Sex differences in binge-like EtOH drinking, corticotropin-releasing hormone and corticosterone: effects of ß-endorphin.

Binge drinking is an increasingly common pattern of risky use associated with numerous health problems, including alcohol use disorders. Because low basal plasma levels of ß-endorphin (ß-E) and an increased ß-E response to alcohol are evident in genetically at-risk human populations, this peptide is thought to contribute to the susceptibility for disordered drinking. Animal models suggest that the effect of ß-E on consumption may be sex-dependent. Here, we studied binge-like EtOH consumption in transgenic mice possessing varying levels of ß-E: wild-type controls with 100% of the peptide (ß-E +/+), heterozygous mice constitutively modified to possess 50% of wild-type levels (ß-E +/-) and mice entirely lacking the capacity to synthesize ß-E (-/-). These three genotypes and both sexes were evaluated in a 4-day, two-bottle choice, drinking in the dark paradigm with limited access to 20% EtOH. ß-E deficiency determined sexually divergent patterns of drinking in that ß-E -/- female mice drank more than their wild-type counterparts, an effect not observed in male mice. ß-E -/- female mice also displayed elevated basal anxiety, plasma corticosterone and corticotropin-releasing hormone mRNA in the extended amygdala, and all of these were normalized by EtOH self-administration. These data suggest that a heightened risk for excessive EtOH consumption in female mice is related to the drug's ability to ameliorate an overactive anxiety/stress-like state. Taken together, our study highlights a critical impact of sex on neuropeptide regulation of EtOH consumption.

Oral self-administration of EtOH: sex-dependent modulation by running wheel access in C57BL/6J mice.

BACKGROUND: The effects of stress, including neuroendocrine and behavioral sequelae aimed at maintaining homeostasis, are associated with increased alcohol consumption. Because both stress and drinking are multifactorial, the mechanisms underlying the relationship are difficult to elucidate. We therefore employed an animal model investigating the influence of blocked access to a running wheel on drinking in C57BL/6J (B6) mice. METHODS: In the first experiment, naïve, adult male and female subjects were individually housed for 2 weeks with 24-hour access to a running wheel and 12% ethanol (EtOH) in a 2-bottle, free choice paradigm. After determining baseline consumption and preference, experimental subjects had the running wheel placed in a locked position for 6 hours, and the EtOH bottle was removed during the first half of this period. Two subsequent experiments, again in adult, naïve B6 mice, examined the influence of locked running wheels on self-administration of 20% EtOH in a limited access paradigm, and blood EtOH concentrations (BECs) were determined on the final day of this protocol. RESULTS: In all 3 studies, using both between- and within-subject analyses, females showed transient yet reliable increases in alcohol drinking during blocked access to a rotating activity, while drinking in male mice was largely insensitive to this manipulation, although both sexes showed appreciable BECs (>130 mg/dl in females and 80 mg/dl in males) following a 2-hour EtOH access period. CONCLUSIONS: These data add to a burgeoning literature suggesting that the factors contributing to excessive alcohol use differ between males and females and that females may be especially sensitive to the influence of wheel manipulation. Elucidating the sex-dependent mechanisms mediating differences in alcohol sensitivity and response is critical to understanding the causes of alcoholism and in developing effective treatments and interventions.

ß-Endorphin influences sedative and ataxic effects of alcohol.

Beta-endorphin (ß-E) is an opioid peptide linked to the behavioral effects of ethanol. For example, ß-E provides negative feedback to inhibit the hypothalamic-pituitary-adrenal (HPA) stress axis, and neuroadaptation of this system to ethanol may facilitate sex differences in disordered drinking. Locomotor sensitivity to ethanol may also influence the risk for addiction; however, the role of ß-E in psychomotor effects of ethanol is not fully understood. We examined the role of ß-E and sex on locomotor effects of ethanol using adult male and female wild-type C57BL/6J and ß-E deficient B6.129S2-Pomctm1Low/J mice in a parallel rod floor apparatus following 0.75 or 2.0 g/kg ethanol. Beginning 15 min after intraperitoneal injection, we recorded foot slips, distance traveled, slips per meter, first instance of immobility, and total time spent off-balance (lying on the floor) over 15 min, and collected blood for analysis of ethanol concentration 60 min after injection. Overall, ß-E deficient mice were more sedated and ataxic following ethanol; at the lower dose they slipped more frequently and had a higher rate of slips per meter traveled. At the higher dose, ß-E deficient mice were predominantly sedated, slipping less frequently, and traveling less, as well as spending more time off-balance and becoming immobile sooner. Genotype interacted with sex in that male ß-E deficient mice slipped more frequently than their female counterparts, suggesting that ß-E may elicit sex-dependent effects of ethanol-induced ataxia. Blood ethanol concentration did not differ between any group, suggesting that behavioral differences result from altered sensitivity to ethanol. Our data support the contention that ß-E modulates the locomotor effects of ethanol and may influence ataxia in a sex-dependent manner. These findings help elucidate the role of ß-E in diverging behavioral responses to ethanol and may aid the development of targeted treatments for alcohol use disorders.

Sensitivity to the initial rewarding effects of alcohol: Influence of age, sex, and ß-endorphin.

BACKGROUND: Alcohol use disorders (AUDs) are widespread, devastating and complex. About 20% of people who consume alcohol develop problem use, accounting for over 5% of worldwide deaths. While numerous animal models have facilitated understanding of the consequences of excessive drinking, translational models allow for experimental manipulation of factors thought to contribute to AUD liability. METHODS: We employ a single-exposure conditioned place preference assay (SE-CPP) to investigate the influence of age, sex and the opioid peptide ß-endorphin (bE) on the initial rewarding effects of ethanol, a strong predictor of AUDs. Adolescent (PND28-35) and adult (PND70-90) male and female, control C57BL/6J and bE-deficient mice were tested following a single injection of 1.5 g/kg of ethanol. Following the SE-CPP test, animals were deeply anesthetized, sacrificed, and perfused, and the brains were subsequently sectioned at 40 microns and processed for immunohistochemical localization of c-fos. One-sample, two-tailed t-tests were used to assess drug preference or aversion and the locomotor effects of alcohol. RESULTS: In general, adults were more sensitive to the effects of alcohol than adolescents, and outcomes depended on sex and bE. For example, among females, adolescents were stimulated by the drug, but insensitive to locomotor effects as adults, while among males, adolescents were insensitive and adults sedated. Wild-type adolescents of both sexes failed to evince initial subjective reward from the drug, but bE-deficient adolescents, and all adult subjects, preferred a context once associated with ethanol over one that had been paired with saline. c-fos immunoreactivity in multiple brain regions was attenuated in bE-deficient animals, though influences of both sex and bE grew stronger with age. CONCLUSIONS: This study demonstrates the utility of the SE-CPP paradigm for elucidating factors that contribute to the liability for AUDs, and supports the growing body of research that shows that sensitivity to the rewarding effects of alcohol changes during the course of development. Our results also suggest that developmental contributions are sex-dependent, and may also depend on the influence of endogenous opioid signaling.

Age, ß-endorphin, and sex dependent effects of maternal separation on locomotor activity, anxiety-like behavior, and alcohol reward.

Introduction: Childhood adversity is pervasive and linked to numerous disadvantages in adulthood, including physical health problems, mental illness, and substance use disorders. Initial sensitivity to the rewarding effects of alcohol predicts the risk of developing an alcohol use disorder, and may be linked to developmental stress. The opioid peptide ß-endorphin (ß-E) regulates the stress response and is also implicated in the risk for excessive alcohol consumption. Methods: We explored the influence of ß-E in an animal model of early life adversity using controlled maternal separation by evaluating changes in locomotor activity, anxiety-like behavior, and the initial rewarding effects of alcohol in a single exposure conditioned place preference paradigm in control C57BL/6J and ß-E deficient ß-E +/+ 0.129S2-Pomc tm1Low/J; ß-E -/- mice. Maternal separation (MS) occurred for 3 h each day from post-natal days (PND) 5-18 in approximately half the subjects. Results: Maternal interactions increased following the separation protocol equally in both genotypes. MS and control subjects were tested as adolescents (PND 26-32) or adults (PND 58-72); the effects of MS were generally more pronounced in older subjects. Adults were more active than adolescents in the open field, and MS decreased activity in adolescent mice but increased it in adults. The increase in adult activity as a result of early life stress depended on both ß-E and sex. ß-E also influenced the effect of maternal separation on anxiety-like behavior in the Elevated Plus Maze. MS promoted rewarding effects of alcohol in male ß-E deficient mice of either age, but had no effect in other groups. Discussion: Taken together, these results suggest that the effects of MS develop over time and are ß-E and sex dependent and may aid understanding of how individual differences influence the impact of adverse childhood experiences.

Beta-endorphin mediates behavioral despair and the effect of ethanol on the tail suspension test in mice.

BACKGROUND: The opioid peptide beta-endorphin (beta-E) is synthesized and released in response to stressful stimuli as well as acute alcohol administration. The release of beta-E following exposure to an inescapable aversive situation may mediate behaviors that contribute to allostasis of the stress response. The present study examines the effects of beta-E on immobility in assays involving inescapable stress, both under basal conditions and after acute administration of EtOH. METHODS: Female and male transgenic mice with varying capacities to translate beta-E were subjected to either the forced swim (FST, Experiment 1) or the tail suspension test (TST, Experiment 2). In Experiment 3, mice were divided into three groups based on hormonal status (male, female-estrous, and female-nonestrous) and injected with either 1 g/kg EtOH or equivolume saline 14 minutes prior to behavioral assessment on the TST. RESULTS: Experiments 1 and 2 demonstrated a direct relationship between beta-E levels and immobility. There were also sex differences in behavior in these tests, with males displaying more immobility than females. A main effect of genotype in Experiment 3 replicated findings in Experiments 1 and 2. There was also an effect of EtOH (increasing immobility) and a significant interaction reflecting a particularly robust effect of the drug in mice with low beta-E. In addition, there were interactions between beta-E, EtOH effects, and hormonal status. CONCLUSIONS: These findings support the contention that beta-E moderates behavioral responses to stressful stimuli and suggest a role for this peptide in coping behavior. Furthermore, the effects of EtOH on the response to stress may be mediated by beta-E. Sex differences in this influence may contribute to sex differences in disease susceptibility and expression.

ß-Endorphin and sex differentially modulate the response to EtOH in a site-specific manner.

The effects of alcohol are multifaceted, involving brain circuits regulating reward, motivation, and stress, frequently via the endogenous opioid, beta-endorphin (ß-E). It is currently unknown how alcohol affects neural circuit activation in females and how ß-E affects ethanol's ability to induce neuronal activation. Therefore, we investigated the impact of acute alcohol treatment on neuronal activation in reward- and stress-related brain circuitry in a sex- and ß-E dependent manner. In this study, male and female control (C57BL/6J; ß-E+/+) and ß-E null (-/-) mice were injected intraperitoneally with 2 g/kg ethanol (EtOH) or saline. Post-injection, animals were sacrificed using ketamine/xylazine and perfused with saline followed by 4% paraformaldehyde. Brain sections (35 µm) were immunohistochemically processed for tyrosine hydroxylase (TH), dopamine's rate-limiting enzyme, and c-fos, a neuronal activation marker. The number of c-fos immunoreactive cell nuclei, TH-immunoreactivity, and TH/c-fos-ir cells were quantified in the nucleus accumbens (NAc), ventral tegmental area (VTA), paraventricular nucleus (PVN), central nucleus of the amygdala (CeA), paraventricular thalamic nucleus (PVA) and Edinger-Westphal nucleus (EW). In females, EtOH increased c-fos expression in the CeA, PVN, EW and NAc shell, while c-fos expression in the VTA, and TH expression in the VTA and NAc, depended on a genotype and treatment interaction. In males, EtOH increased c-fos in the CeA and PVN. EtOH also increased the number of double-labeled cells in the Arc, but only in females. These results suggest that the neurons in females are inherently more sensitive to EtOH, emphasizing the importance of studying the relationship between sex and alcohol addiction.

Influence of beta-Endorphin on anxious behavior in mice: interaction with EtOH.

RATIONALE: The opioid peptide beta-endorphin (beta-E) is synthesized by the pro-opiomelanocortin gene in response to environmental stressors and alcohol administration and is implicated in the behavioral sequelae associated with these stimuli. OBJECTIVES: We sought to determine the influence of beta-E on the stress response by evaluating basal measures of anxiety as well as on EtOH-induced anxiolytic behavior using transgenic mice that differ with respect to beta-E. METHODS: Anxious behavior was evaluated for male and female heterozygous, wild-type, and beta-E knockout mice using the Light-Dark Box and Plus Maze assays. Subsequent tests evaluated behavior 20 min after administration of intraperitoneal saline or EtOH (0.5, 1.0, and 1.5 g/kg). RESULTS: We observed a direct relationship between beta-E levels and the percentage of entries into open arms of the Plus Maze as well as the time spent in either the open arms or the light compartment of the Light-Dark box during basal conditions, suggesting that this peptide normally inhibits anxious behavior. However, mice lacking beta-E demonstrated an exaggerated anxiolytic response to EtOH in these assays. CONCLUSIONS: These data suggest that beta-E moderates the response to stressful stimuli and supports the hypothesis that this peptide influences the behavioral effects of EtOH.

Sex and ß-Endorphin Influence the Effects of Ethanol on Limbic Gabra2 Expression in a Mouse Binge Drinking Model.

Binge drinking is a widespread problem linked to increased risk for alcohol-related complications, including development of alcohol use disorders. In the last decade, binge drinking has increased significantly, specifically in women. Clinically, sexually dimorphic effects of alcohol are well-characterized, however, the underlying mechanisms for these dimorphisms in the physiological and behavioral effects of alcohol are poorly understood. Among its many effects, alcohol consumption reduces anxiety via the inhibitory neurotransmitter GABA, most likely acting upon receptors containing the α-2 subunit (Gabra2). Previous research from our laboratory indicates that female mice lacking the endogenous opioid peptide ß-endorphin (ßE) have an overactive stress axis and enhanced anxiety-like phenotype, coupled with increased binge-like alcohol consumption. Because ßE works via GABA signaling to reduce anxiety, we sought to determine whether sexually dimorphic binge drinking behavior in ßE deficient mice is coupled with differences in CNS Gabra2 expression. To test this hypothesis, we used ßE knock-out mice in a "drinking in the dark" model where adult male and female C57BL/6J controls (ßE +/+) and ßE deficient (ßE -/-; B6.129S2-Pomctm1Low/J) mice were provided with one bottle of 20% ethanol (EtOH) and one of water (EtOH drinkers) or two bottles of water (water drinkers) 3 h into the dark cycle for four consecutive days. Following a binge test on day 4, limbic tissue was collected and frozen for subsequent qRT-PCR analysis of Gabra2 mRNA expression. Water-drinking ßE +/+ females expressed more Gabra2 in central nucleus of the amygdala and the bed nucleus of the stria terminalis than males, but this sex difference was absent in the ßE -/- mice. Genotype alone had no effect on alcohol consumption or drug-induced increase in Gabra2 expression. In contrast, ßE expression had bi-directional effects in females: in wildtypes, Gabra2 mRNA was reduced by binge EtOH consumption, while EtOH increased expression in ßE -/- females to levels commensurate with drug-naïve ßE +/+ females. These results support the contention that ßE plays a role in sexually dimorphic binge-like EtOH consumption, perhaps through differential expression of GABAA α2 subunits in limbic structures known to play key roles in the regulation of stress and anxiety.

Initial subjective reward to alcohol in Sprague-Dawley rats.

Initial subjective response to the rewarding properties of alcohol predicts voluntary consumption and the risk for alcohol use disorders. We assessed the initial subjective reward to alcohol in rats using a single exposure conditioned place preference (SE-CPP) paradigm. Sprague-Dawley rats demonstrate preference for a context paired with a single systemic injection of ethanol (1.0 g/kg, delivered intraperitoneally). However, expression of SE-CPP in males depended on pairing ethanol with the first exposure of two (ethanol; saline) to the conditioning apparatus and procedures, while conditioning day did not appreciably affect SE-CPP in females, consistent with the view that females experience heightened addiction vulnerability. This model offers researchers a high throughput assay for investigating factors that influence alcohol reward and may point the way toward more effective prevention and treatment efforts.

ß-endorphin regulates alcohol consumption induced by exercise restriction in female mice.

Animal models have long been used to study the mechanisms underlying the complex association between alcohol and stress. Female mice prevented from running on a home-cage activity wheel increase voluntary ethanol consumption. ß-endorphin is an endogenous opioid involved in negatively regulating the stress response and has also been implicated in the risk for excessive drinking. The present study investigates the role of ß-endorphin in moderating free-choice consumption of ethanol in response to a blocked activity wheel. Female, transgenic mice with varying levels of the opioid peptide were given daily 2-h access to 20% ethanol with rotations on a running wheel blocked on alternate days. Subjects with low ß-endorphin exhibited enhanced stress sensitivity by self-administering larger quantities of ethanol on days when wheel running was prevented. ß-endorphin levels did not influence voluntary activity on the running wheel. There were genotypic differences in plasma corticosterone levels as well as corticotropin-releasing hormone mRNA content in multiple brain regions associated with the stress response in these free drinking and running subjects. Susceptibility to stress is enhanced in female mice with low levels of ß-endorphin, and better understanding of the role for this opioid in mitigating the response to stressors may aid in the development of interventions and treatments for excessive use of alcohol in women.

The influence of dextromethorphan on morphine analgesia in Swiss Webster mice is sex-specific.

NMDA (N-methyl-d-aspartate) antagonists are known to enhance the analgesic effects of opioids. However, virtually, all studies of this phenomenon have been done using male subjects. Here, the noncompetitive NMDA receptor antagonist dextromethorphan (DEX) was tested over a range of doses (10-200 microg intracerebroventricularly [i.c.v.]) in male and female Swiss Webster mice in combination with 5 mg/kg intraperitoneal (i.p.) morphine. Males exhibited enhanced morphine analgesia following either 100 or 200 microg DEX, but there was no evidence of DEX-mediated potentiation in females at any dose. Instead, DEX attenuated morphine analgesia in females. We also evaluated the effect of 100 microg i.c.v. DEX with different doses of morphine (1, 5 and 10 mg/kg). Again, DEX significantly enhanced morphine analgesia in male mice and attenuated it in females. Next, ovariectomized (OVX) female mice were compared to males following 5 mg/kg i.p. morphine and 100 microg i.c.v. DEX. Male and OVX females exhibited equivalent maximal levels of analgesia following administration of DEX. Morphine analgesia was not enhanced by DEX in sham-treated females and OVX mice with estradiol treatment (5 microg i.p. once daily for 7 days) also did not show DEX enhancement. These experiments demonstrate that the ability of NMDA receptor antagonists to potentiate morphine analgesia is modulated by an estrogen-sensitive mechanism and suggest that sex differences may play a critical role toward a more general understanding of the potentiation of opioid-induced analgesia through NMDA receptor antagonists.

Transgenic studies of pain.

A revolution in molecular biological technology has allowed, for the first time, the study of pain at the level of the gene. The molecular genetic technique currently garnering the most interest is the use of transgenic mice that either overexpress, or do not express, presumably pain-related proteins. This paper reviews the findings of investigations in which a transgenic mouse has been assessed for nociceptive or analgesic sensitivity. As of this writing, 25 different kinds of mutant mice--lacking neurotrophins and their receptors, peripheral mediators of nociception and hyperalgesia, opioids and their receptors, non-opioid transmitter receptors, and intracellular molecules participating in signal transduction--have been produced and tested on behavioral assays of nociception. Results of these studies have been variously confirmatory, contradictory and enlightening compared to conventional investigations. The advantages and limitations of this approach to pain research are discussed.

Modulation of morphine analgesia by site-specific N-methyl-D-aspartate receptor antagonists: dependence on sex, site of antagonism, morphine dose, and time.

Pharmacological blockade of N-methyl-D-aspartate (NMDA) receptors can modulate morphine analgesia in experimental animals and humans. However, this literature is highly inconsistent, with NMDA receptor antagonists variously shown to potentiate, attenuate or produce no effect on morphine analgesic magnitude. A number of factors influencing this modulation have been proposed, but no one has examined such factors simultaneously, and all existing studies in mice were conducted exclusively in male subjects. Thus, the influence of systemic administration of site-specific NMDA receptor antagonists-including dextromethorphan, dextrorphan, MK-801, LY235959, L-701,324, and Ro 25-6981-on morphine analgesia (15-45 mg/kg; 15, 30 and 60 min post-injection) was studied in male and female mice using the 49 degrees C tail-withdrawal test. We found that oral and intraperitoneal dextromethorphan, a low-affinity non-competitive antagonist, dose-dependently potentiated low-dose morphine analgesia but attenuated high-dose morphine analgesia. Dextrorphan and MK-801 were found to potentiate low- but not high-dose morphine analgesia. The competitive glutamate-site antagonist, LY235959, and glycine-site antagonist, L-701,324, potentiated morphine analgesia at all doses. In contrast, the polyamine (NR2B) site antagonist, Ro 25-6981, attenuated morphine analgesia at all doses. Strikingly, the non-competitive antagonists produced no modulation of morphine analgesia whatsoever in female mice, whereas no sex differences were observed using competitive or NR2B antagonists. These findings indicate that NMDA modulation of morphine analgesia is critically influenced by sex, site of antagonism, morphine dose and time after injection. Our data suggest that NMDA antagonism via competitive or glycine site antagonism might result in more reliable clinical effects on morphine analgesia in both sexes.

Quantitative Trait Loci Mapping.

Researchers interested in the physical locations of genes that influence a person's alcohol-related behaviors can use a method known as quantitative trait loci (QTL) mapping to identify the approximate locations of genes in the genome. QTL mapping can use recombinant inbred mouse strains, which are sets of inbred strains derived from cross-breeding the offspring of two genetically distinct parent strains. The inbred strains exhibit different patterns of the parent strains' genes. QTL mapping involves comparing alcohol-related behaviors in these strains and identifying patterns of known genetic markers shared by strains with the same behaviors. The markers allow the identification of probable locations of genes that influence alcohol-related behaviors. These locations can then be verified using other tests, and specific genes can be sought there.

Initial subjective reward: single-exposure conditioned place preference to alcohol in mice.

Most adults consume alcohol with relative impunity, but about 10-20% of users persist (or progress) in their consumption, despite mounting and serious repercussions. Identifying at-risk individuals before neuroadaptative changes associated with chronic use become well ingrained is thus a key step in mitigating and preventing the end stage disease and its devastating impacts. Explaining liability has been impeded, in part, by the absence of animal models for assessing initial sensitivity to the drug's reinforcing properties, an important endophenotype in the trajectory toward excessive drinking. Here we assess the initial rewarding effects of the drug in a novel application of the conditioned place preference paradigm. In contrast to previous studies that have all employed repeated drug administration, we demonstrated a robust preference for a context paired with a single exposure to 1.5 g/kg EtOH in male and female subjects of three strains. This model validates an assay of initial sensitivity to the subjective rewarding effects of alcohol, a widely used drug with multifarious impacts on both brain and society, and provides a new tool for theory-driven endophenotypic pharmacogenetic approaches to understanding and treating addiction.

ß-endorphin modulates the effect of stress on novelty-suppressed feeding.

Although stress is implicated in the pathophysiology of mood and anxiety disorders, not all individuals who suffer stressful life events develop psychopathology. Differential susceptibility to stress may be influenced by genetically mediated differences in hypothalamic-pituitary-adrenal (HPA) axis activity and moderation of the stress response by the opioid peptide ß-endorphin (ß-E). The present study investigated genetic contributions to coping behavior by examining anxious behavior of transgenic mice with varying capacities to synthesize ß-E [B6.129S2-Pomc (tm1Low) /J; regulated by insertion of a premature stop codon into one or both copies of the proopiomelanocortin (POMC) gene], both under normal conditions and following 3 min of forced swim (FS). Ten minutes after this stress exposure or a control manipulation, acutely food-deprived female and male transgenic mice were subjected to a novelty-suppressed feeding (NSF) test, during which their interaction with an almond slice located in the center of an open field box was measured. There was an interaction between genotype and stress for latency to approach the almond and whether or not the almond was approached, such that mice with low or absent ß-E displayed a stronger aversion to novelty-feeding after stress exposure than did mice with normal levels. These data provide evidence for a moderating effect of ß-E on the behavioral response to stress. Genotypic differences in anxious behavior emerged when mice were stressed prior to behavioral assessment, suggesting that ß-E plays a role in coping behavior. These findings indicate that genetic variability in sensitivity of the ß-E system to stress may contribute, at least in part, to heritable differences in stress reactivity as well as vulnerability to stress-related psychopathology.

Locomotor sensitization to EtOH: contribution of ß-Endorphin.

Alcohol use disorders, like all drug addictions, involve a constellation of adaptive changes throughout the brain. Neural activity underlying changes in the rewarding properties of alcohol reflect changes in dopamine transmission in mesolimbic and nigrostriatal pathways and these effects are modulated by endogenous opioids such as ß-Endorphin. In order to study the role of ß-Endorphin in the development of locomotor sensitization to repeated EtOH exposure, we tested transgenic mice that vary in their capacity to synthesize this peptide as a result of constitutive modification of the Pomc gene. Our results indicate that mice deficient in ß-Endorphin show attenuated locomotor activation following an acute injection of EtOH (2.0 g/kg) and, in contrast to wildtype mice, fail to demonstrate locomotor sensitization after 12 days of repeated EtOH injections. These data support the idea that ß-Endorphin modulates the locomotor effects of EtOH and contributes to the neuroadaptive changes associated with chronic use.

Mapping of quantitative trait loci underlying ethanol metabolism in BXD recombinant inbred mouse strains.

BACKGROUND: Genetic factors are well known to play an important role in determining individual differences in the metabolism of ethanol (EtOH), and several specific polymorphic loci have been identified that significantly contribute to the variability of EtOH metabolism in humans. However, these variant genes are either alcohol or aldehyde dehydrogenases, and the identification of new gene products that contribute to variation in alcohol metabolism would be useful. METHODS: To identify quantitative trait loci (QTLs), we correlated variation in polymorphic markers with blood EtOH concentration and the rate of EtOH metabolism (beta) in C57BL/6J and DBA/2J strains and in 25 of their recombinant inbred strains after 2 and 3 g/kg of EtOH intraperitoneally. RESULTS: A QTL associated with beta values for both doses was definitively mapped to the proximal region of chromosome 17, syntenic with human chromosome 6q25-27. Seven to 12 chromosomal regions were provisionally identified for each phenotype; several were associated with 2 or more phenotypes. Each QTL suggests the location of a gene or genes affecting EtOH pharmacokinetics. Candidate genes suggested by these analyses included several whose gene products are known to be induced by EtOH (e.g., superoxide dismutase, glutathione transferase, and cytochrome P450 2E1), as well as several whose gene products have signaling functions likely to contribute to this induction. CONCLUSIONS: These studies provide evidence for the existence of genes affecting EtOH metabolism in multiple chromosomal regions. Future studies will be required to identify the chromosome 17 gene product. Use of other genetic populations, such as B6D2F2 crosses, will be required to determine which of the provisional loci represent true and which represent false-positive associations.

The melanocortin-1 receptor gene mediates female-specific mechanisms of analgesia in mice and humans.

Sex specificity of neural mechanisms modulating nociceptive information has been demonstrated in rodents, and these qualitative sex differences appear to be relevant to analgesia from kappa-opioid receptor agonists, a drug class reported to be clinically effective only in women. Via quantitative trait locus mapping followed by a candidate gene strategy using both mutant mice and pharmacological tools, we now demonstrate that the melanocortin-1 receptor (Mc1r) gene mediates kappa-opioid analgesia in female mice only. This finding suggested that individuals with variants of the human MC1R gene, associated in our species with red hair and fair skin, might also display altered kappa-opioid analgesia. We found that women with two variant MC1R alleles displayed significantly greater analgesia from the kappa-opioid, pentazocine, than all other groups. This study demonstrates an unexpected role for the MC1R gene, verifies that pain modulation in the two sexes involves neurochemically distinct substrates, and represents an example of a direct translation of a pharmacogenetic finding from mouse to human.