Salsolinol Facilitates Glutamatergic Transmission to Dopamine Neurons in the Posterior Ventral Tegmental Area of Rats

Although in vivo evidence indicates that salsolinol, the condensation product of acetaldehyde and dopamine, has properties that may contribute to alcohol abuse, the underlying mechanisms have not been fully elucidated. We have reported previously that salsolinol stimulates dopamine neurons in the posterior ventral tegmental area (p-VTA) partly by reducing inhibitory GABAergic transmission, and that ethanol increases glutamatergic transmission to VTA-dopamine neurons via the activation of dopamine D1 receptors (D1Rs). In this study, we tested the hypothesis that salsolinol stimulates dopamine neurons involving activation of D1Rs. By using whole-cell recordings on p-VTA-dopamine neurons in acute brain slices of rats, we found that salsolinol-induced increase in spike frequency of dopamine neurons was substantially attenuated by DL-2-amino-5-phosphono-valeric acid and 6, 7-dinitroquinoxaline-2, 3-dione, the antagonists of glutamatergic N-Methyl-D-aspartic acid and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Moreover, salsolinol increased the amplitude of evoked excitatory postsynaptic currents (EPSCs) and the frequency but not the amplitude of spontaneous EPSCs. Additionally, SKF83566, a D1R antagonist attenuated the salsolinol-induced facilitation of EPSCs and of spontaneous firing of dopamine neurons. Our data reveal that salsolinol enhances glutamatergic transmission onto dopamine neurons via activation of D1Rs at the glutamatergic afferents in dopamine neurons, which contributes to salsolinol's stimulating effect on p-VTA dopamine neurons. This appears to be a novel mechanism which contributes toward rewarding properties of salsolinol

Adolescent Binge Drinking Leads to Changes in Alcohol Drinking, Anxiety, and Amygdalar Corticotropin Releasing Factor Cells in Adulthood in Male Rats

Heavy episodic drinking early in adolescence is associated with increased risk of addiction and other stress-related disorders later in life. This suggests that adolescent alcohol abuse is an early marker of innate vulnerability and/or binge exposure impacts the developing brain to increase vulnerability to these disorders in adulthood. Animal models are ideal for clarifying the relationship between adolescent and adult alcohol abuse, but we show that methods of involuntary alcohol exposure are not effective. We describe an operant model that uses multiple bouts of intermittent access to sweetened alcohol to elicit voluntary binge alcohol drinking early in adolescence (∼postnatal days 28–42) in genetically heterogeneous male Wistar rats. We next examined the effects of adolescent binge drinking on alcohol drinking and anxiety-like behavior in dependent and non-dependent adult rats, and counted corticotropin-releasing factor (CRF) cell in the lateral portion of the central amygdala (CeA), a region that contributes to regulation of anxiety- and alcohol-related behaviors. Adolescent binge drinking did not alter alcohol drinking under baseline drinking conditions in adulthood. However, alcohol-dependent and non-dependent adult rats with a history of adolescent alcohol binge drinking did exhibit increased alcohol drinking when access to alcohol was intermittent. Adult rats that binged alcohol during adolescence exhibited increased exploration on the open arms of the elevated plus maze (possibly indicating either decreased anxiety or increased impulsivity), an effect that was reversed by a history of alcohol dependence during adulthood. Finally, CRF cell counts were reduced in the lateral CeA of rats with adolescent alcohol binge history, suggesting semi-permanent changes in the limbic stress peptide system with this treatment. These data suggest that voluntary binge drinking during early adolescence produces long-lasting neural and behavioral effects with implications for anxiety and alcohol use disorders

The Novel μ-Opioid Receptor Antagonist GSK1521498 Decreases Both Alcohol Seeking and Drinking: Evidence from a New Preclinical Model of Alcohol Seeking.

Distinct environmental and conditioned stimuli influencing ethanol-associated appetitive and consummatory behaviors may jointly contribute to alcohol addiction. To develop an effective translational animal model that illuminates this interaction, daily seeking responses, maintained by alcohol-associated conditioned stimuli (CSs), need to be dissociated from alcohol drinking behavior. For this, we established a procedure whereby alcohol seeking maintained by alcohol-associated CSs is followed by a period during which rats have the opportunity to drink alcohol. This cue-controlled alcohol-seeking procedure was used to compare the effects of naltrexone and GSK1521498, a novel selective μ-opioid receptor antagonist, on both voluntary alcohol-intake and alcohol-seeking behaviors. Rederived alcohol-preferring, alcohol-nonpreferring, and high-alcohol-drinking replicate 1 line of rats (Indiana University) first received 18 sessions of 24 h home cage access to 10% alcohol and water under a 2-bottle choice procedure. They were trained subsequently to respond instrumentally for access to 15% alcohol under a second-order schedule of reinforcement, in which a prolonged period of alcohol-seeking behavior was maintained by contingent presentations of an alcohol-associated CS acting as a conditioned reinforcer. This seeking period was terminated by 20 min of free alcohol drinking access that achieved significant blood alcohol concentrations. The influence of pretreatment with either naltrexone (0.1-1-3 mg/kg) or GSK1521498 (0.1-1-3 mg/kg) before instrumental sessions was measured on both seeking and drinking behaviors, as well as on drinking in the 2-bottle choice procedure. Naltrexone and GSK1521498 dose-dependently reduced both cue-controlled alcohol seeking and alcohol intake in the instrumental context as well as alcohol intake in the choice procedure. However, GSK1521498 showed significantly greater effectiveness than naltrexone, supporting its potential use for promoting abstinence and preventing relapse in alcohol addiction.The present study was funded by Medical Research Council Programme Grant (no. G1002231) and by GlaxoSmithKline (GSK), which has a commercial interest in GSK1521498. Charles R. Goodlett was funded by a grant from the IUPUI International Development Fund, which supported his sabbatical leave at the University of Cambridge. Maria Pilar Garcia-Pardo was funded by Val+id para investigadores en formación (Conselleria de educacion, Generalitat Valenciana), which also supported her stay at the University of Cambridge (January-April 2014) as a Visiting Student.This is the accepted manuscript. The final version is available from NPG at http://dx.doi.org/10.1038/npp.2015.15

Quantitative trait loci for sensitivity to ethanol intoxication in a C57BL/6J × 129S1/SvImJ inbred mouse cross

Individual variation in sensitivity to acute ethanol (EtOH) challenge is associated with alcohol drinking and is a predictor of alcohol abuse. Previous studies have shown that the C57BL/6J (B6) and 129S1/SvImJ (S1) inbred mouse strains differ in responses on certain measures of acute EtOH intoxication. To gain insight into genetic factors contributing to these differences, we performed quantitative trait locus (QTL) analysis of measures of EtOH-induced ataxia (accelerating rotarod), hypothermia, and loss of righting reflex (LORR) duration in a B6 × S1 F2 population. We confirmed that S1 showed greater EtOH-induced hypothermia (specifically at a high dose) and longer LORR compared to B6. QTL analysis revealed several additive and interacting loci for various phenotypes, as well as examples of genotype interactions with sex. QTLs for different EtOH phenotypes were largely non-overlapping, suggesting separable genetic influences on these behaviors. The most compelling main-effect QTLs were for hypothermia on chromosome 16 and for LORR on chromosomes 4 and 6. Several QTLs overlapped with loci repeatedly linked to EtOH drinking in previous mouse studies. The architecture of the traits we examined was complex but clearly amenable to dissection in future studies. Using integrative genomics strategies, plausible functional and positional candidates may be found. Uncovering candidate genes associated with variation in these phenotypes in this population could ultimately shed light on genetic factors underlying sensitivity to EtOH intoxication and risk for alcoholism in humans

Amygdala 14-3-3ζ as a Novel Modulator of Escalating Alcohol Intake in Mice

Alcoholism is a devastating brain disorder that affects millions of people worldwide. The development of alcoholism is caused by alcohol-induced maladaptive changes in neural circuits involved in emotions, motivation, and decision-making. Because of its involvement in these processes, the amygdala is thought to be a key neural structure involved in alcohol addiction. However, the molecular mechanisms that govern the development of alcoholism are incompletely understood. We have previously shown that in a limited access choice paradigm, C57BL/6J mice progressively escalate their alcohol intake and display important behavioral characteristic of alcohol addiction, in that they become insensitive to quinine-induced adulteration of alcohol. This study used the limited access choice paradigm to study gene expression changes in the amygdala during the escalation to high alcohol consumption in C57BL/6J mice. Microarray analysis revealed that changes in gene expression occurred predominantly after one week, i.e. during the initial escalation of alcohol intake. One gene that stood out from our analysis was the adapter protein 14-3-3ζ, which was up-regulated during the transition from low to high alcohol intake. Independent qPCR analysis confirmed the up-regulation of amygdala 14-3-3ζ during the escalation of alcohol intake. Subsequently, we found that local knockdown of 14-3-3ζ in the amygdala, using RNA interference, dramatically augmented alcohol intake. In addition, knockdown of amygdala 14-3-3ζ promoted the development of inflexible alcohol drinking, as apparent from insensitivity to quinine adulteration of alcohol. This study identifies amygdala 14-3-3ζ as a novel key modulator that is engaged during escalation of alcohol use

Genetic Dissection of Acute Ethanol Responsive Gene Networks in Prefrontal Cortex: Functional and Mechanistic Implications

Background Individual differences in initial sensitivity to ethanol are strongly related to the heritable risk of alcoholism in humans. To elucidate key molecular networks that modulate ethanol sensitivity we performed the first systems genetics analysis of ethanol-responsive gene expression in brain regions of the mesocorticolimbic reward circuit (prefrontal cortex, nucleus accumbens, and ventral midbrain) across a highly diverse family of 27 isogenic mouse strains (BXD panel) before and after treatment with ethanol. Results Acute ethanol altered the expression of ~2,750 genes in one or more regions and 400 transcripts were jointly modulated in all three. Ethanol-responsive gene networks were extracted with a powerful graph theoretical method that efficiently summarized ethanol\u27s effects. These networks correlated with acute behavioral responses to ethanol and other drugs of abuse. As predicted, networks were heavily populated by genes controlling synaptic transmission and neuroplasticity. Several of the most densely interconnected network hubs, including Kcnma1 and Gsk3β, are known to influence behavioral or physiological responses to ethanol, validating our overall approach. Other major hub genes like Grm3, Pten and Nrg3 represent novel targets of ethanol effects. Networks were under strong genetic control by variants that we mapped to a small number of chromosomal loci. Using a novel combination of genetic, bioinformatic and network-based approaches, we identified high priority cis-regulatory candidate genes, including Scn1b,Gria1, Sncb and Nell2. Conclusions The ethanol-responsive gene networks identified here represent a previously uncharacterized intermediate phenotype between DNA variation and ethanol sensitivity in mice. Networks involved in synaptic transmission were strongly regulated by ethanol and could contribute to behavioral plasticity seen with chronic ethanol. Our novel finding that hub genes and a small number of loci exert major influence over the ethanol response of gene networks could have important implications for future studies regarding the mechanisms and treatment of alcohol use disorders

Convergent functional genomics of anxiety disorders: translational identification of genes, biomarkers, pathways and mechanisms

Anxiety disorders are prevalent and disabling yet understudied from a genetic standpoint, compared with other major psychiatric disorders such as bipolar disorder and schizophrenia. The fact that they are more common, diverse and perceived as embedded in normal life may explain this relative oversight. In addition, as for other psychiatric disorders, there are technical challenges related to the identification and validation of candidate genes and peripheral biomarkers. Human studies, particularly genetic ones, are susceptible to the issue of being underpowered, because of genetic heterogeneity, the effect of variable environmental exposure on gene expression, and difficulty of accrual of large, well phenotyped cohorts. Animal model gene expression studies, in a genetically homogeneous and experimentally tractable setting, can avoid artifacts and provide sensitivity of detection. Subsequent translational integration of the animal model datasets with human genetic and gene expression datasets can ensure cross-validatory power and specificity for illness. We have used a pharmacogenomic mouse model (involving treatments with an anxiogenic drug—yohimbine, and an anti-anxiety drug—diazepam) as a discovery engine for identification of anxiety candidate genes as well as potential blood biomarkers. Gene expression changes in key brain regions for anxiety (prefrontal cortex, amygdala and hippocampus) and blood were analyzed using a convergent functional genomics (CFG) approach, which integrates our new data with published human and animal model data, as a translational strategy of cross-matching and prioritizing findings. Our work identifies top candidate genes (such as FOS, GABBR1, NR4A2, DRD1, ADORA2A, QKI, RGS2, PTGDS, HSPA1B, DYNLL2, CCKBR and DBP), brain–blood biomarkers (such as FOS, QKI and HSPA1B), pathways (such as cAMP signaling) and mechanisms for anxiety disorders—notably signal transduction and reactivity to environment, with a prominent role for the hippocampus. Overall, this work complements our previous similar work (on bipolar mood disorders and schizophrenia) conducted over the last decade. It concludes our programmatic first pass mapping of the genomic landscape of the triad of major psychiatric disorder domains using CFG, and permitted us to uncover the significant genetic overlap between anxiety and these other major psychiatric disorders, notably the under-appreciated overlap with schizophrenia. PDE10A, TAC1 and other genes uncovered by our work provide a molecular basis for the frequently observed clinical co-morbidity and interdependence between anxiety and other major psychiatric disorders, and suggest schizo-anxiety as a possible new nosological domain

Convergent functional genomic studies of omega-3 fatty acids in stress reactivity, bipolar disorder and alcoholism

Omega-3 fatty acids have been proposed as an adjuvant treatment option in psychiatric disorders. Given their other health benefits and their relative lack of toxicity, teratogenicity and side effects, they may be particularly useful in children and in females of child-bearing age, especially during pregnancy and postpartum. A comprehensive mechanistic understanding of their effects is needed. Here we report translational studies demonstrating the phenotypic normalization and gene expression effects of dietary omega-3 fatty acids, specifically docosahexaenoic acid (DHA), in a stress-reactive knockout mouse model of bipolar disorder and co-morbid alcoholism, using a bioinformatic convergent functional genomics approach integrating animal model and human data to prioritize disease-relevant genes. Additionally, to validate at a behavioral level the novel observed effects on decreasing alcohol consumption, we also tested the effects of DHA in an independent animal model, alcohol-preferring (P) rats, a well-established animal model of alcoholism. Our studies uncover sex differences, brain region-specific effects and blood biomarkers that may underpin the effects of DHA. Of note, DHA modulates some of the same genes targeted by current psychotropic medications, as well as increases myelin-related gene expression. Myelin-related gene expression decrease is a common, if nonspecific, denominator of neuropsychiatric disorders. In conclusion, our work supports the potential utility of omega-3 fatty acids, specifically DHA, for a spectrum of psychiatric disorders such as stress disorders, bipolar disorder, alcoholism and beyond