Publications

2018
Zhao Q, Fritz M, Pfefferbaum A, Sullivan EV, Pohl KM, Zahr NM. Jacobian maps reveal under-reported brain regions sensitive to extreme binge ethanol intoxication in the rat. Frontiers in Neuroanatomy [Internet]. 2018;12 :108. Publisher's VersionAbstract
Individuals aged 12-20 years drink 11% of all alcohol consumed in the United States with more than 90% consumed in the form of bingedrinking. Early onset alcohol use is a strong predictor of future alcohol dependence. The study of the effects of excessive alcohol use on the human brain is hampered by limited information regarding the quantity and frequency of exposure to alcohol. Animal models can control for age at alcohol exposure onset and enable isolation of neural substrates of exposure to different patterns and quantities of ethanol (EtOH). As with humans, a frequently used binge exposure model is thought to produce dependence and affect predominantly corticolimbic brainregions. in vivo neuroimaging enables animals models to be examined longitudinally, allowing for each animal to serve as its own control. Accordingly, we conducted 3 magnetic resonance imaging (MRI) sessions (baseline, binge, recovery) to track structure throughout the brains of wild type Wistar rats to test the hypothesis that binge EtOH exposure affects specific brain regions in addition to corticolimbic circuitry. Voxel-based comparisons of 13 EtOH- vs. 12 water- exposed animals identified significant thalamic shrinkage and lateral ventricular enlargement as occurring with EtOH exposure, but recovering with a week of abstinence. By contrast, pretectal nuclei and superior and inferior colliculi shrank in response to binge EtOH treatment but did not recover with abstinence. These results identify brainstem structures that have been relatively underreported but are relevant for localizing neurocircuitry relevant to the dynamic course of alcoholism.
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 [Internet]. 2018;133 :470-480. Publisher's VersionAbstract
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.
Shillinglaw JE, Morrisett RA, Mangieri RA. Ethanol modulates glutamatergic transmission and NMDAR-mediated synaptic plasticity in the agranular insular cortex. Frontiers in Pharmacology [Internet]. 2018;9 :1458. Publisher's VersionAbstract
The agranular insular cortex (AIC) has recently been investigated by the alcohol field because of its connectivity to and modulatory control over limbic and brainstem regions implicated in alcohol use disorder (AUD), and because it has shown involvement in animal models of alcohol drinking. Despite evidence of AIC involvement in AUD, there has not yet been an examination of whether ethanol modulatesglutamatergic and γ-amino-butyric acid (GABA)ergic synaptic transmission and plasticity in the AIC. Characterizing how the synaptictransmission and plasticity states of AIC cortical processing neurons are modulated by acute ethanol will likely reveal the molecular targets by which chronic ethanol alters AIC function as alcohol drinking transitions from controlled to problematic. Therefore, we collected brain slices from ethanol-naïve adult male mice, obtained whole-cell recording configuration in layer 2/3 AIC pyramidal neurons, and bath-applied ethanolat pharmacologically relevant concentrations during electrophysiological assays of glutamatergic and GABAergic synaptic transmission and plasticity. We found that ethanol inhibited electrically evoked N-methyl-D-aspartate receptor (NMDAR)-mediated excitatory post-synapticcurrents (EPSCs) in a concentration-related fashion, and had little effect on evoked α-amino-3-hydrox-5-methylisoxazole-4-propionic acid-type receptor (AMPAR)-mediated EPSCs. Ethanol had no effect on spontaneous excitatory post-synaptic currents (sEPSCs) or inhibitory GABAAR-mediated post-synaptic currents (sIPSCs). We found that synaptic conditioning (low-frequency stimulation for 15 min at 1 Hz) induced a form of long-term depression (LTD) of evoked AMPAR-mediated EPSCs. The ability to induce LTD was inhibited by a non-selective NMDAR antagonist (DL-2-amino-5-phosphonovaleric acid), and also by acute, intoxicating concentrations of ethanol. Taken together these data suggest that the glutamate, but not GABA system in the AIC is uniquely sensitive to ethanol, and that in particular NMDAR-mediatedprocesses in the AIC may be disrupted by pharmacologically relevant concentrations of ethanol.
Savarese A, Lasek AW. Regulation of anxiety-like behavior and Crhr1 expression in the basolateral amygdala by LMO3. Psychoneuroendocrinology [Internet]. 2018;92 :13-20. Publisher's VersionAbstract
The LIM domain only protein LMO3 is a transcriptional regulator that has been shown to regulate several behavioral responses to alcohol. Specifically, Lmo3 null (Lmo3Z) mice consume more ethanol in a binge-drinking test and show enhanced ethanol-induced sedation. Due to the high comorbidity of alcohol use and anxiety, we investigated anxiety-like behavior in Lmo3Z mice. Lmo3Z mice spent more time in the open arms of the elevated plus maze compared with their wild-type littermates, but the effect was confounded by reduced locomotor activity. To verify the anxiety phenotype in the Lmo3Z mice, we tested them for novelty-induced hypophagia and found that they also showed reduced anxiety in this test. We next explored the mechanism by which LMO3 might regulate anxiety by measuring mRNA and protein levels of corticotropin releasing factor (encoded by the Crh gene) and its receptor type 1 (Crhr1) in Lmo3Z mice. Reduced Crhr1 mRNA and protein was evident in the basolateral amygdala (BLA) of Lmo3Z mice. To examine whether Lmo3 in the amygdala is important for anxiety-like behavior, we locally reduced Lmo3 expression in the BLA of wild type mice using a lentiviral vector expressing a short hairpin RNA targeting the Lmo3 transcript. Mice with Lmo3 knockdown in the BLA exhibited decreased anxiety-like behavior relative to control mice. These results suggest that Lmo3 promotes anxiety-like behavior specifically in the BLA, possibly by altering Crhr1 expression. This study is the first to support a role for Lmo3 in anxiety-like behavior.
Rompala GR, Simons A, Kihle B, Homanics GE. Paternal preconception chronic variable stress confers attenuated ethanol drinking behavior selectively to male offspring in a pre-stress environment dependent manner. Frontiers in Behavioral Neuroscience [Internet]. 2018;12 :257. Publisher's VersionAbstract

Stress-related psychiatric disorders such as major depression are strongly associated with alcohol abuse and alcohol use disorder. Recently, many epidemiological and preclinical studies suggest that chronic stress prior to conception has cross-generational effects on the behaviorand physiological response to stress in subsequent generations. Thus, we hypothesized that chronic stress may also affect ethanol drinkingbehaviors in the next generation. In the first cohort of mice, we found that paternal preconception chronic variable stress significantly reduced both two-bottle choice and binge-like ethanol drinking selectively in male offspring. However, these results were not replicated in a second cohort that were tested under experimental conditions that were nearly identical, except for one notable difference. Cohort 1 offspring were derived from in-house C57BL/6J sires that were born in the animal vivarium at the University of Pittsburgh whereas cohort 2 offspring were derived from C57BL/6J sires shipped directly from the vendor. Therefore, a third cohort that included both in-house and vendor born sires was analyzed. Consistent with the first two cohorts, we observed a significant interaction between chronic stress and sire-source with only stressed sires that were born in-house able to impart reduced ethanol drinking behaviors to male offspring. Overall, these results demonstrate that paternal preconception stress can impact ethanol drinking behavior in males of the next generation. These studies provide additional support for a recently recognized role of the paternal preconception environment in shaping ethanol drinking behavior.

Nasseef MT, GA D, Mechling AE, Harsan LA, MM C, Kieffer BL, Darcq E. Deformation-based morphometry MRI reveals brain structural modifications in living mu opioid receptor knockout mice. Frontiers in Psychiatry. 2018;9 :643.Abstract
Mu opioid receptor (MOR) activation facilitates reward processing and reduces pain, and brain networks underlying these effects are under intense investigation. Mice lacking the MOR gene (MOR KO mice) show lower drug and social reward, enhanced pain sensitivity and altered emotional responses. Our previous neuroimaging analysis using Resting-state (Rs) functional Magnetic Resonance Imaging (fMRI) showed significant alterations of functional connectivity (FC) within reward/aversion networks in these mice, in agreement with their behavioral deficits. Here we further used a structural MRI approach to determine whether volumetric alterations also occur in MOR KO mice. We acquired anatomical images using a 7-Tesla MRI scanner and measured deformation-based morphometry (DBM) for each voxel in subjects from MOR KO and control groups. Our analysis shows marked anatomical differences in mutant animals. We observed both local volumetric contraction (striatum, nucleus accumbens, bed nucleus of the stria terminalis, hippocampus, hypothalamus and periacqueducal gray) and expansion (prefrontal cortex, amygdala, habenula, and periacqueducal gray) at voxel level. Volumetric modifications occurred mainly in MOR-enriched regions and across reward/aversion centers, consistent with our prior FC findings. Specifically, several regions with volume differences corresponded to components showing highest FC changes in our previous Rs-fMRI study, suggesting a possible function-structure relationship in MOR KO-related brain differences. In conclusion, both Rs-fMRI and volumetric MRI in live MOR KO mice concur to disclose functional and structural whole-brain level mechanisms that likely drive MOR-controlled behaviors in animals, and may translate to MOR-associated endophenotypes or disease in humans.
Mayfield RD, Harris RA. Persistence of drug memories: melting transcriptomes. Biological Psychiatry [Internet]. 2018;(84) :860-861. Publisher's VersionAbstract
One of the defining characteristics of drug dependence is relapse after a period of abstinence. Cue-induced drug craving and relapse risk not only persist during abstinence, but increase over time, a phenomenon termed incubation (1). This progressive increase in risk factors likely requires enduring “memory-like” changes in synaptic plasticity, but there has been little exploration of the underlying molecular mechanisms. It is assumed that this remodeling of the brain is initiated by changes in gene expression (2). The artwork by Salvador Dali titled The Disintegration of the Persistence of Memory offers a symbolic illustration of how extrinsic factors shape the transcriptome over time (https://www.wikiart.org/en/salvador-dali/the-disintegration-of-the-persistence-of-memory). Viewed from this perspective, the bricks in the painting evoke the assembly of RNA sequencing reads into a transcriptome in a surrealist landscape that implies a relationship to memories and time, while the water simulates the depth of changes by drugs of abuse. These persistent influences produce a transcriptome melting pot.
Liu K, Yu J, Zhao J, Zhou Y, Xiong N, Xu J, Wang T, Bell RL, Qing H, Lin Z. (AZI2)3'UTR Is a new SLC6A3 downregulator associated with an epistatic protection against substance use disorders. Molecular Neurobiology [Internet]. 2018;55 :5611-5622. Publisher's VersionAbstract

Regulated activity of SLC6A3, which encodes the human dopamine transporter (DAT), contributes to diseases such as substance abuse disorders (SUDs); however, the exact transcription mechanism remains poorly understood. Here, we used a common genetic variant of the gene, intron 1 DNP1B sequence, as bait to screen and clone a new transcriptional activity, AZI23'UTR, for SLC6A3. AZI23'UTR is a 3' untranslated region (3'UTR) of the human 5-Azacytidine Induced 2 gene (AZI2) but appeared to be transcribed independently of AZI2. Found to be present in both human cell nuclei and dopamine neurons, this RNA was shown to downregulate promoter activity through a variant-dependent mechanism in vitro. Both reduced RNA density ratio of AZI23'UTR/AZI2 and increased DAT mRNA levels were found in ethanol-naive alcohol-preferring rats. Secondary analysis of dbGaP GWAS datasets (Genome-Wide Association Studies based on the database of Genotypes and Phenotypes) revealed significant interactions between regions upstream of AZI23'UTR and SLC6A3 in SUDs. Jointly, our data suggest that AZI23'UTR confers variant-dependent transcriptional regulation of SLC6A3, a potential risk factor for SUDs.

Lasek AW, Chen H, Chen WY. Releasing addiction memories trapped in perineuronal nets. Trends in Genetics [Internet]. 2018;34 :197-208. Publisher's VersionAbstract
Drug addiction can be conceptualized on a basic level as maladaptive learning and memory. Addictive substances elicit changes in brain circuitry involved in reward, cognition, and emotional state, leading to the formation and persistence of strong drug-associated memories that lead to craving and relapse. Recently, perineuronal nets (PNNs), extracellular matrix structures surrounding neurons, have emerged as regulators of learning, memory, and addiction behaviors. PNNs do not just provide structural support to neurons, but are dynamically remodeled in an experience-dependent manner by metalloproteinases. They function in various brain regions through constituent proteins such as brevican that are implicated in neural plasticity. Understanding the function of PNN components in memory processes may lead to new therapeutic approaches to treating addiction.
McCarthy GM, Farris SP, Blednov YA, Harris RA, Mayfield RD. Microglial-specific transcriptome changes following chronic alcohol consumption. Neuropharmacology [Internet]. 2018;128 :416-424. Publisher's VersionAbstract

Microglia are fundamentally important immune cells within the central nervous system (CNS) that respond to environmental challenges to maintain normal physiological processes. Alterations in steady-state cellular function and over-activation of microglia can facilitate the initiation and progression of neuropathological conditions such as Alzheimer's disease, Multiple Sclerosis, and Major Depressive Disorder. Alcohol consumption disrupts signaling pathways including both innate and adaptive immune responses that are necessary for CNS homeostasis. Coordinate expression of these genes is not ascertained from an admixture of CNS cell-types, underscoring the importance of examining isolated cellular populations to reveal systematic gene expression changes arising from mature microglia. Unbiased RNA-Seq profiling was used to identify gene expression changes in isolated prefrontal cortical microglia in response to recurring bouts of voluntary alcohol drinking behavior. The voluntary ethanol paradigm utilizes long-term consumption ethanol that results in escalated alcohol intake and altered cortical plasticity that is seen in humans. Gene coexpression analysis identified a coordinately regulated group of genes, unique to microglia, that collectively are associated with alcohol consumption. Genes within this group are involved in toll-like receptor signaling and transforming growth factor beta signaling. Network connectivity of this group identified Siglech as a putative hub gene and highlighted the potential importance of proteases in the microglial response to chronic ethanol. In conclusion, we identified a distinctive microglial gene expression signature for neuroimmune responses related to alcohol consumption that provides valuable insight into microglia-specific changes underlying the development of substance abuse, and possibly other CNS disorders.

You C, Savarese A, Vandegrift BJ, He D, Pandey SC, Lasek AW, Brodie MS. Ethanol acts on KCNK13 potassium channels in the ventral tegmental area to increase firing rate and modulate binge-like drinking. Neuropharmacology [Internet]. 2018;144 :29-36. Publisher's VersionAbstract
Alcohol excitation of the ventral tegmental area (VTA) is important in neurobiological processes related to the development of alcoholism. The ionotropic receptors on VTA neurons that mediate ethanol-induced excitation have not been identified. Quinidine blocks ethanol excitation of VTA neurons, and blockade of two-pore potassium channels is among the actions of quinidine. Therefore two-pore potassium channels in the VTA may be potential targets for the action of ethanol. Here, we explored whether ethanol activation of VTA neurons is mediated by the two-pore potassium channel KCNK13. Extracellular recordings of the response of VTA neurons to ethanol were performed in combination with knockdown of Kcnk13 using a short hairpin RNA (shRNA) in C57BL/6 J mice. Real-time PCR and immunohistochemistry were used to examine expression of this channel in the VTA. Finally, the role of KCNK13 in binge-like drinking was examined in the drinking in the dark test after knockdown of the channel. Kcnk13 expression in the VTA was increased by acute ethanol exposure. Ethanol-induced excitation of VTA neurons was selectively reduced by shRNA targeting Kcnk13. Importantly, knockdown of Kcnk13 in the VTA resulted in increased alcohol drinking. These results are consistent with the idea that ethanol stimulates VTA neurons at least in part by inhibiting KCNK13, a specific two-pore potassium channel, and that KCNK13 can control both VTA neuronal activity and binge drinking. KCNK13 is a novel alcohol-sensitive molecular target and may be amenable to the development of pharmacotherapies for alcoholism treatment.
Varodayan FP, Khom S, Patel RR, Steinman MQ, Hedges DM, Oleata CS, Homanics GE, Roberto M, Bajo M. Role of TLR4 in the modulation of central amygdala GABA transmission by CRF following restraint stress. Alcohol and Alcoholism [Internet]. 2018;53 :642-649. Publisher's VersionAbstract

AIMS:

Stress induces neuroimmune responses via Toll-like receptor 4 (TLR4) activation. Here, we investigated the role of TLR4 in the effects of the stress peptide corticotropin-releasing factor (CRF) on GABAergic transmission in the central nucleus of the amygdala (CeA) followingrestraint stress.

METHODS:

Tlr4 knock out (KO) and wild-type rats were exposed to no stress (naïve), a single restraint stress (1 h) or repeated restraintstress (1 h per day for 3 consecutive days). After 1 h recovery from the final stress session, whole-cell patch-clamp electrophysiology was used to investigate the effects of CRF (200 nM) on CeA GABAA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs).

RESULTS:

TLR4 does not regulate baseline GABAergic transmission in the CeA of naive and stress-treated animals. However, CRFsignificantly increased the mean sIPSC frequencies (indicating enhanced GABA release) across all genotypes and stress treatments, except for the Tlr4 KO rats that experienced repeated restraint stress.

CONCLUSIONS:

Overall, our results suggest a limited role for TLR4 in CRF's modulation of CeA GABAergic synapses in naïve and single stress rats, though TLR4-deficient rats that experienced repeated psychological stress exhibit a blunted CRF cellular response.

SHORT SUMMARY:

TLR4 has a limited role in CRF's activation of the CeA under basal conditions, but interacts with the CRF system to regulate GABAergic synapse function in animals that experience repeated psychological stress.

Savarese AM, Lasek AW. Transcriptional regulators as targets for alcohol pharmacotherapies. Handbook of Experimental Pharmacology [Internet]. 2018;248 :505-533. Publisher's VersionAbstract

Alcohol use disorder (AUD) is a chronic relapsing brain disease that currently afflicts over 15 million adults in the United States. Despite its prevalence, there are only three FDA-approved medications for AUD treatment, all of which show limited efficacy. Because of their ability to alter expression of a large number of genes, often with great cell-type and brain-region specificity, transcription factors and epigenetic modifiers serve as promising new targets for the development of AUD treatments aimed at the neural circuitry that underlies chronic alcoholabuse. In this chapter, we will discuss transcriptional regulators that can be targeted pharmacologically and have shown some efficacy in attenuating alcohol consumption when targeted. Specifically, the transcription factors cyclic AMP-responsive element binding protein (CREB), peroxisome proliferator-activated receptors (PPARs), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and glucocorticoid receptor (GR), as well as the epigenetic enzymes, the DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), will be discussed.

Maggio SE, Saunders MA, Nixon K, Prendergast MA, Zheng G, Crooks PA, Dwoskin LP, Bell RL, Bardo MT. An improved model of ethanol and nicotine co-use in female P rats: effects of naltrexone, varenicline, and the selective nicotinic α6β2* antagonist r-bPiDI. Drug and Alcohol Dependence [Internet]. 2018;193 :154-161. Publisher's VersionAbstract

Background Although pharmacotherapies are available for alcohol (EtOH) or tobacco use disorders individually, it may be possible to develop a single pharmacotherapy to treat heavy drinking tobacco smokers by capitalizing on the commonalities in their mechanisms of action. Methods Female alcohol-preferring (P) rats were trained for EtOH drinking and nicotine self-administration in two phases: (1) EtOH alone (0 vs. 15% EtOH, 2-bottle choice) and (2) concomitant access, during which EtOH access continued with access to nicotine (0.03 mg/kg/infusion, i.v.) using a 2-lever choice procedure (active vs. inactive lever) in which the fixed ratio (FR) requirement was gradually increased to FR30. When stable co-use was obtained, rats were pretreated with varying doses of naltrexone, varenicline, or r-bPiDI, an α6β2* subtype-selective nicotinic acetylcholine receptor antagonist shown previously to reduce nicotine self-administration. Results While EtOH intake was initially suppressed in phase 2 (co-use), pharmacologically relevant intake for both substances was achieved by raising the "price" of nicotine to FR30. In phase 2, naltrexone decreased EtOH and water consumption but not nicotine intake; in contrast, naltrexone in phase 1 (EtOH only) did not significantly alter EtOH intake. Varenicline and r-bPiDI in phase 2 both decreased nicotine self-administration and inactive lever pressing, but neither altered EtOH or water consumption. Conclusions These results indicate that increasing the "price" of nicotine increases EtOH intake during co-use. Additionally, the efficacy of naltrexone, varenicline, and r-bPiDI was specific to either EtOH or nicotine, with no efficacy for co-use. Nevertheless, future studies on combining these treatments may reveal synergistic efficacy.

Jin C, Decker AM, Makhijani VH, Besheer J, Darcq E, Kieffer BL, Maitra R. Discovery of a potent, selective, and brain-penetrant small molecule that activates the orphan receptor GPR88 and reduces alcohol intake. Journal of Medicinal Chemistry [Internet]. 2018;61 :6748-6758. Publisher's VersionAbstract
The orphan G-protein-coupled receptor GPR88 is highly expressed in the striatum. Studies using GPR88 knockout mice have suggested that the receptor is implicated in alcohol seeking and drinking behaviors. To date, the biological effects of GPR88 activation are still unknown due to the lack of a potent and selective agonist appropriate for in vivo investigation. In this study, we report the discovery of the first potent, selective, and brain-penetrant GPR88 agonist RTI-13951-33 (6). RTI-13951-33 exhibited an EC50 of 25 nM in an in vitro cAMP functional assay and had no significant off-target activity at 38 GPCRs, ion channels, and neurotransmitter transporters that were tested. RTI-13951-33 displayed enhanced aqueous solubility compared to (1 R,2 R)-2-PCCA (2) and had favorable pharmacokinetic properties for behavioral assessment. Finally, RTI-13951-33 significantly reduced alcohol self-administration and alcohol intake in a dose-dependent manner without effects on locomotion and sucrose self-administration in rats when administered intraperitoneally.
Hilderbrand ER, Lasek AW. Studying sex differences in animal models of addiction: an emphasis on alcohol-related behaviors. ACS Chemical Neuroscience [Internet]. 2018;9 :1907-1916. Publisher's VersionAbstract
Animal models are essential for understanding the biological factors that contribute to drug and alcohol addiction and discovering new pharmacotherapies to treat these disorders. Alcohol (ethanol) is the most commonly abused drug in the world, and as the prevalence of alcohol use disorder (AUD) increases, so does the need for effective pharmacotherapies. In particular, treatments with high efficacy in the growing number of female AUD sufferers are needed. Female animals remain underrepresented in biomedical research and sex differencesin the brain's response to alcohol are poorly understood. To help bridge the gender gap in addiction research, this Review discusses strategies that researchers can use to examine sex differences in the context of several common animal models of AUD. Self-administration, two-bottle choice, drinking in the dark, and conditioned place preference are discussed, with a focus on the role of estrogen as a mediator of sex differences in alcohol-related behaviors.
Ferguson LB, Zhang L, Wang S, Bridges C, Harris RA, Ponomarev I. Peroxisome proliferator activated receptor agonists modulate transposable element expression in brain and liver. Frontiers in Molecular Neuroscience [Internet]. 2018;11 :331. Publisher's VersionAbstract
Peroxisome proliferator activated receptors (PPARs) are nuclear hormone receptors that act as transcription factors in response to endogenous lipid messengers. The fibrates and thiazolidinediones are synthetic PPAR agonists used clinically to treat dyslipidemia and Type 2 Diabetes Mellitus, respectively, but also improve symptoms of several other diseases. Transposable elements (TEs), repetitive sequences in mammalian genomes, are implicated in many of the same conditions for which PPAR agonists are therapeutic, including neurodegeneration, schizophrenia, and drug addiction. We tested the hypothesis that there is a link between actions of PPAR agonists and TE expression. We developed an innovative application of microarray data by mapping Illumina mouse WG-6 microarray probes to areas of the mouse genome that contain TEs. Using this information, we assessed the effects of systemic administration of three PPAR agonists with different PPAR subtype selectivity: fenofibrate, tesaglitazar, and bezafibrate, on TE probe expression in mouse brain [prefrontal cortex (PFC) and amygdala] and liver. We found that fenofibrate, and bezafibrate to a lesser extent, up-regulated probes mapped to retrotransposons: Short-Interspersed Elements (SINEs) and Long-Interspersed Elements (LINEs), in the PFC. Conversely, all PPAR agonists down-regulated LINEs and tesaglitazar and bezafibrate also down-regulated SINEs in liver. We built gene coexpression networks that partitioned the diverse transcriptional response to PPAR agonists into groups of probes with highly correlated expression patterns (modules). Most of the differentially expressed retrotransposons were within the same module, suggesting coordinated regulation of their expression, possibly by PPAR signaling. One TE module was conserved across tissues and was enriched with genes whose products participate in epigenetic regulation, suggesting that PPAR agonists affect TE expression via epigenetic mechanisms. Other enriched functional categories included phenotypes related to embryonic development and learning and memory, suggesting functional links between these biological processes and TE expression. In summary, these findings suggest mechanistic relationships between retrotransposons and PPAR agonists and provide a basis for future exploration of their functional roles in brain and liver.
Farris SP, Riley BP, Williams RW, Mulligan MK, Miles MF, Lopez MF, Hitzemann R, Iancu OD, Colville A, Walter NAR, et al. Cross-species molecular dissection across alcohol behavioral domains. . Alcohol [Internet]. 2018;72 :19-31. Publisher's VersionAbstract
This review summarizes the proceedings of a symposium presented at the "Alcoholism and Stress: A Framework for Future Treatment Strategies" conference held in Volterra, Italy on May 9-12, 2017. Psychiatric diseases, including alcohol-use disorders (AUDs), are influenced through complex interactions of genes, neurobiological pathways, and environmental influences. A better understanding of the common neurobiological mechanisms underlying an AUD necessitates an integrative approach, involving a systematic assessment of diverse species and phenotype measures. As part of the World Congress on Stress and Alcoholism, this symposium provided a detailed account of current strategies to identify mechanisms underlying the development and progression of AUDs. Dr. Sean Farris discussed the integration and organization of transcriptome and postmortem human brain data to identify brain regional- and cell type-specific differences related to excessive alcohol consumption that are conserved across species. Dr. Brien Riley presented the results of a genome-wide association study of DSM-IV alcohol dependence; although replication of genetic associations with alcohol phenotypes in humans remains challenging, model organism studies show that COL6A3, KLF12, and RYR3 affect behavioral responses to ethanol, and provide substantial evidence for their role in human alcohol-related traits. Dr. Rob Williams expanded upon the systematic characterization of extensive genetic-genomic resources for quantifying and clarifying phenotypes across species that are relevant to precision medicine in human disease. The symposium concluded with Dr. Robert Hitzemann's description of transcriptome studies in a mouse model selectively bred for high alcohol ("binge-like") consumption and a non-human primate model of long-term alcohol consumption. Together, the different components of this session provided an overview of systems-based approaches that are pioneering the experimental prioritization and validation of novel genes and gene networks linked with a range of behavioral phenotypes associated with stress and AUDs.
Erickson EK, Farris SP, Blednov YA, Mayfield RD, Harris RA. Astrocyte-specific transcriptome responses to chronic ethanol consumption. Pharmacogenomics Journal [Internet]. 2018;18 :578-589. Publisher's VersionAbstract

Astrocytes play critical roles in central nervous system (CNS) homeostasis and are implicated in the pathogenesis of neurological and psychiatric conditions, including drug dependence. Little is known about the effects of chronic ethanol consumption on astrocyte gene expression. To address this gap in knowledge, we performed transcriptome-wide RNA sequencing of astrocytes isolated from the prefrontal cortex (PFC) of mice following chronic ethanol consumption. Differential expression analysis revealed ethanol-induced changes unique to astrocytes that were not identified in total homogenate preparations. Astrocyte-specific gene expression revealed calcium-related signaling and regulation of extracellular matrix genes as responses to chronic ethanol use. These findings emphasize the importance of investigating expression changes in specific cellular populations to define molecular consequences of chronic ethanol consumption in mammalian brain.

Deehan GAJ, Hauser SR, Getachew B, Waeiss RA, Engleman EA, Knight CP, McBride WJ, Truitt WA, Bell RL, Rodd ZA. Selective breeding for high alcohol consumption and response to nicotine: locomotor activity, dopaminergic in the mesolimbic system, and innate genetic differences in male and female alcohol-preferring, non-preferring, and replicate lines of high-alcohol. Psychopharmacology [Internet]. 2018;235 :2755-2769. Publisher's VersionAbstract

RATIONALE:

There is evidence for a common genetic link between alcohol and nicotine dependence. Rodents selectively bred for high alcohol consumption/responsivity are also more likely to self-administer nicotine than controls.

OBJECTIVES:

The experiments examined the response to systemic nicotine, the effects of nicotine within the drug reward pathway, and innate expression of nicotine-related genes in a brain region regulating drug reward/self-administration in multiple lines of rats selectively bred for high and low alcohol consumption.

METHODS:

The experiments examined the effects of systemic administration of nicotine on locomotor activity, the effects of nicotineadministered directly into the (posterior ventral tegmental area; pVTA) on dopamine (DA) release in the nucleus accumbens shell (AcbSh), and innate mRNA levels of acetylcholine receptor genes in the pVTA were determined in 6 selectively bred high/low alcohol consuming and Wistar rat lines.

RESULTS:

The high alcohol-consuming rat lines had greater nicotine-induced locomotor activity compared to low alcohol-consuming rat lines. Microinjections of nicotine into the pVTA resulted in DA release in the AcbSh with the dose response curves for high alcohol-consuming ratsshifted leftward and upward. Genetic analysis of the pVTA indicated P rats expressed higher levels of α2 and β4.

CONCLUSION:

Selective breeding for high alcohol preference resulted in a genetically divergent behavioral and neurobiological sensitivity to nicotine. The observed behavioral and neurochemical differences between the rat lines would predict an increased likelihood of nicotinereinforcement. The data support the hypothesis of a common genetic basis for drug addiction and identifies potential receptor targets.

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