Adolescent Intermittent ethanol exposure enhances ethanol activation of the nucleus accumbens while blunting the prefrontal cortex responses in adult rat

The brain continues to develop through adolescence when excessive alcohol consumption is prevalent in humans. We hypothesized that binge drinking doses of ethanol during adolescence will cause changes in brain ethanol responses that persist into adulthood. To test this hypothesis Wistar rats were treated with an adolescent intermittent ethanol (AIE; 5 g/kg, i.g. 2 days on–2 days off; P25–P54) model of underage drinking followed by 25 days of abstinence during maturation to young adulthood (P80). Using markers of neuronal activation c-Fos, EGR1, and phophorylated extracellar signal regulated kinase (pERK1/2), adult responses to a moderate and binge drinking ethanol challenge, e.g., 2 or 4 g/kg, were determined. Adult rats showed dose dependent increases in neuronal activation markers in multiple brain regions during ethanol challenge. Brain regional responses correlated are consistent with anatomical connections. AIE led to marked decreases in adult ethanol PFC (prefrontal cortex) and blunted responses in the amygdala. Binge drinking doses led to the nucleus accumbens (NAc) activation that correlated with the ventral tegmental area (VTA) activation. In contrast to other brain regions, AIE enhanced the adult NAc response to binge drinking doses. These studies suggest that adolescent alcohol exposure causes long-lasting changes in brain responses to alcohol that persist into adulthood

Adolescent binge drinking increases expression of the danger signal receptor agonist HMGB1 and toll-like receptors in the adult prefrontal cortex

Adolescence is a critical developmental stage of life during which the prefrontal cortex (PFC) matures, and binge drinking and alcohol abuse are common. Recent studies have found that ethanol increases neuroinflammation via upregulated high-mobility group box 1 (HMGB1) signaling through Toll-like receptors (TLRs). HMGB1/TLR ‘danger signaling’ induces multiple brain innate immune genes that could alter brain function. To determine whether adolescent binge drinking persistently increases innate immune gene expression in the PFC, rats (P25–P55) were exposed to adolescent intermittent ethanol (AIE [5.0 g/kg, 2-day on/2-day off schedule]). On P56, HMGB1/TLR danger signaling was assessed using immunohistochemistry (i.e., +immunoreactivity [+IR]). In a separate group of subjects, spatial and reversal learning on the Barnes maze was assessed in early adulthood (P64–P75), and HMGB1/TLR danger signaling was measured using immunohistochemistry for +IR and RT-PCR for mRNA in adulthood (P80). Immunohistochemical assessment at P56 and 24 days later at P80 revealed increased frontal cortical HMGB1, TLR4, and TLR3 in the AIE-treated rats. Adolescent intermittent ethanol treatment did not alter adult spatial learning on the Barnes maze, but did cause reversal learning deficits and increased perseverative behavior. Barnes maze deficits correlated with the expression of danger signal receptors in the PFC. Taken together, these findings provide evidence that adolescent binge drinking leads to persistent upregulation of innate immune danger signaling in the adult PFC that correlates with adult neurocognitive dysfunction

Induction of innate immune genes in brain create the neurobiology of addiction

Addiction occurs through repeated abuse of drugs that progressively reduce behavioral control and cognitive flexibility while increasing limbic negative emotion. Recent discoveries indicate neuroimmune signaling underlies addiction and co-morbid depression. Low threshold microglia undergo progressive stages of innate immune activation involving astrocytes and neurons with repeated drug abuse, stress, and/or cell damage signals. Increased brain NF-κB transcription of proinflammatory chemokines, cytokines, oxidases, proteases, TLR and other genes create loops amplifying NF-κB transcription and innate immune target gene expression. Human post-mortem alcoholic brain has increased NF-κB and NF-κB target gene message, increased microglial markers and chemokine-MCP1. Polymorphisms of human NF-κB1 and other innate immune genes contribute to genetic risk for alcoholism. Animal transgenic and genetic studies link NF-κB innate immune gene expression to alcohol drinking. Human drug addicts show deficits in behavioral flexibility modeled pre-clinically using reversal learning. Binge alcohol, chronic cocaine, and lesions link addiction neurobiology to frontal cortex, neuroimmune signaling and loss of behavioral flexibility. Addiction also involves increasing limbic negative emotion and depression-like behavior that is reflected in hippocampal neurogenesis. Innate immune activation parallels loss of neurogenesis and increased depression-like behavior. Protection against loss of neurogenesis and negative affect by anti-oxidant, anti-inflammatory, anti-depressant, opiate antagonist and abstinence from ethanol dependence link limbic affect to changes in innate immune signaling. The hypothesis that innate immune gene induction underlies addiction and affective disorders creates new targets for therapy

Periadolescent ethanol vapor exposure persistently reduces measures of hippocampal neurogenesis that are associated with behavioral outcomes in adulthood

Excessive alcohol consumption is prevalent among adolescents and may result in lasting neurobehavioral consequences. The use of animal models to study adolescent alcohol exposure has the advantage of allowing for the control necessary in order to evaluate the effects of ethanol on the brain and separate such effects from genetic background and other environmental insults. In the present study the effects of moderate ethanol vapor exposure, during adolescence, on measures of neurogenesis and behavioral measures were evaluated at two different times following ethanol withdrawal, in adulthood. The two groups of Wistar rats were both exposed to intermittent ethanol vapor (14 hrs on/10 hrs off/day) for 35–36 days from PD 23-PD 58 (average blood ethanol concentration (BEC): 163 mg%). In the first group, after rats were withdrawal from vapor they were subsequently assessed for locomotor activity, conflict behavior in the open field, and behaviors in the forced swim test and then sacrificed at 72 days of age. The second group of rats were withdrawn from vapor and injected for 5 days with Bromo-deoxy-Uridine (BrdU). Over the next 8 weeks they were also assessed for locomotor activity, conflict behavior in the open field, and behaviors in the forced swim test and then sacrificed at 113/114 days of age. All rats were perfused for histochemical analyses. Ethanol vapor exposed rats displayed hypoactivity in tests of locomotion and less anxiety-like and/or more “disinhibitory” behavior in the open field conflict. Quantitative analyses of immunoreactivity revealed a significant reduction in measures of neurogenesis, progenitor proliferation, as indexed by doublecortin (DCX), Ki67, and increased markers of cell death as indexed by cleaved caspase-3, and Fluoro-Jade at 72 days, and decreases in doublecortin (DCX), and increases in cleaved caspase-3 at 114 days in the ethanol vapor exposed rats. Progenitor survival, as assessed by BrdU+, was reduced in the vapor exposed animals that were sacrificed at 114 days. The reduction seen in DCX labeled in cell counts was significantly correlated with hypoactivity at 24 hours after withdrawal as well as less anxiety-like and/or more “disinhibitory” behavior in the open field conflict test at 2 and 8 weeks following termination of vapor exposure. These studies demonstrate that behavioral measures of disinhibitory behavior correlated with decreases in neurogenesis are all significantly and persistently impacted by periadolescent ethanol exposure and withdrawal in Wistar rats

NADPH oxidase and reactive oxygen species contribute to alcohol-induced microglial activation and neurodegeneration

<p>Abstract</p> <p>Background</p> <p>Activation of microglia causes the production of proinflammatory factors and upregulation of NADPH oxidase (NOX) that form reactive oxygen species (ROS) that lead to neurodegeneration. Previously, we reported that 10 daily doses of ethanol treatment induced innate immune genes in brain. In the present study, we investigate the effects of chronic ethanol on activation of NOX and release of ROS, and their contribution to ethanol neurotoxicity.</p> <p>Methods</p> <p>Male C57BL/6 and NF-κB enhanced GFP mice were treated intragastrically with water or ethanol (5 g/kg, i.g., 25% ethanol w/v) daily for 10 days. The effects of chronic ethanol on cell death markers (activated caspase-3 and Fluoro-Jade B), microglial morphology, NOX, ROS and NF-κB were examined using real-time PCR, immunohistochemistry and hydroethidine histochemistry. Also, Fluoro-Jade B staining and NOX gp91^phoximmunohistochemistry were performed in the orbitofrontal cortex (OFC) of human postmortem alcoholic brain and human moderate drinking control brain.</p> <p>Results</p> <p>Ethanol treatment of C57BL/6 mice showed increased markers of neuronal death: activated caspase-3 and Fluoro-Jade B positive staining with Neu-N (a neuronal marker) labeling in cortex and dentate gyrus. The OFC of human post-mortem alcoholic brain also showed significantly more Fluoro-Jade B positive cells colocalized with Neu-N, a neuronal marker, compared to the OFC of human moderate drinking control brain, suggesting increased neuronal death in the OFC of human alcoholic brain. Iba1 and GFAP immunohistochemistry showed activated morphology of microglia and astrocytes in ethanol-treated mouse brain. Ethanol treatment increased NF-κB transcription and increased NOX gp91^phoxat 24 hr after the last ethanol treatment that remained elevated at 1 week. The OFC of human postmortem alcoholic brain also had significant increases in the number of gp91^phox+ immunoreactive (IR) cells that are colocalized with neuronal, microglial and astrocyte markers. In mouse brain ethanol increased gp91^phoxexpression coincided with increased production of O₂^-and O₂^-- derived oxidants. Diphenyleneiodonium (DPI), a NOX inhibitor, reduced markers of neurodegeneration, ROS and microglial activation.</p> <p>Conclusions</p> <p>Ethanol activation of microglia and astrocytes, induction of NOX and production of ROS contribute to chronic ethanol-induced neurotoxicity. NOX-ROS and NF-κB signaling pathways play important roles in chronic ethanol-induced neuroinflammation and neurodegeneration.</p

Periadolescent ethanol exposure reduces adult forebrain ChAT+IR neurons: correlation with behavioral pathology

Substance abuse typically begins in adolescence; therefore, the impact of alcohol during this critical time in brain development is of particular importance. Epidemiological data indicate that excessive alcohol consumption is prevalent among adolescents and may have lasting neurobehavioral consequences. Loss of cholinergic input to the forebrain has been demonstrated following fetal alcohol exposure and in adults with Wernicke-Korsakoff syndrome. In the present study, immunohistochemistry for choline acetyltransferase (ChAT) was determined to assess forebrain cholinergic neurons (Ch1–4), and behavioral changes following periadolescent alcohol exposure. Wistar rats were exposed to intermittent ethanol vapor (14 hrs on/10 hrs off/day) for 35 days from PD 22-PD 57 (average blood alcohol concentration (BAC): 163 mg%). Rats were withdrawn from vapor and assessed for locomotor activity, startle response, conflict behavior in the open field, and immobility in the forced swim test, as adults. Rats were then sacrificed at day 71/72 and perfused for histochemical analyses. Ethanol vapor exposed rats displayed: increased locomotor activity 8 hrs after the termination of vapor delivery for that 24 hr period at day 10 and day 20 of alcohol vapor exposure, significant reductions in the amplitude of their responses to prepulse stimuli during the startle paradigm at 24 hrs withdrawal, and at two weeks following withdrawal, less anxiety-like and/or more “disinhibitory” behavior in the open field conflict, and more immobility in the forced swim test. Quantitative analyses of ChAT immunoreactivity revealed a significant reduction in cell counts in the Ch1–2 and Ch3–4 regions of the basal forebrain in ethanol vapor exposed rats. This reduction in cell counts was significantly correlated with less anxiety-like and/or more “disinhibitory” behavior in the open field conflict test. These studies demonstrate that behavioral measures of arousal, affective state, disinhibitory behavior and ChAT+IR, are all significantly impacted by periadolescent ethanol exposure and withdrawal in Wistar rats

The effect of pre-shot routines on golf wedge shot performance

The objective of this study was to examine the effect of pre-performance routines among golfers of low skill and non-golfers on wedge golf shot performance. The intervention strategies involved a physical skill and cognitive-behavioral routine program, as well as a physical skills-only program. Performance was measured on a pre-intervention test, postintervention test, and following a period of time without treatment, and involved wedge shots being played from distances of 40, 50, and 60 m from a target. Participants in this study (N = 68) were assigned to either a golfer or non-golfer group. Participants in the treatment groups attended 2 practice sessions per week during the acquisition phase. A variable practice design was incorporated during the intervention phase. Non-golfers in both intervention groups improved performance following the acquisition phase and maintained these levels of performance in the retention test. Greater improvements in performance were found in the non-golfer physical skills and cognitive-behavioral routine group. The non-golfer physical skills and cognitive-behavioral routine group was the only group to realize significant improvements in performance when comparing initial test performance measures to post-intervention and retention test performance measures across all test distances. Although the golfer treatment groups had consistent improvement in performance measures following the intervention phase, these improvements did not reach statistical significance in the majority of cases

A two-stage genome-wide association study of sporadic amyotrophic lateral sclerosis

The cause of sporadic amyotrophic lateral sclerosis (ALS) is largely unknown, but genetic factors are thought to play a significant role in determining susceptibility to motor neuron degeneration. To identify genetic variants altering risk of ALS, we undertook a two-stage genome-wide association study (GWAS): we followed our initial GWAS of 545 066 SNPs in 553 individuals with ALS and 2338 controls by testing the 7600 most associated SNPs from the first stage in three independent cohorts consisting of 2160 cases and 3008 controls. None of the SNPs selected for replication exceeded the Bonferroni threshold for significance. The two most significantly associated SNPs, rs2708909 and rs2708851 [odds ratio (OR) = 1.17 and 1.18, and P-values = 6.98 x 10–7 and 1.16 x 10–6], were located on chromosome 7p13.3 within a 175 kb linkage disequilibrium block containing the SUNC1, HUS1 and C7orf57 genes. These associations did not achieve genome-wide significance in the original cohort and failed to replicate in an additional independent cohort of 989 US cases and 327 controls (OR = 1.18 and 1.19, P-values = 0.08 and 0.06, respectively). Thus, we chose to cautiously interpret our data as hypothesis-generating requiring additional confirmation, especially as all previously reported loci for ALS have failed to replicate successfully. Indeed, the three loci (FGGY, ITPR2 and DPP6) identified in previous GWAS of sporadic ALS were not significantly associated with disease in our study. Our findings suggest that ALS is more genetically and clinically heterogeneous than previously recognized. Genotype data from our study have been made available online to facilitate such future endeavors

Development and validation of a rapid method for the detection of latrunculol A in plasma

Latrunculol A is a recently discovered 6,7-dihydroxy analog of the potent actin inhibitor latrunculin A. Latrunculol A has exhibited greater cytotoxicity than latrunculin A against both murine and human colon tumor cell lines in vitro. Currently, there are no reports regarding the bioavailability of latrunculol A in vivo. This study was undertaken as a prelude to pharmacokinetic assessments and it is the first work where bioavailability of latrunculol A was studied. In the present work, a simple plasma preparation and a rapid HPLC method have been developed. Mouse plasma containing latrunculol A was first treated by acetonitrile and then centrifuged at 14,000 rpm at 4 °C for 25 min. The supernatant was injected in an HPLC system comprising a Waters Symmetry NH2 column, a mobile phase of acetonitrile/water (95/5, v/v), a flow rate of 1.0 mL/min, at 220 nm. The method was validated by parameters including a good linear correlation, a limit of quantification of 9 ng/mL, and a good precision with a coefficient variation of 1.65, 1.86, and 1.26% for 20, 400, and 800 ng/mL, respectively. With this simple method, excellent separation and sensitivity of latrunculol A are achieved, thus allowing a rapid analysis of the plasma samples for absorption, distribution, and metabolism studies

Discovery of a Dusty Ring in the Coalsack: A Dense Core Caught in the Act of Formation?

We present a new infrared extinction study of Globule 2, the most opaque molecular cloud core in the Coalsack complex. Using deep near-infrared imaging observations obtained with the ESO NTT we are able to examine the structure of the globule in significantly greater detail than previously possible. We find the most prominent structural feature of this globule to be a strong central ring of dust column density which was not evident in lower resolution studies of this cloud. This ring represents a region of high density and pressure that is likely a transient structure. For a spherical cloud geometry the ring would correspond to a dense inner shell of high pressure that could not be in dynamical equilibrium with its surroundings since there appear to be no sources of pressure in the central regions of the cloud that could support the shell against gravity and prevent its inward implosion. The timescale for the inward collapse of the ring would be less than 2 x 10^5 years, suggesting that this globule is in an extremely early stage of evolution, and perhaps caught in the process of forming a centrally condensed dense core or Bok globule. Outside its central regions the globule displays a well-behaved density profile whose shape is very similar to that of a stable Bonnor-Ebert sphere. Using SEST we also obtained a C18O spectrum toward the center of the cloud. The CO observation indicates that the globule is a gravitationally bound object. Analysis of the CO line profile reveals significant non-thermal gas motions likely due to turbulence. As a whole the globule may be evolving to a global state of quasi-static dynamical equilibrium in which thermal and turbulent pressure balance gravity.Comment: Contains 21 pages and 5 figures. Due to space limitations in astroph, figures 1, 3 & 5 are submitted as separate .jpg files. Full resolution figures and text can be downloaded from http://cfa-www.harvard.edu/~clada/preprints.htm