Involvement of Purinergic P2X4 Receptors in Alcohol Intake of High-Alcohol-Drinking (HAD) Rats

Background: The P2X4 receptor is thought to be involved in regulating alcohol-consuming behaviors and ethanol (EtOH) has been reported to inhibit P2X4 receptors. Ivermectin is an anti-parasitic agent that acts as a positive allosteric modulator of the P2X4 receptor. The current study examined the effects of systemically- and centrally-administered ivermectin on alcohol drinking of replicate lines of high-alcohol-drinking (HAD-1/HAD-2) rats, and the effects of lentiviral-delivered short-hairpin RNAs (shRNAs) targeting P2rx4 on EtOH intake of female HAD2 rats. Method: For the 1st experiment, adult male HAD-1 & HAD-2 rats were given 24-hr free-choice access to 15% EtOH vs. water. Dose-response effects of ivermectin (1.5 to 7.5 mg/kg i.p.) on EtOH intake were determined; the effects of ivermectin were then examined for 2% w/v sucrose intake over 5 consecutive days. In the 2nd experiment, female HAD-2 rats were trained to consume 15% EtOH under 2-hr limited access conditions, and dose-response effects of intracerebroventricular (ICV) administration of ivermectin (0.5 to 2.0 μg) were determined over 5 consecutive days. The 3rd experiment determined the effects of microinfusion of a lentivirus expressing P2rx4 shRNAs into the posterior ventral tegmental area (VTA) on 24-hr EtOH free-choice drinking of female HAD-2 rats. Results: The highest i.p. dose of ivermectin reduced alcohol drinking (30-45%) in both rat lines, but did not alter sucrose intake. HAD-2 rats appeared to be more sensitive than HAD1 rats to the effects of ivermectin. ICV administration of ivermectin reduced 2-hr limited access intake (∼35%) of femal

Reduced Levels of mGlu2 Receptors within the Prelimbic Cortex Are Not Associated with Elevated Glutamate Transmission or High Alcohol Drinking

Background A Grm2 cys407* stop codon mutation, which results in a loss of the metabotropic glutamate 2 (mGlu2) receptor protein, was identified as being associated with high alcohol drinking by alcohol-preferring (P) rats. The objectives of the current study were to characterize the effects of reduced levels of mGlu2 receptors on glutamate transmission and alcohol drinking. Methods Quantitative no-net-flux microdialysis was used to test the hypothesis that basal extracellular glutamate levels in the prelimbic (PL) cortex and nucleus accumbens shell (NACsh) will be higher in P than Wistar rats. A lentiviral-delivered short-hairpin RNA (shRNA)-mediated knockdown was used to test the hypothesis that reduced levels of mGlu2 receptors within the PL cortex will increase voluntary alcohol drinking by Wistar rats. A linear regression analysis was used to test the hypothesis that there will be a significant correlation between the Grm2 cys407* mutation and level of alcohol intake. Results Extracellular glutamate concentrations within the PL cortex (3.6 ± 0.6 vs. 6.4 ± 0.6 μM) and NACsh (3.2 ± 0.4 vs. 6.6 ± 0.6 μM) were significantly lower in female P than female Wistar rats. Western blot detected the presence of mGlu2 receptors in these regions of female Wistar rats, but not female P rats. Micro-infusion of shRNAs into the PL cortex significantly reduced local mGlu2 receptor levels (by 40%), but did not alter voluntary alcohol drinking in male Wistar rats. In addition, there was no significant correlation between the Grm2 mutation and alcohol intake in 36 rodent lines (r = 0.29, p > 0.05). Conclusions Collectively, these results suggest a lack of association between the loss of mGlu2 receptors and glutamate transmission in the NACsh and PL cortex of female P rats, and between the level of mGlu2 receptors in the PL cortex and alcohol drinking of male Wistar rats

Cis‐acting allele specific expression (ASE) differences induced by alcohol and impacted by sex as well as parental genotype of origin

Background Alcohol use disorders (AUDs) are influenced by complex interactions between the genetics of the individual and their environment. We have previously identified hundreds of polygenic genetic variants between the selectively bred high and low alcohol drinking (HAD and LAD) rat lines. Here we report allele specific expression (ASE) differences, between the HAD2 and LAD2 rat lines. Methods The HAD2 and LAD2 rats which have been sequenced were reciprocally crossed to generate 10 litters of F1 progeny. For 5 of these litters, the sire was HAD2; and, for the other 5 litters, the sire was a LAD2. From these 10 litters, two males and two females were picked from each F1 litter (N = 40 total). The F1‐pups were divided, with balancing for sex and direction of cross, into an alcohol (15%) vs a water control group. Alcohol‐drinking started in the middle of adolescence (~PND 35) and lasted 9 weeks. At the end of these treatments, rats were euthanized, the nucleus accumbens was dissected, and RNA was processed for RNA‐sequencing and ASE analyses. Results Analyses revealed that adolescent ethanol drinking, individual ethanol drinking levels, parentage, and sex‐of‐animal affected ASEs of about 300 genes. The identified genes included those associated with ethanol metabolism (e.g., Aldh2); neuromodulatory function [e.g., Cckbr, Slc6a7, and Slc1a1]; ion channel activity (e.g., Kcnc3); as well as other synaptic and epigenetic function. Conclusion These data indicate that ethanol drinking differentially amplified paternal vs maternal allelic contribution to the transcriptome. We hypothesize that this was due, at least in part, to ethanol‐induced changes in cis‐regulation of polymorphisms previously identified between the HAD2 and LAD2 rat lines. This report highlights the complexity of gene‐by‐environment interactions mediating a genetic predisposition for, and/or the active development of, alcohol use disorders

Reduction of alcohol drinking of alcohol-preferring (P) and high-alcohol drinking (HAD1) rats by targeting phosphodiesterase-4 (PDE4)

RATIONALE: Phosphodiesterase-4 (PDE4) and neuroimmune signaling have been posited to regulate alcohol drinking. OBJECTIVES: This study evaluated the involvement of PDE4 and Il22ra2 on ethanol (EtOH) intake by alcohol-preferring (P) and high-alcohol-drinking (HAD1) rats. METHODS: Exp 1 determined the dose-response effects of PDE4 inhibitors, rolipram, and Ro 20-1724, on 2 h/day free-choice EtOH intake by adult P and HAD1 rats. Exps 2-3 examined the effects of repeated administration with the PDE4 inhibitors on EtOH or sucrose intake and locomotor behavior. Exp 4 determined Pde4-associated gene expression differences in subregions of the extended amygdala, between high- and low-alcohol-consuming rat lines. Exp 5 evaluated the effects of infusing short hairpin RNA to knock down Il22ra2 in the nucleus accumbens (NAc) shell on a 24-h free-choice EtOH drinking by P rats. RESULTS: Administration of rolipram or Ro 20-1724 reduced EtOH intake by P rats; Ro 20-1724 reduced EtOH intake by HAD1 rats. Repeated rolipram or Ro 20-1724 exposure reduced EtOH intake by P and HAD1 rats. PDE4 inhibition induced motor impairment during the first hour of EtOH intake by P rats. Higher gene expression levels for PDE4A were found in the NAc shell of P vs NP rats. ShRNAs targeting Il22ra2 in the NAc shell significantly reduced chronic EtOH intake. CONCLUSIONS: PDE4 and neuroinflammatory/immune signaling pathways could represent molecular targets for the treatment of alcohol use disorders in genetically predisposed subjects. This study underscores the importance of testing compounds over multiple days and rat lines when determining efficacy to disrupt excessive alcohol intake

Utility of Two iPhone Device Apps in Assessing Heart Rate at Rest and During Activity

Heart rate (HR) is a critical physiological variable used for prescribing exercise, assessing fitness level and tracking fitness improvements. Electrocardiography (ECG) stands as the criterion measure of HR. While recent development of HR-detecting mobile device applications (apps) has made evaluating HR more convenient; their degree of accuracy is unknown. Therefore, the purpose of this current study was to examine the accuracy and reliability of two-iPhone applications to detect HR at rest and during low-intensity exercise conditions. Eighteen female and 22 male subjects (26 + 9.5 yrs) were prepped for simultaneous detection of HR via three methods: ECG and two HR-detecting apps. App 1, a camera-based app called Azumio Instant Heart Rate (CAM), was used by placement of a finger over the camera lens of the mobile device. App 2, a microphone-based app called Heart Monitor by Bluespark, was employed via placement of an external microphone over the radial pulse. The participants underwent a series of 5-minute stages: seated rest followed by cycle then treadmill walking at low intensities. HR was recorded concurrently, at several time intervals from the three methods once a steady-state HR was reached. The means of the three devices were compared via ANOVA with the significance level set, a priori, at 0.05. Correlation analysis was employed to investigate relationships between the apps and ECG. No statistical difference was found between the CAM and ECG HR (p \u3e 0.05) during the resting and cycle stages. However, during the treadmill phase, there was a significant difference (p = 0.018) between CAM and ECG. Nevertheless, there was a significant (p \u3c 0.05), positive correlation between CAM and ECG under the resting, cycle and treadmill conditions (r = .966, r = .984, r = .877, respectively). Significant differences (p \u3c 0.05) were found for each condition when comparing ECG and MIC HR. Data also revealed poor correlations (p \u3e 0.05; r between -.004 and -.136) between MIC and ECG. The utility of CAM and MIC-based apps to detect HR remains in question as evidence appears to indicate exercise mode and app specificity. Caution should be shown when using these devices. The CAM-based app may accurately detect HR during resting and seated cycling but not during treadmill activity. The MIC-based app is not recommended for use in any condition. Of note, statistical significance may not mitigate usefulness when considering the accuracy of palpation. Additional research is necessary

A Two-Stage Cluster Sampling Method Using Gridded Population Data, a GIS, and Google Earth(TM) Imagery in a Population-Based Mortality Survey in Iraq

BACKGROUND: Mortality estimates can measure and monitor the impacts of conflict on a population, guide humanitarian efforts, and help to better understand the public health impacts of conflict. Vital statistics registration and surveillance systems are rarely functional in conflict settings, posing a challenge of estimating mortality using retrospective population-based surveys. RESULTS: We present a two-stage cluster sampling method for application in population-based mortality surveys. The sampling method utilizes gridded population data and a geographic information system (GIS) to select clusters in the first sampling stage and Google Earth TM imagery and sampling grids to select households in the second sampling stage. The sampling method is implemented in a household mortality study in Iraq in 2011. Factors affecting feasibility and methodological quality are described. CONCLUSION: Sampling is a challenge in retrospective population-based mortality studies and alternatives that improve on the conventional approaches are needed. The sampling strategy presented here was designed to generate a representative sample of the Iraqi population while reducing the potential for bias and considering the context specific challenges of the study setting. This sampling strategy, or variations on it, are adaptable and should be considered and tested in other conflict settings

Benign Conduction Abnormalities in Response to Acute, Moderately-High, Simulated Altitude Exposure

Acclimatization to altitude can improve endurance performance above levels achieved solely by training at sea level. There is natural limitation in the applicability of employing terrestrial altitude training – namely proximity. A simple, non-cumbersome method of simulating altitude is desirable to many types of endurance athletes. The Alto2Lab (Pharma Pacific Inc.), consisting of primarily a breathing tube and silo stack, has shown some potential in this role. There is a lack of evidence regarding whether simulated altitude exposure triggers abnormal cardiovascular responses. The aim of this study was to provide initial evidence of cardiac changes associated with usage patterns that follow distributor guidelines. Twenty-five participants (mean age 29 ± 10.7; 16 males; 9 females) volunteered for the study. Subjects underwent a baseline ECG recording followed by ECG recording during sham (4-5 mins), hypoxia (~6 mins), and recovery (3-4 mins) phases. The sham phase consisted of subjects breathing normoxive air through a foam-filled silo system. The sham stack mimicked the look and feel of the silo system used to produce hypoxia with the difference being a single, soda lime-filled silo. A recovery phase followed hypoxia. Pulse oximetry (SpO2) was used to assess oxygen saturation. Cochran’s Q was employed to test the frequencies of responses across the phases. An independent, blinded, experienced clinician (DK) analyzed the recordings. Two subjects were removed from the final analysis (inability to finish the protocol, baseline right bundle branch block). All subjects demonstrated an increase in heart rate (mean = +16.8 ± 8.0) during the hypoxia (mean oxygen saturation = 82 ± 4.1%) phase. No ECG ischemic changes were seen across any of the phases. Benign conduction abnormalities (sinus arrhythmia = 9; junctional rhythms = 4) occurred with some regularity during hypoxia. These abnormalities occurred with less frequency during the sham and recovery phases. It is possible that an altered breathing pattern or an inadequate washout period between phases might account for these findings. Overall, there was no significant relationship between the heart response and phase (p = .375). While the Alto2Lab did not produce any ECG changes indicative of an ischemic response, the present study used a small sample of healthy, recreationally-active participants. A larger study employing patients among higher risk categories would provide data that is not currently present in the literature and to which this trial cannot speak

Ly alpha emitting galaxies as early stages in galaxy formation

We present optical spectroscopy of two samples of GALEX grism selected Ly alpha emitters (LAEs): one at z=0.195-0.44 and the other at z=0.65-1.25. We have also observed a comparison sample of galaxies in the same redshift intervals with the same UV magnitude distributions but with no detected Ly alpha. We use the optical spectroscopy to eliminate active galactic nuclei (AGNs) and to obtain the optical emission-line properties of the samples. We compare the luminosities of the LAEs in the two redshift intervals and show that there is dramatic evolution in the maximum Ly alpha luminosity over z=0-1. Focusing on the z=0.195-0.44 samples alone, we show that there are tightly defined relations between all of the galaxy parameters and the rest-frame equivalent width (EW) of H alpha. The higher EW(H alpha) sources all have lower metallicities, bluer colors, smaller sizes, and less extinction, consistent with their being in the early stages of the galaxy formation process. We find that 75 +- 12% of the LAEs have EW(H alpha)>100 Angstrom, and, conversely, that 31 +/- 3% of galaxies with EW(H alpha)>100 Angstrom are LAEs. We correct the broadband magnitudes for the emission-line contributions and use spectral synthesis fits to estimate the ages of the galaxies. We find a median age of 1.1x10^{8} yr for the LAE sample and 1.4x10^{9} yr for the UV-continuum sample without detected Ly alpha. The median metallicity of the LAE sample is 12+log(O/H)=8.24, or about 0.4 dex lower than the UV-continuum sample.Comment: to be published in the Astrophysical Journa

Burden of Cardiovascular Risk Factors Over Time and Arterial Stiffness in Youth With Type 1 Diabetes Mellitus: The SEARCH for Diabetes in Youth Study

Background: The incidence of type 1 diabetes mellitus (T1DM) in children is increasing, resulting in higher burden of cardiovascular diseases due to diabetes mellitus–related vascular dysfunction. Methods and Results: We examined cardiovascular risk factors (CVRFs) and arterial parameters in 1809 youth with T1DM. Demographics, anthropometrics, blood pressure, and laboratory data were collected at T1DM onset and 5 years later. Pulse wave velocity and augmentation index were collected with tonometry. ANOVA or chi�square tests were used to test for differences in measures of arterial parameters by CVRF. Area under the curve of CVRFs was entered in general linear models to explore determinants of accelerate vascular aging. Participants at the time of arterial measurement were 17.6±4.5 years old, 50% female, 76% non�Hispanic white, and duration of T1DM was 7.8±1.9 years. Glycemic control was poor (glycated hemoglobin, 9.1±1.8%). All arterial parameters were higher in participants with glycated hemoglobin ≥9% and pulse wave velocity was higher with lower insulin sensitivity or longer duration of diabetes mellitus. Differences in arterial parameters were found by sex, age, and presence of obesity, hypertension, or dyslipidemia. In multivariable models, higher glycated hemoglobin, lower insulin sensitivity, body mass index, blood pressure, and lipid areas under the curve were associated with accelerated vascular aging. Conclusions: In young people with T1DM, persistent poor glycemic control and higher levels of traditional CVRFs are independently associated with arterial aging. Improving glycemic control and interventions to lower CVRFs may prevent future cardiovascular events in young individuals with T1DM

Western Bumble Bee: Declines in the Continental United States and Range-Wide Information Gaps

In recent decades, many bumble bee species have declined due to changes in habitat, climate, and pressures from pathogens, pesticides, and introduced species. The western bumble bee (Bombus occidentalis), once common throughout western North America, is a species of concern and will be considered for listing by the U.S. Fish and Wildlife Service (USFWS) under the Endangered Species Act (ESA). We attempt to improve alignment of data collection and research with USFWS needs to consider redundancy, resiliency, and representation in the upcoming species status assessment. We reviewed existing data and literature on B. occidentalis, highlighting information gaps and priority topics for research. Priorities include increased knowledge of trends, basic information on several life‐history stages, and improved understanding of the relative and interacting effects of stressors on population trends, especially the effects of pathogens, pesticides, climate change, and habitat loss. An understanding of how and where geographic range extent has changed for the two subspecies of B. occidentalis is also needed. We outline data that could be easily collected in other research projects that would increase their utility for understanding range‐wide trends of bumble bees. We modeled the overall trend in occupancy from 1998 to 2018 of Bombus occidentalis within the continental United States using existing data. The probability of local occupancy declined by 93% over 21 yr from 0.81 (95% CRI = 0.43, 0.98) in 1998 to 0.06 (95% CRI = 0.02, 0.16) in 2018. The decline in occupancy varied spatially by landcover and other environmental factors. Detection rates vary in both space and time, but peak detection across the continental United States occurs in mid‐July. We found considerable spatial gaps in recent sampling, with limited sampling in many regions, including most of Alaska, northwestern Canada, and the southwestern United States. We therefore propose a sampling design to address these gaps to best inform the ESA species status assessment through improved assessment of how the spatial distribution of stressors influences occupancy changes. Finally, we request involvement via data sharing, participation in occupancy sampling with repeated visits to distributed survey sites, and complementary research to address priorities outlined in this paper