Selena E. Bartlett

Intermittent Access to 20% Ethanol Induces High Ethanol Consumption in Long–Evans and Wistar Rats

BACKGROUND There has been some difficulty getting standard laboratory rats to voluntarily consume large amounts of ethanol without the use of initiation procedures. It has previously been shown that standard laboratory rats will voluntarily consume high levels of ethanol if given intermittent-access to 20% ethanol in a 2-bottle-choice setting [Wise, Psychopharmacologia 29 (1973), 203]. In this study, we have further characterized this drinking model. METHODS Ethanol-naïve Long-Evans rats were given intermittent-access to 20% ethanol (three 24-hour sessions per week). No sucrose fading was needed and water was always available ad libitum. Ethanol consumption, preference, and long-term drinking behaviors were investigated. Furthermore, to pharmacologically validate the intermittent-access 20% ethanol drinking paradigm, the efficacy of acamprosate and naltrexone in decreasing ethanol consumption were compared with those of groups given continuous-access to 10 or 20% ethanol, respectively. Additionally, ethanol consumption was investigated in Wistar and out-bred alcohol preferring (P) rats following intermittent-access to 20% ethanol. RESULTS The intermittent-access 20% ethanol 2-bottle-choice drinking paradigm led standard laboratory rats to escalate their ethanol intake over the first 5 to 6 drinking sessions, reaching stable baseline consumption of high amounts of ethanol (Long-Evans: 5.1 +/- 0.6; Wistar: 5.8 +/- 0.8 g/kg/24 h, respectively). Furthermore, the cycles of excessive drinking and abstinence led to an increase in ethanol preference and increased efficacy of both acamprosate and naltrexone in Long-Evans rats. P-rats initiate drinking at a higher level than both Long-Evans and Wistar rats using the intermittent-access 20% ethanol paradigm and showed a trend toward a further escalation in ethanol intake over time (mean ethanol intake: 6.3 +/- 0.8 g/kg/24 h). CONCLUSION Standard laboratory rats will voluntarily consume ethanol using the intermittent-access 20% ethanol drinking paradigm without the use of any initiation procedures. This model promises to be a valuable tool in the alcohol research field.

Varenicline, an α4β2 nicotinic acetylcholine receptor partial agonist, selectively decreases ethanol consumption and seeking

Alcohol dependence is a disease that impacts millions of individuals worldwide. There has been some progress with pharmacotherapy for alcohol-dependent individuals; however, there remains a critical need for the development of novel and additional therapeutic approaches. Alcohol and nicotine are commonly abused together, and there is evidence that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in both alcohol and nicotine dependence. Varenicline, a partial agonist at the α4β2 nAChRs, reduces nicotine intake and was recently approved as a smoking cessation aid. We have investigated the role of varenicline in the modulation of ethanol consumption and seeking using three different animal models of drinking. We show that acute administration of varenicline, in doses reported to reduce nicotine reward, selectively reduced ethanol but not sucrose seeking using an operant self-administration drinking paradigm and also decreased voluntary ethanol but not water consumption in animals chronically exposed to ethanol for 2 months before varenicline treatment. Furthermore, chronic varenicline administration decreased ethanol consumption, which did not result in a rebound increase in ethanol intake when the varenicline was no longer administered. The data suggest that the α4β2 nAChRs may play a role in ethanol-seeking behaviors in animals chronically exposed to ethanol. The selectivity of varenicline in decreasing ethanol consumption combined with its reported safety profile and mild side effects in humans suggest that varenicline may prove to be a treatment for alcohol dependence.

The excitatory effects of morphine-3-glucuronide are attenuated by LY274614, a competitive NMDA receptor antagonist, and by midazolam, an agonist at the benzodiazepine site on the GABAA receptor complex

Administration of morphine-3-glucuronide (M3G) by the intracerebroventricular (i.c.v.) route in doses of 2-8 micrograms produced a marked dose-dependent behavioural excitation in adult Sprague-Dawley rats. When LY274614 (1-50 ng i.c.v.) or midazolam maleate (25-50 micrograms i.c.v.) was administered 20 min prior to a maximal excitatory dose of M3G (7 micrograms), all excitatory behaviours were reduced. In contrast, when LY274614 (200 ng i.c.v.) was given after M3G (7 micrograms), it did not reduce all of the excitatory behaviours. Since we have also shown in in vitro binding studies that M3G has very low affinity for the known binding sites on the N-methyl-D-aspartate (NMDA) and the gamma-amino-butyric acid (GABAA) receptor complexes (1), the results of this study suggest that the anti-convulsant compounds, LY274614 and midazolam, are functional antagonists of the excitatory effects of M3G. LY274614 (1-50 ng i.c.v.) and midazolam (25-50 micrograms i.c.v.) did not produce significant behavioural excitation and phenyclidine (PCP)-type effects were not observed. This contrasts with the NMDA receptor antagonists CGS19755 and MK801, where PCP-type effects have been reported to interfere with behavioural assessment. Morphine hydrochloride in a maximal analgesic dose (50 micrograms i.c.v.), did not reduce the excitation score of a maximal excitatory dose of M3G (7 micrograms), lending support to the view that M3Gs excitatory effects are elicited through a non-opioid mechanism.

Partial agonists of the α3β4* neuronal nicotinic acetylcholine receptor reduce ethanol consumption and seeking in rats.

Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) have a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3–CHRNA5–CHRNB4 encoding the α3, α5, and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high-affinity partial agonists at α3β4 nAChRs, CP-601932, and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations, suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2* nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies, implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.

Varenicline, a partial agonist at neuronal nicotinic acetylcholine receptors, reduces nicotine-induced increases in 20% ethanol operant self-administration in Sprague-Dawley rats

Alcohol and nicotine use disorders are often treated as separate diseases, despite evidence that approximately 80–90% of alcohol dependent individuals are also heavy smokers. Both nicotine and ethanol have been shown to interact with neuronal nicotinic acetylcholine receptors (nAChRs), suggesting these receptors are a common biological target for the effects of nicotine and ethanol in the brain. There are few studies that have examined the effects of co‐administered nicotine and ethanol on the activity of nAChRs in rodents. In the present study, we show that Sprague‐Dawley rats, a strain often used for nicotine studies but not as often for voluntary ethanol intake studies, will consume 20% ethanol using both the intermittent‐access two‐bottle‐choice and operant self‐administration models without the need for sucrose fading. We show that nicotine (0.2 mg/kg and 0.8 mg/kg, s.c.) significantly increases operant 20% ethanol self‐administration and varenicline (2 mg/kg, s.c), a partial agonist at nAChRs, significantly decreases operant ethanol self‐administration and nicotine‐induced increases in ethanol self‐administration. This suggests that nAChRs play an important role in increasing ethanol self‐administration and that varenicline may be an efficacious treatment for alcohol and nicotine co‐dependencies.

Happyhour, a Ste20 Family Kinase, Implicates EGFR Signaling in Ethanol-Induced Behaviors

The consequences of alcohol use disorders (AUDs) are devastating to individuals and society, yet few treatments are currently available. To identify genes regulating the behavioral effects of ethanol, we conducted a genetic screen in Drosophila and identified a mutant, happyhour (hppy), due to its increased resistance to the sedative effects of ethanol. Hppy protein shows strong homology to mammalian Ste20 family kinases of the GCK-1 subfamily. Genetic and biochemical experiments revealed that the epidermal growth factor (EGF)-signaling pathway regulates ethanol sensitivity in Drosophila and that Hppy functions as an inhibitor of the pathway. Acute pharmacological inhibition of the EGF receptor (EGFR) in adult animals altered acute ethanol sensitivity in both flies and mice and reduced ethanol consumption in a preclinical rat model of alcoholism. Inhibitors of the EGFR or components of its signaling pathway are thus potential pharmacotherapies for AUDs.

Molecular mechanisms regulating the retrograde axonal transport of neurotrophins.

Neurotrophins are released from target tissues following neural innervation and bind to specific receptors situated on the nerve terminal plasma membrane. The neurotrophin-receptor complex undergoes retrograde axonal transport towards the cell soma, where it signals to the nucleus. This process allows neurotrophins to perform their numerous functions, which include the promotion of neuronal survival and the outgrowth of axons towards certain target tissues. The molecular events controlling each of the components of retrograde axonal transport are beginning to become defined. There is good evidence for the participation of phosphatidylinositol 3-kinase, phosphatidylinositol 4-kinase and the actin cytoskeleton in neurotrophin retrograde axonal transport in vivo. It also appears that the retrograde motor protein dynein mediates the retrograde axonal transport in vivo of neurotrophins such as nerve growth factor. This review discusses the role of the neurotrophin receptors in binding and axonal transport, the endocytic processes required for neurotrophin internalization, the targeting and trafficking of neurotrophins, and the propagation of neurotrophin-induced signals along the axon.

Neuronal Nicotinic Acetylcholine Receptors as Pharmacotherapeutic Targets for the Treatment of Alcohol Use Disorders

Alcohol use disorders (AUDs) are complex, and developing effective treatments will require the combination of novel medications and cognitive behavioral therapy approaches. Epidemiological studies have shown there is a high correlation between alcohol consumption and tobacco use, and the prevalence of smoking in alcoholics is as high as 80% compared to about 30% for the general population. Both preclinical and clinical data provide evidence that nicotine administration increases alcohol intake and non-specific nicotinic receptor antagonists reduce alcohol-mediated behaviors. As nicotine interacts specifically with the neuronal nicotinic acetylcholine receptor (nAChR) system, this suggests that nAChRs play an important role in the behavioral effects of alcohol. In this review, we discuss the importance of nAChRs for the treatment of AUDs and argue that the use of FDA approved nAChR ligands, such as varenicline and mecamylamine, approved as smoking cessation aids may prove to be valuable treatments for AUDs. We also address the importance of combining effective medications with behavioral therapy for the treatment of alcohol dependent individuals.

Ghrelin receptor (GHS-R1A) antagonism suppresses both operant alcohol self-administration and high alcohol consumption in rats.

The mechanisms involved in alcohol use disorders are complex. It has been shown that ghrelin is an important signal for the control of body weight homeostasis, preferably by interacting with hypothalamic circuits, as well as for drug reward by activating the mesolimbic dopamine system. The ghrelin receptor (GHS‐R1A) has been shown to be required for alcohol‐induced reward. Additionally, ghrelin increases and GHR‐R1A antagonists reduce moderate alcohol consumption in mice, and a single nucleotide polymorphism in the GHS‐R1A gene has been associated with high alcohol consumption in humans. However, the role of central ghrelin signaling in high alcohol consumption is not known. Therefore, the role of GHS‐R1A in operant self‐administration of alcohol in rats as well as for high alcohol consumption in Long‐Evans rats and in alcohol preferring [Alko alcohol (AA)] rats was studied here. In the present study, the GHS‐R1A antagonist, JMV2959, was found to reduce the operant self‐administration of alcohol in rats and to decrease high alcohol intake in Long‐Evans rats as well as in AA rats. These results suggest that the ghrelin receptor signaling system, specifically GHS‐R1A, is required for operant self‐administration of alcohol and for high alcohol intake in rats. Therefore, the GHS‐R1A may be a therapeutic target for treatment of addictive behaviors, such as alcohol dependence.

Hypertolerance to morphine in Gzα-deficient mice

Abstract Our laboratory has generated a mouse deficient in the alpha (α) subunit of the G protein, Gz, (Gzα) gene and we have examined the involvement of Gzα in spinal and supraspinal analgesia and tolerance mechanisms. Spinal analgesia was tested by the response times to heat or cold tail flick times in a water bath at 50°C or −5°C and supraspinal analgesia was tested by the times for paw licking and jumping from a plate at 52°C or 0.5°C. Tolerance to morphine was induced in wild type and Gzα-deficient mice over a 5 day period and the behavioral tests were performed daily. The tail flick reaction times to both hot and cold stimuli did not differ between the wild type and Gzα-deficient mice. Analysis of the reaction times from the hot and cold plate tests showed the Gzα-deficient mice developed tolerance to morphine to a greater degree and at a faster rate than wild type mice. Opioid binding assays were performed on synaptic membranes prepared from naive and morphine tolerant wild type and Gzα-deficient brains. No changes in the affinity of morphine for its receptor or in the density of μ and δ opioid receptors were found between the two groups of mice in the naive or morphine tolerant state. This indicates that the absence of Gzα does not affect opioid receptor affinity or receptor up or down regulation. Our results suggest that the presence of Gzα delays the development of morphine tolerance and represents a possible therapeutic target for improving the clinical use of morphine.