Publications

2018
Renteria R, Buske TR, Morrisett RA. Long-term subregion-specific encoding of enhanced ethanol intake by D1DR medium spiny neurons of the nucleus accumbens. Addiction Biology. 2018;23 :689-698.Abstract
While the risks of maternal alcohol abuse during pregnancy are well-established, several preclinical studies suggest that chronic preconception alcohol consumption by either parent may also have significance consequences for offspring health and development. Notably, since isogenic male mice used in these studies are not involved in gestation or rearing of offspring, the cross-generational effects of paternal alcohol exposure suggest a germline-based epigenetic mechanism. Many recent studies have demonstrated that the effects of paternal environmental exposures such as stress or malnutrition can be transmitted to the next generation via alterations to small noncoding RNAs in sperm. Therefore, we used high throughput sequencing to examine the effect of preconception ethanol on small noncoding RNAs in sperm. We found that chronic intermittent ethanol exposure altered several small noncoding RNAs from three of the major small RNA classes in sperm, tRNA-derived small RNA (tDR), mitochondrial small RNA, and microRNA. Six of the ethanol-responsive small noncoding RNAs were evaluated with RT-qPCR on a separate cohort of mice and five of the six were confirmed to be altered by chronic ethanol exposure, supporting the validity of the sequencing results. In addition to altered sperm RNA abundance, chronic ethanol exposure affected post-transcriptional modifications to sperm small noncoding RNAs, increasing two nucleoside modifications previously identified in mitochondrial tRNA. Furthermore, we found that chronic ethanol reduced epididymal expression of a tRNA methyltransferase, Nsun2, known to directly regulate tDR biogenesis. Finally, ethanol-responsive sperm tDR are similarly altered in extracellular vesicles of the epididymis (i.e., epididymosomes), supporting the hypothesis that alterations to sperm tDR emerge in the epididymis and that epididymosomes are the primary source of small noncoding RNAs in sperm. These results add chronic ethanol to the growing list of paternal exposures that can affect small noncoding RNA abundance and nucleoside modifications in sperm. As small noncoding RNAs in sperm have been shown to causally induce heritable phenotypes in offspring, additional research is warranted to understand the potential effects of ethanol-responsive sperm small noncoding RNAs on offspring health and development.
Rompala GR, Mounier A, Wolfe CM, Lin Q, Lefterov I, Homanics GE. Heavy chronic intermittent ethanol exposure alters small noncoding RNAs in mouse sperm and epididymosomes. Frontiers in Genetics. 2018;9 :32.Abstract
While the risks of maternal alcohol abuse during pregnancy are well-established, several preclinical studies suggest that chronic preconception alcohol consumption by either parent may also have significance consequences for offspring health and development. Notably, since isogenic male mice used in these studies are not involved in gestation or rearing of offspring, the cross-generational effects of paternal alcohol exposure suggest a germline-based epigenetic mechanism. Many recent studies have demonstrated that the effects of paternal environmental exposures such as stress or malnutrition can be transmitted to the next generation via alterations to small noncoding RNAs in sperm. Therefore, we used high throughput sequencing to examine the effect of preconception ethanol on small noncoding RNAs in sperm. We found that chronic intermittent ethanol exposure altered several small noncoding RNAs from three of the major small RNA classes in sperm, tRNA-derived small RNA (tDR), mitochondrial small RNA, and microRNA. Six of the ethanol-responsive small noncoding RNAs were evaluated with RT-qPCR on a separate cohort of mice and five of the six were confirmed to be altered by chronic ethanol exposure, supporting the validity of the sequencing results. In addition to altered sperm RNA abundance, chronic ethanol exposure affected post-transcriptional modifications to sperm small noncoding RNAs, increasing two nucleoside modifications previously identified in mitochondrial tRNA. Furthermore, we found that chronic ethanol reduced epididymal expression of a tRNA methyltransferase, Nsun2, known to directly regulate tDR biogenesis. Finally, ethanol-responsive sperm tDR are similarly altered in extracellular vesicles of the epididymis (i.e., epididymosomes), supporting the hypothesis that alterations to sperm tDR emerge in the epididymis and that epididymosomes are the primary source of small noncoding RNAs in sperm. These results add chronic ethanol to the growing list of paternal exposures that can affect small noncoding RNA abundance and nucleoside modifications in sperm. As small noncoding RNAs in sperm have been shown to causally induce heritable phenotypes in offspring, additional research is warranted to understand the potential effects of ethanol-responsive sperm small noncoding RNAs on offspring health and development.
Satta R, Hilderbrand ER, Lasek AW. Ovarian hormones contribute to high levels of binge-like drinking by female mice. Alcoholism: Clinical and Experimental Research. 2018;42 :286-294.Abstract

BACKGROUND:

Recently, the incidence of binge drinking by women has increased. Binge drinking is detrimental to women's health, yet the biological mechanisms that promote excessive drinking by women are not well understood. One method of assessing binge-like ethanol (EtOH) consumption in mice is the drinking in the dark (DID) test, in which mice drink sufficient EtOH to achieve intoxication. In this study, we directly compared male, female, and ovariectomized (OVX) mice for DID and tested whether 17β-estradiol (E2) contributes to DID. We also measured whether DID varies throughout the estrous cycle and whether repeated intermittent DID impacts the estrous cycle.

METHODS:

Male, female, and OVX C57BL/6J mice were tested for DID for 2 hours per day on days 1 to 3 and for 4 hours on day 4 using a single bottle containing 20% EtOH. To measure the effects of E2 on DID, OVX mice were treated with estradiol benzoate (EB) or vehicle daily starting 2 weeks prior to the drinking test and throughout the DID procedure. In a separate group of experiments, EtOH consumption and estrous cycle phase were measured in freely cycling mice that were drinking EtOH or water 5 days per week for 2 or 6 weeks.

RESULTS:

Female mice consumed more EtOH than male and OVX mice. Treatment with EB increased EtOH consumption by OVX mice compared with vehicle-treated controls. However, EtOH intake did not vary across the estrous cycle, nor did long-term DID alter the estrous cycle.

CONCLUSIONS:

These results demonstrate that ovarian hormones, specifically E2, contribute to increased EtOH consumption by female mice in the DID test. Although ovarian hormones contribute to this behavior, EtOH consumption is not affected by estrous cycle phase in freely cycling mice. This study provides a framework for understanding the factors that contribute to binge drinking in females.

Varodayan FP, Sidhu H, Kreifeldt M, Roberto M, Contet C. Morphological and functional evidence of increased excitatory signaling in the prelimbic cortex during ethanol withdrawal. Neuropharmacology. 2018;133 :470-480.Abstract
Excessive alcohol consumption in humans induces deficits in decision making and emotional processing, which indicates a dysfunction of the prefrontal cortex (PFC). The present study aimed to determine the impact of chronic intermittent ethanol (CIE) inhalation on mouse medial PFC pyramidal neurons. Data were collected 6–8 days into withdrawal from 7 weeks of CIE exposure, a time point when mice exhibit behavioral symptoms of withdrawal. We found that spine maturity in prelimbic (PL) layer 2/3 neurons was increased, while dendritic spines in PL layer 5 neurons or infralimbic (IL) neurons were not affected. Corroborating these morphological observations, CIE enhanced glutamatergic transmission in PL layer 2/3 pyramidal neurons, but not IL layer 2/3 neurons. Contrary to our predictions, these cellular alterations were associated with improved, rather than impaired, performance in reversal learning and strategy switching tasks in the Barnes maze at an earlier stage of chronic ethanol exposure (5–7 days withdrawal from 3 to 4 weeks of CIE), which could result from the anxiety-like behavior associated with ethanol withdrawal. Altogether, this study adds to a growing body of literature indicating that glutamatergic activity in the PFC is upregulated following chronic ethanol exposure, and identifies PL layer 2/3 pyramidal neurons as a sensitive target of synaptic remodeling. It also indicates that the Barnes maze is not suitable to detect deficits in cognitive flexibility in CIE-withdrawn mice.
2017
Mahnke AH, Miranda RC, GE. H. Epigenetic mediators and consequences of excessive alcohol consumption. Alcohol [Internet]. 2017;60 :1-6. Publisher's VersionAbstract
Alcohol has pleiotropic effects across multiple organ systems, including brain, cardio-vascularendocrine, immune, musculoskeletal, and gastrointestinal systems. Moreover, some effects, such as intoxication, can be brief, but others, such as the development of Alcohol Use Disorders (AUDs), cardiovascular disease, and liver damage, can persist for a lifetime. Effects resulting in encoding of endocrine and other dysfunctions can also persist across generations. This complexity creates a barrier to the creation of therapeutics and discovery of biomarkers. However, we know that environmental factors, which can include drugs of abuse such as alcohol, can have short- and long-term effects on gene expression through epigenetic mechanisms (Holliday, 2006Shukla et al., 2008). Epigenetic mechanisms affect the transcription and translation of many genes simultaneously. Therefore, by understanding the mechanics of these epigenetic changes, we will have the ability to craft powerful new therapeutics to offset negative effects of alcohol exposure.
Le Berre AP, Fama R, EV S. Executive functions, memory, and social cognitive deficits and recovery in chronic alcoholism: a critical review to inform future research. Alcoholism: Clinical and Experimental Research [Internet]. 2017;41 :1432-1443. Publisher's VersionAbstract

Alcoholism is a complex and dynamic disease, punctuated by periods of abstinence and relapse, and influenced by a multitude of vulnerability factors. Chronic excessive alcohol consumption is associated with cognitive deficits, ranging from mild to severe, in executivefunctions, memory, and metacognitive abilities, with associated impairment in emotional processes and social cognition. These deficits can compromise efforts in initiating and sustaining abstinence by hampering efficacy of clinical treatment and can obstruct efforts in enabling good decision making success in interpersonal/social interactions, and awareness of cognitive and behavioral dysfunctions. Despite evidence for differences in recovery levels of selective cognitive processes, certain deficits can persist even with prolonged sobriety. Herein is presented a review of alcohol-related cognitive impairments affecting component processes of executive functioning, memory, and the recently investigated cognitive domains of metamemory, social cognition, and emotional processing; also considered are trajectories of cognitiverecovery with abstinence. Finally, in the spirit of critical review, limitations of current knowledge are noted and avenues for new researchefforts are proposed that focus on (i) the interaction among emotion-cognition processes and identification of vulnerability factors contributing to the development of emotional and social processing deficits and (ii) the time line of cognitive recovery by tracking alcoholism's dynamic course of sobriety and relapse. Knowledge about the heterochronicity of cognitive recovery in alcoholism has the potential of indicating at which points during recovery intervention may be most beneficial.

Ji X, Saha S, Gao G, Lasek AW, Homanics GE, Guildford M, Tapper AR, GE. M. The sodium channel β4 auxiliary subunit selectively controls long-term depression in core nucleus accumbens medium spiny neurons. Frontiers in Cellular Neuroscience [Internet]. 2017;11 :17. Publisher's VersionAbstract

Voltage-gated sodium channels are essential for generating the initial rapid depolarization of neuronal membrane potential during action potentials (APs) that enable cell-to-cell communication, the propagation of signals throughout the brain, and the induction of synaptic plasticity. Although all brain neurons express one or several variants coding for the core pore-forming sodium channel α subunit, the expression of the β (β1-4) auxiliary subunits varies greatly. Of particular interest is the β4 subunit, encoded by the Scn4b gene, that is highly expressed in dorsal and ventral (i.e., nucleus accumbens - NAc) striata compared to other brain regions, and that endows sodium channels with unique gating properties. However, its role on neuronal activity, synaptic plasticity, and behaviors related to drugs of abuse remains poorly understood. Combining whole-cell patch-clamp recordings with two-photon calcium imaging in Scn4b knockout (KO) and knockdown mice, we found that Scn4b altered the properties of APs in core accumbens medium spiny neurons (MSNs). These alterations are associated with a reduction of the probability of MSNs to evoke spike-timing-dependent long-term depression (tLTD) and a reduced ability of backpropagating APs to evoke dendritic calcium transients. In contrast, long-term potentiation (tLTP) remained unaffected. Interestingly, we also showed that amphetamine-induced locomotor activity was significantly reduced in male Scn4b KO mice compared to wild-type controls. Taken together, these data indicate that the Scn4b subunit selectively controls tLTD by modulating dendritic calcium transients evoked by backpropagating APs.

Hitzemann R, Oberbeck D, Iancu O, Darakjian P, McWeeney S, Spence S, Schlumbohm J, Metten P, Crabbe J. Alignment of the transcriptome with individual variation in animals selectively bred for High Drinking-In-the-Dark (HDID). Alcohol [Internet]. 2017;60 :115-120. Publisher's VersionAbstract

Among animals at risk for excessive ethanol consumption such as the HDID selected mouse lines, there is considerable individual variation in the amount of ethanol consumed and the associated blood ethanol concentrations (BECs). For the HDID lines, this variation occurs even though the residual genetic variation associated with the DID phenotype has been largely exhausted and thus is most likely associated with epigenetic factors. Here we focus on the question of whether the genes associated with individual variation in HDID-1 mice are different from those associated with selection (risk) (Iancu et al., 2013). Thirty-three HDID-1 mice were phenotyped for their BECs at the end of a standard DID trial, were sacrificed 3 weeks later, and RNA-Seq was used to analyze the striatal transcriptome. The data obtained illustrate that there is considerable overlap of the risk and variation gene sets, both focused on the fine-tuning of synaptic plasticity.

Hashimoto JG, Gavin DP, Wiren KM, Crabbe JC, M. G. Prefrontal cortex expression of chromatin modifier genes in male WSP and WSR mice changes across ethanol dependence, withdrawal, and abstinence. Alcohol [Internet]. 2017;60 :83-94. Publisher's VersionAbstract
Alcohol-use disorder (AUD) is a relapsing disorder associated with excessive ethanol consumption. Recent studies support the involvement of epigenetic mechanisms in the development of AUD. Studies carried out so far have focused on a few specific epigenetic modifications. The goal of this project was to investigate gene expression changes of epigenetic regulators that mediate a broad array of chromatinmodifications after chronic alcohol exposure, chronic alcohol exposure followed by 8 h withdrawal, and chronic alcohol exposure followed by 21 days of abstinence in Withdrawal-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) selected mouse lines. We found that chronic vapor exposure to highly intoxicating levels of ethanol alters the expression of several chromatin remodeling genes measured by quantitative PCR array analyses. The identified effects were independent of selected lines, which, however, displayed baseline differences in epigenetic gene expression. We reported dysregulation in the expression of genes involved in histone acetylation, deacetylation, lysine and arginine methylation and ubiquitinationhylation during chronic ethanol exposure and withdrawal, but not after 21 days of abstinence. Ethanol-induced changes are consistent with decreased histone acetylation and with decreased deposition of the permissive ubiquitination mark H2BK120ub, associated with reduced transcription. On the other hand, ethanol-induced changes in the expression of genes involved in histone lysine methylation are consistent with increased transcription. The net result of these modifications on gene expression is likely to depend on the combination of the specific histone tail modifications present at a given time on a given promoter. Since alcohol does not modulate gene expression unidirectionally, it is not surprising that alcohol does not unidirectionally alter chromatin structure toward a closed or open state, as suggested by the results of this study.
Dutton JW 3rd, Chen H, You C, Brodie MS, AW. L. Anaplastic lymphoma kinase regulates binge-like drinking and dopamine receptor sensitivity in the ventral tegmental area. Addiction Biology [Internet]. 2017;22 :665-678. Publisher's VersionAbstract

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase associated with alcohol dependence in humans and behavioral responses to ethanol in mice. To characterize the ability of ALK to control ethanol consumption, we treated mice with the ALK inhibitors TAE684 or alectinib before testing them for binge-like drinking using the drinking in the dark protocol. Mice treated with ALK inhibitors drank less ethanol than controls. In addition, TAE684 treatment abolished ethanol conditioned place preference, indicating that ALK regulates the rewarding properties of ethanol. Because the ventral tegmental area (VTA) is a key brain region involved in the rewarding effects of ethanol, we determined if Alk expression in the VTA is important for binge-like ethanol consumption. Mice expressing a short hairpin ribonucleic acid targeting Alk in the VTA drank less ethanol compared with controls. ALK is expressed on dopamine (DA) neurons in the VTA, suggesting that ALK might regulate their firing properties. Extracellular recordings of putative DA neurons in VTA slices demonstrated that ALK inhibition did not affect the ability of ethanol to stimulate, or DA to inhibit, the firing of DA neurons. However, inhibiting ALK attenuated the time-dependent reversal of inhibition produced by moderate concentrations of DA, suggesting that ALK affects DA D2 autoreceptor (D2R) desensitization. Altered desensitization of the D2R changes the firing of DA neurons and is predicted to affect DA levels and alcohol drinking. These data support the possibility that ALK might be a novel target of pharmacotherapy for reducing excessive alcohol consumption.

Dir AL, Bell RL, Adams ZW, LA. H. Gender differences in risk factors for adolescent binge drinking and implications for intervention and prevention. Frontiers in Psychiatry [Internet]. 2017;8 :289. Publisher's VersionAbstract
Alcohol use, particularly binge drinking (BD), is a major public health concern among adolescents. Recent national data show that the gendergap in alcohol use is lessening, and BD among girls is rising. Considering the increase in BD among adolescent girls, as well as females' increased risk of experiencing more severe biopsychosocial negative effects and consequences from BD, the current review sought to examine gender differences in risk factors for BD. The review highlights gender differences in (1) developmental-related neurobiological vulnerability to BD, (2) psychiatric comorbidity and risk phenotypes for BD, and (3) social-related risk factors for BD among adolescents, as well as considerations for BD prevention and intervention. Most of the information gleaned thus far has come from preclinical research. However, it is expected that, with recent advances in clinical imaging technology, neurobiological effects observed in lower mammals will be confirmed in humans and vice versa. A synthesis of the literature highlights that males and females experience unique neurobiological paths of development, and although there is debate regarding the specific nature of these differences, literature suggests that these differences in turn influence gender differences in psychiatric comorbidity and risk for BD. For one, girls are more susceptible to stress, depression, and other internalizing behaviors and, in turn, these symptoms contribute to their risk for BD. On the other hand, males, given gender differencesacross the lifespan as well as gender differences in development, are driven by an externalizing phenotype for risk of BD, in part, due to unique paths of neurobiological development that occur across adolescence. With respect to social domains, although social and peer influences are important for both adolescent males and females, there are gender differences. For example, girls may be more sensitive to pressure from peers to fit in and impress others, while male gender role stereotypes regarding BD may be more of a risk factor for boys. Given these unique differences in male and female risk for BD, further research exploring risk factors, as well as tailoring intervention and prevention, is necessary. Although recent research has tailored substance use intervention to target males and females, more literature on gender considerations in treatment for prevention and intervention of BD in particular is warranted.
Ding ZM, Ingraham CM, Hauser SR, Lasek AW, Bell RL, WJ. MB. Reduced Levels of mGlu2 Receptors within the prelimbic cortex are not associated with elevated glutamate transmission or high alcohol drinking. Alcoholism: Clinical and Experimental Research [Internet]. 2017;41 :1896-1906. Publisher's VersionAbstract

BACKGROUND:

A Grm2 cys407* stop codon mutation, which results in a loss of the metabotropic glutamate 2 (mGlu2) receptor protein, was identified as being associated with high alcohol drinking by alcohol-preferring (P) rats. The objectives of the current study were to characterize the effects of reduced levels of mGlu2 receptors on glutamate transmission and alcohol drinking.

METHODS:

Quantitative no-net-flux microdialysis was used to test the hypothesis that basal extracellular glutamate levels in the prelimbic(PL) cortex and nucleus accumbens shell (NACsh) will be higher in P than Wistar rats. A lentiviral-delivered short-hairpin RNA (shRNA)-mediated knockdown was used to test the hypothesis that reduced levels of mGlu2 receptors within the PL cortex will increase voluntary alcohol drinking by Wistar rats. A linear regression analysis was used to test the hypothesis that there will be a significant correlation between the Grm2 cys407* mutation and level of alcohol intake.

RESULTS:

Extracellular glutamate concentrations within the PL cortex (3.6 ± 0.6 vs. 6.4 ± 0.6 μM) and NACsh (3.2 ± 0.4 vs. 6.6 ± 0.6 μM) were significantly lower in female P than female Wistar rats. Western blot detected the presence of mGlu2 receptors in these regions of female Wistar rats, but not female P rats. Micro-infusion of shRNAs into the PL cortex significantly reduced local mGlu2 receptor levels (by 40%), but did not alter voluntary alcohol drinking in male Wistar rats. In addition, there was no significant correlation between the Grm2 mutation and alcohol intake in 36 rodent lines (r = 0.29, p > 0.05).

CONCLUSIONS:

Collectively, these results suggest a lack of association between the loss of mGlu2 receptors and glutamate transmission in the NACsh and PL cortex of female P rats, and between the level of mGlu2 receptors in the PL cortex and alcohol drinking of male Wistar rats.

Crabbe JC. High Drinking in the Dark (HDID) mice are sensitive to the effects of some clinically relevant drugs to reduce binge-like drinking. Pharmacology Biochemistry and Behavior [Internet]. 2017;169 :55-62. Publisher's VersionAbstract
There is a serious public health need for better understanding of alcohol use disorder disease mechanisms and for improved treatments. At this writing, only three drugs are approved by the Food and Drug Administration as medications to treat alcohol use disorders - disulfiram, naltrexone, and acamprosate. Binge drinking is a form of abusive alcohol drinking defined by the NIAAA as a drinkingto blood alcohol levels (BALs)>0.08% during a period of approximately 2h. To model genetic risk for binge-like drinking, we have used selective breeding to create a unique animal model, High Drinking in the Dark (HDID) mice. Behavioral characterization of HDID mice has revealed that HDID mice exhibit behavioral impairment after drinking, withdrawal after a single binge-drinking session, and escalate their intake in response to induction of successive cycles of dependence. Notably, HDID mice do not exhibit altered tastant preference or alcohol clearance rates. We therefore asked whether drugs of known clinical relevance could modulate binge-like ethanol drinking in HDID mice, reasoning that this characterization of HDID responses should inform future use of this genetic animal model for screening and development of novel potential therapeutics.
Colville AM, Iancu OD, Oberbeck DL, Darakjian P, Zheng CL, Walter NA, Harrington CA, Searles RP, McWeeney S, RJ. H. Effects of selection for ethanol preference on gene expression in the nucleus accumbens of HS-CC mice. Genes Brain and Behavior [Internet]. 2017;16 (4) :462-471. Publisher's VersionAbstract

Previous studies on changes in murine brain gene expression associated with the selection for ethanol preference have used F2 intercross or heterogeneous stock (HS) founders, derived from standard laboratory strains. However, these populations represent only a small proportion of the genetic variance available in Mus musculus. To investigate a wider range of genetic diversity, we selected mice for ethanol preferenceusing an HS derived from the eight strains of the collaborative cross. These HS mice were selectively bred (four generations) for high and low ethanol preference. The nucleus accumbens shell of naive S4 mice was interrogated using RNA sequencing (RNA-Seq). Gene networks were constructed using the weighted gene coexpression network analysis assessing both coexpression and cosplicing. Selection targeted one of the network coexpression modules (greenyellow) that was significantly enriched in genes associated with receptor signaling activity including Chrna7, Grin2a, Htr2a and Oprd1. Connectivity in the module as measured by changes in the hub nodes was significantly reduced in the low preference line. Of particular interest was the observation that selection had marked effects on a large number of cell adhesion molecules, including cadherins and protocadherins. In addition, the coexpression data showed that selection had marked effects on long non-coding RNA hub nodes. Analysis of the cosplicing network data showed a significant effect of selection on a large cluster of Ras GTPase-binding genes including Cdkl5, Cyfip1, Ndrg1, Sod1 and Stxbp5. These data in part support the earlier observation that preference is linked to Ras/Mapk pathways.

Chen H, He D, AW. L. Midkine in the mouse ventral tegmental area limits ethanol intake and Ccl2 gene expression. Genes Brain and Behavior [Internet]. 2017;16 :699-708. Publisher's VersionAbstract
Midkine (MDK) is a cytokine and neurotrophic factor that is more highly expressed in the brains of alcoholics and in mice predisposed to drink large amounts of ethanol, suggesting that MDK may regulate ethanol consumption. Here we measured ethanol consumption in male and female Mdk knockout (-/-) mice using the two-bottle choice and the drinking in the dark (DID) tests. We found that Mdk -/- mice consumed significantly more ethanol than wild-type controls in both tests. To determine if MDK acts in the ventral tegmental area (VTA) to regulate ethanol consumption, we delivered lentivirus expressing a Mdk shRNA into the VTA of male C57BL/6J mice to locally knockdown Mdk and performed the DID test. Mice expressing a Mdk shRNA in the VTA consumed more ethanol than mice expressing a control non-targeting shRNA, demonstrating that the VTA is one site in the brain through which MDK acts to regulate ethanol consumption. Since MDK also controls the expression of inflammatory cytokines in other organs, we examined gene expression of interleukin-1 beta (Il1b), tumor necrosis factor alpha (Tnfα) and the chemokine (C-C motif) ligand 2 (Ccl2) in the VTA of Mdk -/- mice and in mice expressing Mdk shRNA in the VTA. Expression of Ccl2 was elevated in the VTA of Mdk -/- mice and in mice expressing Mdk shRNA in the VTA. These results demonstrate that MDK functions in the VTA to limit ethanol consumption and levels of CCL2, a chemokine known to increase ethanol consumption.
Borghese CM, Herman M, Snell LD, Lawrence KJ, Lee HY, Backos DS, Vanderlinden LA, RA H, M R, Hoffman PL, et al. Novel molecule exhibiting selective affinity for GABAA receptor subtypes. Scientific Reports [Internet]. 2017;7 :6230. Publisher's VersionAbstract
Aminoquinoline derivatives were evaluated against a panel of receptors/channels/transporters in radioligand binding experiments. One of these derivatives (DCUK-OEt) displayed micromolar affinity for brain γ-aminobutyric acid type A (GABAA) receptors. DCUK-OEt was shown to be a positive allosteric modulator (PAM) of GABA currents with α1β2γ2, α1β3γ2, α5β3γ2 and α1β3δ GABAA receptors, while having no significant PAM effect on αβ receptors or α1β1γ2, α1β2γ1, α4β3γ2 or α4β3δ receptors. DCUK-OEt modulation of α1β2γ2 GABAA receptors was not blocked by flumazenil. The subunit requirements for DCUK-OEt actions distinguished DCUK-OEt from other currently known modulators of GABA function (e.g., anesthetics, neurosteroids or ethanol). Simulated docking of DCUK-OEt at the GABAA receptorsuggested that its binding site may be at the α + β- subunit interface. In slices of the central amygdala, DCUK-OEt acted primarily on extrasynaptic GABAA receptors containing the α1 subunit and generated increases in extrasynaptic "tonic" current with no significant effect on phasic responses to GABA. DCUK-OEt is a novel chemical structure acting as a PAM at particular GABAA receptors. Given that neurons in the central amygdala responding to DCUK-OEt were recently identified as relevant for alcohol dependence, DCUK-OEt should be further evaluated for the treatment of alcoholism.
Blednov YA, Borghese CM, Ruiz CI, Cullins MA, Da Costa A, Osterndorff-Kahanek EA, Homanics GE, RA. H. Mutation of the inhibitory ethanol site in GABA(A) ρ1 receptors promotes tolerance to ethanol-induced motor incoordination. Neuropharmacology [Internet]. 2017;123 :201-209. Publisher's VersionAbstract
Genes encoding the ρ1/2 subunits of GABAA receptors have been associated with alcohol (ethanol) dependence in humans, and ρ1 was also shown to regulate some of the behavioral effects of ethanol in animal models. Ethanol inhibits GABA-mediated responses in wild-type (WT) ρ1, but not ρ1(T6'Y) mutant receptors expressed in Xenopus laevis oocytes, indicating the presence of an inhibitory site for ethanol in the second transmembrane helix. In this study, we found that ρ1(T6'Y) receptors expressed in oocytes display overall normal responses to GABA, the endogenous GABA modulator (zinc), and partial agonists (β-alanine and taurine). We generated ρ1 (T6'Y) knockin (KI) mice using CRISPR/Cas9 to test the behavioral importance of the inhibitory actions of ethanol on this receptor. Both ρ1 KI and knockout (KO) mice showed faster recovery from acute ethanol-induced motor incoordination compared to WT mice. Both KI and KO mutant strains also showed increased tolerance to motor impairment produced by ethanol. The KI mice did not differ from WT mice in other behavioral actions, including ethanol intake and preference, conditioned taste aversion to ethanol, and duration of ethanol-induced loss of righting reflex. WT and KI mice did not differ in levels of ρ1 or ρ2 mRNA in cerebellum or in ethanol clearance. Our findings indicate that the inhibitory site for ethanol in GABAA ρ1 receptors regulates acute functional tolerance to moderate ethanol intoxication. We note that low sensitivity to alcohol intoxication has been linked to risk for development of alcohol dependence in humans.
Blednov YA, Black M, Benavidez JM, Da Costa A, Mayfield J, RA. H. Sedative and motor incoordination effects of ethanol in mice lacking CD14, TLR2, TLR4, or MyD88. Alcoholism: Clinical and Experimental Research [Internet]. 2017;41 (3) :531-540. Publisher's VersionAbstract

BACKGROUND:

In our companion article, we examined the role of MyD88-dependent signaling in ethanol (EtOH) consumption in mice lacking key components of this inflammatory pathway and observed differential effects on drinking. Here, we studied the role of these same signaling components in the acute sedative, intoxicating, and physiological effects of EtOH. Toll-like receptor 4 (TLR4) has been reported to strongly reduce the duration of EtOH-induced sedation, although most studies do not support its direct involvement in EtOH consumption. We examined TLR4 and other MyD88 pathway molecules to determine signaling specificity in acute EtOH-related behaviors. We also studied other GABAergic sedatives to gauge the EtOH specificity and potential role for GABA in EtOH's sedative and intoxicating effects in the mutant mice.

METHODS:

Loss of righting reflex (LORR) and recovery from motor incoordination were studied following acute injection of EtOH or other sedative drugs in male and female control C57BL/6J mice versus mice lacking CD14, TLR2, TLR4 (C57BL/10ScN), or MyD88. We also examined EtOH-induced hypothermia and blood EtOH clearance in these mice.

RESULTS:

Male and female mice lacking TLR4 or MyD88 showed reduced duration of EtOH-induced LORR and faster recovery from EtOH-induced motor incoordination in the rotarod test. MyD88 knockout mice had slightly faster recovery from EtOH-induced hypothermia compared to control mice. None of the mutants differed from control mice in the rate of blood EtOH clearance. All of the mutants showed similar decreases in the duration of gaboxadol-induced LORR, but only mice lacking TLR4 were less sensitive to the sedative effects of pentobarbital. Faster recovery from diazepam-induced motor impairment was observed in CD14, TLR4, and MyD88 null mice of both sexes.

CONCLUSIONS:

TLR4 and MyD88 were key mediators of the sedative and intoxicating effects of EtOH and GABAergic sedatives, indicating a strong influence of TLR4-MyD88 signaling on GABAergic function. Despite the involvement of TLR4 in EtOH's acute behaviors, it did not regulate EtOH consumption in any drinking model as shown in our companion article. Collectively, our studies demonstrate differential effects of TLR-MyD88 components in the acute versus chronic actions of EtOH.

Copyright © 2017 by the Research Society on Alcoholism

Bell RL, Hauser SR, Liang T, Sari Y, Maldonado-Devincci A, ZA. R. Rat animal models for screening medications to treat alcohol use disorders. Neuropharmacology [Internet]. 2017;122 :201-243. Publisher's VersionAbstract
The purpose of this review is to present animal research models that can be used to screen and/or repurpose medications for the treatment of alcohol abuse and dependence. The focus will be on rats and in particular selectively bred rats. Brief introductions discuss various aspects of the clinical picture, which provide characteristics of individuals with alcohol use disorders (AUDs) to model in animals. Following this, multiple selectively bred rat lines will be described and evaluated in the context of animal models used to screen medications to treat AUDs. Next, common behavioral tests for drug efficacy will be discussed particularly as they relate to stages in the addiction cycle. Tables highlighting studies that have tested the effects of compounds using the respective techniques are included. Wherever possible the Tables are organized chronologically in ascending order to describe changes in the focus of research on AUDs over time. In general, high ethanol-consuming selectively bred rats have been used to test a wide range of compounds. Older studies usually followed neurobiological findings in the selected lines that supported an association with a propensity for high ethanol intake. Most of these tests evaluated the compound's effects on the maintenance of ethanol drinking. Very few compounds have been tested during ethanol-seeking and/or relapse and fewer still have assessed their effects during the acquisition of AUDs. Overall, while a substantial number of neurotransmitter and neuromodulatory system targets have been assessed; the roles of sex- and age-of-animal, as well as the acquisition of AUDs, ethanol-seeking and relapse continue to be factors and behaviors needing further study. This article is part of the Special Issue entitled "Alcoholism".
Arefin TM, Mechling AE, Meirsman AC, Bienert T, Hübner NS, Lee HL, Ben Hamida S, Ehrlich A, Roquet D, Hennig J, et al. Remodeling of sensorimotor brain connectivity in Gpr88-deficient mice. Brain Connectivity [Internet]. 2017;7 (8) :526-540. Publisher's VersionAbstract
Recent studies have demonstrated that orchestrated gene activity and expression support synchronous activity of brain networks. However, there is a paucity of information on the consequences of single gene function on overall brain functional organization and connectivity and how this translates at the behavioral level. In this study, we combined mouse mutagenesis with functional and structural magnetic resonance imaging (MRI) to determine whether targeted inactivation of a single gene would modify whole-brain connectivity in live animals. The targeted gene encodes GPR88 (G protein-coupled receptor 88), an orphan G protein-coupled receptor enriched in the striatum and previously linked to behavioral traits relevant to neuropsychiatric disorders. Connectivity analysis of Gpr88-deficient mice revealed extensive remodeling of intracortical and cortico-subcortical networks. Most prominent modifications were observed at the level of retrosplenial cortex connectivity, central to the default mode network (DMN) whose alteration is considered a hallmark of many psychiatric conditions. Next, somatosensory and motor cortical networks were most affected. These modifications directly relate to sensorimotor gating deficiency reported in mutant animals and also likely underlie their hyperactivity phenotype. Finally, we identified alterations within hippocampal and dorsal striatum functional connectivity, most relevant to a specific learning deficit that we previously reported in Gpr88-/- animals. In addition, amygdala connectivity with cortex and striatum was weakened, perhaps underlying the risk-taking behavior of these animals. This is the first evidence demonstrating that GPR88 activity shapes the mouse brain functional and structural connectome. The concordance between connectivityalterations and behavior deficits observed in Gpr88-deficient mice suggests a role for GPR88 in brain communication.

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