neck cancer. Thus, we sought to analyze the cumulative consumption of coffee as a protective factor in the genesis of head and neck cancer in Brazil, one of the main coffee producing countries, from January 2011 to February 2017. We carried out a case-control study in 5 referral centers for head and neck cancer with 839 cases and 842 non-cancer hospital controls matched by sex, data collection center and age group. The results of logistic regression analysis showed that the cumulative consumption of >2 cups of coffee per day is an important protective factor (OR: 0.73, 95% CI: 0.5-0.9) against head and neck cancer. Smoking increased the risk by 22 times (OR: 22.19; 95% CI: 13.7-35.8) in individuals who smoke more than 50 packs per year, and the habit of ingesting more than 155 ml of alcohol per day represented approximately twice as high risk (OR: 2.20; 95% CI: 1.4-3.4). In summary, this study suggests that coffee consumption is associated with a lower chance of head and neck cancer.

Changes in Alcohol Consumption among Different Population Groups during the SARS-CoV-2 Pandemic: Outcomes of the Slovenian Cross-Sectional National Survey (SI-PANDA).

BACKGROUND: Slovenia ranks amongst the countries with the highest recorded alcohol consumption. The mortality rate attributed to alcohol-related causes of death in Slovenia also exceeds the EU average. The aim of our research was to confirm the changes in alcohol consumption in Slovenia during the SARS-CoV-2 virus pandemic on a representative sample and to identify vulnerable groups at higher risk of increasing alcohol consumption. METHODS: Two consecutive data collections of the National Survey on the Impact of the Pandemic on Life, each in different epidemiological situations, were conducted. A structured questionnaire was used to monitor the number of alcoholic beverages consumed during the pandemic, compared to the time before the pandemic. RESULTS: The majority of the population did not change the number of alcoholic beverages consumed, and among those with changes, there were significantly more of those who drank less than those who drank more. Among respondents who drank a greater number of alcoholic beverages, statistically significantly higher proportions were found in younger age groups, people with post-secondary vocational education or higher, and people with a higher probability of mental health problems. CONCLUSIONS: During the pandemic crisis, we need to pay special attention to vulnerable groups that are at higher risk of increasing alcohol use.

Mephedrone and Alcohol Interactions in Humans.

Mephedrone (4-MMC, mephedrone) is a synthetic cathinone derivative included in the class of new psychoactive substances. It is commonly used simultaneously with alcohol (ethanol). The aim of the present study was to evaluate the interactions on subjective, cardiovascular and hormone effects and pharmacokinetics between mephedrone and alcohol in humans. Eleven male volunteers participated as outpatients in four experimental sessions in a double-blind, randomized, cross-over, and placebo-controlled clinical trial. Participants received a single oral dose of 200 mg of mephedrone plus 0.8 g/kg of alcohol (combination condition); 200 mg of mephedrone plus placebo alcohol (mephedrone condition); placebo mephedrone plus 0.8 g/kg of ethanol (alcohol condition); and placebo mephedrone plus placebo alcohol (placebo condition). Outcome variables included physiological (blood pressure, heart rate, temperature, and pupil diameter), psychomotor (Maddox wing), subjective (visual analogue scales, Addiction Research Center Inventory 49 item short form, and Valoración de los Efectos Subjetivos de Sustancias con Potencial de Abuso questionnaire), and pharmacokinetic parameters (mephedrone and ethanol concentrations). The study was registered in ClinicalTrials.gov, number NCT02294266. The mephedrone and alcohol combination produced an increase in the cardiovascular effects of mephedrone and induced a more intense feeling of euphoria and well-being in comparison to the two drugs alone. Mephedrone reduced the sedative effects produced by alcohol. These results are similar to those obtained when other psychostimulants such as amphetamines and 3,4-methylenedioxymethamphetamine are combined simultaneously with alcohol. The abuse liability of mephedrone combined with alcohol is greater than that induced by mephedrone alone.

Genetic deletion of the circadian clock transcription factor BMAL1 and chronic alcohol consumption differentially alter hepatic glycogen in mice.

Multiple metabolic pathways exhibit time-of-day-dependent rhythms that are controlled by the molecular circadian clock. We have shown that chronic alcohol is capable of altering the molecular clock and diurnal oscillations in several elements of hepatic glycogen metabolism ( 19 , 44 ). Herein, we sought to determine whether genetic disruption of the hepatocyte clock differentially impacts hepatic glycogen content in chronic alcohol-fed mice. Male hepatocyte-specific BMAL1 knockout (HBK) and littermate controls were fed control or alcohol-containing diets for 5 wk to alter hepatic glycogen content. Glycogen displayed a significant diurnal rhythm in livers of control genotype mice fed the control diet. While rhythmic, alcohol significantly altered the diurnal oscillation of glycogen in livers of control genotype mice. The glycogen rhythm was mildly altered in livers of control-fed HBK mice. Importantly, glycogen content was arrhythmic in livers of alcohol-fed HBK mice. Consistent with these changes in hepatic glycogen content, we observed that some glycogen and glucose metabolism genes were differentially altered by chronic alcohol consumption in livers of HBK and littermate control mice. Diurnal rhythms in glycogen synthase (mRNA and protein) were significantly altered by alcohol feeding and clock disruption. Alcohol consumption significantly altered Gck, Glut2, and Ppp1r3g rhythms in livers of control genotype mice, with diurnal rhythms of Pklr, Glut2, Ppp1r3c, and Ppp1r3g further disrupted (dampened or arrhythmic) in livers of HBK mice. Taken together, these findings show that chronic alcohol consumption and hepatocyte clock disruption differentially influence the diurnal rhythm of glycogen and various key glycogen metabolism-related genes in the liver. NEW & NOTEWORTHY We report that circadian clock disruption exacerbates alcohol-mediated alterations in hepatic glycogen. We observed differential responsiveness in diurnal rhythms of glycogen and glycogen metabolism genes and proteins in livers of hepatocyte-specific BMAL1 knockout and littermate control mice fed alcohol. Our findings provide new insights into potential mechanisms by which alcohol alters glycogen, an important energy source for liver and other organs.

CRF modulates glutamate transmission in the central amygdala of naïve and ethanol-dependent rats.

Corticotropin-releasing factor (CRF) signaling in the central nucleus of the amygdala (CeA) is hypothesized to drive the development of alcohol dependence, as it regulates ethanol intake and several anxiogenic behaviors linked to withdrawal. Excitatory glutamatergic neurotransmission contributes to alcohol reinforcement, tolerance and dependence. Therefore, in this study we used in vitro slice electrophysiology to investigate the effects of CRF and its receptor subtype (CRF1 and CRF2) antagonists on both evoked and spontaneous action potential-independent glutamatergic transmission in the CeA of naive and ethanol-dependent Sprague-Dawley rats. We found that CRF (25-200 nM) concentration-dependently diminished evoked compound excitatory postsynaptic potentials (EPSPs), but increased miniature excitatory postsynaptic current (mEPSC) frequencies similarly in CeA neurons of both naïve and ethanol-dependent rats, indicating reduced evoked glutamatergic responses and enhanced vesicular glutamate release, respectively. This CRF-induced vesicular glutamate release was prevented by the CRF1/2 antagonist (Astressin B) and the CRF1 antagonist (R121919), but not by the CRF2 antagonist (Astressin 2B). Similarly, CRF's effects on evoked glutamatergic responses were completely blocked by CRF1 antagonism, but only slightly decreased in the presence of the CRF2 antagonist. Moreover, CRF1 antagonism reveals a tonic facilitation of vesicular glutamate, whereas the CRF2 antagonism revealed a tonic inhibition of vesicular glutamate release. Collectively our data show that CRF primarily acts at presynaptic CRF1 to produce opposite effects on CeA evoked and spontaneous glutamate release and that the CRF system modulates CeA glutamatergic synapses throughout the development of alcohol dependence.

P/Q-type voltage-gated calcium channels mediate the ethanol and CRF sensitivity of central amygdala GABAergic synapses.

The central amygdala (CeA) GABAergic system is hypothesized to drive the development of alcohol dependence, due to its pivotal roles in the reinforcing actions of alcohol and the expression of negative emotion, anxiety and stress. Recent work has also identified an important role for the CeA corticotropin-releasing factor (CRF) system in the interaction between anxiety/stress and alcohol dependence. We have previously shown that acute alcohol and CRF each increase action potential-independent GABA release in the CeA via their actions at presynaptic CRF type 1 receptors (CRF1s); however, the shared mechanism employed by these two compounds requires further investigation. Here we report that acute alcohol interacts with the CRF/CRF1 system, such that CRF and alcohol act via presynaptic CRF1s and P/Q-type voltage-gated calcium channels to promote vesicular GABA release and that both compounds occlude the effects of each other at these synapses. Chronic alcohol exposure does not alter P/Q-type voltage-gated calcium channel membrane abundance or this CRF1/P/Q-type voltage-gated calcium channel mechanism of acute alcohol-induced GABA release, indicating that alcohol engages this molecular mechanism at CeA GABAergic synapses throughout the transition to dependence. Thus, P/Q-type voltage-gated calcium channels, like CRF1s, are key regulators of the effects of alcohol on GABAergic signaling in the CeA.

Alcohol Dependence Disrupts Amygdalar L-Type Voltage-Gated Calcium Channel Mechanisms.

L-type voltage-gated calcium channels (LTCCs) are implicated in several psychiatric disorders that are comorbid with alcoholism and involve amygdala dysfunction. Within the amygdala, the central nucleus (CeA) is critical in acute alcohol's reinforcing actions, and its dysregulation in human alcoholics drives their negative emotional state and motivation to drink. Here we investigated the specific role of CeA LTCCs in the effects of acute alcohol at the molecular, cellular physiology, and behavioral levels, and their potential neuroadaptation in alcohol-dependent rats. Alcohol increases CeA activity (neuronal firing rates and GABA release) in naive rats by engaging LTCCs, and intra-CeA LTCC blockade reduces alcohol intake in nondependent rats. Alcohol dependence reduces CeA LTCC membrane abundance and disrupts this LTCC-based mechanism; instead, corticotropin-releasing factor type 1 receptors (CRF1s) mediate alcohol's effects on CeA activity and drive the escalated alcohol intake of alcohol-dependent rats. Collectively, our data indicate that alcohol dependence functionally alters the molecular mechanisms underlying the CeA's response to alcohol (from LTCC- to CRF1-driven). This mechanistic switch contributes to and reflects the prominent role of the CeA in the negative emotional state that drives excessive drinking.SIGNIFICANCE STATEMENT The central amygdala (CeA) plays a critical role in the development of alcohol dependence. As a result, much preclinical alcohol research aims to identify relevant CeA neuroadaptions that promote the transition to dependence. Here we report that acute alcohol increases CeA neuronal activity in naive rats by engaging L-type calcium channels (LTCCs) and that intra-CeA LTCC blockade reduces alcohol intake in nondependent rats. Alcohol dependence disrupts this LTCC-based mechanism; instead, corticotropin-releasing factor type 1 receptors (CRF1s) mediate alcohol's effects on CeA activity and drive the escalated alcohol intake of alcohol-dependent rats. This switch reflects the important role of the CeA in the pathophysiology of alcohol dependence and represents a new potential avenue for therapeutic intervention during the transition period.

Synaptic targets: Chronic alcohol actions.

Alcohol acts on numerous cellular and molecular targets to regulate neuronal communication within the brain. Chronic alcohol exposure and acute withdrawal generate prominent neuroadaptations at synapses, including compensatory effects on the expression, localization and function of synaptic proteins, channels and receptors. The present article reviews the literature describing the synaptic effects of chronic alcohol exposure and their relevance for synaptic transmission in the central nervous system. This review is not meant to be comprehensive, but rather to highlight the effects that have been observed most consistently and that are thought to contribute to the development of alcohol dependence and the negative aspects of withdrawal. Specifically, we will focus on the major excitatory and inhibitory neurotransmitters in the brain, glutamate and GABA, respectively, and how their neuroadaptations after chronic alcohol exposure contributes to alcohol reinforcement, dependence and withdrawal. This article is part of the Special Issue entitled "Alcoholism".

Prenatal Alcohol Exposure and Pair Feeding Differentially Impact Puberty and Reproductive Development in Female Rats: Role of the Kisspeptin System.

BACKGROUND: Reproductive maturation is initiated with the onset of puberty, which activates the hypothalamic-pituitary-gonadal axis and coincidences with increased expression of the hormone kisspeptin within the hypothalamus. Maturational events are sensitive to environmental factors, including alcohol, which is known to delay reproductive development. We hypothesized that, similar to alcohol's adverse effects during reproductive maturation, prenatal alcohol exposure (PAE) would alter pubertal markers, sex hormone profiles, and kisspeptin expression in the hypothalamus. METHODS: Female offspring from control (C), pair-fed (PF), and PAE groups were sacrificed prior to puberty onset (postnatal day [PND] 30), during puberty [PND 35], or in adulthood [PND 65]. Estradiol (E2 ), progesterone (P4 ), prolactin, and luteinizing hormone levels, and Kiss1 mRNA expression were measured in the arcuate (ARC) and anteroventral periventricular (AVPV) nuclei of the hypothalamus. Pubertal markers (vaginal opening [VO], uterus/body wt ratio) were assessed. RESULTS: Our findings indicate that (i) PAE inhibits the expected increases in E2 levels with age and delays maturational increases of P4 levels; (ii) PAE and pair feeding have similar adverse effects on VO and uterus/body wt ratio; (iii) differential relationships between PRL and P4 suggest that different mechanisms may underlie delayed maturation in PAE and PF; that is, PF females have low PRL levels and no increase in P4 with age, whereas PAE animals, despite low PRL, show the expected age-related increase in P4 ; and (iv) there is higher mean density of Kiss1 mRNA in the ARC of adult PAE females and altered Kiss1 expression in the AVPV of both PAE and PF females. CONCLUSIONS: PAE and pair feeding have some overlapping but important differential effects on hormonal profiles and Kiss1 mRNA expression during reproductive development. Preadolescent alterations in Kiss1 expression in the AVPV and ARC, which may change the balance of function in these 2 nuclei, may differentially contribute to delayed reproductive maturation in PAE and PF compared to C females.

The Effect and Underlying Mechanism of Ethanol on Intracellular H(+) -Equivalent Membrane Transporters in Human Aorta Smooth Muscle Cells.

BACKGROUND: The presence of intracellular pH (pHi ) regulators, including Na(+) -H(+) exchanger (NHE), Na(+) -HCO3- co-transporter (NBC), Cl(-) /OH(-) exchanger (CHE), and Cl(-) /HCO3- exchanger (AE), have been confirmed in many mammalian cells. Alcohol consumption is associated with increased risk of cardiovascular disorder. The aims of the study were to identify the possible transmembrane pHi regulators and to explore the effects of ethanol (EtOH) (10 to 300 mM) on the resting pHi and pHi regulators in human aorta smooth muscle cells (HASMCs). METHODS: HASMCs were obtained from patients undergoing heart transplant. The pHi was measured by microspectrofluorimetry with the pH-sensitive dye, BCECF-AM. RESULTS: The following results are obtained. (i) In cultured HASMCs, the resting pHi was 7.19 ± 0.04 and 7.13 ± 0.02 for HEPES- and CO2 /HCO3--buffered solution, respectively. (ii) Two different Na(+) -dependent acid-equivalent extruders, including NHE and Na(+) -coupled HCO3- transporter, functionally coexisted. (iii) Two different Cl(-) -dependent acid loaders (CHE and AE) were functionally identified. (iv) EtOH induced a biphasic, concentration-dependent change in resting pHi (+0.25 pH unit at 100 mM but only +0.05 pH unit at 300 mM) in bicarbonate-buffered solution, while caused a concentration-dependent decrease in resting pHi (-0.06 pH unit at 300 mM) in HEPES-buffered solution. (v) The effect of EtOH on NHE activity was also biphasic: increase of 40% at lower concentration of 10 mM, followed by decrease of 30% at higher concentration of 300 mM. (vi) The increase in Na(+) -coupled HCO3- transporter activity by EtOH was concentration dependent. (vii) The effect of EtOH on CHE and AE activities was both biphasic: increase of ~25% at 30 mM, followed by decrease of 10 to 25% at 100 mM, and finally increase of 15 to 20% at 300 mM. CONCLUSIONS: This study demonstrated that 2 acid extruders and 2 acid loaders coexisted functionally in HASMCs and that EtOH induced a biphasic, concentration-dependent change in resting pHi by altering the activity of the 2 acid extruders, NHE and Na(+) -coupled HCO3- transporter, and the 2 acid loaders, CHE and AE.

Exposure to Chronic Mild Stress Differentially Alters Corticotropin-Releasing Hormone and Arginine Vasopressin mRNA Expression in the Stress-Responsive Neurocircuitry of Male and Female Rats Prenatally Exposed to Alcohol.

BACKGROUND: Prenatal alcohol exposure (PAE) results in dysregulation of the offspring hypothalamic-pituitary-adrenal (HPA) axis, increasing sensitivity to stressors and vulnerability to stress-related disorders. We have previously shown that exposure to chronic mild stress (CMS) in adulthood significantly increases anxiety-like behaviors (elevated plus maze) in PAE males and females compared to controls. To explore neurobiological mechanisms linking HPA dysregulation and altered anxiety-like behavior, we investigated neuropeptide (corticotropin-releasing hormone [CRH] and arginine vasopressin [AVP]) expression in brain areas involved in the stress neurocircuitry of animals from this previous behavioral study. METHODS: Adult PAE, pair-fed (PF), and ad libitum fed control (C) male and female offspring exposed to CMS or remaining undisturbed (non-CMS) were terminated 30 minutes following behavioral testing. RESULTS: In the paraventricular nucleus, CMS increased CRH mRNA levels in PAE compared to PF and C males and increased AVP mRNA levels in PAE compared to C males, with no differential effects for CRH or AVP in females. In the central nucleus of the amygdala, there was an increase in CRH mRNA expression overall, regardless of CMS condition or sex, in PAE compared to C animals. Moreover, in PF males, CMS increased AVP mRNA levels in the paraventricular nucleus, resulting in a decreased CRH/AVP ratio compared to PAE males, and decreased amygdala CRH mRNA compared to that in the non-CMS condition. CONCLUSIONS: CMS differentially altered central HPA peptide expression in PAE and PF animals compared to their control counterparts, with a possible shift toward greater CRH mediation of HPA regulation in PAE males, and greater AVP mediation of HPA regulation in PF males. However, changes in CRH and AVP expression do not align fully with the anxiogenic profile observed in our previous behavior study, suggesting that other neuronal substrates and limbic forebrain regions also contribute to increased anxiety-like behavior following CMS.

Relative fluid novelty differentially alters the time course of limited-access ethanol and water intake in selectively bred high-alcohol-preferring mice.

BACKGROUND: The influence of previous alcohol (ethanol [EtOH])-drinking experience on increasing the rate and amount of future EtOH consumption might be a genetically regulated phenomenon critical to the development and maintenance of repeated excessive EtOH abuse. We have recently found evidence supporting this view, wherein inbred C57BL/6J (B6) mice develop progressive increases in the rate of binge EtOH consumption over repeated drinking-in-the-dark (DID) EtOH access sessions (i.e., "front loading"). The primary goal of this study was to evaluate identical parameters in high-alcohol-preferring (HAP) mice to determine whether similar temporal alterations in limited-access EtOH drinking develop in a population selected for high EtOH preference/intake under continuous (24-hour) access conditions. METHODS: Using specialized volumetric drinking devices, HAP mice received 14 daily 2-hour DID EtOH or water access sessions. A subset of these mice was then given 1 day access to the opposite assigned fluid on day 15. Home cage locomotor activity was recorded concomitantly on each day of these studies. The possibility of behavioral/metabolic tolerance was evaluated on day 16 using experimenter-administered EtOH. RESULTS: The amount of EtOH consumed within the first 15 minutes of access increased markedly over days. However, in contrast to previous observations in B6 mice, EtOH front loading was also observed on day 15 in mice that only had previous DID experience with water. Furthermore, a decrease in the amount of water consumed within the first 15 minutes of access compared to animals given repeated water access was observed on day 15 in mice with 14 previous days of EtOH access. CONCLUSIONS: These data further illustrate the complexity and importance of the temporal aspects of limited-access EtOH consumption and suggest that previous procedural/fluid experience in HAP mice selectively alters the time course of EtOH and water consumption.

Operant alcohol self-administration in dependent rats: focus on the vapor model.

Alcoholism (alcohol dependence) is characterized by a compulsion to seek and ingest alcohol (ethanol), loss of control over intake, and the emergence of a negative emotional state during withdrawal. Animal models are critical in promoting our knowledge of the neurobiological mechanisms underlying alcohol dependence. Here, we review the studies involving operant alcohol self-administration in rat models of alcohol dependence and withdrawal with the focus on the alcohol vapor model. In 1996, the first articles were published reporting that rats made dependent on alcohol by exposure to alcohol vapors displayed increased operant alcohol self-administration during acute withdrawal compared with nondependent rats (i.e., not exposed to alcohol vapors). Since then, it has been repeatedly demonstrated that this model reliably produces physical and motivational symptoms of alcohol dependence. The functional roles of various systems implicated in stress and reward, including opioids, dopamine, corticotropin-releasing factor (CRF), glucocorticoids, neuropeptide Y (NPY), γ-aminobutyric acid (GABA), norepinephrine, and cannabinoids, have been investigated in the context of alcohol dependence. The combination of models of alcohol withdrawal and dependence with operant self-administration constitutes an excellent tool to investigate the neurobiology of alcoholism. In fact, this work has helped lay the groundwork for several ongoing clinical trials for alcohol dependence. Advantages and limitations of this model are discussed, with an emphasis on what future directions of great importance could be.