Publications

2016
Amanda M. Barkley-Levenson, Andrey E. Ryabinin, and John C. Crabbe. “Neuropeptide Y response to alcohol is altered in nucleus accumbens of mice selectively bred for drinking to intoxication.” Behavioural Brain Research, 302, Pp. 160–170. Abstract
The High Drinking in the Dark (HDID) mice have been selectively bred for drinking to intoxicating blood alcohol levels and represent a genetic model of risk for binge-like drinking. Presently, little is known about the specific genetic factors that promote excessive intake in these mice. Previous studies have identified neuropeptide Y (NPY) as a potential target for modulating alcohol intake. NPY expression differs in some rodent lines that have been selected for high and low alcohol drinking phenotypes, as well as inbred mouse strains that differ in alcohol preference. Alcohol drinking and alcohol withdrawal also produce differential effects on NPY expression in the brain. Here, we assessed brain NPY protein levels in HDID mice of two replicates of selection and control heterogeneous stock (HS) mice at baseline (water drinking) and after binge-like alcohol drinking to determine whether selection is associated with differences in NPY expression and its sensitivity to alcohol. NPY levels did not differ between HDID and HS mice in any brain region in the water-drinking animals. HS mice showed a reduction in NPY levels in the nucleus accumbens (NAc) - especially in the shell - in ethanol-drinking animals vs. water-drinking controls. However, HDID mice showed a blunted NPY response to alcohol in the NAc core and shell compared to HS mice. These findings suggest that the NPY response to alcohol has been altered by selection for drinking to intoxication in a region-specific manner. Thus, the NPY system may represent a potential target for altering binge-like alcohol drinking in these mice.
Bin Qiu, Richard L. Bell, Yong Cao, Lingling Zhang, Robert B. Stewart, Tamara Graves, Lawrence Lumeng, Weidong Yong, and Tiebing Liang. “Npy deletion in an alcohol non-preferring rat model elicits differential effects on alcohol consumption and body weight.” Journal of Genetics and Genomics = Yi Chuan Xue Bao, 43, 7, Pp. 421–430. Abstract
Neuropeptide Y (NPY) is widely expressed in the central nervous system and influences many physiological processes. It is located within the rat quantitative trait locus (QTL) for alcohol preference on chromosome 4. Alcohol-nonpreferring (NP) rats consume very little alcohol, but have significantly higher NPY expression in the brain than alcohol-preferring (P) rats. We capitalized on this phenotypic difference by creating an Npy knockout (KO) rat using the inbred NP background to evaluate NPY effects on alcohol consumption. Zinc finger nuclease (ZNF) technology was applied, resulting in a 26-bp deletion in the Npy gene. RT-PCR, Western blotting and immunohistochemistry confirmed the absence of Npy mRNA and protein in KO rats. Alcohol consumption was increased in Npy(+/-) but not Npy(-/-) rats, while Npy(-/-) rats displayed significantly lower body weight when compared to Npy(+/+) rats. In whole brain tissue, expression levels of Npy-related and other alcohol-associated genes, Npy1r, Npy2r, Npy5r, Agrp, Mc3r, Mc4r, Crh and Crh1r, were significantly greater in Npy(-/-) rats, whereas Pomc and Crhr2 expressions were highest in Npy(+/-) rats. These findings suggest that the NPY-system works in close coordination with the melanocortin (MC) and corticotropin-releasing hormone (CRH) systems to modulate alcohol intake and body weight.
Gerald A. Deehan, Christopher P. Knight, R. Aaron Waeiss, Eric A. Engleman, Jamie E. Toalston, William J. McBride, Sheketha R. Hauser, and Zachary A. Rodd. “Peripheral Administration of Ethanol Results in a Correlated Increase in Dopamine and Serotonin Within the Posterior Ventral Tegmental Area.” Alcohol and Alcoholism (Oxford, Oxfordshire), 51, 5, Pp. 535–540. Abstract
AIMS: Two critical neurotransmitter systems regulating ethanol (EtOH) reward are serotonin (5-HT) and dopamine (DA). Within the posterior ventral tegmental area (pVTA), 5-HT receptors have been shown to regulate DA neuronal activity. Increased pVTA neuronal activity has been linked to drug reinforcement. The current experiment sought to determine the effect of EtOH on 5-HT and DA levels within the pVTA. METHODS: Wistar rats were implanted with cannula aimed at the pVTA. Neurochemical levels were determined using standard microdialysis procedures with concentric probes. Rats were randomly assigned to one of the five groups (n = 41; 7-9 per group) that were treated with 0-3.0 g/kg EtOH (intraperitoneally). RESULTS: Ethanol produced increased extracellular DA levels in the pVTA that resembled an inverted U-shape dose-response curve with peak levels (\textasciitilde200% of baseline) at the 2.25 g/kg dose. The increase in DA levels was observed for an extended period of time (\textasciitilde100 minutes). The effects of EtOH on extracellular 5-HT levels in the pVTA also resembled an inverted U-shape dose-response curve. However, increased 5-HT levels were only observed during the initial post-injection sample. The increases in extracellular DA and 5-HT levels were significantly correlated. CONCLUSION: The data indicate intraperitoneal EtOH administration stimulated the release of both 5-HT and DA within the pVTA, the levels of which were significantly correlated. Overall, the current findings suggest that the ability of EtOH to stimulate DA activity within the mesolimbic system may be modulated by increases in 5-HT release within the pVTA. SHORT SUMMARY: Two critical neurotransmitter systems regulating ethanol reward are serotonin and dopamine. The current experiment determined that intraperitoneal ethanol administration increased serotonin and dopamine levels within the pVTA (levels were significantly correlated). The current findings suggest the ability of EtOH to stimulate serotonin and dopamine activity within the mesolimbic system.
Yuri A. Blednov, Mendy Black, Jillian M. Benavidez, Eleni E. Stamatakis, and R. Adron Harris. “PPAR Agonists: I. Role of Receptor Subunits in Alcohol Consumption in Male and Female Mice.” Alcoholism, Clinical and Experimental Research, 40, 3, Pp. 553–562. Abstract
BACKGROUND: Several peroxisome proliferator-activated receptor (PPAR) agonists reduce voluntary alcohol consumption in rodent models, and evidence suggests that PPARα and γ subunits play an important role in this effect. To define the subunit dependence of this action, we tested selective PPARα and α/γ agonists and antagonists in addition to null mutant mice lacking PPARα. METHODS: The effects of fenofibrate (PPARα agonist) and tesaglitazar (PPARα/γ agonist) on continuous and intermittent 2-bottle choice drinking tests were examined in male and female wild-type mice and in male mice lacking PPARα. We compared the ability of MK886 (PPARα antagonist) and GW9662 (PPARγ antagonist) to inhibit the effects of fenofibrate and tesaglitazar in wild-type mice. The estrogen receptor antagonist, tamoxifen, can inhibit PPARγ-dependent transcription and was also studied in male and female mice. RESULTS: Fenofibrate and tesaglitazar reduced ethanol (EtOH) consumption and preference in wild-type mice, but these effects were not observed in mice lacking PPARα. MK886 inhibited the action of fenofibrate, but not tesaglitazer, while GW9662 did not inhibit either agonist. The PPAR agonists were more effective in male mice compared to females, and drinking in the continuous 2-bottle choice test was more sensitive to fenofibrate and tesaglitazar compared to drinking in the intermittent access test. Tamoxifen also reduced EtOH consumption in male mice and this action was inhibited by GW9662, but not MK886, suggesting that it acts by activation of PPARγ. CONCLUSIONS: Our study using selective PPAR agonists, antagonists, and null mutant mice indicates a key role for PPARα in mediating reduced EtOH consumption by fenofibrate and tesaglitazar.
Yuri A. Blednov, Mendy Black, Jillian M. Benavidez, Eleni E. Stamatakis, and R. Adron Harris. “PPAR Agonists: II. Fenofibrate and Tesaglitazar Alter Behaviors Related to Voluntary Alcohol Consumption.” Alcoholism, Clinical and Experimental Research, 40, 3, Pp. 563–571. Abstract
BACKGROUND: In the accompanying article, we showed that activation of peroxisome proliferator-activated receptor alpha (PPARα) signaling by fenofibrate and tesaglitazar decreases ethanol (EtOH) consumption in mice. In this study, we determined the role of these PPAR agonists in EtOH-related behaviors and other actions that may be important in regulating EtOH consumption. METHODS: The effects of fenofibrate (150 mg/kg) and tesaglitazar (1.5 mg/kg) were examined on the following responses in male and female C57BL/6J (B6) and B6 × 129S4 mice: preference for saccharin, EtOH-induced conditioned place preference (CPP), conditioned taste aversion (CTA), loss of righting reflex, and withdrawal, acoustic startle reflex, response to novelty, and EtOH clearance. Because the B6 inbred strain usually displays weak EtOH-induced CPP and weak EtOH-induced acute withdrawal, B6 × 129S4 mice were also studied. RESULTS: Fenofibrate and tesaglitazar decreased the novelty response and increased acute EtOH withdrawal severity, and fenofibrate increased EtOH-induced CTA. Two important factors for EtOH consumption (saccharin preference and EtOH-induced CPP) were not altered by fenofibrate or tesaglitazar. EtOH clearance was increased by both fenofibrate and tesaglitazar. Response to novelty, acute withdrawal, and EtOH clearance show sex differences and could contribute to the reduced EtOH consumption following fenofibrate administration. CONCLUSIONS: These studies indicate the complexity of EtOH-dependent and EtOH-independent behaviors that are altered by PPAR agonists and provide evidence for novel behavioral actions of these drugs that may contribute to PPAR-mediated effects on alcohol drinking.
A. E. Ryabinin and C. M. Hostetler. “Prairie Voles as a Model to Screen Medications for the Treatment of Alcoholism and Addictions.” International Review of Neurobiology, 126, Pp. 403–421. Abstract
Most preclinical studies of medications to treat addictions are performed in mice and rats. These two rodent species belong to one phylogenetic subfamily, which narrows the likelihood of identifying potential mechanisms regulating addictions in other species, ie, humans. Expanding the genetic diversity of organisms modeling alcohol and drug abuse enhances our ability to screen for medications to treat addiction. Recently, research laboratories adapted the prairie vole model to study mechanisms of alcohol and drugs of abuse. This development not only expanded the diversity of genotypes used to screen medications, but also enhanced capabilities of such screens. Prairie voles belong to 3-5% of mammalian species exhibiting social monogamy. This unusual trait is reflected in their ability to form lasting long-term affiliations between adult individuals. The prairie vole animal model has high predictive validity for mechanisms regulating human social behaviors. In addition, these animals exhibit high alcohol intake and preference. In laboratory settings, prairie voles are used to model social influences on drug reward and alcohol consumption as well as effects of addictive substances on social bonding. As a result, this species can be adapted to screen medications whose effectiveness could be (a) resistant to social influences promoting excessive drug taking, (b) dependent on the presence of social support, and (c) medications affecting harmful social consequences of alcohol and drug abuse. This report reviews the literature on studies of alcohol and psychostimulants in prairie voles and discusses capabilities of this animal model as a screen for novel medications to treat alcoholism and addictions.
John C. Crabbe. “Progress With Nonhuman Animal Models of Addiction.” Journal of Studies on Alcohol and Drugs, 77, 5, Pp. 696–699. Abstract
Nonhuman animals have been major contributors to the science of the genetics of addiction. Given the explosion of interest in genetics, it is fair to ask, are we making reasonable progress toward our goals with animal models? I will argue that our goals are changing and that overall progress has been steady and seems likely to continue apace. Genetics tools have developed almost incredibly rapidly, enabling both more reductionist and more synthetic or integrative approaches. I believe that these approaches to making progress have been unbalanced in biomedical science, favoring reductionism, particularly in animal genetics. I argue that substantial, novel progress is also likely to come in the other direction, toward synthesis and abstraction. Another area in which future progress with genetic animal models seems poised to contribute more is the reconciliation of human and animal phenotypes, or consilience. The inherent power of the genetic animal models could be more profitably exploited. In the end, animal research has continued to provide novel insights about how genes influence individual differences in addiction risk and consequences. The rules of the genetics game are changing so fast that it is hard to remember how comparatively little we knew even a generation ago. Rather than worry about whether we have been wasting time and resources asking the questions we have been, we should look to the future and see if we can come up with some new ones. The valuable findings from the past will endure, and the sidetracks will be forgotten.
Debra K. Cozzoli, Justin Courson, Charlotte Rostock, Rianne R. Campbell, Melissa G. Wroten, Hadley McGregor, Amanda L. Caruana, Bailey W. Miller, Jia-Hua Hu, Ping Wu Zhang, Bo Xiao, Paul F. Worley, John C. Crabbe, Deborah A. Finn, and Karen K. Szumlinski. “Protein Kinase C Epsilon Activity in the Nucleus Accumbens and Central Nucleus of the Amygdala Mediates Binge Alcohol Consumption.” Biological Psychiatry, 79, 6, Pp. 443–451. Abstract
BACKGROUND: Protein kinase C epsilon (PKCε) is emerging as a potential target for the development of pharmacotherapies to treat alcohol use disorders, yet little is known regarding how a history of a highly prevalent form of drinking, binge alcohol intake, influences enzyme priming or the functional relevance of kinase activity for excessive alcohol intake. METHODS: Immunoblotting was employed on tissue from subregions of the nucleus accumbens (NAc) and the amygdala to examine both idiopathic and binge drinking-induced changes in constitutive PKCε priming. The functional relevance of PKCε translocation for binge drinking and determination of potential upstream signaling pathways involved were investigated using neuropharmacologic approaches within the context of two distinct binge drinking procedures, drinking in the dark and scheduled high alcohol consumption. RESULTS: Binge alcohol drinking elevated p(Ser729)-PKCε levels in both the NAc and the central nucleus of the amygdala (CeA). Moreover, immunoblotting studies of selectively bred and transgenic mouse lines revealed a positive correlation between the propensity to binge drink alcohol and constitutive p(Ser729)-PKCε levels in the NAc and CeA. Finally, neuropharmacologic inhibition of PKCε translocation within both regions reduced binge alcohol consumption in a manner requiring intact group 1 metabotropic glutamate receptors, Homer2, phospholipase C, and/or phosphotidylinositide-3 kinase function. CONCLUSIONS: Taken together, these data indicate that PKCε signaling in both the NAc and CeA is a major contributor to binge alcohol drinking and to the genetic propensity to consume excessive amounts of alcohol.
Shafiqur Rahman, Eric A. Engleman, and Richard L. Bell. “Recent Advances in Nicotinic Receptor Signaling in Alcohol Abuse and Alcoholism.” Progress in Molecular Biology and Translational Science, 137, Pp. 183–201. Abstract
Alcohol is the most commonly abused legal substance and alcoholism is a serious public health problem. It is a leading cause of preventable death in the world. The cellular and molecular mechanisms of alcohol reward and addiction are still not well understood. Emerging evidence indicates that unlike other drugs of abuse, such as nicotine, cocaine, or opioids, alcohol targets numerous channel proteins, receptor molecules, and signaling pathways in the brain. Previously, research has identified brain nicotinic acetylcholine receptors (nAChRs), a heterogeneous family of pentameric ligand-gated cation channels expressed in the mammalian brain, as critical molecular targets for alcohol abuse and dependence. Genetic variations encoding nAChR subunits have been shown to increase the vulnerability to develop alcohol dependence. Here, we review recent insights into the rewarding effects of alcohol, as they pertain to different nAChR subtypes, associated signaling molecules, and pathways that contribute to the molecular mechanisms of alcoholism and/or comorbid brain disorders. Understanding these cellular changes and molecular underpinnings may be useful for the advancement of brain nicotinic-cholinergic mechanisms, and will lead to a better translational and therapeutic outcome for alcoholism and/or comorbid conditions.
John C. Crabbe. “Reproducibility of Experiments with Laboratory Animals: What Should We Do Now?.” Alcoholism, Clinical and Experimental Research, 40, 11, Pp. 2305–2308.
Ma HY, Xu J, Liu X, Zhu Y, Gao B, Karin M, Tsukamoto H, Jeste DV, Grant I, Roberts AJ, Contet C, Geoffroy C, Zheng B, and Brenner D. “The role of IL-17 signaling in regulation of the liver-brain axis and intestinal permeability in alcoholic liver disease..” Current Pathobiology Reports, 4, Pp. 27-35. Publisher's Version Abstract

Alcoholic liver disease (ALD) progresses from a normal liver, to steatosis, steatohepatitis, fibrosis and hepatocellular carcinoma (HCC). Despite intensive studies, the pathogenesis of ALD is poorly understood, in part due to a lack of suitable animal models which mimic the stages of ALD progression. Furthermore, the role of IL-17 in ALD has not been evaluated. We and others have recently demonstrated that IL-17 signaling plays a critical role in development of liver fibrosis and cancer. Here we summarize the most recent evidence supporting the roleof IL-17 in ALD. As a result of a collaborative effort of Drs. Karin, Gao, Tsukamoto and Kisseleva, we developed several improved models of ALD in mice: 1) chronic-plus-binge model that mimics early stages of steatohepatitis, 2) intragastric ethanol feeding model that mimics alcoholic steatohepatitis and fibrosis, and 3) diethylnitrosamine (DEN)+alcohol model that mimics alcoholic liver cancer. These models might provide new insights into the mechanism of IL-17 signaling in ALD and help identify novel therapeutic targets.

Dana Most, Courtney Leiter, Yuri A. Blednov, R. Adron Harris, and R. Dayne Mayfield. “Synaptic microRNAs Coordinately Regulate Synaptic mRNAs: Perturbation by Chronic Alcohol Consumption.” Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology, 41, 2, Pp. 538–548. Abstract
Local translation of mRNAs in the synapse has a major role in synaptic structure and function. Chronic alcohol use causes persistent changes in synaptic mRNA expression, possibly mediated by microRNAs localized in the synapse. We profiled the transcriptome of synaptoneurosomes (SN) obtained from the amygdala of mice that consumed 20% ethanol (alcohol) in a 30-day continuous two-bottle choice test to identify the microRNAs that target alcohol-induced mRNAs. SN are membrane vesicles containing pre- and post-synaptic compartments of neurons and astroglia and are a unique model for studying the synaptic transcriptome. We previously showed that chronic alcohol regulates mRNA expression in a coordinated manner. Here, we examine microRNAs and mRNAs from the same samples to define alcohol-responsive synaptic microRNAs and their predicted interactions with targeted mRNAs. The aim of the study was to identify the microRNA-mRNA synaptic interactions that are altered by alcohol. This was accomplished by comparing the effect of alcohol in SN and total homogenate preparations from the same samples. We used a combination of unbiased bioinformatic methods (differential expression, correlation, co-expression, microRNA-mRNA target prediction, co-targeting, and cell type-specific analyses) to identify key alcohol-sensitive microRNAs. Prediction analysis showed that a subset of alcohol-responsive microRNAs was predicted to target many alcohol-responsive mRNAs, providing a bidirectional analysis for identifying microRNA-mRNA interactions. We found microRNAs and mRNAs with overlapping patterns of expression that correlated with alcohol consumption. Cell type-specific analysis revealed that a significant number of alcohol-responsive mRNAs and microRNAs were unique to glutamate neurons and were predicted to target each other. Chronic alcohol consumption appears to perturb the coordinated microRNA regulation of mRNAs in SN, a mechanism that may explain the aberrations in synaptic plasticity affecting the alcoholic brain.
Ruben V. Hernandez, Alana C. Puro, Jessica C. Manos, Salvador Huitron-Resendiz, Kenneth C. Reyes, Kevin Liu, Khanh Vo, Amanda J. Roberts, and Donna L. Gruol. “Transgenic mice with increased astrocyte expression of IL-6 show altered effects of acute ethanol on synaptic function.” Neuropharmacology, 103, Pp. 27–43. Abstract
A growing body of evidence has revealed that resident cells of the central nervous system (CNS), and particularly the glial cells, comprise a neuroimmune system that serves a number of functions in the normal CNS and during adverse conditions. Cells of the neuroimmune system regulate CNS functions through the production of signaling factors, referred to as neuroimmune factors. Recent studies show that ethanol can activate cells of the neuroimmune system, resulting in the elevated production of neuroimmune factors, including the cytokine interleukin-6 (IL-6). Here we analyzed the consequences of this CNS action of ethanol using transgenic mice that express elevated levels of IL-6 through increased astrocyte expression (IL-6-tg) to model the increased IL-6 expression that occurs with ethanol use. Results show that increased IL-6 expression induces neuroadaptive changes that alter the effects of ethanol. In hippocampal slices from non-transgenic (non-tg) littermate control mice, synaptically evoked dendritic field excitatory postsynaptic potential (fEPSP) and somatic population spike (PS) at the Schaffer collateral to CA1 pyramidal neuron synapse were reduced by acute ethanol (20 or 60 mM). In contrast, acute ethanol enhanced the fEPSP and PS in hippocampal slices from IL-6 tg mice. Long-term synaptic plasticity of the fEPSP (i.e., LTP) showed the expected dose-dependent reduction by acute ethanol in non-tg hippocampal slices, whereas LTP in the IL-6 tg hippocampal slices was resistant to this depressive effect of acute ethanol. Consistent with altered effects of acute ethanol on synaptic function in the IL-6 tg mice, EEG recordings showed a higher level of CNS activity in the IL-6 tg mice than in the non-tg mice during the period of withdrawal from an acute high dose of ethanol. These results suggest a potential role for neuroadaptive effects of ethanol-induced astrocyte production of IL-6 as a mediator or modulator of the actions of ethanol on the CNS, including persistent changes in CNS function that contribute to cognitive dysfunction and the development of alcohol dependence.
Ruben V. Hernandez, Alana C. Puro, Jessica C. Manos, Salvador Huitron-Resendiz, Kenneth C. Reyes, Kevin Liu, Khanh Vo, Amanda J. Roberts, and Donna L. Gruol. “Transgenic mice with increased astrocyte expression of IL-6 show altered effects of acute ethanol on synaptic function.” Neuropharmacology, 103, Pp. 27–43. Abstract
A growing body of evidence has revealed that resident cells of the central nervous system (CNS), and particularly the glial cells, comprise a neuroimmune system that serves a number of functions in the normal CNS and during adverse conditions. Cells of the neuroimmune system regulate CNS functions through the production of signaling factors, referred to as neuroimmune factors. Recent studies show that ethanol can activate cells of the neuroimmune system, resulting in the elevated production of neuroimmune factors, including the cytokine interleukin-6 (IL-6). Here we analyzed the consequences of this CNS action of ethanol using transgenic mice that express elevated levels of IL-6 through increased astrocyte expression (IL-6-tg) to model the increased IL-6 expression that occurs with ethanol use. Results show that increased IL-6 expression induces neuroadaptive changes that alter the effects of ethanol. In hippocampal slices from non-transgenic (non-tg) littermate control mice, synaptically evoked dendritic field excitatory postsynaptic potential (fEPSP) and somatic population spike (PS) at the Schaffer collateral to CA1 pyramidal neuron synapse were reduced by acute ethanol (20 or 60 mM). In contrast, acute ethanol enhanced the fEPSP and PS in hippocampal slices from IL-6 tg mice. Long-term synaptic plasticity of the fEPSP (i.e., LTP) showed the expected dose-dependent reduction by acute ethanol in non-tg hippocampal slices, whereas LTP in the IL-6 tg hippocampal slices was resistant to this depressive effect of acute ethanol. Consistent with altered effects of acute ethanol on synaptic function in the IL-6 tg mice, EEG recordings showed a higher level of CNS activity in the IL-6 tg mice than in the non-tg mice during the period of withdrawal from an acute high dose of ethanol. These results suggest a potential role for neuroadaptive effects of ethanol-induced astrocyte production of IL-6 as a mediator or modulator of the actions of ethanol on the CNS, including persistent changes in CNS function that contribute to cognitive dysfunction and the development of alcohol dependence.
Natalie M. Zahr, Torsten Rohlfing, Dirk Mayer, Richard Luong, Edith V. Sullivan, and Adolf Pfefferbaum. “Transient CNS responses to repeated binge ethanol treatment.” Addiction Biology, 21, 6, Pp. 1199–1216. Abstract
The effects of ethanol (EtOH) on in vivo magnetic resonance (MR)-detectable brain measures across repeated exposures have not previously been reported. Of 28 rats weighing 340.66 ± 21.93 g at baseline, 15 were assigned to an EtOH group and 13 to a control group. Animals were exposed to five cycles of 4 days of intragastric (EtOH or dextrose) treatment and 10 days of recovery. Rats in both groups had structural MR imaging and whole-brain MR spectroscopy (MRS) scans at baseline, immediately following each binge period and after each recovery period (total = 11 scans per rat). Blood alcohol level at each of the five binge periods was \textasciitilde300 mg/dl. Blood drawn at the end of the experiment did not show group differences for thiamine or its phosphate derivatives. Postmortem liver histopathology provided no evidence for hepatic steatosis, alcoholic hepatitis or alcoholic cirrhosis. Cerebrospinal fluid volumes of the lateral ventricles and cisterns showed enlargement with each binge EtOH exposure but recovery with each abstinence period. Similarly, changes in MRS metabolite levels were transient: levels of N-acetylaspartate and total creatine decreased, while those of choline-containing compounds and the combined resonance from glutamate and glutamine increased with each binge EtOH exposure cycle and then recovered during each abstinence period. Changes in response to EtOH were in expected directions based on previous single-binge EtOH exposure experiments, but the current MR findings do not provide support for accruing changes with repeated binge EtOH exposure.
R. Renteria, Z. M. Jeanes, R. A. Mangieri, E. Y. Maier, D. M. Kircher, T. R. Buske, and R. A. Morrisett. “Using In Vitro Electrophysiology to Screen Medications: Accumbal Plasticity as an Engram of Alcohol Dependence.” International Review of Neurobiology, 126, Pp. 441–465. Abstract
The nucleus accumbens (NAc) is a central component of the mesocorticolimbic reward system. Increasing evidence strongly implicates long-term synaptic neuroadaptations in glutamatergic excitatory activity of the NAc shell and/or core medium spiny neurons in response to chronic drug and alcohol exposure. Such neuroadaptations likely play a critical role in the development and expression of drug-seeking behaviors. We have observed unique cell-type-specific bidirectional changes in NAc synaptic plasticity (metaplasticity) following acute and chronic intermittent ethanol exposure. Other investigators have also previously observed similar metaplasticity in the NAc following exposure to psychostimulants, opiates, and amazingly, even following an anhedonia-inducing experience. Considering that the proteome of the postsynaptic density likely contains hundreds of biochemicals, proteins and other components and regulators, we believe that there is a large number of potential molecular sites through which accumbal metaplasticity may be involved in chronic alcohol abuse. Many of our companion laboratories are now engaged in identifying and screening medications targeting candidate genes and its products previously linked to maladaptive alcohol phenotypes. We hypothesize that if manipulation of such target genes and their products change NAc plasticity, then that observation constitutes an important validation step for the development of novel therapeutics to treat alcohol dependence.
2015
Mary-Louise Risher, Hannah G. Sexton, W. Christopher Risher, Wilkie A. Wilson, Rebekah L. Fleming, Roger D. Madison, Scott D. Moore, Cagla Eroglu, and H. Scott Swartzwelder. “Adolescent Intermittent Alcohol Exposure: Dysregulation of Thrombospondins and Synapse Formation are Associated with Decreased Neuronal Density in the Adult Hippocampus.” Alcoholism, Clinical and Experimental Research, 39, 12, Pp. 2403–2413. Abstract
BACKGROUND: Adolescent intermittent alcohol exposure (AIE) has profound effects on neuronal function. We have previously shown that AIE causes aberrant hippocampal structure and function that persists into adulthood. However, the possible contributions of astrocytes and their signaling factors remain largely unexplored. We investigated the acute and enduring effects of AIE on astrocytic reactivity and signaling on synaptic expression in the hippocampus, including the impact of the thrombospondin (TSP) family of astrocyte-secreted synaptogenic factors and their neuronal receptor, alpha2delta-1 (α2δ-1). Our hypothesis is that some of the influences of AIE on neuronal function may be secondary to direct effects on astrocytes. METHODS: We conducted Western blot analysis on TSPs 1 to 4 and α2δ-1 from whole hippocampal lysates 24 hours after the 4th and 10th doses of AIE, then 24 days after the last dose (in adulthood). We used immunohistochemistry to assess astrocyte reactivity (i.e., morphology) and synaptogenesis (i.e., colocalization of pre- and postsynaptic puncta). RESULTS: Adolescent AIE reduced α2δ-1 expression, and colocalized pre- and postsynaptic puncta after the fourth ethanol (EtOH) dose. By the 10th dose, increased TSP2 levels were accompanied by an increase in colocalized pre- and postsynaptic puncta, while α2δ-1 returned to control levels. Twenty-four days after the last EtOH dose (i.e., adulthood), TSP2, TSP4, and α2δ-1 expression were all elevated. Astrocyte reactivity, indicated by increased astrocytic volume and area, was also observed at that time. CONCLUSIONS: Repeated EtOH exposure during adolescence results in long-term changes in specific astrocyte signaling proteins and their neuronal synaptogenic receptor. Continued signaling by these traditionally developmental factors in adulthood may represent a compensatory mechanism whereby astrocytes reopen the synaptogenic window and repair lost connectivity, and consequently contribute to the enduring maladaptive structural and functional abnormalities previously observed in the hippocampus after AIE.
Mary-Louise Risher, Rebekah L. Fleming, W. Christopher Risher, K. M. Miller, Rebecca C. Klein, Tiffany Wills, Shawn K. Acheson, Scott D. Moore, Wilkie A. Wilson, Cagla Eroglu, and H. S. Swartzwelder. “Adolescent intermittent alcohol exposure: persistence of structural and functional hippocampal abnormalities into adulthood.” Alcoholism, Clinical and Experimental Research, 39, 6, Pp. 989–997. Abstract
BACKGROUND: Human adolescence is a crucial stage of neurological development during which ethanol (EtOH) consumption is often at its highest. Alcohol abuse during adolescence may render individuals at heightened risk for subsequent alcohol abuse disorders, cognitive dysfunction, or other neurological impairments by irreversibly altering long-term brain function. To test this possibility, we modeled adolescent alcohol abuse (i.e., intermittent EtOH exposure during adolescence [AIE]) in rats to determine whether adolescent exposure to alcohol leads to long-term structural and functional changes that are manifested in adult neuronal circuitry. METHODS: We specifically focused on hippocampal area CA1, a brain region associated with learning and memory. Using electrophysiological, immunohistochemical, and neuroanatomical approaches, we measured post-AIE changes in synaptic plasticity, dendritic spine morphology, and synaptic structure in adulthood. RESULTS: We found that AIE-pretreated adult rats manifest robust long-term potentiation, induced at stimulus intensities lower than those required in controls, suggesting a state of enhanced synaptic plasticity. Moreover, AIE resulted in an increased number of dendritic spines with characteristics typical of immaturity. Immunohistochemistry-based analysis of synaptic structures indicated a significant decrease in the number of co-localized pre- and postsynaptic puncta. This decrease is driven by an overall decrease in 2 postsynaptic density proteins, PSD-95 and SAP102. CONCLUSIONS: Taken together, these findings reveal that repeated alcohol exposure during adolescence results in enduring structural and functional abnormalities in the hippocampus. These synaptic changes in the hippocampal circuits may help to explain learning-related behavioral changes in adult animals preexposed to AIE.
James M. Reno, Neha Thakore, Rueben Gonzales, Timothy Schallert, Richard L. Bell, W. Todd Maddox, and Christine L. Duvauchelle. “Alcohol-preferring P rats emit spontaneous 22-28 kHz ultrasonic vocalizations that are altered by acute and chronic alcohol experience.” Alcoholism, Clinical and Experimental Research, 39, 5, Pp. 843–852. Abstract
BACKGROUND: Emotional states are often thought to drive excessive alcohol intake and influence the development of alcohol use disorders. To gain insight into affective properties associated with excessive alcohol intake, we utilized ultrasonic vocalization (USV) detection and analyses to characterize the emotional phenotype of selectively bred alcohol-preferring (P) rats; an established animal model of excessive alcohol intake. USVs emitted by rodents have been convincingly associated with positive (50-55 kHz frequency-modulated [FM]) and negative (22-28 kHz) affective states. Therefore, we hypothesized that 50-55 and 22-28 kHz USV emission patterns in P rats would reveal a unique emotional phenotype sensitive to alcohol experience. METHODS: 50-55 kHz FM and 22-28 kHz USVs elicited from male P rats were assessed during access to water, 15 and 30% EtOH (v/v). Ethanol (EtOH; n = 12) or water only (Control; n = 4) across 8 weeks of daily drinking-in-the-dark (DID) sessions. RESULTS: Spontaneous 22-28 kHz USVs are emitted by alcohol-naïve P rats and are enhanced by alcohol experience. During DID sessions when alcohol was not available (e.g., "EtOH OFF" intervals), significantly more 22-28 kHz than 50-55 kHz USVs were elicited, while significantly more 50-55 kHz FM than 22-28 kHz USVs were emitted when alcohol was available (e.g., "EtOH ON" intervals). In addition, USV acoustic property analyses revealed chronic effects of alcohol experience on 22-28 kHz USV mean frequency, indicative of lasting alcohol-mediated alterations to neural substrates underlying emotional response. CONCLUSIONS: Our findings demonstrate that acute and chronic effects of alcohol exposure are reflected in changes in 22-28 and 50-55 kHz FM USV counts and acoustic patterns. These data support the notion that initiation and maintenance of alcohol intake in P rats may be due to a unique, alcohol-responsive emotional phenotype and further suggest that spontaneous 22-28 kHz USVs serve as behavioral markers for excessive drinking vulnerability.
Sunil Goodwani, P. S. S. Rao, Richard L. Bell, and Youssef Sari. “Amoxicillin and amoxicillin/clavulanate reduce ethanol intake and increase GLT-1 expression as well as AKT phosphorylation in mesocorticolimbic regions.” Brain Research, 1622, Pp. 397–408. Abstract
Studies have shown that administration of the β-lactam antibiotic ceftriaxone (CEF) attenuates ethanol consumption and cocaine seeking behavior as well as prevents ethanol-induced downregulation of glutamate transporter 1 (GLT-1) expression in central reward brain regions. However, it is not known if these effects are compound-specific. Therefore, the present study examined the effects of two other β-lactam antibiotics, amoxicillin (AMOX) and amoxicillin/clavulanate (Augmentin, AUG), on ethanol drinking, as well as GLT-1 and phosphorylated-AKT (pAKT) levels in the nucleus accumbens (Acb) and medial prefrontal cortex (mPFC) of alcohol-preferring (P) rats. P rats were exposed to free-choice of ethanol (15% and 30%) for five weeks and were given five consecutive daily i.p. injections of saline vehicle, 100 mg/kg AMOX or 100mg/kg AUG. Both compounds significantly decreased ethanol intake and significantly increased GLT-1 expression in the Acb. AUG also increased GLT-1 expression in the mPFC. Results for changes in pAKT levels matched those for GLT-1, indicating that β-lactam antibiotic-induced reductions in ethanol intake are negatively associated with increases in GLT-1 and pAKT levels within two critical brains regions mediating drug reward and reinforcement. These findings add to a growing literature that pharmacological increases in GLT-1 expression are associated with decreases in ethanol intake and suggest that one mechanism mediating this effect may be increased phosphorylation of AKT. Thus, GLT-1 and pAKT may serve as molecular targets for the treatment of alcohol and drug abuse/dependence.

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