Leon G. Coleman

Adolescent Binge Drinking Alters Adult Brain Neurotransmitter Gene Expression, Behavior, Brain Regional Volumes, and Neurochemistry in Mice

BACKGROUND Binge drinking is common in human adolescents. The adolescent brain is undergoing structural maturation and has a unique sensitivity to alcohol neurotoxicity. Therefore, adolescent binge ethanol may have long-term effects on the adult brain that alter brain structure and behaviors that are relevant to alcohol-use disorders. METHODS To determine whether adolescent ethanol (AE) binge drinking alters the adult brain, male C57BL/6 mice were treated with either water or ethanol during adolescence (5 g/kg/d, i.g., postnatal days P28 to P37) and assessed during adulthood (P60 to P88). An array of neurotransmitter-specific genes, behavioral tests (i.e., reversal learning, prepulse inhibition, and open field), and postmortem brain structure using magnetic resonance imaging (MRI) and immunohistochemistry, were employed to assess persistent alterations in adult brain. RESULTS At P38, 24 hours after AE binge, many neurotransmitter genes, particularly cholinergic and dopaminergic, were reduced by ethanol treatment. Interestingly, dopamine receptor type 4 mRNA was reduced and confirmed using immunohistochemistry. Normal control maturation (P38 to P88) resulted in decreased neurotransmitter mRNA, e.g., an average decrease of 56%. Following AE treatment, adults showed greater gene expression reductions than controls, averaging 73%. Adult spatial learning assessed in the Morris water maze was not changed by AE treatment, but reversal learning experiments revealed deficits. Assessment of adult brain region volumes using MRI indicated that the olfactory bulb and basal forebrain were smaller in adults following AE. Immunohistochemical analyses found reduced basal forebrain area and fewer basal forebrain cholinergic neurons. CONCLUSIONS Adolescent binge ethanol treatment reduces adult neurotransmitter gene expression, particularly cholinergic genes, reduces basal forebrain and olfactory bulb volumes, and causes a reduction in the density of basal forebrain acetylcholine neurons. Loss of cholinergic neurons and forebrain structure could underlie adult reversal learning deficits following adolescent binge drinking.

Adolescent binge ethanol treatment alters adult brain regional volumes, cortical extracellular matrix protein and behavioral flexibility

Adolescents binge drink more than any other age group, increasing risk of disrupting the development of the frontal cortex. We hypothesized that adolescent binge drinking would lead to persistent alterations in adulthood. In this study, we modeled adolescent weekend underage binge-drinking, using adolescent mice (post-natal days [P] 28-37). The adolescent intermittent binge ethanol (AIE) treatment includes 6 binge intragastric doses of ethanol in an intermittent pattern across adolescence. Assessments were conducted in adulthood following extended abstinence to determine if there were persistent changes in adults. Reversal learning, open field and other behavioral assessments as well as brain structure using magnetic imaging and immunohistochemistry were determined. We found that AIE did not impact adult Barnes Maze learning. However, AIE did cause reversal learning deficits in adults. AIE also caused structural changes in the adult brain. AIE was associated with adulthood volume enlargements in specific brain regions without changes in total brain volume. Enlarged regions included the orbitofrontal cortex (OFC, 4%), cerebellum (4.5%), thalamus (2%), internal capsule (10%) and genu of the corpus callosum (7%). The enlarged OFC volume in adults after AIE is consistent with previous imaging studies in human adolescents. AIE treatment was associated with significant increases in the expression of several extracellular matrix (ECM) proteins in the adult OFC including WFA (55%), Brevican (32%), Neurocan (105%), Tenacin-C (25%), and HABP (5%). These findings are consistent with AIE causing persistent changes in brain structure that could contribute to a lack of behavioral flexibility.

Deficits in adult prefrontal cortex neurons and behavior following early post-natal NMDA antagonist treatment

The prefrontal cortex (PFC) is associated with higher cognitive functions including attention and working memory and has been implicated in the regulation of impulsivity as well as the pathology of complex mental illnesses. N-methyl D-aspartate (NMDA) antagonist treatment with dizocilpine induces cell death which is greatest in the frontal cortex on post-natal day seven (P7), however the long-term structural and behavioral effects of this treatment are unknown. This study investigates both the acute neurotoxicity of P7 dizocilpine and the persistent effects of this treatment on pyramidal cells and parvalbumin interneurons in the adult PFC, a brain region involved in the regulation of impulsivity. Dizocilpine treatment on P7 increased cleaved caspase-3 immunoreactivity (IR) in the PFC on P8. In adult mice (P82), P7 dizocilpine treatment resulted in 50% fewer parvalbumin-positive interneurons (p<0.01) and 42% fewer layer V pyramidal neurons (p<0.01) in the PFC. Double immunohistochemistry revealed cleaved caspase-3 IR in both GAD67 IR interneurons and GAD67 (-) neurons. Following dizocilpine treatment at P7, adults showed reduced time in the center of the open field suggesting increased anxiety-like behavior. These findings indicate that early brain insults affecting glutamatergic neurotransmission lead to persistent brain pathology that could contribute to impulsivity and cognitive dysfunction.

Postnatal day 7 ethanol treatment causes persistent reductions in adult mouse brain volume and cortical neurons with sex specific effects on neurogenesis

Ethanol treatment on postnatal day seven (P7) causes robust brain cell death and is a model of late gestational alcohol exposure (Ikonomidou et al., 2000). To investigate the long-term effects of P7 ethanol treatment on adult brain, mice received either two doses of saline or ethanol on P7 (2.5 g/kg, s.c., 2 h apart) and were assessed as adults (P82) for brain volume (using postmortem MRI) and cellular architecture (using immunohistochemistry). Adult mice that received P7 ethanol had reduced MRI total brain volume (4%) with multiple brain regions being reduced in both males and females. Immunohistochemistry indicated reduced frontal cortical parvalbumin immunoreactive (PV + IR) interneurons (18-33%) and reduced Cux1+IR layer II pyramidal neurons (15%) in both sexes. Interestingly, markers of adult hippocampal neurogenesis differed between sexes, with only ethanol treated males showing increased doublecortin and Ki67 expression (52 and 57% respectively) in the dentate gyrus, consistent with increased neurogenesis compared to controls. These findings suggest that P7 ethanol treatment causes persistent reductions in adult brain volume and frontal cortical neurons in both males and females. Increased adult neurogenesis in males, but not females, is consistent with differential adaptive responses to P7 ethanol toxicity between the sexes. One day of ethanol exposure, e.g. P7, causes persistent adult brain dysmorphology.

Toll-like receptor signaling and stages of addiction

BackgroundAthina Markou and her colleagues discovered persistent changes in adult behavior following adolescent exposure to ethanol or nicotine consistent with increased risk for developing addiction. Building on Dr. Markou’s important work and that of others in the field, researchers at the Bowles Center for Alcohol Studies have found that persistent changes in behavior following adolescent stress or alcohol exposure may be linked to induction of immune signaling in brain.AimThis study aims to illuminate the critical interrelationship of the innate immune system (e.g., toll-like receptors [TLRs], high-mobility group box 1 [HMGB1]) in the neurobiology of addiction.MethodThis study reviews the relevant research regarding the relationship between the innate immune system and addiction.ConclusionEmerging evidence indicates that TLRs in brain, particularly those on microglia, respond to endogenous innate immune agonists such as HMGB1 and microRNAs (miRNAs). Multiple TLRs, HMGB1, and miRNAs are induced in the brain by stress, alcohol, and other drugs of abuse and are increased in the postmortem human alcoholic brain. Enhanced TLR-innate immune signaling in brain leads to epigenetic modifications, alterations in synaptic plasticity, and loss of neuronal cell populations, which contribute to cognitive and emotive dysfunctions. Addiction involves progressive stages of drug binges and intoxication, withdrawal-negative affect, and ultimately compulsive drug use and abuse. Toll-like receptor signaling within cortical-limbic circuits is modified by alcohol and stress in a manner consistent with promoting progression through the stages of addiction.

Microglial-derived miRNA let-7 and HMGB1 contribute to ethanol-induced neurotoxicity via TLR7

BackgroundToll-like receptor (TLR) signaling is emerging as an important component of neurodegeneration. TLR7 senses viral RNA and certain endogenous miRNAs to initiate innate immune responses leading to neurodegeneration. Alcoholism is associated with hippocampal degeneration, with preclinical studies linking ethanol-induced neurodegeneration with central innate immune induction and TLR activation. The endogenous miRNA let-7b binds TLR7 to cause neurodegeneration.MethodsTLR7 and other immune markers were assessed in postmortem human hippocampal tissue that was obtained from the New South Wales Tissue Bank. Rat hippocampal-entorhinal cortex (HEC) slice culture was used to assess specific effects of ethanol on TLR7, let-7b, and microvesicles.ResultsWe report here that hippocampal tissue from postmortem human alcoholic brains shows increased expression of TLR7 and increased microglial activation. Using HEC slice culture, we found that ethanol induces TLR7 and let-7b expression. Ethanol caused TLR7-associated neuroimmune gene induction and initiated the release let-7b in microvesicles (MVs), enhancing TLR7-mediated neurotoxicity. Further, ethanol increased let-7b binding to the danger signaling molecule high mobility group box-1 (HMGB1) in MVs, while reducing let-7 binding to classical chaperone protein argonaute (Ago2). Flow cytometric analysis of MVs from HEC media and analysis of MVs from brain cell culture lines found that microglia were the primary source of let-7b and HMGB1-containing MVs.ConclusionsOur results identify that ethanol induces neuroimmune pathology involving the release of let-7b/HMGB1 complexes in microglia-derived microvesicles. This contributes to hippocampal neurodegeneration and may play a role in the pathology of alcoholism.

Innate Immune Signaling and Alcohol Use Disorders

Innate immune signaling is an important feature in the pathology of alcohol use disorders. Alcohol abuse causes persistent innate immune activation in the brain. This is seen in postmortem human alcoholic brain specimens, as well as in primate and rodent models of alcohol consumption. Further, in vitro models of alcohol exposure in neurons and glia also demonstrate innate immune activation. The activation of the innate immune system seems to be important in the development of alcohol use pathology, as anti-immune therapies reduce pathology and ethanol self-administration in rodent models. Further, innate immune activation has been identified in each of the stages of addiction: binge/intoxication, withdrawal/negative affect, and preoccupation/craving. This suggests that innate immune activation may play a role both in the development and maintenance of alcoholic pathology. In this chapter, we discuss the known contributions of innate immune signaling in the pathology of alcohol use disorders, and present potential therapeutic interventions that may be beneficial for alcohol use disorders.

HMGB1/IL-1β complexes in plasma microvesicles modulate immune responses to burn injury

Modulating immune responses to sepsis and trauma remain one of the most difficult challenges in modern medicine. Large burn injuries (LBI) are a severe form of trauma associated with sepsis, immune impairment, and mortality. Immune dysfunction after LBI is complex, involving both enhanced and impaired immune activation. The release of Damage-Associated Molecular Patterns (DAMPs), such as HMGB1, and cytokines (e.g. IL-1β) creates an environment of immune dysfunction often leading to end organ failure and death. Both HMGB1 and IL-1β have been found to play critical roles in sepsis and post-burn immune dysfunction. HMGB1 and IL-1β have been shown previously to form potent complexes in vitro. We recently identified the presence of HMGB1/IL-1β heterocomplexes in human tissue. We now find HMGB1/IL-1β complexes in human and mouse plasma, and identify a synergistic role of HMGB1/IL-1β complexes in post-burn immune dysfunction. In both humans and mice, we found that HMGB1 was enriched in plasma microvesicles (MVs) after LBI. HMGB1 was found form complexes with IL-1β. Using flow cytometry of mouse plasma MVs, we identified an increase in an HMGB1+/IL-1β+ MVs. Using co-IP, HMGB1 was found to bind the pro-form of IL-1β in mouse and human plasma. Pro-IL-1β, which is traditionally considered inactive, became active when complexed with HMGB1. Human THP-1 monocytes treated with HMGB1-pro-IL-1β complexes showed increased transcription of LBI associated cytokines IL-6 and IFNβ along with suppression of iNOS, mimicking findings associated with LBI. These findings identify that HMGB1/IL-1β complexes released after burn injuries can modulate immune responses, and microvesicles are identified as a novel reservoir for these immune mediators. These complexes might serve as novel immune targets for the treatment of systemic immune responses due to LBI or other causes of sepsis.

HMGB1/IL-1β complexes regulate neuroimmune responses in alcoholism

Neuroimmune activation is a key feature of the pathologies of numerous psychiatric disorders including alcoholism, depression, and anxiety. Both HMGB1 and IL-1β have been implicated in brain disorders. Previous studies find HMGB1 andIL-1β form heterocomplexes in vitro with enhanced immune responses, lead to our hypothesis that HMGB1 and IL-1β heterocomplexes formed in vivo to contribute to the pathology of alcoholism. HMGB1/IL-1β heterocomplexes were prepared in vitro and found to potentiate IL-1β receptor proinflammatory gene induction compared to IL-1β alone in hippocampal brain slice culture. These HMGB1/IL-1β complexes were found to be increased in post-mortem human alcoholic hippocampus by co-immunoprecipiation. In mice, acute binge ethanol induced both HMGB1 and IL-1β in the brain and plasma. HMGB1 and IL-1β complexes were found only in mouse brain, with confocal microscopy revealing an ethanol-induced HMGB1 and IL-1β cytoplasmic co-localization. Surprisingly, IL-1β was found primarily in neurons. Studies in hippocampal brain slice culture found ethanol increased HMGB1/IL-1β complexes in the media. These studies suggest a novel neuroimmune mechanism in the pathology of alcoholism. Immunogenic HMGB1/IL-1β complexes represent a novel target for immune modulatory therapy in alcohol use disorders, and should be investigated in other psychiatric diseases that involve a neuroimmune component.