Jenica D. Tapocik

microRNA-206 in Rat Medial Prefrontal Cortex Regulates BDNF Expression and Alcohol Drinking

Escalation of voluntary alcohol consumption is a hallmark of alcoholism, but its neural substrates remain unknown. In rats, escalation occurs following prolonged exposure to cycles of alcohol intoxication, and is associated with persistent, wide-ranging changes in gene expression within the medial prefrontal cortex (mPFC). Here, we examined whether induction of microRNA (miR) 206 in mPFC contributes to escalated alcohol consumption. Following up on a microarray screen, quantitative real-time reverse transcription PCR (qPCR) confirmed that a history of dependence results in persistent (>3weeks) up-regulation of miR-206 expression in the mPFC, but not in the ventral tegmental area, amygdala, or nucleus accumbens. Viral-mediated overexpression of miR-206 in the mPFC of nondependent rats reproduced the escalation of alcohol self-administration seen following a history of dependence and significantly inhibited BDNF expression. Bioinformatic analysis identified three conserved target sites for miR-206 in the 3′-UTR of the rat BDNF transcript. Accordingly, BDNF was downregulated in post-dependent rats on microarray analysis, and this was confirmed by qPCR. In vitro, BDNF expression was repressed by miR-206 but not miR-9 in a 3′-UTR reporter assay, confirming BDNF as a functional target of miR-206. Mutation analysis showed that repression was dependent on the presence of all three miR-206 target sites in the BDNF 3′-UTR. Inhibition of miR-206 expression in differentiated rat cortical primary neurons significantly increased secreted levels of BDNF. In conclusion, recruitment of miR-206 in the mPFC contributes to escalated alcohol consumption following a history of dependence, with BDNF as a possible mediator of its action.

Coordinated dysregulation of mRNAs and microRNAs in the rat medial prefrontal cortex following a history of alcohol dependence

Long-term changes in brain gene expression have been identified in alcohol dependence, but underlying mechanisms remain unknown. Here, we examined the potential role of microRNAs (miRNAs) for persistent gene expression changes in the rat medial prefrontal cortex (mPFC) after a history of alcohol dependence. Two-bottle free-choice alcohol consumption increased following 7-week exposure to intermittent alcohol intoxication. A bioinformatic approach using microarray analysis, quantitative PCR (qPCR), bioinformatic analysis and microRNA–messenger RNA (mRNA) integrative analysis identified expression patterns indicative of a disruption in synaptic processes and neuroplasticity. About 41 rat miRNAs and 165 mRNAs in the mPFC were significantly altered after chronic alcohol exposure. A subset of the miRNAs and mRNAs was confirmed by qPCR. Gene ontology categories of differential expression pointed to functional processes commonly associated with neurotransmission, neuroadaptation and synaptic plasticity. microRNA–mRNA expression pairing identified 33 miRNAs putatively targeting 89 mRNAs suggesting transcriptional networks involved in axonal guidance and neurotransmitter signaling. Our results demonstrate a significant shift in microRNA expression patterns in the mPFC following a history of dependence. Owing to their global regulation of multiple downstream target transcripts, miRNAs may have a pivotal role in the reorganization of synaptic connections and long-term neuroadaptations in alcohol dependence. MicroRNA-mediated alterations of transcriptional networks may be involved in disrupted prefrontal control over alcohol drinking observed in alcoholic patients.

Loss of metabotropic glutamate receptor 2 escalates alcohol consumption

Significance Gene identification for complex behavioral traits, alcoholism in particular, has been largely unsuccessful, in part because of the rarity of many causative variants and the heterogeneity and small effect size of the causal loci. Artificially selected animals may be valuable in gene identification. We identified a causal role of metabotropic glutamate receptor 2 (mGluR2) in altered alcohol preference via genomic sequencing of selectively bred rats, linkage analysis in the F2 rats, and function validation in mGluR2 null mice. Our findings represent a valuable contribution to understanding the neurobiology of alcoholism and a strategy for gene identification for complex behavior traits. Identification of genes influencing complex traits is hampered by genetic heterogeneity, the modest effect size of many alleles, and the likely involvement of rare and uncommon alleles. Etiologic complexity can be simplified in model organisms. By genomic sequencing, linkage analysis, and functional validation, we identified that genetic variation of Grm2, which encodes metabotropic glutamate receptor 2 (mGluR2), alters alcohol preference in animal models. Selectively bred alcohol-preferring (P) rats are homozygous for a Grm2 stop codon (Grm2 *407) that leads to largely uncompensated loss of mGluR2. mGluR2 receptor expression was absent, synaptic glutamate transmission was impaired, and expression of genes involved in synaptic function was altered. Grm2 *407 was linked to increased alcohol consumption and preference in F2 rats generated by intercrossing inbred P and nonpreferring rats. Pharmacologic blockade of mGluR2 escalated alcohol self-administration in Wistar rats, the parental strain of P and nonpreferring rats. The causal role of mGluR2 in altered alcohol preference was further supported by elevated alcohol consumption in Grm2 −/− mice. Together, these data point to mGluR2 as an origin of alcohol preference and a potential therapeutic target.

DNA methylation in the medial prefrontal cortex regulates alcohol-induced behavior and plasticity.

Recent studies have suggested an association between alcoholism and DNA methylation, a mechanism that can mediate long-lasting changes in gene transcription. Here, we examined the contribution of DNA methylation to the long-term behavioral and molecular changes induced by a history of alcohol dependence. In search of mechanisms underlying persistent rather than acute dependence-induced neuroadaptations, we studied the role of DNA methylation regulating medial prefrontal cortex (mPFC) gene expression and alcohol-related behaviors in rats 3 weeks into abstinence following alcohol dependence. Postdependent rats showed escalated alcohol intake, which was associated with increased DNA methylation as well as decreased expression of genes encoding synaptic proteins involved in neurotransmitter release in the mPFC. Infusion of the DNA methyltransferase inhibitor RG108 prevented both escalation of alcohol consumption and dependence-induced downregulation of 4 of the 7 transcripts modified in postdependent rats. Specifically, RG108 treatment directly reversed both downregulation of synaptotagmin 2 (Syt2) gene expression and hypermethylation on CpG#5 of its first exon. Lentiviral inhibition of Syt2 expression in the mPFC increased aversion-resistant alcohol drinking, supporting a mechanistic role of Syt2 in compulsive-like behavior. Our findings identified a functional role of DNA methylation in alcohol dependence-like behavioral phenotypes and a candidate gene network that may mediate its effects. Together, these data provide novel evidence for DNA methyltransferases as potential therapeutic targets in alcoholism.

Identification of Candidate Genes and Gene Networks Specifically Associated with Analgesic Tolerance to Morphine

Chronic morphine administration may alter the expression of hundreds to thousands of genes. However, only a subset of these genes is likely involved in analgesic tolerance. In this report, we used a behavior genetics strategy to identify candidate genes specifically linked to the development of morphine tolerance. Two inbred genotypes [C57BL/6J (B6), DBA2/J (D2)] and two reciprocal congenic genotypes (B6D2, D2B6) with the proximal region of chromosome 10 (Chr10) introgressed into opposing backgrounds served as the behavior genetic filter. Tolerance after therapeutically relevant doses of morphine developed most rapidly in the B6 followed by the B6D2 genotype and did not develop in the D2 mice and only slightly in the D2B6 animals indicating a strong influence of the proximal region of Chr10 in the development of tolerance. Gene expression profiling and pattern matching identified 64, 53, 86, and 123 predisposition genes and 81, 96, 106, and 82 tolerance genes in the periaqueductal gray (PAG), prefrontal cortex, temporal lobe, and ventral striatum, respectively. A potential gene network was identified in the PAG in which 19 of the 34 genes were strongly associated with tolerance. Eleven of the network genes were found to reside in quantitative trait loci previously associated with morphine-related behaviors, whereas seven were predictive of tolerance (morphine-naive condition). Overall, the genes modified by chronic morphine administration show a strong presence in canonical pathways representative of neuroadaptation. A potentially significant role for the micro-RNA and epigenetic mechanisms in response to chronic administration of pharmacologically relevant doses of morphine was highlighted by candidate genes Dicer and H19.

FKBP5 Moderates Alcohol Withdrawal Severity: Human Genetic Association and Functional Validation in Knockout Mice

Alcohol withdrawal is associated with hypothalamic–pituitary–adrenal (HPA) axis dysfunction. The FKBP5 gene codes for a co-chaperone, FK506-binding protein 5, that exerts negative feedback on HPA axis function. This study aimed to examine the effects of single-nucleotide polymorphisms (SNPs) of the FKBP5 gene in humans and the effect of Fkbp5 gene deletion in mice on alcohol withdrawal severity. We genotyped six FKBP5 SNPs (rs3800373, rs9296158, rs3777747, rs9380524, rs1360780, and rs9470080) in 399 alcohol-dependent inpatients with alcohol consumption 48 h before admission and recorded scores from the Clinical Institute Withdrawal Assessment-Alcohol revised (CIWA-Ar). Fkbp5 gene knockout (KO) and wild-type (WT) mice were assessed for alcohol withdrawal using handling-induced convulsions (HICs) following both acute and chronic alcohol exposure. We found the minor alleles of rs3800373 (G), rs9296158 (A), rs1360780 (T), and rs9470080 (T) were significantly associated with lower CIWA-Ar scores whereas the minor alleles of rs3777747 (G) and rs9380524 (A) were associated with higher scores. The haplotype-based analyses also showed an association with alcohol withdrawal severity. Fkbp5 KO mice showed significantly greater HICs during withdrawal from chronic alcohol exposure compared with WT controls. This study is the first to show a genetic effect of FKBP5 on the severity of alcohol withdrawal syndrome. In mice, the absence of the Fkbp5 gene enhances sensitivity to alcohol withdrawal. We suggest that FKBP5 variants may trigger different adaptive changes in HPA axis regulation during alcohol withdrawal with concomitant effects on withdrawal severity.

A Novel Brain Penetrant NPS Receptor Antagonist, NCGC00185684, Blocks Alcohol- Induced ERK-Phosphorylation in the Central Amygdala and Decreases Operant Alcohol Self- Administration in Rats

The Neuropeptide S receptor, a Gs/Gq-coupled GPCR expressed in brain regions involved in mediating drug reward, has recently emerged as a candidate therapeutic target in addictive disorders. Here, we describe the in vitro and in vivo pharmacology of a novel, selective and brain penetrant NPSR antagonist with nanomolar affinity for the NPSR, NCGC00185684. In vitro, NCGC00185684 shows biased antagonist properties, and preferentially blocks ERK-phosphorylation over intracellular cAMP or calcium responses to NPS. In vivo, systemic NCGC00185684 blocks alcohol-induced ERK-phosphorylation in the rat central amygdala, a region involved in regulation of alcohol intake. NCGC00185684 also decreases operant alcohol self-administration, and lowers motivation for alcohol reward as measured using progressive ratio responding. These effects are behaviorally specific, in that they are observed at doses that do not influence locomotor activity or reinstatement responding following extinction. Together, these data provide an initial validation of the NPSR as a therapeutic target in alcoholism.

Tacr1 Gene Variation and Neurokinin 1 Receptor Expression Is Associated with Antagonist Efficacy in Genetically Selected Alcohol-Preferring Rats

BACKGROUND Genetic deletion or antagonism of the neurokinin 1 receptor (NK1R) decreases alcohol intake, alcohol reward, and stress-induced alcohol relapse in rodents, while TACR1 variation is associated with alcoholism in humans. METHODS We used L822429, a specific antagonist with high affinity for the rat NK1R, and examined whether sensitivity to NK1R blockade is altered in alcohol-preferring (P) rats. Operant alcohol self-administration and progressive ratio responding were analyzed in P-rats and their founder Wistar line. We also analyzed Tacr1 expression and binding and sequenced the Tacr1 promoter from both lines. RESULTS Systemic L822429 decreased alcohol self-administration in P-rats but did not affect the lower rates of alcohol self-administration in Wistar rats. Tacr1 expression was elevated in the prefrontal cortex and the amygdala of P-rats. In central amygdala, elevated Tacr1 expression was accompanied by elevated NK1R binding. Central amygdala (but not prefrontal cortex) infusion of L822429 replicated the systemic antagonist effects on alcohol self-administration in P-rats. All P-rats, but only 18% of their founder Wistar population, were CC homozygous for a-1372G/C single nucleotide polymorphism. In silico analysis indicated that the Tacr1-1372 genotype could modulate binding of the transcription factors GATA-2 and E2F-1. Electromobility shift and luciferase reporter assays suggested that the-1372C allele confers increased transcription factor binding and transcription. CONCLUSIONS Genetic variation at the Tacr1 locus may contribute to elevated rates of alcohol self-administration, while at the same time increasing sensitivity to NK1R antagonist treatment.

Neuroplasticity, axonal guidance and micro‐RNA genes are associated with morphine self‐administration behavior

Neuroadaptations in the ventral striatum (VS) and ventral midbrain (VMB) following chronic opioid administration are thought to contribute to the pathogenesis and persistence of opiate addiction. In order to identify candidate genes involved in these neuroadaptations, we utilized a behavior‐genetics strategy designed to associate contingent intravenous drug self‐administration with specific patterns of gene expression in inbred mice differentially predisposed to the rewarding effects of morphine. In a Yoked‐control paradigm, C57BL/6J mice showed clear morphine‐reinforced behavior, whereas DBA/2J mice did not. Moreover, the Yoked‐control paradigm revealed the powerful consequences of self‐administration versus passive administration at the level of gene expression. Morphine self‐administration in the C57BL/6J mice uniquely up‐ or down‐regulated 237 genes in the VS and 131 genes in the VMB. Interestingly, only a handful of the C57BL/6J self‐administration genes (<3%) exhibited a similar expression pattern in the DBA/2J mice. Hence, specific sets of genes could be confidently assigned to regional effects of morphine in a contingent‐ and genotype‐dependent manner. Bioinformatics analysis revealed that neuroplasticity, axonal guidance and micro‐RNAs (miRNAs) were among the key themes associated with drug self‐administration. Noteworthy were the primary miRNA genes H19 and micro‐RNA containing gene (Mirg), processed, respectively, to mature miRNAs miR‐675 and miR‐154, because they are prime candidates to mediate network‐like changes in responses to chronic drug administration. These miRNAs have postulated roles in dopaminergic neuron differentiation and mu‐opioid receptor regulation. The strategic approach designed to focus on reinforcement‐associated genes provides new insight into the role of neuroplasticity pathways and miRNAs in drug addiction.

Neurokinin-1 receptor antagonism attenuates neuronal activity triggered by stress-induced reinstatement of alcohol seeking

Substance P (SP) and its cognate neurokinin-1 receptor (NK1R) are involved in alcohol-related behaviors. We have previously reported that NK1R antagonism attenuates stress-induced reinstatement of alcohol seeking and suppresses escalated alcohol self-administration, but does not affect primary reinforcement or cue-induced reinstatement. Here, we administered an NK1R antagonist or vehicle prior to footshock-induced reinstatement of alcohol seeking, and mapped the resulting neuronal activation using Fos immunohistochemistry. As expected, vehicle treated animals exposed to footshock showed induction of Fos immunoreactivity in several regions of the brain stress circuitry, including the amygdala (AMG), nucleus accumbens (NAC), dorsal raphe nucleus (DR), prefrontal cortex (PFC), and bed nucleus of the stria terminalis (BNST). NK1R antagonism selectively suppressed the stress-induced increase in Fos in the DR and NAC shell. In the DR, Fos-induction by stress largely overlapped with tryptophan hydroxylase (TrpH), indicating activation of serotonergic neurons. Of NAC shell neurons activated during stress-induced reinstatement of alcohol seeking, about 30% co-expressed dynorphin (DYN), while 70% co-expressed enkephalin (ENK). Few (<1%) activated NAC shell neurons coexpressed choline acetyltransferase (ChAT), which labels the cholinergic interneurons of this region. Infusion of the NK1R antagonist L822429 into the NAC shell blocked stress-induced reinstatement of alcohol seeking. In contrast, L822429 infusion into the DR had no effect, suggesting that the influence of NK1R signaling on neuronal activity in the DR is indirect. Taken together, our results outline a potential pathway through which endogenous NK1R activation mediates stress-induced alcohol seeking.