Adaptor protein complex 2 in the orbitofrontal cortex predicts alcohol use disorder.

Alcohol use disorder (AUD) is a life-threatening disease characterized by compulsive drinking, cognitive deficits, and social impairment that continue despite negative consequences. The inability of individuals with AUD to regulate drinking may involve functional deficits in cortical areas that normally balance actions that have aspects of both reward and risk. Among these, the orbitofrontal cortex (OFC) is critically involved in goal-directed behavior and is thought to maintain a representation of reward value that guides decision making. In the present study, we analyzed post-mortem OFC brain samples collected from age- and sex-matched control subjects and those with AUD using proteomics, bioinformatics, machine learning, and reverse genetics approaches. Of the 4,500+ total unique proteins identified in the proteomics screen, there were 47 proteins that differed significantly by sex that were enriched in processes regulating extracellular matrix and axonal structure. Gene ontology enrichment analysis revealed that proteins differentially expressed in AUD cases were involved in synaptic and mitochondrial function, as well as transmembrane transporter activity. Alcohol-sensitive OFC proteins also mapped to abnormal social behaviors and social interactions. Machine learning analysis of the post-mortem OFC proteome revealed dysregulation of presynaptic (e.g., AP2A1) and mitochondrial proteins that predicted the occurrence and severity of AUD. Using a reverse genetics approach to validate a target protein, we found that prefrontal Ap2a1 expression significantly correlated with voluntary alcohol drinking in male and female genetically diverse mouse strains. Moreover, recombinant inbred strains that inherited the C57BL/6J allele at the Ap2a1 interval consumed higher amounts of alcohol than those that inherited the DBA/2J allele. Together, these findings highlight the impact of excessive alcohol consumption on the human OFC proteome and identify important cross-species cortical mechanisms and proteins that control drinking in individuals with AUD.

The histone methyltransferase G9a mediates stress-regulated alcohol drinking.

The epigenetic enzyme G9a is a histone methyltransferase that dimethylates lysine 9 on histone H3 (H3K9me2), and in the adult nucleus accumbens (NAc), G9a regulates multiple behaviors associated with substance use disorder. We show here that chronic intermittent ethanol (CIE) exposure in male mice reduced both G9a and H3K9me2 levels in the adult NAc, but not dorsal striatum. Viral-mediated reduction of G9a in the NAc had no effects on baseline volitional ethanol drinking or escalated alcohol drinking produced by CIE exposure; however, NAc G9a was required for stress-regulated changes in ethanol drinking, including potentiated alcohol drinking produced by activation of the kappa-opioid receptor. In addition, we observed that chronic systemic administration of a G9a inhibitor, UNC0642, also blocked stress-potentiated alcohol drinking. Together, our findings suggest that chronic alcohol use, similar to other abused substances, produces a NAc-selective reduction in G9a levels that serves to limit stress-regulated alcohol drinking. Moreover, our findings suggest that pharmacological inhibition of G9a might provide a novel therapeutic approach to treat stress-induced alcohol drinking, which is a major trigger of relapse in individuals suffering from AUD.

Adolescent Intermittent Ethanol (AIE) Enhances the Dopaminergic Response to Ethanol within the Mesolimbic Pathway during Adulthood: Alterations in Cholinergic/Dopaminergic Genes Expression in the Nucleus Accumbens Shell.

A consistent preclinical finding is that exposure to alcohol during adolescence produces a persistent hyperdopaminergic state during adulthood. The current experiments determine that effects of Adolescent Intermittent Ethanol (AIE) on the adult neurochemical response to EtOH administered directly into the mesolimbic dopamine system, alterations in dendritic spine and gene expression within the nucleus accumbens shell (AcbSh), and if treatment with the HDACII inhibitor TSA could normalize the consequences of AIE. Rats were exposed to the AIE (4 g/kg ig; 3 days a week) or water (CON) during adolescence, and all testing occurred during adulthood. CON and AIE rats were microinjected with EtOH directly into the posterior VTA and dopamine and glutamate levels were recorded in the AcbSh. Separate groups of AIE and CON rats were sacrificed during adulthood and Taqman arrays and dendritic spine morphology assessments were performed. The data indicated that exposure to AIE resulted in a significant leftward and upward shift in the dose-response curve for an increase in dopamine in the AcbSh following EtOH microinjection into the posterior VTA. Taqman array indicated that AIE exposure affected the expression of target genes (Chrna7, Impact, Chrna5). The data indicated no alterations in dendritic spine morphology in the AcbSh or any alteration in AIE effects by TSA administration. Binge-like EtOH exposure during adolescence enhances the response to acute ethanol challenge in adulthood, demonstrating that AIE produces a hyperdopaminergic mesolimbic system in both male and female Wistar rats. The neuroadaptations induced by AIE in the AcbSh could be part of the biological basis of the observed negative consequences of adolescent binge-like alcohol exposure on adult drug self-administration behaviors.

The Abused Inhalant Toluene Impairs Medial Prefrontal Cortex Activity and Risk/Reward Decision-Making during a Probabilistic Discounting Task.

Inhalant (e.g., toluene) misuse is linked to behavioral and cognitive deficits in humans, yet preclinical studies of the effect of inhalants on higher-order cognition are limited. We addressed this gap in the literature by examining the effect of toluene vapor exposure on risk/reward decision-making in male and female Sprague-Dawley rats using a probabilistic discounting task. In this task, rodents chose a risky/large reward or a safe/small reward, with the odds of risky reinforcement descending or ascending throughout the test session. We observed a dose-dependent, sex-independent deficit in behavioral flexibility during probabilistic discounting caused by acute toluene exposure. Rats exposed to toluene vapor during adolescence and tested as adults performed comparably to air-treated controls and were susceptible to the effects of an acute toluene challenge. These behavioral flexibility deficits observed suggests dysfunctional medial prefrontal cortex (mPFC) activity. To address this hypothesis, we virally expressed the genetically encoded calcium sensor GCaMP6f in glutamatergic mPFC neurons and monitored calcium transients in real-time using in vivo fiber photometry. mPFC activity peaked before either lever press during free-choice trials in toluene- and air-treated animals. During forced-choice trials, GCaMP6f transients shifted from pre-risky to pre-safe choice, an effect mitigated by acute toluene exposure. mPFC activity decreased during rewarded trials, with larger decreases following risky/large wins compared with safe/small wins. Toluene-treated animals also had decreased mPFC activity during rewarded trials, but there was no distinction between risky/large wins and safe/small wins. These results provide physiological evidence for mPFC-dependent behavioral deficits caused by toluene.SIGNIFICANCE STATEMENT Inhalants (e.g., toluene) are an understudied class of drugs of abuse that cause devastating behavioral and cognitive deficits in humans. Understanding the neurobiological interactions of toluene vapor using animal models is important for developing effective treatment strategies for inhalant addicts. Here we find that toluene vapor reduces behavioral flexibility in rodents making risk/reward-based decisions. The medial prefrontal cortex (mPFC) drives behavioral flexibility during this type of decision-making and we show that toluene reduces the ability of mPFC neurons to track optimal choices as reward probabilities change. Toluene also reduces these neurons' ability to distinguish between small and large rewards. A combination of these factors likely leads to the impaired performance in probabilistic discounting following acute toluene exposure.

Dynamic c-Fos changes in mouse brain during acute and protracted withdrawal from chronic intermittent ethanol exposure and relapse drinking.

Alcohol dependence promotes neuroadaptations in numerous brain areas, leading to escalated drinking and enhanced relapse vulnerability. We previously developed a mouse model of ethanol dependence and relapse drinking in which repeated cycles of chronic intermittent ethanol (CIE) vapor exposure drive a significant escalation of voluntary ethanol drinking. In the current study, we used this model to evaluate changes in neuronal activity (as indexed by c-Fos expression) throughout acute and protracted withdrawal from CIE (combined with or without a history of ethanol drinking). We analyzed c-Fos protein expression in 29 brain regions in mice sacrificed 2, 10, 26, and 74 hours or 7 days after withdrawal from 5 cycles of CIE. Results revealed dynamic time- and brain region-dependent changes in c-Fos activity over the time course of withdrawal from CIE exposure, as compared with nondependent air-exposed control mice, beginning with markedly low expression levels upon removal from the ethanol vapor chambers (2 hours), reflecting intoxication. c-Fos expression was enhanced during acute CIE withdrawal (10 and 26 hours), followed by widespread reductions at the beginning of protracted withdrawal (74 hours) in several brain areas. Persistent reductions in c-Fos expression were observed during prolonged withdrawal (7 days) in prelimbic cortex, nucleus accumbens shell, dorsomedial striatum, paraventricular nucleus of thalamus, and ventral subiculum. A history of ethanol drinking altered acute CIE withdrawal effects and caused widespread reductions in c-Fos that persisted during extended abstinence even without CIE exposure. These data indicate that ethanol dependence and relapse drinking drive long-lasting neuroadaptations in several brain regions.

Mechanisms of Persistent Neurobiological Changes Following Adolescent Alcohol Exposure: NADIA Consortium Findings.

The Neurobiology of Adolescent Drinking in Adulthood (NADIA) Consortium has focused on the impact of adolescent binge drinking on brain development, particularly on effects that persist into adulthood. Adolescent binge drinking is common, and while many factors contribute to human brain development and alcohol use during adolescence, animal models are critical for understanding the specific consequences of alcohol exposure during this developmental period and the underlying mechanisms. Using adolescent intermittent ethanol (AIE) exposure models, NADIA investigators identified long-lasting AIE-induced changes in adult behavior that are consistent with observations in humans, such as increased alcohol drinking, increased anxiety (particularly social anxiety), increased impulsivity, reduced behavioral flexibility, impaired memory, disrupted sleep, and altered responses to alcohol. These behavioral changes are associated with multiple molecular, cellular, and physiological alterations in the brain that persist long after AIE exposure. At the molecular level, AIE results in long-lasting changes in neuroimmune/trophic factor balance and epigenetic-microRNA (miRNA) signaling across glia and neurons. At the cellular level, AIE history is associated in adulthood with reduced expression of cholinergic, serotonergic, and dopaminergic neuron markers, attenuated cortical thickness, decreased neurogenesis, and altered dendritic spine and glial morphology. This constellation of molecular and cellular adaptations to AIE likely contributes to observed alterations in neurophysiology, measured by synaptic physiology, EEG patterns, and functional connectivity. Many of these AIE-induced brain changes replicate findings seen in postmortem brains of humans with alcohol use disorder (AUD). NADIA researchers are now elucidating mechanisms of these adaptations. Emerging data demonstrate that exercise, antiinflammatory drugs, anticholinesterases, histone deacetylase inhibitors, and other pharmacological compounds are able to prevent (administered during AIE) and/or reverse (given after AIE) AIE-induced pathology in adulthood. These studies support hypotheses that adolescent binge drinking increases risk of adult hazardous drinking and influences brain development, and may provide insight into novel therapeutic targets for AIE-induced neuropathology and AUDs.

Prefrontal Cortex KCa2 Channels Regulate mGlu5-Dependent Plasticity and Extinction of Alcohol-Seeking Behavior.

Identifying novel treatments that facilitate extinction learning could enhance cue-exposure therapy and reduce high relapse rates in alcoholics. Activation of mGlu5 receptors in the infralimbic prefrontal cortex (IL-PFC) facilitates learning during extinction of cue-conditioned alcohol-seeking behavior. Small-conductance calcium-activated potassium (KCa2) channels have also been implicated in extinction learning of fear memories, and mGlu5 receptor activation can reduce KCa2 channel function. Using a combination of electrophysiological, pharmacological, and behavioral approaches, this study examined KCa2 channels as a novel target to facilitate extinction of alcohol-seeking behavior in rats. This study also explored related neuronal and synaptic mechanisms within the IL-PFC that underlie mGlu5-dependent enhancement of extinction learning. Using whole-cell patch-clamp electrophysiology, activation of mGlu5 in ex vivo slices significantly reduced KCa2 channel currents in layer V IL-PFC pyramidal neurons, confirming functional downregulation of KCa2 channel activity by mGlu5 receptors. Additionally, positive modulation of KCa2 channels prevented mGlu5 receptor-dependent facilitation of long-term potentiation in the IL-PFC. Systemic and intra-IL-PFC treatment with apamin (KCa2 channel allosteric inhibitor) significantly enhanced extinction of alcohol-seeking behavior across multiple extinction sessions, an effect that persisted for 3 weeks, but was not observed after apamin microinfusions into the prelimbic PFC. Positive modulation of IL-PFC KCa2 channels significantly attenuated mGlu5-dependent facilitation of alcohol cue-conditioned extinction learning. These data suggest that mGlu5-dependent facilitation of extinction learning and synaptic plasticity in the IL-PFC involves functional inhibition of KCa2 channels. Moreover, these findings demonstrate that KCa2 channels are a novel target to facilitate long-lasting extinction of alcohol-seeking behavior.SIGNIFICANCE STATEMENT Alcohol use disorder is a chronic relapsing disorder that is associated with compulsive alcohol-seeking behavior. One of the main causes of alcohol relapse is the craving caused by environmental cues that are associated with alcohol. These cues are formed by normal learning and memory principles, and the understanding of the brain mechanisms that help form these associations can lead to the development of drugs and/or behavior therapies that reduce the impact that these cues have on relapse in alcoholics.

Orbitofrontal Neuroadaptations and Cross-Species Synaptic Biomarkers in Heavy-Drinking Macaques.

Cognitive impairments, uncontrolled drinking, and neuropathological cortical changes characterize alcohol use disorder. Dysfunction of the orbitofrontal cortex (OFC), a critical cortical subregion that controls learning, decision-making, and prediction of reward outcomes, contributes to executive cognitive function deficits in alcoholic individuals. Electrophysiological and quantitative synaptomics techniques were used to test the hypothesis that heavy drinking produces neuroadaptations in the macaque OFC. Integrative bioinformatics and reverse genetic approaches were used to identify and validate synaptic proteins with novel links to heavy drinking in BXD mice. In drinking monkeys, evoked firing of OFC pyramidal neurons was reduced, whereas the amplitude and frequency of postsynaptic currents were enhanced compared with controls. Bath application of alcohol reduced evoked firing in neurons from control monkeys, but not drinking monkeys. Profiling of the OFC synaptome identified alcohol-sensitive proteins that control glutamate release (e.g., SV2A, synaptogyrin-1) and postsynaptic signaling (e.g., GluA1, PRRT2) with no changes in synaptic GABAergic proteins. Western blot analysis confirmed the increase in GluA1 expression in drinking monkeys. An exploratory analysis of the OFC synaptome found cross-species genetic links to alcohol intake in discrete proteins (e.g., C2CD2L, DIRAS2) that discriminated between low- and heavy-drinking monkeys. Validation studies revealed that BXD mouse strains with the D allele at the C2cd2l interval drank less alcohol than B allele strains. Thus, by profiling of the OFC synaptome, we identified changes in proteins controlling glutamate release and postsynaptic signaling and discovered several proteins related to heavy drinking that have potential as novel targets for treating alcohol use disorder.SIGNIFICANCE STATEMENT Clinical research identified cognitive deficits in alcoholic individuals as a risk factor for relapse, and alcoholic individuals display deficits on cognitive tasks that are dependent upon the orbitofrontal cortex (OFC). To identify neurobiological mechanisms that underpin OFC dysfunction, this study used electrophysiology and integrative synaptomics in a translational nonhuman primate model of heavy alcohol consumption. We found adaptations in synaptic proteins that control glutamatergic signaling in chronically drinking monkeys. Our functional genomic exploratory analyses identified proteins with genetic links to alcohol and cocaine intake across mice, monkeys, and humans. Future work is necessary to determine whether targeting these novel targets reduces excessive and harmful levels of alcohol drinking.

Donepezil Reverses Dendritic Spine Morphology Adaptations and Fmr1 Epigenetic Modifications in Hippocampus of Adult Rats After Adolescent Alcohol Exposure.

BACKGROUND: Adolescent intermittent ethanol (AIE) exposure produces persistent impairments in cholinergic and epigenetic signaling and alters markers of synapses in the hippocampal formation, effects that are thought to drive hippocampal dysfunction in adult rodents. Donepezil (Aricept), a cholinesterase inhibitor, is used clinically to ameliorate memory-related cognitive deficits. Given that donepezil also prevents morphological impairment in preclinical models of neuropsychiatric disorders, we investigated the ability of donepezil to reverse morphological and epigenetic adaptations in the hippocampus of adult rats exposed to AIE. Because of the known relationship between dendritic spine density and morphology with the fragile X mental retardation 1 (Fmr1) gene, we also assessed Fmr1 expression and its epigenetic regulation in hippocampus after AIE and donepezil pretreatment. METHODS: Adolescent rats were administered intermittent ethanol for 16 days starting on postnatal day 30. Rats were treated with donepezil (2.5 mg/kg) once a day for 4 days starting 20 days after the completion of AIE exposure. Brains were dissected out after the fourth donepezil dose, and spine analysis was completed in dentate gyrus granule neurons. A separate cohort of rats, treated identically, was used for molecular studies. RESULTS: AIE exposure significantly reduced dendritic spine density and altered morphological characteristics of subclasses of dendritic spines. AIE exposure also increased mRNA levels and H3-K27 acetylation occupancy of the Fmr1 gene in hippocampus. Treatment of AIE-exposed adult rats with donepezil reversed both the dendritic spine adaptations and epigenetic modifications and expression of Fmr1. CONCLUSIONS: These findings indicate that AIE produces long-lasting decreases in dendritic spine density and changes in Fmr1 gene expression in the hippocampal formation, suggesting morphological and epigenetic mechanisms underlying previously reported behavioral deficits after AIE. The reversal of these effects by subchronic, post-AIE donepezil treatment indicates that these AIE effects can be reversed by up-regulating cholinergic function.

Chronic Alcohol, Intrinsic Excitability, and Potassium Channels: Neuroadaptations and Drinking Behavior.

Neural mechanisms underlying alcohol use disorder remain elusive, and this lack of understanding has slowed the development of efficacious treatment strategies for reducing relapse rates and prolonging abstinence. While synaptic adaptations produced by chronic alcohol exposure have been extensively characterized in a variety of brain regions, changes in intrinsic excitability of critical projection neurons are understudied. Accumulating evidence suggests that prolonged alcohol drinking and alcohol dependence produce plasticity of intrinsic excitability as measured by changes in evoked action potential firing and after-hyperpolarization amplitude. In this chapter, we describe functional changes in cell firing of projection neurons after long-term alcohol exposure that occur across species and in multiple brain regions. Adaptations in calcium-activated (KCa2), voltage-dependent (KV7), and G protein-coupled inwardly rectifying (Kir3 or GIRK) potassium channels that regulate the evoked firing and after-hyperpolarization parallel functional changes in intrinsic excitability induced by chronic alcohol. Moreover, there are strong genetic links between alcohol-related behaviors and genes encoding KCa2, KV7, and GIRK channels, and pharmacologically targeting these channels reduces alcohol consumption and alcohol-related behaviors. Together, these studies demonstrate that chronic alcohol drinking produces adaptations in KCa2, KV7, and GIRK channels leading to impaired regulation of the after-hyperpolarization and aberrant cell firing. Correcting the deficit in the after-hyperpolarization with positive modulators of KCa2 and KV7 channels and altering the GIRK channel binding pocket to block the access of alcohol represent a potentially highly effective pharmacological approach that can restore changes in intrinsic excitability and reduce alcohol consumption in affected individuals.

Correction to: Chronic Alcohol, Intrinsic Excitability, and Potassium Channels: Neuroadaptations and Drinking Behavior.

In section 8.1 on the 10th line in first paragraph the reference citation Mateos-Aparicio et al. 2014 is incorrect.

FMRP Mediates Chronic Ethanol-Induced Changes in NMDA, Kv4.2, and KChIP3 Expression in the Hippocampus.

BACKGROUND: Exposure to chronic ethanol (EtOH) results in changes in the expression of proteins that regulate neuronal excitability. This study examined whether chronic EtOH alters the hippocampal expression and function of fragile X mental retardation protein (FMRP) and the role of FMRP in the modulation of chronic EtOH-induced changes in the expression of NMDA receptors and Kv4.2 channels. METHODS: For in vivo studies, C57BL/6J mice underwent a chronic intermittent EtOH (CIE) vapor exposure procedure. After CIE, hippocampal tissue was collected and subjected to immunoblot blot analysis of NMDA receptor subunits (GluN1, GluN2B), Kv4.2, and its accessory protein KChIP3. For in vitro studies, hippocampal slice cultures were exposed to 75 mM EtOH for 8 days. Following EtOH exposure, mRNAs bound to FMRP was measured. In a separate set of studies, cultures were exposed to an inhibitor of S6K1 (PF-4708671 [PF], 6 µM) in order to assess whether EtOH-induced homeostatic changes in protein expression depend upon changes in FMRP activity. RESULTS: Immunoblot blot analysis revealed increases in GluN1 and GluN2B but reductions in Kv4.2 and KChIP3. Analysis of mRNAs bound to FMRP revealed a similar bidirectional change observed as reduction of GluN2B and increase in Kv4.2 and KChIP3 mRNA transcripts. Analysis of FMRP further revealed that while chronic EtOH did not alter the expression of FMRP, it significantly increased phosphorylation of FMRP at the S499 residue that is known to critically regulate its activity. Inhibition of S6K1 prevented the chronic EtOH-induced increase in phospho-FMRP and changes in NMDA subunits, Kv4.2, and KChIP3. In contrast, PF had no effect in the absence of alcohol, indicating it was specific for the chronic EtOH-induced changes. CONCLUSIONS: These findings demonstrate that chronic EtOH exposure enhances translational control of plasticity-related proteins by FMRP, and that S6K1 and FMRP activities are required for expression of chronic EtOH-induced homeostatic plasticity at glutamatergic synapses in the hippocampus.

Chronic intermittent ethanol exposure and withdrawal leads to adaptations in nucleus accumbens core postsynaptic density proteome and dendritic spines.

Alcohol use disorder is a chronic relapsing brain disease characterized by the loss of ability to control alcohol (ethanol) intake despite knowledge of detrimental health or personal consequences. Clinical and pre-clinical models provide strong evidence for chronic ethanol-associated alterations in glutamatergic signaling and impaired synaptic plasticity in the nucleus accumbens (NAc). However, the neural mechanisms that contribute to aberrant glutamatergic signaling in ethanol-dependent individuals in this critical brain structure remain unknown. Using an unbiased proteomic approach, we investigated the effects of chronic intermittent ethanol (CIE) exposure on neuroadaptations in postsynaptic density (PSD)-enriched proteins in the NAc of ethanol-dependent mice. Compared with controls, CIE exposure significantly changed expression levels of 50 proteins in the PSD-enriched fraction. Systems biology and functional annotation analyses demonstrated that the dysregulated proteins are expressed at tetrapartite synapses and critically regulate cellular morphology. To confirm this latter finding, the density and morphology of dendritic spines were examined in the NAc core of ethanol-dependent mice. We found that CIE exposure and withdrawal differentially altered dendrite diameter and dendritic spine density and morphology. Through the use of quantitative proteomics and functional annotation, these series of experiments demonstrate that ethanol dependence produces neuroadaptations in proteins that modify dendritic spine morphology. In addition, these studies identified novel PSD-related proteins that contribute to the neurobiological mechanisms of ethanol dependence that drive maladaptive structural plasticity of NAc neurons.

Kv7 channels in the nucleus accumbens are altered by chronic drinking and are targets for reducing alcohol consumption.

Alcohol use disorders (AUDs) are a major public health issue and produce enormous societal and economic burdens. Current Food and Drug Administration (FDA)-approved pharmacotherapies for treating AUDs suffer from deleterious side effects and are only effective in a subset of individuals. It is therefore essential to find improved medications for the management of AUDs. Emerging evidence suggests that anticonvulsants are a promising class of drugs for treating individuals with AUDs. In these studies, we used integrative functional genomics to demonstrate that genes that encode Kv7 channels (i.e. Kcnq2/3) are related to alcohol (ethanol) consumption, preference and acceptance in rodents. We then tested the ability of the FDA-approved anticonvulsant retigabine, a Kv7 channel opener, to reduce voluntary ethanol consumption of Wistar rats in a two-bottle choice intermittent alcohol access paradigm. Systemic administration and microinjections of retigabine into the nucleus accumbens significantly reduced alcohol drinking, and retigabine was more effective at reducing intake in high- versus low-drinking populations of Wistar rats. Prolonged voluntary drinking increased the sensitivity to the proconvulsant effects of pharmacological blockade of Kv7 channels and altered surface trafficking and SUMOylation patterns of Kv7.2 channels in the nucleus accumbens. These data implicate Kcnq2/3 in the regulation of ethanol drinking and demonstrate that long-term drinking produces neuroadaptations in Kv7 channels. In addition, these results have identified retigabine as a potential pharmacotherapy for treating AUDs and Kv7 channels as a novel therapeutic target for reducing heavy drinking.

Chronic alcohol disrupts dopamine receptor activity and the cognitive function of the medial prefrontal cortex.

Dopamine (DA) receptors in the medial prefrontal cortex (mPFC) exert powerful effects on cognition by modulating the balance between excitatory and inhibitory neurotransmission. The present study examined the impact of chronic intermittent ethanol (CIE) exposure on cognitive function and DA receptor-mediated neurotransmission in the rat mPFC. Consistent with alterations in executive function in alcoholics, CIE-exposed rats exhibited deficits in behavioral flexibility in an operant set-shifting task. Since alterations in dopaminergic neurotransmission in the mPFC have been implicated in a number of behavioral disorders including addiction, studies were then performed in the adult acute slice preparation to examine changes in DA receptor function in the mPFC following CIE exposure. In slices obtained from control rats, DA receptor stimulation was observed to exert complex actions on neuronal firing and synaptic neurotransmission that were not only dependent upon the particular receptor subtype but also whether it was a pyramidal cell or a fast-spiking interneuron. In contrast to slices from control rats, there was a near complete loss of the modulatory actions of D2/D4 receptors on cell firing and neurotransmission in slices obtained immediately, 1 and 4 weeks after the last day of CIE exposure. This loss did not appear to be associated with changes in receptor expression. In contrast, CIE exposure did not alter D1 receptor function or mGluR1 modulation of firing. These studies are consistent with the suggestion that chronic alcohol exposure disrupts cognitive function at least in part through disruption of D2 and D4 receptor signaling in mPFC.

Optogenetic stimulation of MCH neurons increases sleep.

Melanin concentrating hormone (MCH) is a cyclic neuropeptide present in the hypothalamus of all vertebrates. MCH is implicated in a number of behaviors but direct evidence is lacking. To selectively stimulate the MCH neurons the gene for the light-sensitive cation channel, channelrhodopsin-2, was inserted into the MCH neurons of wild-type mice. Three weeks later MCH neurons were stimulated for 1 min every 5 min for 24 h. A 10 Hz stimulation at the start of the night hastened sleep onset, reduced length of wake bouts by 50%, increased total time in non-REM and REM sleep at night, and increased sleep intensity during the day cycle. Sleep induction at a circadian time when all of the arousal neurons are active indicates that MCH stimulation can powerfully counteract the combined wake-promoting signal of the arousal neurons. This could be potentially useful in treatment of insomnia.

Adolescent alcohol exposure alters GABAA receptor subunit expression in adult hippocampus.

BACKGROUND: The long-term consequences of adolescent alcohol abuse that persist into adulthood are poorly understood and have not been widely investigated. We have shown that intermittent exposure to alcohol during adolescence decreased the amplitude of GABAA receptor (GABAA R)-mediated tonic currents in hippocampal dentate granule cells in adulthood. The aim of this study was to investigate the enduring effects of chronic intermittent alcohol exposure during adolescence or adulthood on the expression of hippocampal GABAA Rs. METHODS: We used a previously characterized tissue fractionation method to isolate detergent resistant membranes and soluble fractions, followed by Western blots to measure GABAA R protein expression. We also measured mRNA levels of GABAA R subunits using quantitative real-time polymerase chain reaction. RESULTS: Although the protein levels of α1-, α4-, and δ-GABAA R subunits remained stable between postnatal day (PD) 30 (early adolescence) and PD71 (adulthood), the α5-GABAA R subunit was reduced across that period. In rats that were subjected to adolescent intermittent ethanol (AIE) exposure between PD30 and PD46, there was a significant reduction in the protein levels of the δ-GABAA R, in the absence of any changes in mRNA levels, at 48 hours and 26 days after the last ethanol (EtOH) exposure. Protein levels of the α4-GABAA R subunit were significantly reduced, but mRNA levels were increased, 26 days (but not 48 hours) after the last AIE exposure. Protein levels of α5-GABAA R were not changed by AIE, but mRNA levels were reduced at 48 hours but normalized 26 days after AIE. In contrast to the effects of AIE, chronic intermittent ethanol (CIE) exposure during adulthood had no effect on expression of any of the GABAA R subunits examined. CONCLUSIONS: AIE produced both short- and long-term alterations of GABAA R subunits mRNA and protein expression in the hippocampus, whereas CIE produced no long-lasting effects on those measures. The observed reduction of protein levels of the δ-GABAA R, specifically, is consistent with previously reported altered hippocampal GABAA R-mediated electrophysiological responses after AIE. The absence of effects of CIE underscores the emerging view of adolescence as a time of distinctive vulnerability to the enduring effects of repeated EtOH exposure.

Stream denitrification across biomes and its response to anthropogenic nitrate loading.

Anthropogenic addition of bioavailable nitrogen to the biosphere is increasing and terrestrial ecosystems are becoming increasingly nitrogen-saturated, causing more bioavailable nitrogen to enter groundwater and surface waters. Large-scale nitrogen budgets show that an average of about 20-25 per cent of the nitrogen added to the biosphere is exported from rivers to the ocean or inland basins, indicating that substantial sinks for nitrogen must exist in the landscape. Streams and rivers may themselves be important sinks for bioavailable nitrogen owing to their hydrological connections with terrestrial systems, high rates of biological activity, and streambed sediment environments that favour microbial denitrification. Here we present data from nitrogen stable isotope tracer experiments across 72 streams and 8 regions representing several biomes. We show that total biotic uptake and denitrification of nitrate increase with stream nitrate concentration, but that the efficiency of biotic uptake and denitrification declines as concentration increases, reducing the proportion of in-stream nitrate that is removed from transport. Our data suggest that the total uptake of nitrate is related to ecosystem photosynthesis and that denitrification is related to ecosystem respiration. In addition, we use a stream network model to demonstrate that excess nitrate in streams elicits a disproportionate increase in the fraction of nitrate that is exported to receiving waters and reduces the relative role of small versus large streams as nitrate sinks.

Nitrous oxide emission from denitrification in stream and river networks.

Nitrous oxide (N(2)O) is a potent greenhouse gas that contributes to climate change and stratospheric ozone destruction. Anthropogenic nitrogen (N) loading to river networks is a potentially important source of N(2)O via microbial denitrification that converts N to N(2)O and dinitrogen (N(2)). The fraction of denitrified N that escapes as N(2)O rather than N(2) (i.e., the N(2)O yield) is an important determinant of how much N(2)O is produced by river networks, but little is known about the N(2)O yield in flowing waters. Here, we present the results of whole-stream (15)N-tracer additions conducted in 72 headwater streams draining multiple land-use types across the United States. We found that stream denitrification produces N(2)O at rates that increase with stream water nitrate (NO(3)(-)) concentrations, but that <1% of denitrified N is converted to N(2)O. Unlike some previous studies, we found no relationship between the N(2)O yield and stream water NO(3)(-). We suggest that increased stream NO(3)(-) loading stimulates denitrification and concomitant N(2)O production, but does not increase the N(2)O yield. In our study, most streams were sources of N(2)O to the atmosphere and the highest emission rates were observed in streams draining urban basins. Using a global river network model, we estimate that microbial N transformations (e.g., denitrification and nitrification) convert at least 0.68 Tg·y(-1) of anthropogenic N inputs to N(2)O in river networks, equivalent to 10% of the global anthropogenic N(2)O emission rate. This estimate of stream and river N(2)O emissions is three times greater than estimated by the Intergovernmental Panel on Climate Change.

Constitutive knockout of the membrane cytoskeleton protein beta adducin decreases mushroom spine density in the nucleus accumbens but does not prevent spine remodeling in response to cocaine.

The adducin family of proteins associates with the actin cytoskeleton in a calcium-dependent manner. Beta adducin (ßAdd) is involved in synaptic plasticity in the hippocampus; however, the role of ßAdd in synaptic plasticity in other brain areas is unknown. Using diolistic labeling with the lipophilic dye DiI, we found that the density of mature mushroom-shaped spines was significantly decreased in the nucleus accumbens (NAc) in brain slices from ßAdd-knockout (KO) mice as compared to their wildtype (WT) siblings. The effect of 10 days of daily cocaine (15 mg/kg) administration on NAc spine number and locomotor behavior was also measured in ßAdd WT and KO mice. As expected, there was a significant increase in overall spine density in NAc slices from cocaine-treated WT mice at this time-point; however, there was a greater increase in the density of mushroom spines in ßAdd-KO animals following chronic cocaine administration than in WT. In addition, ßAdd-KO mice showed elevated locomotor activity in response to cocaine treatment compared to WT siblings. These results indicate that ßAdd is required for stabilising mature spines under basal conditions in the NAc, but that lack of this protein does not prevent synaptic remodeling following repeated cocaine administration. In addition, these data are consistent with previous studies suggesting that ßAdd may normally be involved in stabilising spines once drug- or experience-dependent remodeling has occurred.