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Browsing by Author "Lasek, Amy W."
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Item Genetic and Pharmacologic Manipulation of TLR4 Has Minimal Impact on Ethanol Consumption in Rodents(Society for Neuroscience, 2017-02-01) Harris, R. Adron; Bajo, Michal; Bell, Richard L.; Blednov, Yuri A.; Varodayan, Florence P.; Truitt, Jay M.; de Guglielmo, Giordano; Lasek, Amy W.; Logrip, Marian L.; Vendruscolo, Leandro F.; Roberts, Amanda J.; Roberts, Edward; George, Olivier; Mayfield, Jody; Billiar, Timothy R.; Hackam, David J.; Mayfield, R. Dayne; Koob, George F.; Roberto, Marisa; Homanics, Gregg E.; Psychiatry, School of MedicineToll-like receptor 4 (TLR4) is a critical component of innate immune signaling and has been implicated in alcohol responses in preclinical and clinical models. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium tested the hypothesis that TLR4 mediates excessive ethanol drinking using the following models: (1) Tlr4 knock-out (KO) rats, (2) selective knockdown of Tlr4 mRNA in mouse nucleus accumbens (NAc), and (3) injection of the TLR4 antagonist (+)-naloxone in mice. Lipopolysaccharide (LPS) decreased food/water intake and body weight in ethanol-naive and ethanol-trained wild-type (WT), but not Tlr4 KO rats. There were no consistent genotypic differences in two-bottle choice chronic ethanol intake or operant self-administration in rats before or after dependence. In mice, (+)-naloxone did not decrease drinking-in-the-dark and only modestly inhibited dependence-driven consumption at the highest dose. Tlr4 knockdown in mouse NAc did not decrease drinking in the two-bottle choice continuous or intermittent access tests. However, the latency to ethanol-induced loss of righting reflex increased and the duration decreased in KO versus WT rats. In rat central amygdala neurons, deletion of Tlr4 altered GABAA receptor function, but not GABA release. Although there were no genotype differences in acute ethanol effects before or after chronic intermittent ethanol exposure, genotype differences were observed after LPS exposure. Using different species and sexes, different methods to inhibit TLR4 signaling, and different ethanol consumption tests, our comprehensive studies indicate that TLR4 may play a role in ethanol-induced sedation and GABAA receptor function, but does not regulate excessive drinking directly and would not be an effective therapeutic target., SIGNIFICANCE STATEMENT Toll-like receptor 4 (TLR4) is a key mediator of innate immune signaling and has been implicated in alcohol responses in animal models and human alcoholics. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium participated in the first comprehensive study across multiple laboratories to test the hypothesis that TLR4 regulates excessive alcohol consumption in different species and different models of chronic, dependence-driven, and binge-like drinking. Although TLR4 was not a critical determinant of excessive drinking, it was important in the acute sedative effects of alcohol. Current research efforts are directed at determining which neuroimmune pathways mediate excessive alcohol drinking and these findings will help to prioritize relevant pathways and potential therapeutic targets.Item Involvement of Purinergic P2X4 Receptors in Alcohol Intake of High-Alcohol-Drinking (HAD) Rats(Wiley, 2015-10) Franklin, Kelle M.; Hauser, Sheketha R.; Lasek, Amy W.; Bell, Richard L.; McBride, William J.; Department of Psychiatry, IU School of MedicineBackground: The P2X4 receptor is thought to be involved in regulating alcohol-consuming behaviors and ethanol (EtOH) has been reported to inhibit P2X4 receptors. Ivermectin is an anti-parasitic agent that acts as a positive allosteric modulator of the P2X4 receptor. The current study examined the effects of systemically- and centrally-administered ivermectin on alcohol drinking of replicate lines of high-alcohol-drinking (HAD-1/HAD-2) rats, and the effects of lentiviral-delivered short-hairpin RNAs (shRNAs) targeting P2rx4 on EtOH intake of female HAD2 rats. Method: For the 1st experiment, adult male HAD-1 & HAD-2 rats were given 24-hr free-choice access to 15% EtOH vs. water. Dose-response effects of ivermectin (1.5 to 7.5 mg/kg i.p.) on EtOH intake were determined; the effects of ivermectin were then examined for 2% w/v sucrose intake over 5 consecutive days. In the 2nd experiment, female HAD-2 rats were trained to consume 15% EtOH under 2-hr limited access conditions, and dose-response effects of intracerebroventricular (ICV) administration of ivermectin (0.5 to 2.0 μg) were determined over 5 consecutive days. The 3rd experiment determined the effects of microinfusion of a lentivirus expressing P2rx4 shRNAs into the posterior ventral tegmental area (VTA) on 24-hr EtOH free-choice drinking of female HAD-2 rats. Results: The highest i.p. dose of ivermectin reduced alcohol drinking (30-45%) in both rat lines, but did not alter sucrose intake. HAD-2 rats appeared to be more sensitive than HAD1 rats to the effects of ivermectin. ICV administration of ivermectin reduced 2-hr limited access intake (∼35%) of femaleItem Reduced Levels of mGlu2 Receptors within the Prelimbic Cortex Are Not Associated with Elevated Glutamate Transmission or High Alcohol Drinking(Wiley, 2017-11) Ding, Zheng-Ming; Ingraham, Cynthia M.; Hauser, Sheketha R.; Lasek, Amy W.; Bell, Richard L.; McBride, William J.; Department of Psychiatry, School of MedicineBackground 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.Item Reduction of alcohol drinking of alcohol-preferring (P) and high-alcohol drinking (HAD1) rats by targeting phosphodiesterase-4 (PDE4)(Springer-Verlag, 2015-07) Franklin, Kelle M.; Hauser, Sheketha R.; Lasek, Amy W.; McClintick, Jeanette; Ding, Zheng-Ming; McBride, William J.; Bell, Richard L.; Department of Psychiatry, IU School of MedicineRATIONALE: Phosphodiesterase-4 (PDE4) and neuroimmune signaling have been posited to regulate alcohol drinking. OBJECTIVES: This study evaluated the involvement of PDE4 and Il22ra2 on ethanol (EtOH) intake by alcohol-preferring (P) and high-alcohol-drinking (HAD1) rats. METHODS: Exp 1 determined the dose-response effects of PDE4 inhibitors, rolipram, and Ro 20-1724, on 2 h/day free-choice EtOH intake by adult P and HAD1 rats. Exps 2-3 examined the effects of repeated administration with the PDE4 inhibitors on EtOH or sucrose intake and locomotor behavior. Exp 4 determined Pde4-associated gene expression differences in subregions of the extended amygdala, between high- and low-alcohol-consuming rat lines. Exp 5 evaluated the effects of infusing short hairpin RNA to knock down Il22ra2 in the nucleus accumbens (NAc) shell on a 24-h free-choice EtOH drinking by P rats. RESULTS: Administration of rolipram or Ro 20-1724 reduced EtOH intake by P rats; Ro 20-1724 reduced EtOH intake by HAD1 rats. Repeated rolipram or Ro 20-1724 exposure reduced EtOH intake by P and HAD1 rats. PDE4 inhibition induced motor impairment during the first hour of EtOH intake by P rats. Higher gene expression levels for PDE4A were found in the NAc shell of P vs NP rats. ShRNAs targeting Il22ra2 in the NAc shell significantly reduced chronic EtOH intake. CONCLUSIONS: PDE4 and neuroinflammatory/immune signaling pathways could represent molecular targets for the treatment of alcohol use disorders in genetically predisposed subjects. This study underscores the importance of testing compounds over multiple days and rat lines when determining efficacy to disrupt excessive alcohol intake.