Jose Manuel Lerma-Cabrera

Assessment of Voluntary Ethanol Consumption and the Effects of a Melanocortin (MC) Receptor Agonist on Ethanol Intake in Mutant C57BL/6J Mice Lacking the MC‐4 Receptor

BACKGROUND The melanocortin (MC) system is composed of peptides that are cleaved from the polypeptide precursor proopiomelanocortin (POMC). Recent evidence shows that chronic exposure to ethanol significantly blunts central MC peptide immunoreactivity and MC receptor (MCR) agonists protect against high ethanol intake characteristic of C57BL/6J mice. Here, we assessed the role of the MC-4 receptor (MC4R) in voluntary ethanol intake and in modulating the effects of the nonselective MCR agonist melanotan-II (MTII) on ethanol consumption. METHODS To assess the role of the MC4R, MC4R knockout (Mc4r(-/-) ) and littermate wild-type (Mc4r(+/+) ) mice on a C57BL/6J background were used. Voluntary ethanol (3, 5, 8, 10, 15, and 20%, v/v) and water intake were assessed using standard two-bottle procedures. In separate experiments, Mc4r(-/-) and Mc4r(+/+) mice were given intracerebroventricular (i.c.v.) infusion of MTII (0, 0.5, or 1.0 μg/1 μl) or intraperitoneal (i.p.) injection of MTII (0 or 5 mg/kg/5 ml). The effects of MTII (0 or 0.5 μg/1 μl, i.c.v.) on 10% sucrose and 0.15% saccharin intake were assessed in C57BL/6J mice. RESULTS Mc4r(-/-) mice showed normal consumption of ethanol over all concentrations tested. I.c.v. infusion of MTII significantly reduced ethanol drinking in Mc4r(+/+) mice, but failed to influence ethanol intake in Mc4r(-/-) mice. When administered in an i.p. injection, MTII significantly reduced ethanol drinking in both Mc4r(-/-) and Mc4r(+/+) mice. MTII attenuated consumption of caloric (ethanol, sucrose, and food) and noncaloric (saccharin) reinforcers. CONCLUSIONS When given centrally, the MCR agonist MTII reduced ethanol drinking by signaling through the MC4R. On the other hand, MTII-induced reduction of ethanol drinking did not require the MC4R when administered peripherally. Together, the present observations show that the MC4R is necessary for the central actions of MCR agonists on ethanol drinking and that MTII blunts the consumption natural reinforcers, regardless of caloric content, in addition to ethanol.

Binge-like consumption of caloric and non-caloric palatable substances in ad libitum-fed C57BL/6J mice: pharmacological and molecular evidence of orexin involvement.

The orexin (OX) system has been implicated in food-reinforced behavior, food-seeking and food overconsumption. Recent evidence suggests that OX signaling might influence consumption of palatable foods with high reinforcing value depending upon the caloric status of the animal. The present study evaluates from a pharmacological and a molecular approach the contribution of OX to excessive binge-like consumption of highly preferred palatable substances (sucrose and saccharin) in ad libitum-fed C57BL/6J mice. The main findings of this study are: (1) intraperitoneal (ip) injection of SB-334867 (10, 20 or 30mg/kg), a selective OXR1 antagonist, significantly decreased binge-like consumption of sucrose (10%, w/v) and saccharin (0.15%, w/v) during the test day in a Drinking in the Dark procedure in ad libitum-fed animals, without evidence of any significant alteration of locomotor activity. (2) Four repetitive, 2-h daily episodes of sucrose and saccharin (but not water) binge-like drinking significantly dampened OX mRNA expression in the LH. Present findings show for the first time a role for OXR1 signaling in binge-like consumption of palatable substances in animals under no caloric needs. Targeting OXR1 could represent a novel pharmacological approach to treat binge-eating episodes.

The partial reinforcement extinction effect (PREE) in female Roman high- (RHA-I) and low-avoidance (RLA-I) rats.

The present experiment was designed with the goal of studying the partial reinforcement extinction effect (PREE) in female inbred Roman high- (RHA-I) and low-avoidance (RLA-I) rats. Two groups of RHA-I and two of RLA-I food-deprived animals were placed in a straight alley where they were partially or continuously reinforced. Once the animals reached the acquisition criterion, they were exposed to an extinction phase where the reinforcement was omitted. During the extinction phase RHA-I animals continuously reinforced during acquisition exhibited more resistance to extinction than their RLA-I counterparts, whereas only RLA-I rats partially reinforced during acquisition showed an increased resistance to extinction in comparison to continuously reinforced control RLA-I rats, this PREE being absent in RHA-I animals. These results are discussed within the framework of PREE theories that account for this effect by using emotional mechanisms, as pertains to the repeatedly observed RHA-RLA differences in emotional reactivity.

Control of food intake by MC4-R signaling in the lateral hypothalamus, nucleus accumbens shell and ventral tegmental area: interactions with ethanol.

The melanocortin system is involved in animal models of obesity and anorexia-cachexia and MC4 receptors (MC4-R) are currently a target system for the development of drugs aimed to treat obesity and eating disorders in humans. Previous evidence suggest that feeding peptides might lack their orexigenic activity while stimulate ethanol intake. The present study comparatively evaluated food intake (4-h interval) in Sprague-Dawley (SD) rats drinking ethanol (6% w/v, 2 bottle choice paradigm) (EE group) and ethanol-naïve (EN) rats in response to bilateral infusion of the selective MC4-R antagonist HS014 (0, 0.02 or 0.05 μg/0.5 μl/site) or the selective MC4-R agonist cyclo(NH-CH(2)-CH(2)-CO-His-d-Phe-Arg-Trp-Glu)-NH(2) (0, 0.75 or 1.5 μg/0.5 μl/site), into the lateral hypothalamus (LH), the nucleus accumbens (NAc), or the ventral tegmental area (VTA). The main findings in the study are: (1) LH-infusions of the MC4-R antagonist increased and the agonist reduced feeding and total calories consumed, while ethanol intake remained unaltered. (2) NAc- and VTA-infusions of the selective agonist reduced food, ethanol and total calories intake. (3) NAc- and VTA-infusions of the MC4-R antagonist increased feeding in EN rats, but not in EE animals which showed a mild increase in ethanol intake, while total calories consumed remained unaltered. Present data show that having ethanol available reduces feeding elicited by NAc and VTA-MC4-R blockade. Additionally, while MC4-R signaling in the LH appears to modulate homeostatic aspects of feeding, it may contribute to non-homeostatic aspects of ingestive behaviors in the VTA and the NAc.

Adolescent binge-like ethanol exposure reduces basal α-MSH expression in the hypothalamus and the amygdala of adult rats

Melanocortins (MC) are central peptides that have been implicated in the modulation of ethanol consumption. There is experimental evidence that chronic ethanol exposure reduces α-MSH expression in the limbic and hypothalamic brain regions and alters central pro-opiomelanocortin (POMC) mRNA activity in adult rats. Adolescence is a critical developmental period of high vulnerability in which ethanol exposure alters corticotropin releasing factor, neuropeptide Y, substance P and neurokinin neuropeptide activities, all of which have key roles in ethanol consumption. Given the involvement of MC and the endogenous inverse agonist AgRP in ethanol drinking, here we evaluate whether a binge-like pattern of ethanol treatment during adolescence has a relevant impact on basal and/or ethanol-stimulated α-MSH and AgRP activities during adulthood. To this end, adolescent Sprague-Dawley rats (beginning at PND25) were pre-treated with either saline (SP group) or binge-like ethanol exposure (BEP group; 3.0 g/kg given in intraperitoneal (i.p.) injections) of one injection per day over two consecutive days, followed by 2 days without injections, repeated for a total of 8 injections. Following 25 ethanol-free days, we evaluated α-MSH and AgRP immunoreactivity (IR) in the limbic and hypothalamic nuclei of adult rats (PND63) in response to ethanol (1.5 or 3.0 g/kgi.p.) and saline. We found that binge-like ethanol exposure during adolescence significantly reduced basal α-MSH IR in the central nucleus of the amygdala (CeA), the arcuate nucleus (Arc) and the paraventricular nucleus of the hypothalamus (PVN) during adulthood. Additionally, acute ethanol elicited AgRP IR in the Arc. Rats given the adolescent ethanol treatment required higher doses of ethanol than saline-treated rats to express AgRP. In light of previous evidence that endogenous MC and AgRP regulate ethanol intake through MC-receptor signaling, we speculate that the α-MSH and AgRP disturbances induced by binge-like ethanol exposure during adolescence may contribute to excessive ethanol consumption during adulthood.

Ethanol-Induced Increase of Agouti-Related Protein (AgRP) Immunoreactivity in the Arcuate Nucleus of the Hypothalamus of C57BL/6J, but not 129/SvJ, Inbred Mice

BACKGROUND The melanocortin (MC) system is composed of peptides that are cleaved from the polypeptide precursor, pro-opiomelanocortin (POMC). Previous research has shown that MC receptor (MCR) agonists reduce, and MCR antagonists increase, ethanol consumption in rats and mice. Consistently, genetic deletion of the endogenous MCR antagonist, agouti-related protein (AgRP), causes reductions of ethanol-reinforced lever pressing and binge-like ethanol drinking in C57BL/6J mice. Ethanol also has direct effects on the central MC system, as chronic exposure to an ethanol-containing diet causes significant reductions of alpha-melanocyte stimulating hormone (alpha-MSH) immunoreactivity in specific brain regions of Sprague-Dawley rats. Together, these observations suggest that the central MC system modulates neurobiological responses to ethanol. To further characterize the role of the MC system in responses to ethanol, here we compared AgRP and alpha-MSH immunoreactivity in response to an acute injection of saline or ethanol between high ethanol drinking C57BL/6J mice and moderate ethanol drinking 129/SvJ mice. METHODS Mice received an intraperitoneal (i.p.) injection of ethanol (1.5 g/kg or 3.5 g/kg; mixed in 0.9% saline) or an equivolume of 0.9% saline. Two hours after injection, animals were sacrificed and their brains were processed for AgRP and alpha-MSH immunoreactivity. RESULTS Results indicated that acute ethanol administration triggered a dose-dependent increase in AgRP immunoreactivity in the arcuate (ARC) of C57BL/6J mice, an effect that was not evident in the 129/SvJ strain. Although acute administration of ethanol did not influence alpha-MSH immunoreactivity, C57BL/6J mice had significantly greater overall alpha-MSH immunoreactivity in the ARC, dorsomedial, and lateral regions of the hypothalamus relative to the 129/SvJ strain. In contrast, C57BL/6J mice displayed significantly lower alpha-MSH immunoreactivity in the medial amygdala. CONCLUSIONS The results show that acute ethanol exposure has direct effects on endogenous AgRP activity in ethanol preferring C57BL/6J mice. It is suggested that ethanol-induced increases in AgRP may be part of a positive feedback system that stimulates excessive binge-like ethanol drinking in C57BL/6J mice. Inherent differences in alpha-MSH immunoreactivity may contribute to differences in neurobiological responses to ethanol that are characteristically observed between the C57BL/6J and 129/SvJ inbred strains of mice.

Orexin receptor 1 signaling contributes to ethanol binge-like drinking: Pharmacological and molecular evidence.

Orexins (OX) have been recently implicated in ethanol seeking and self-administration. A few recent studies have provided additional evidence that OX receptor antagonists effectively reduce voluntary ethanol consumption in subjects spontaneously showing high levels of ethanol intake. The present study further evaluates the contribution of OXR1 to excessive binge-like drinking of ethanol in ad libitum-fed C57BL/6J mice from a pharmacological and molecular approach. The main findings in the study are: (1) Icv administration of SB-334867 (3 μg/μl) blunted ethanol (20% v/v), but not saccharin (0.15% w/v) binge-like drinking in a drinking in the dark procedure, without any alteration of chow consumption or total calories ingested; (2) Icv administration of SB-334867 (3 μg/μl) increased the latency to recover the righting reflex after a sedative dose of ethanol without any significant alteration in ethanol peripheral metabolism; (3) four repetitive, 2-h daily episodes of saccharin, but not ethanol binge-like drinking blunted OXR1 mRNA expression in the lateral hypothalamus. Present findings extend the current knowledge pointing to a role for OX signaling in ethanol sedation, which might partially explain the inhibitory effect of OXR1 antagonists on ethanol consumption. Combined pharmacological and molecular data suggesting the contribution of OXR1 in ethanol binge-drinking leading us to propose the idea that targeting OXR1 could represent a novel pharmacological approach to control binge-consumption episodes of ethanol in vulnerable organisms failing to spontaneously reduce OX activity.

MC4-R signaling within the nucleus accumbens shell, but not the lateral hypothalamus, modulates ethanol palatability in rats

The Melanocortin (MC) system is one of the crucial neuropeptidergic systems that modulate energy balance. The roles of endogenous MC and MC-4 receptor (MC4-R) signaling within the hypothalamus in the control of homeostatic aspects of feeding are well established. Additional evidence points to a key role for the central MC system in ethanol consumption. Recently, we have shown that nucleus accumbens (NAc), but not lateral hypothalamic (LH), infusion of a selective MC4-R agonist decreases ethanol consumption. Given that MC signaling might contribute to non-homeostatic aspects of feeding within limbic circuits, we assessed here whether MC4-R signaling within the NAc and the lateral hypothalamus (LH) alters normal ingestive hedonic and/or aversive responses to ethanol in rats as measured by a taste reactivity test. Adult male Sprague-Dawley rats were given NAc- or LH- bilateral infusion of the selective MC4-R agonist cyclo (NH-CH(2)-CH(2)-CO-His-D-Phe-Arg-Trp-Glu)-NH(2) (0, 0.75 or 1.5μg/0.5μl/site) and following 30 min, the animals received 1 ml of ethanol solution (6% w/v) intraoral for 1 minute and aversive and hedonic behaviors were recorded. We found that NAc-, but not LH-administration, of a selective MC4-R agonist decreased total duration of hedonic reactions and significantly increased aversive reactions relative to saline-infused animals which support the hypothesis that MC signaling within the NAc may contribute to ethanol consumption by modulating non-homeostatic aspects (palatability) of intake.

Repeated Binge-Like Ethanol Administration During Adolescence cause Decreased C-Fos Immunoreactivity in Amygdala and Arcuate Nucleus in Adult Sprague-Dawley Rats

El consumo en atracon durante la adolescencia esta asociado con neurotoxicidad y con el riesgo de desarrollar un trastorno en el uso de alcohol. Diversos estudios muestran que la administracion aguda y cronica de alcohol en ratas adultas altera la expresion de c-fos, un marcador indirecto de actividad celular, en diferentes areas cerebrales. Nosotros evaluamos si el patron de consumo de alcohol en atracon durante la adolescencia tiene un impacto en la actividad basal de c-fos en esas regiones activadas por el alcohol. Utilizamos ratas Sprague-Dawley en su dia post-natal 25 (PND25) tratadas con suero salino (grupo SP) o con etanol tipo atracon (grupo BEP) durante dos dias consecutivos, en intervalos de 48 h, durante 14 dias (PND25- PND38). En la edad adulta (PND63) y despues de 25 dias sin etanol, evaluamos la inmunorreactividad para c-fos en respuesta a una administracion aguda de suero salino o etanol (1,5 o 3,0 g/kg) en diferentes regiones cerebrales. La administracion de alcohol incremento de manera dosis-dependiente la actividad de c-fos en el nucleo paraventricular del hipotalamo. Ademas la exposicion a etanol tipo atracon durante la adolescencia disminuyo la actividad basal de c-fos en la adultez en el nucleo central de la amigdala y en el nucleo arqueado del hipotalamo. Concluimos que el consumo de alcohol en atracon durante la adolescencia causa problemas a largo plazo en la actividad basal de regiones cerebrales implicadas en el consumo de alcohol.

Interaction Between Exposure to Neurotoxicants and Drug Abuse

Humans are continuously exposed to a variety of environmental neurotoxicants. Over the past 30 years, at least 100,000 chemicals, including pesticides, food additives, drugs, and cosmetics, have been registered for commercial use in the United States (Muir & Howard, 2006). Twenty years ago, about 750 chemicals had shown neurotoxic effects in laboratory animals (Anger, 1984). Actually, the number is thought to exceed a thousand, although no authoritative estimate of the real number of neurotoxicants is available (Grandjeand & Landrigan, 2006).