Mice deficient in corticotropin-releasing factor receptor type 2 exhibit normal ethanol-associated behaviors

BACKGROUND: Stress is believed to influence alcohol use and relapse in alcoholics. Animal studies suggest an interaction between corticotropin-releasing factor (CRF) and its receptors and the behavioral effects and consumption of alcohol. The objective of these studies was to examine the effect of corticotropin-releasing factor receptor type 2 (CRF2) on ethanol consumption, conditioned taste aversion, sedation, and hypothermia. METHODS: CRF2-null mutant or knock-out (KO), and wild-type (WT) mice were used to assess consumption of increasing concentrations of ethanol in a two-bottle, 24-hr test and during daily limited-access sessions. Ethanol-induced conditioned taste aversion (CTA), loss of righting reflex (LORR), hypothermia, and ethanol metabolism kinetics were also examined in the CRF2 KO and WT mice. RESULTS: CRF2 KO mice did not differ from WT mice in sensitivity to ethanol-induced CTA, LORR, hypothermia, or ethanol metabolism kinetics. There was no genotypic difference in ethanol intake or preference in the 24-hr, two-bottle choice procedure, and only modestly reduced consumption of the 7.5 and 10% ethanol solutions in KO versus WT mice in the limited-access procedure. CONCLUSIONS: CRF2 deficiency had little effect on several ethanol-associated behaviors in CRF2-null mutant compared with WT mice, suggesting that this receptor does not have a primary role in modulating these behaviors. Evidence of a role for this receptor in neural circuits subserving stress-coping behaviors suggest that future studies should focus on the role of endogenous CRF2 in ethanol-associated behaviors in mice that are stressed or withdrawing from dependence on ethanol.     

Immunocytochemical identification of low-affinity NTS2 neurotensin receptors in parietal cells of human gastric mucosa

The biological effects of neurotensin (NT) are mediated by two distinct G protein-coupled receptors, NTS(1) and NTS(2). Although it is well established that neurotensin inhibits gastric acid secretion in man, the plasma membrane receptor mediating these effects has not been visualized yet. We developed and characterized a novel antipeptide antibody to the carboxy-terminal region of the human NTS(2) receptor. The cellular and subcellular distribution of NTS(2) receptors was evaluated in various human gastrointestinal tissues. Specificity of the antiserum was demonstrated by (1) detection of a broadband migrating at M(r) 90 000-100 000 in Western blots of membranes from NTS(2)-expressing tissues; (2) cell-surface staining of NTS(2)-transfected cells; (3) translocation of NTS(2) receptor immunostaining after agonist exposure; and (4) abolition of tissue immunostaining by preadsorbtion of the antibody with its immunizing peptide. In the gastrointestinal tract, NTS(2) receptor immunoreactivity was highly abundant in parietal cells of the gastric mucosa, in neuroendocrine cells of the stomach small and large intestine, and in cells of the exocrine pancreas. NTS(2) receptors were clearly located in the plasma membrane and uniformly present on nearly all target cells. The presence of NTS(2) receptors was rarely detected in human tumors. This is the first localization of NTS(2) receptors in human formalin-fixed, paraffin-embedded tissues at the cellular level. The abundant expression of low-affinity NTS(2) receptors on the plasma membrane of human parietal cells provides a morphological substrate for the direct inhibition of gastric acid secretion observed after i.v. administration of neurotensin.     

The cyclic AMP/protein kinase A signal transduction pathway modulates tolerance to sedative and hypothermic effects of ethanol

Background: An expanding body of literature indicates the important role of the cAMP/PKA signaling pathway in establishing initial sensitivity to alcohol as well as being involved in certain forms of tolerance to ethanol. The use of mice with heterozygous inactivation of the Gnas gene encoding Gsα allowed us to explore the relationship between tolerance to ethanol and cAMP/PKA signaling. Methods: Mice with the targeted disruption of one Gsα allele were compared with wild‐type littermates in their initial sensitivity to ethanol‐induced sedation and hypothermia and then monitored for the development of tolerance during two subsequent bouts of intoxication. Components of the cAMP/PKA signaling pathway were analyzed in ethanol‐naïve mice and again following the development of tolerance to ethanol to better understand the contribution of this signaling pathway to the acquisition of tolerance. Results: During the initial exposure to ethanol, mice with the targeted disruption of one Gsα allele (Gnas) were more sensitive to the sedative effects of ethanol compared with wild‐type littermates. Wild‐type mice developed within‐session tolerance to ethanol‐induced hypothermia whereas Gnas mice did not. Following the subsequent ethanol treatments, wild‐type mice developed between‐session tolerance to the sedative effects of ethanol to a greater degree than mice with heterozygous inactivation of the Gnas gene. The development of tolerance to the sedative effects of ethanol was accompanied by increased expression of phospho‐CREB in the cerebellum, hippocampus, and frontal cortex. No changes in phospho‐CREB expression were detected in these brain regions in mice with heterozygous inactivation of the Gnas gene. Conclusion: The results show that cAMP/PKA signal transduction modulates sensitivity to sedative and hypothermic effects of ethanol. This signal transduction system also influences the acquisition of within‐session and between‐session tolerance. The mechanism through which cAMP/PKA signaling modulates the development of tolerance remains to be elucidated but may involve changes in phospho‐CREB expression.     

Corticotropin-releasing factor acting on corticotropin-releasing factor receptor type 1 is critical for binge alcohol drinking in mice

Background: The corticotropin‐releasing factor (CRF) system has been implicated in the regulation of alcohol consumption. However, previous mouse knockout (KO) studies using continuous ethanol access have failed to conclusively confirm this. Recent studies have shown that CRF receptor type 1 (CRFR1) antagonists attenuate alcohol intake in the limited access “drinking in the dark” (DID) model of binge drinking. To avoid the potential nonspecific effects of antagonists, in this study, we tested alcohol drinking in CRFR1, CRFR2, CRF, and urocortin 1 (Ucn1) KO and corresponding wild‐type (WT) littermates using the DID paradigm. Methods: On days 1 to 3, the CRFR1, CRFR2, Ucn1, and CRF KO mice and their respective WT littermates were provided with 20% ethanol or 10% sucrose for 2 hours with water available at all other times. On day 4, access to ethanol or sucrose was increased to 4 hours. At the end of each drinking session, the volume of ethanol consumed was recorded, and at the conclusion of the last session, blood was also collected for blood ethanol concentration (BEC) analysis. Results: CRFR1 KO mice had lower alcohol intakes and BECs and higher intakes of sucrose compared with WTs. In contrast, CRFR2 KO mice, while having reduced intakes initially, had similar alcohol intakes on days 2 to 4 and similar BECs as the WTs. To determine the ligand responsible, Ucn1 and CRF KO and WT mice were tested next. While Ucn1 KOs had similar alcohol intakes and BECs to their WTs, CRF KO mice showed reduced alcohol consumption and lower BECs compared with WTs. Conclusions: Our results confirm that CRFR1 plays a key role in binge drinking and identify CRF as the ligand critically involved in excessive alcohol consumption.     

Alcohol-Induced Tolerance and Physical Dependence in Mice With Ethanol Insensitive α1 GABAA Receptors

Background: Although many people consume alcohol (ethanol), it remains unknown why some become addicted. Elucidating the molecular mechanisms of tolerance and physical dependence (withdrawal) may provide insight into alcohol addiction. While the exact molecular mechanisms of ethanol action are unclear, γ‐aminobutyric acid type A receptors (GABA_A‐Rs) have been extensively implicated in ethanol action. The α1 GABA_A‐R subunit is associated with tolerance and physical dependence, but its exact role remains unknown. In this report, we tested the hypothesis that α1‐GABA_A‐Rs mediate in part these effects of ethanol. Methods: Ethanol‐induced behavioral responses related to tolerance and physical dependence were investigated in knockin (KI) mice that have ethanol‐insensitive α1 GABA_A‐Rs and wildtype (WT) controls. Acute functional tolerance (AFT) was assessed using the stationary dowel and loss of righting reflex (LORR) assays. Chronic tolerance was assessed on the LORR, fixed speed rotarod, hypothermia, and radiant tail‐flick assays following 10 consecutive days of ethanol exposure. Withdrawal‐related hyperexcitability was assessed by handling‐induced convulsions following 3 cycles of ethanol vapor exposure/withdrawal. Immunoblots were used to assess α1 protein levels. Results: Compared with controls, KI mice displayed decreased AFT and chronic tolerance to ethanol‐induced motor ataxia, and also displayed heightened ethanol‐withdrawal hyperexcitability. No differences between WT and KI mice were seen in other ethanol‐induced behavioral measures. Following chronic exposure to ethanol, control mice displayed reductions in α1 protein levels, but KIs did not. Conclusions: We conclude that α1‐GABA_A‐Rs play a role in tolerance to ethanol‐induced motor ataxia and withdrawal‐related hyperexcitability. However, other aspects of behavioral tolerance and physical dependence do not rely on α1‐containing GABA_A‐Rs.     

Neurotensin and Ethanol Interactions on Hypothermia and Locomotor Activity in LS and SS Mice

Ethanol, i.p., produced a greater dose-dependent hypothermia in long sleep (LS) than in short sleep (SS) mice with significant decreases in rectal temperature observed only at doses greater than 3 g/kg, i.p. Likewise, at doses of 1 to 2 g/kg ethanol, i.p., these lines of mice differ markedly in locomotor activity. Neurotensin (NT), intracerebroventricular (I.C.V.), induced a similar hypothermia in both SS and LS mice at doses greater than 0.02 microgram. Doses of ethanol (1.0 g/kg) or NT (0.005 microgram, i.c.v.) that failed to cause hypothermia when administered separately produced a pronounced hypothermia when administered together. Potentiation of NT and ethanol-induced hypothermia was greater in SS than in LS mice. Sensitivity to NT-induced hypothermia was greater following i.c.v. administration than by infusion into the nucleus accumbens (NA) or the ventral tegmental area (VTA). Neurotensin, i.c.v. or intra-NA, markedly inhibited ethanol-induced increase in locomotor activity in both SS and LS mice; however, NT, intra-VTA, did not alter the effects of ethanol on locomotor activity. The results suggest that NT and ethanol act in a synergistic manner on specific neuronal processes mediating thermoregulation and spontaneous motor activity.     

GDNF is an Endogenous Negative Regulator of Ethanol-Mediated Reward and of Ethanol Consumption After a Period of Abstinence

Background:  We previously found that activation of the glial cell line-derived neurotrophic factor (GDNF) pathway in the ventral tegmental area (VTA) reduces ethanol-drinking behaviors. In this study, we set out to assess the contribution of endogenous GDNF or its receptor GFRα1 to the regulation of ethanol-related behaviors.
Methods:  GDNF and GFRα1 heterozygote mice (HET) and their wild-type littermate controls (WT) were used for the studies. Ethanol-induced hyperlocomotion, sensitization, and conditioned place preference (CPP), as well as ethanol consumption before and after a period of abstinence were evaluated. Blood ethanol concentration (BEC) was also measured.
Results:  We observed no differences between the GDNF HET and WT mice in the level of locomotor activity or in sensitization to ethanol-induced hyperlocomotion after systemic injection of a nonhypnotic dose of ethanol and in BEC. However, GDNF and GFRα1 mice exhibited increased place preference to ethanol as compared with their WT littermates. The levels of voluntary ethanol or quinine consumption were similar in the GDNF HET and WT mice, however, a small but significant increase in saccharin intake was observed in the GDNF HET mice. No changes were detected in voluntary ethanol, saccharin or quinine consumption of GFRα1 HET mice as compared with their WT littermates. Interestingly, however, both the GDNF and GFRα1 HET mice consumed much larger quantities of ethanol after a period of abstinence from ethanol as compared with their WT littermates. Furthermore, the increase in ethanol consumption after abstinence was found to be specific for ethanol as similar levels of saccharin intake were measured in the GDNF and GFRα1 HET and WT mice after abstinence.
Conclusions:  Our results suggest that endogenous GDNF negatively regulates the rewarding effect of ethanol and ethanol-drinking behaviors after a period of abstinence.     

Ghrelin Receptor Antagonism Decreases Alcohol Consumption and Activation of Perioculomotor Urocortin‐Containing Neurons

Background: The current therapies for alcohol abuse disorders are not effective in all patients, and continued development of pharmacotherapies is needed. One approach that has generated recent interest is the antagonism of ghrelin receptors. Ghrelin is a gut‐derived peptide important in energy homeostasis and regulation of hunger. Recent studies have implicated ghrelin in alcoholism, showing altered plasma ghrelin levels in alcoholic patients as well as reduced intakes of alcohol in ghrelin receptor knockout mice and in mice treated with ghrelin receptor antagonists. The aim of this study was to determine the neuroanatomical locus/loci of the effect of ghrelin receptor antagonism on alcohol consumption using the ghrelin receptor antagonist, D‐Lys3‐GHRP‐6. Methods: In Experiment 1 , male C57BL/6J mice were injected with saline 3 hours into the dark cycle and allowed access to 15% (v/v) ethanol or water for 2 hours in a 2‐bottle choice experiment. On test day, the mice were injected with either saline or 400 nmol of the ghrelin receptor antagonist, D‐Lys3‐GHRP‐6, and allowed to drink 15% ethanol or water for 4 hours. The preference for alcohol and alcohol intake were determined. In Experiment 2 , the same procedure was followed as in Experiment 1 but mice were only allowed access to a single bottle of 20% ethanol (v/v), and alcohol intake was determined. Blood ethanol levels were analyzed, and immunohistochemistry for c‐Fos was carried out to investigate changes in neural activity. To further elucidate the mechanism by which D‐Lys3‐GHRP‐6 affects alcohol intake, in Experiment 3 , the effect of D‐Lys3‐GHRP‐6 on the neural activation induced by intraperitoneal ethanol was investigated. For the c‐Fos studies, brain regions containing ghrelin receptors were analyzed, i.e. the perioculomotor urocortin population of neurons (pIIIu), the ventral tegmental area (VTA), and the arcuate nucleus (Arc). In Experiment 4 , to test if blood ethanol concentrations were affected by D‐Lys3‐GHRP‐6, blood samples were taken at 2 time‐points after D‐Lys3‐GHRP‐6 pretreatment and systemic ethanol administration. Results: In Experiment 1 , D‐Lys3‐GHRP‐6 reduced preference to alcohol and in a follow‐up experiment ( Experiment 2 ) also dramatically reduced alcohol intake when compared to saline‐treated mice. The resulting blood ethanol concentrations were lower in mice treated with the ghrelin receptor antagonist. Immunohistochemistry for c‐Fos showed fewer immunopositive cells in the pIIIu of the antagonist‐treated mice but no difference was seen in the VTA or Arc. In Experiment 3 , D‐Lys3‐GHRP‐6 reduced the induction of c‐Fos by intraperitoneal ethanol in the pIIIu but had no effect in the VTA. In the Arc, there was a significant increase in the number of c‐Fos immunopositive cells after D‐Lys3‐GHRP‐6 administration, but the antagonist had no effect on ethanol‐induced expression of c‐Fos. D‐Lys3‐GHRP‐6‐pretreatment also did not affect the blood ethanol concentrations observed after a systemic injection of ethanol when compared to saline‐pretreated mice ( Experiment 4 ). Conclusions: These findings indicate that the action of ghrelin on the regulation of alcohol consumption may occur via the pIIIu.     

Lesions of the extended amygdala in C57BL/6J mice do not block the intermittent ethanol vapor-induced increase in ethanol consumption

Background: The central extended amygdala (cEA) which includes the central nucleus of the amygdala (CeA) and the lateral posterior bed nucleus of the stria terminalis (BNSTLP), has been proposed to play a key role in excessive ethanol consumption in humans (Koob and Le Moal, 2005 Nat Neurosci 8:1442). To examine this relationship, we used a murine model of ethanol dependence (Becker and Lopez, 2004 Alcohol Clin Exp Res 28:1829; Lopez and Becker, 2005 Psychopharmacology (Berl) 181:688) and compared animals with sham lesions and electrolytic lesions of the CeA and BNSTLP. Methods: Male C57BL/6J (B6) mice were first acclimated to a limited‐access 2‐bottle‐choice preference procedure. The access period began 3 hours into the dark phase of the light‐dark cycle and continued for 2 hours. Once acclimated (1 week), mice underwent chronic exposure to and intermittent withdrawal from ethanol vapor. The animals were then retested in the limited‐access 2‐bottle‐choice preference procedure. In some experiments, electrolytic and sham lesions of the CeA or BNSTLP were performed prior to initiating the 2‐bottle choice procedure. Results: In a series of 5 preliminary experiments, mice were randomly assigned either to the standard intermittent ethanol vapor procedure or to the standard procedure but with air in the vapor chamber (control). The air‐control procedure produced no change in ethanol intake when compared to baseline consumption. In contrast, intermittent ethanol vapor exposure increased ethanol consumption by almost 50%. The increase in consumption was associated with an increase in total fluid volume consumed and no change in ethanol preference. Lesions of both the BNSTLP and CeA significantly decreased baseline ethanol consumption, the former by decreasing fluid consumption and the latter by decreasing ethanol preference. Intermittent ethanol vapor exposure significantly increased consumption in both the BNSTLP‐ and CeA‐lesioned animals, largely by increasing the total volume of fluid consumed. Conclusions: The results obtained clearly demonstrate that the cEA has a role in the regulation of ethanol consumption in the limited‐access procedure. However, neither lesions of the CeA nor BNSTLP prevented the intermittent ethanol vapor‐induced increase in consumption. These data do not preclude some role of the cEA in the increased ethanol consumption following intermittent ethanol vapor exposure, but would suggest that other brain regions also must have a significant influence.     

Restraint stress and ethanol consumption in two mouse strains

Background: This study examined the interaction between restraint stress and ethanol drinking in mice that consume low and high amounts of ethanol. Methods: Two strains of mice (129SVEV and C57BL/6J) underwent 1 hour of restraint stress twice per day for 4 days in the presence of a CRF‐1 receptor antagonist, a glucocorticoid receptor antagonist or vehicle. Ethanol preference and consumption were assessed using a two bottle choice design. In another study, mice were implanted with pellets containing corticosterone; ethanol preference and consumption were assessed using a two bottle choice design. Results: Restraint stress significantly increased ethanol preference and consumption in 129SVEV mice but not in C57BL/6J mice. Then 129SVEV mice underwent the identical stress procedure; however, mice received either the CRF‐1 receptor antagonist, R121919 (15 or 20 mg/kg, ip) or vehicle 30 minutes prior to stress. R121919 did not block the stress‐induced change in ethanol preference despite causing a significant blunting in the HPA axis. Negative results were also obtained using the CRF‐1 receptor antagonist, Antalarmin (20 mg/kg, ip). In another study, 129SVEV mice were administered either the glucocorticoid receptor antagonist Mifepristone (25, 50 or 100 μg/kg, ip) or vehicle under the same procedure. Mifepristone did not alter ethanol preference. Moreover, the three receptor antagonist did not alter nonstress ethanol consumption either. In the last study, both mouse strains underwent active or sham adrenalectomy, then pellets containing corticosterone or placebo were implanted and preference for ethanol versus water was tested. Corticosterone administration decreased ethanol consumption in a strain‐dependent manner. Conclusion: These data show the restraint model for stress can modestly increase ethanol consumption in 129SVEV mice but not in C57BL/6J mice. Pharmacologic manipulation of CRF and corticosterone did not blunt baseline or stress‐induced change in ethanol preference nor did administration of corticosterone mimic the effects of restraint stress on ethanol consumption. These findings suggest the mechanism responsible for increasing ethanol consumption in this model is independent of the HPA axis and extra‐hypothalamic CRF.     

Tolerance to ethanol's ataxic effects and alterations in ethanol-induced locomotion following repeated binge-like ethanol intake using the DID model

Background: Tolerance to the behavioral and subjective effects of alcohol (ethanol) is thought to be a major predictive factor for the development of alcoholism. Evidence from rodent models has supported this view with those animals most likely to develop tolerance generally drinking and preferring ethanol more so than those resistant to it. Despite this evidence, very little is known about the behavioral relationships between ethanol‐induced tolerance and consumption. The goal of this study was to evaluate the development of tolerance to the ataxic effects of ethanol using a mouse model of binge‐like intake dubbed “Drinking in the Dark” (DID; Physiol Behav 2005, 84:53–63). We hypothesized that mice would become tolerant to the ataxic effects of ethanol as this behavior is known to be altered at the blood ethanol concentrations reached using this model (≥80 mg/dl). Methods: To evaluate this, we gave daily DID ethanol or water access sessions to male C57BL/6J (B6) mice and monitored ataxia (and in some cases locomotion) at various time points. Results: In general, mice given 14 consecutive days of ethanol access displayed tolerance to the ataxic effects of ethanol compared to water‐drinking controls. These effects were coupled with alterations in locomotor behavior and in some cases differences in ethanol pharmacokinetics. Conclusions: Thus, we can conclude that tolerance to the behavioral effects of binge‐like ethanol intake might play a key role in the daily maintenance of this behavior and that these effects may be evidence of important neuroadaptations involved in the development of alcoholism.     

Confirmation of Correlations and Common Quantitative Trait Loci Between Neurotensin Receptor Density and Hypnotic Sensitivity to Ethanol

BACKGROUND: In previous studies, genetic correlations were observed between hypnotic sensitivity to ethanol and high-affinity neurotensin receptor (NTS1) binding. Provisional quantitative trait loci (QTLs) were identified for these traits, and some of these QTLs were found on common chromosomal regions. In continued efforts to examine the relationship between NTS1 binding capacity and hypnotic sensitivity to ethanol, studies were designed to confirm correlations between NTS1 densities in the brain, duration of ethanol-induced loss of righting reflex (LORR), and blood ethanol concentrations at regain of righting reflex (BECRR). Another purpose of the study was to confirm QTLs for these traits. METHODS: ILS X ISS F2 mice and HAS X LAS F2 rats as well as the progenitors were tested for LORR, BECRR, and NTS1 densities. Phenotypic correlations were calculated between LORR and BECRR and between these measures and NTS1 densities in striatum from both mice and rats. The F2 mice were genotyped by using polymorphic markers for five previously reported QTLs for LORR to confirm QTLs for BECRR and NTS1 densities in striatum, ventral midbrain, and frontal cortex. RESULTS: Phenotypic correlations were found between LORR and BECRR (r = -0.66 to -0.74, p < 10(-9)) and between these measures and NTS1 densities in striatum (r = 0.28-0.38, p < 10(-2)) from both mice and rats. QTLs for LORR and BECRR (lod score = 2-6) were found in common regions of chromosomes 1, 2, and 15. By using the combined results from a previous LSXSS RI study and the current results, a suggestive QTL (lod score = 3.1) for striatal NTS1 receptor densities was found on chromosome 15 at approximately 60 cM, in the same region as the chromosome 15 LORR/BECRR QTL. CONCLUSIONS: The results are in agreement with previously reported correlations and QTLs for NTS1 receptor densities and measures of hypnotic sensitivity to ethanol in mice and extend those correlations to another species, the rat. These findings support a role for NTS1 in genetically mediated differences in hypnotic sensitivity to ethanol.     

Mutant mice lacking the gamma isoform of protein kinase C show decreased behavioral actions of ethanol and altered function of gamma-aminobutyrate type A receptors.

Calcium/phospholipid-dependent protein kinase (protein kinase C, PKC) has been suggested to play a role in the sensitivity of gamma-aminobutyrate type A (GABAA) receptors to ethanol. We tested a line of null mutant mice that lacks the gamma isoform of PKC (PKC gamma) to determine the role of this brain-specific isoenzyme in ethanol sensitivity. We found that the mutation reduced the amount of PKC gamma immunoreactivity in cerebellum to undetectable levels without altering the levels of the alpha, beta I, or beta II isoforms of PKC. The mutant mice display reduced sensitivity to the effects of ethanol on loss of righting reflex and hypothermia but show normal responses to flunitrazepam or pentobarbital. Likewise, GABAA receptor function of isolated brain membranes showed that the mutation abolished the action of ethanol but did not alter actions of flunitrazepam or pentobarbital. These studies show the unique interactions of ethanol with GABAA receptors and suggest protein kinase isoenzymes as possible determinants of genetic differences in response to ethanol.     

Involvement of the beta-endorphin neurons of the hypothalamic arcuate nucleus in ethanol-induced place preference conditioning in mice

Background: Increasing evidence indicates that mu‐ and delta‐opioid receptors are decisively involved in the retrieval of memories underlying conditioned effects of ethanol. The precise mechanism by which these receptors participate in such effects remains unclear. Given the important role of the proopiomelanocortin (POMc)‐derived opioid peptide beta‐endorphin, an endogenous mu‐ and delta‐opioid receptor agonist, in some of the behavioral effects of ethanol, we hypothesized that beta‐endorphin would also be involved in ethanol conditioning. Methods: In this study, we treated female Swiss mice with estradiol valerate (EV), which induces a neurotoxic lesion of the beta‐endorphin neurons of the hypothalamic arcuate nucleus (ArcN). These mice were compared to saline‐treated controls to investigate the role of beta‐endorphin in the acquisition, extinction, and reinstatement of ethanol (0 or 2 g/kg; intraperitoneally)‐induced conditioned place preference (CPP). Results: Immunohistochemical analyses confirmed a decreased number of POMc‐containing neurons of the ArcN with EV treatment. EV did not affect the acquisition or reinstatement of ethanol‐induced CPP, but facilitated its extinction. Behavioral sensitization to ethanol, seen during the conditioning days, was not present in EV‐treated animals. Conclusions: The present data suggest that ArcN beta‐endorphins are involved in the retrieval of conditioned memories of ethanol and are implicated in the processes that underlie extinction of ethanol‐cue associations. Results also reveal a dissociated neurobiology supporting behavioral sensitization to ethanol and its conditioning properties, as a beta‐endorphin deficit affected sensitization to ethanol, while leaving acquisition and reinstatement of ethanol‐induced CPP unaffected.     

Ethanol-Induced Conditioned Taste Aversion in BXD Recombinant Inbred Mice

Genetic differences in sensitivity to ethanol's aversive effects may play an important role in the development of alcohol-seeking behavior and alcoholism. The present study examined the development of ethanol-induced conditioned taste aversion in 20 BXD/Ty recombinant inbred strains of mice and their progenitor inbred strains, C57BL/6J (B6) and DBA/2J (D2). Adult male mice were given 1-hr access to a saccharin-flavored solution every 48 hr for 12 days. After all but the first and last saccharin access periods, they received ethanol injections (0, 2, or 4 g/kg, ip). Separate groups of unpaired control mice received 4 g/kg of ethanol 1 hr after water access. Saline control mice were also used for examining preference across a wide range of saccharin concentrations (0.019 to 4.864% w/v). As expected, saccharin consumption during taste conditioning declined over conditioning trials in a dose-dependent manner, indicating development of ethanol-induced conditioned taste aversion. Correlational analyses using strain means from recently published papers indicated no significant genetic correlation between taste conditioning and two phenotypes thought to reflect ethanol reinforcement or reward (ethanol drinking, conditioned place preference). However, there were significant genetic correlations between taste conditioning at the high dose and sensitivity to ethanol-induced hypothermia, rotarod ataxia, and acute withdrawal. Quantitative trait locus (QTL) analyses of strain means indicated that taste aversion was associated (p 0.01) with genetic markers on nine chromosomes (1, 2, 3, 4, 6, 7, 9,11, and 17). These QTLs were located near several candidate genes, including genes encoding several different acetylcholine receptor subunits, the 6 opioid receptor, and two serotonin receptors (lB and 1D). QTLs for saccharin preference were located on several of the same chromosomes (2,3,4,6, and 11). Two of these saccharin QTLs overlap candidate genes influencing sensitivity to sweet or bitter taste stimuli. In general, these findings support the conclusion that multiple genes influence ethanol-induced conditioned taste aversion. Some of these genes appear to influence taste sensitivity, whereas others appear to mediate sensitivity to aversive pharmacological effects of ethanol.     

Decreased Pulmonary Inflammation Following Ethanol and Burn Injury in Mice Deficient in TLR4 but not TLR2 Signaling

Background: Clinical and laboratory evidence suggests that alcohol consumption prior to burn injury leads to dysregulated immune function and subsequent higher rates of morbidity and mortality. Our laboratory previously observed higher levels of pro‐inflammatory cytokines and leukocyte infiltration in the lungs of mice following ethanol and burn injury. To understand the mechanism of the increased inflammatory response, we looked at different signaling initiators of inflammation including toll‐like receptors 2 and 4 (TLR2 and 4) pathways. Methods: Wild‐type, TLR2, and TLR4 knockout mice were treated with vehicle or a single binge dose of ethanol (1.11 g/kg) and subsequently given a sham or burn injury. Twenty‐four hours postinjury, systemic and pulmonary levels of pro‐inflammatory cytokines were quantified, and differences in neutrophil infiltration were determined by histological examination. Results: Higher numbers of neutrophils were observed in the lungs of wild‐type mice following the combined insult of ethanol and burn injury relative to either injury alone. This increase in leukocyte accumulation was absent in the TLR4 knockout mice. Circulating levels of IL‐6 and tumor necrosis factor‐α were also elevated in wild‐type mice but not in TLR4 knockout mice. Consistent with these findings, pulmonary levels of KC and IL‐6 were increased in wild‐type mice following burn and ethanol compared to burn injury alone as well as to their TLR4 knockout counterparts. In contrast, TLR2 knockout mice displayed similar levels, to wild‐type mice, of neutrophil infiltration as well as IL‐6 and KC in the lung. Conclusions: These data suggest that TLR4 signaling is a crucial contributory component in the exuberant inflammation after ethanol and burn injury. However, TLR2 does not appear to play a vital role in the aberrant pulmonary inflammation.     

Galanin Knockout Mice Show Disturbances in Ethanol Consumption and Expression of Hypothalamic Peptides That Stimulate Ethanol Intake

Background: There is growing evidence suggesting that hypothalamic galanin (GAL), which is known to stimulate intake of a fat‐rich diet, has a role in promoting the consumption of ethanol. The present study further examined this possibility in GAL knockout (GALKO) mice. Methods: Two groups of female and male GALKO mice, compared to wild‐type (WT) controls, were trained to voluntarily drink increasing concentrations of ethanol, while maintained on lab chow and water. They were examined in terms of their daily ethanol intake and preference, acute consumption of a high‐fat diet, preference for flavored solutions, and expression of different peptides shown to stimulate ethanol intake. Results: In the GALKO mice compared to WT, the results revealed: (i) a 35 to 45% decrease in ethanol intake and preference, which was evident only at the highest (15%) ethanol concentration, was stronger in female than in male mice, and was seen with comparisons to littermate as well as nonlittermate WT mice; (ii) a 48% decrease in acute intake of a fat‐rich diet, again stronger in female than male mice; (iii) no difference in consumption of sucrose or quinine solutions in preference tests; (iv) a total loss of GAL mRNA in the hypothalamic paraventricular nucleus (PVN) of female and male mice; and (v) a gender‐specific change in mRNA levels of peptides in the perifornical lateral hypothalamus (PFLH), orexin and melanin‐concentrating hormone, which are known to stimulate ethanol and food intake and were markedly decreased in females while increased in males. Conclusions: These results provide strong support for a physiological role of PVN GAL in stimulating the consumption of ethanol, as well as a fat‐rich diet. Ablation of the GAL gene produced a behavioral phenotype, particularly in females, which may reflect the functional relationship of galanin to ovarian steroids. It also altered the peptides in the PFLH, with their reduced expression contributing to the larger behavioral effects observed in females and their increased expression attenuating these effects in males.     

Gender and age at drinking onset affect voluntary alcohol consumption but neither the alcohol deprivation effect nor the response to stress in mice

Background: Epidemiological studies suggest that initiation of alcohol drinking at an early age is associated with an increased risk of developing an alcohol use disorder later in life. Nevertheless, relatively few studies using animal models have investigated the relationship between age of onset of drinking and ethanol drinking patterns in adulthood. Besides age at drinking onset, other factors such as gender could also affect the pattern of development of alcohol consumption. In rodents, many studies have shown that females drink more than males. However, even if it is assumed that hormonal changes occurring at puberty could explain these differences, only one study performed in rats has investigated the emergence of sex‐specific alcohol drinking patterns in adolescence and the transition from adolescence to adulthood. The aim of the present study was to compare the acquisition of voluntary alcohol consumption, relapse‐like drinking (the Alcohol Deprivation Effect—ADE) and stress‐induced alcohol drinking in male and female outbred mice that acquired alcohol consumption during adolescence or adulthood. Methods: Separate groups of naïve female and male WSC‐1 mice aged ± 28 days (adolescents) or ±70 days (adults) were given ad libitum access to water and 6% ethanol solution for 8 weeks (1st to 8th week) before undergoing a 2‐week deprivation phase (9th and 10th week). After the deprivation period, 2‐bottle preference testing (ethanol vs. water) resumed for 3 weeks (11th to 13th). During the 13th week, all animals were subjected to restraint stress for 2 consecutive days. Results: Over the entire time course of the experiment, ethanol intake and preference increased in females (both adults and adolescents). Adolescent animals (both females and males) showed a transient increase in alcohol consumption and preference compared to adults. However, by the end of continuous alcohol exposure (when all mice were adults), ethanol intake was not affected by age at drinking onset. A deprivation phase was followed by a rise in ethanol intake (ADE) that was not affected by sex or age. Finally, stress did not alter alcohol self‐administration either during or after its occurrence. Conclusions: Emergence of greater alcohol consumption in adult females does not seem to be limited to a specific developmental period (i.e., puberty). Age of voluntary drinking onset (adolescence vs. adulthood) does not affect eventual alcohol intake in adult WSC‐1 mice and does not modify the transient increase in ethanol consumption after alcohol deprivation.     

Combined Scopolamine and Ethanol Treatment Results in a Locomotor Stimulant Response Suggestive of Synergism That is Not Blocked by Dopamine Receptor Antagonists

Background: Muscarinic acetylcholine receptors (mAChRs) are well positioned to mediate ethanol’s stimulant effects. To investigate this possibility, we examined the effects of scopolamine, a receptor subtype nonselective mAChR antagonist, on ethanol‐induced stimulation in genotypes highly sensitive to this effect of ethanol. We also investigated whether the dopamine D1‐like receptor antagonist, SCH‐23390 or the dopamine D2‐like receptor antagonist, haloperidol, could block the extreme stimulant response found following co‐administration of scopolamine and ethanol. Methods: Scopolamine (0, 0.0625, 0.125, 0.25, or 0.5 mg/kg) was given 10 minutes prior to saline or ethanol (0.75 to 2 g/kg) to female FAST (Experiment I) or DBA/2J (Experiment II) mice that were then tested for locomotion for 30 minutes. In Experiments III and IV, respectively, SCH‐23390 (0, 0.015, or 0.03 mg/kg) was given 10 minutes prior, and haloperidol (0, 0.08, or 0.16 mg/kg) was given 2 minutes prior, to scopolamine (0 or 0.5 mg/kg), followed 10 minutes later by saline or ethanol (1.5 g/kg) and female DBA/2J mice were tested for locomotion for 30 minutes. Results: FAST and DBA/2J mice displayed a robust enhancement of the locomotor effects of ethanol following pretreatment with scopolamine that was suggestive of synergism. SCH‐23390 had no effect on the response to the scopolamine + ethanol drug combination, nor did it attenuate ethanol‐ or scopolamine‐induced locomotor activity. Haloperidol, while attenuating the effects of ethanol, was not able to block the effects of scopolamine or the robust response to the scopolamine‐ethanol drug combination. Conclusions: These results suggest that while muscarinic receptor antagonism robustly enhances acute locomotor stimulation to ethanol, dopamine receptors are not involved in the super‐additive interaction of scopolamine and ethanol treatment. They also suggest that in addition to cautions regarding the use of alcohol when scopolamine is clinically prescribed due to enhanced sedative effects, enhanced stimulation may also be a concern.