RESUMEN
Acetylcholine is the endogenous agonist for the neuronal nicotinic acetylcholine receptor (nAChR) system, which is involved in attention, memory, affective behaviours and substance use disorders. Brain nAChRs are highly diverse with 11 different subunits that can form multiple receptor subtypes, each with distinct receptor and pharmacological properties. Different neuronal cell types can also express different nAChR subtypes, resulting in highly complex cholinergic signalling. Identifying which nAChR subunit transcripts are expressed in cell types can provide an indication of which nAChR combinations are possible and which receptor subtypes may be most pharmacologically relevant to target. In addition to differences in expression across cell types, nAChRs also undergo changes in expression levels from adolescence to adulthood. In this study, we used fluorescent in situ hybridization to identify and quantify the expression of α4, α5, α6, ß2 and ß3 nAChR subunit transcripts in dopaminergic, GABAergic, glutamatergic and noradrenergic neurons and astrocytes in the ventral tegmental area (VTA) and locus coeruleus (LC) in adult and adolescent, male and female C57BL/6J mice. There were distinct differences in the pattern of nAChR subunit transcript expression between the two brain regions. LC noradrenergic neurons had high prevalence of α6, ß2 and ß3 expression, with very low expression of α4, suggesting the α6(non-α4)ß2ß3 receptor as a main subtype in these neurons. VTA astrocytes from adult mice showed greater prevalence of α5, α6, ß2 and ß3 transcript compared with adolescent mice. These data highlight the complex nAChR expression patterns across brain region and cell type.
RESUMEN
BACKGROUND AND PURPOSE: Drugs of abuse, including alcohol, increase dopamine in the mesocorticolimbic system via actions on dopamine neurons in the ventral tegmental area (VTA). Increased dopamine transmission can activate inhibitory G protein signalling pathways in VTA dopamine neurons, including those controlled by GABAB and D2 receptors. Members of the R7 subfamily of regulator of G protein signalling (RGS) proteins can regulate inhibitory G protein signalling, but their influence on VTA dopamine neurons is unclear. Here, we investigated the influence of RGS6, an R7 RGS family memberthat has been implicated in the regulation of alcohol consumption in mice, on inhibitory G protein signalling in VTA dopamine neurons. EXPERIMENTAL APPROACH: We used molecular, electrophysiological and genetic approaches to probe the impact of RGS6 on inhibitory G protein signalling in VTA dopamine neurons and on binge-like alcohol consumption in mice. KEY RESULTS: RGS6 is expressed in adult mouse VTA dopamine neurons and it modulates inhibitory G protein signalling in a receptor-dependent manner, tempering D2 receptor-induced somatodendritic currents and accelerating deactivation of synaptically evoked GABAB receptor-dependent responses. RGS6-/- mice exhibit diminished binge-like alcohol consumption, a phenotype replicated in female (but not male) mice lacking RGS6 selectively in VTA dopamine neurons. CONCLUSIONS AND IMPLICATIONS: RGS6 negatively regulates GABAB - and D2 receptor-dependent inhibitory G protein signalling pathways in mouse VTA dopamine neurons and exerts a sex-dependent positive influence on binge-like alcohol consumption in adult mice. As such, RGS6 may represent a new diagnostic and/or therapeutic target for alcohol use disorder.
Asunto(s)
Dopamina , Neuronas Dopaminérgicas , Animales , Femenino , Ratones , Consumo de Bebidas Alcohólicas , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Transducción de Señal , Área Tegmental Ventral/metabolismo , MasculinoRESUMEN
The prevailing view is that enhancement of dopamine (DA) transmission in the mesolimbic system, consisting of DA neurons in the ventral tegmental area (VTA) that project to the nucleus accumbens (NAc), underlies the reward properties of ethanol (EtOH) and nicotine (NIC). We have shown previously that EtOH and NIC modulation of DA release in the NAc is mediated by α6-containing nicotinic acetylcholine receptors (α6*-nAChRs), that α6*-nAChRs mediate low-dose EtOH effects on VTA GABA neurons and EtOH preference, and that α6*-nAChRs may be a molecular target for low-dose EtOH. However, the most sensitive target for reward-relevant EtOH modulation of mesolimbic DA transmission and the involvement of α6*-nAChRs in the mesolimbic DA reward system remains to be elucidated. The aim of this study was to evaluate EtOH effects on GABAergic modulation of VTA GABA neurons and VTA GABAergic input to cholinergic interneurons (CINs) in the NAc. Low-dose EtOH enhanced GABAergic input to VTA GABA neurons that was blocked by knockdown of α6*-nAChRs. Knockdown was achieved either by α6-miRNA injected into the VTA of VGAT-Cre/GAD67-GFP mice or by superfusion of the α-conotoxin MII[H9A;L15A] (MII). Superfusion of MII blocked EtOH inhibition of mIPSCs in NAc CINs. Concomitantly, EtOH enhanced CIN firing rate, which was blocked by knockdown of α6*-nAChRs with α6-miRNA injected into the VTA of VGAT-Cre/GAD67-GFP mice. The firing rate of CINs was not enhanced by EtOH in EtOH-dependent mice, and low-frequency stimulation (LFS; 1 Hz, 240 pulses) caused inhibitory long-term depression at this synapse (VTA-NAc CIN-iLTD) which was blocked by knockdown of α6*-nAChR and MII. Ethanol inhibition of CIN-mediated evoked DA release in the NAc was blocked by MII. Taken together, these findings suggest that α6*-nAChRs in the VTA-NAc pathway are sensitive to low-dose EtOH and play a role in plasticity associated with chronic EtOH.
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MicroARNs , Receptores Nicotínicos , Ratones , Animales , Receptores Nicotínicos/metabolismo , Núcleo Accumbens/metabolismo , Nicotina/farmacología , Transmisión Sináptica , Área Tegmental Ventral/metabolismo , Etanol/farmacología , Colinérgicos/farmacología , Interneuronas/metabolismo , MicroARNs/metabolismoRESUMEN
Previous studies indicate that moderate-to-high ethanol (EtOH) concentrations enhance dopamine (DA) neurotransmission in the mesolimbic DA system from the ventral tegmental area (VTA) and projecting to the nucleus accumbens core (NAc). However, voltammetry studies demonstrate that moderate-to-high EtOH concentrations decrease evoked DA release at NAc terminals. The involvement of γ-aminobutyric acid (GABA) receptors (GABAA Rs), glycine (GLY) receptors (GLYRs) and cholinergic interneurons (CINs) in mediating EtOH inhibition of evoked NAc DA release were examined. Fast scan cyclic voltammetry, electrophysiology, optogenetics and immunohistochemistry techniques were used to evaluate the effects of acute and chronic EtOH exposure on DA release and CIN activity in C57/BL6, CD-1, transgenic mice and δ-subunit knockout (KO) mice (δ-/-). Ethanol decreased DA release in mice with an IC50 of 80 mM ex vivo and 2.0 g/kg in vivo. GABA and GLY decreased evoked DA release at 1-10 mM. Typical GABAA R agonists inhibited DA release at high concentrations. Typical GABAA R antagonists had minimal effects on EtOH inhibition of evoked DA release. However, EtOH inhibition of DA release was blocked by the α4 ß3 δ GABAA R antagonist Ro15-4513, the GLYR antagonist strychnine and by the GABA ρ1 (Rho-1) antagonist TPMPA (10 µM) and reduced significantly in GABAA R δ-/- mice. Rho-1 expression was observed in CINs. Ethanol inhibited GABAergic synaptic input to CINs from the VTA and enhanced firing rate, both of which were blocked by TPMPA. Results herein suggest that EtOH inhibition of DA release in the NAc is modulated by GLYRs and atypical GABAA Rs on CINs containing δ- and Rho-subunits.
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Dopamina/metabolismo , Etanol/farmacología , Núcleo Accumbens/efectos de los fármacos , Receptores de GABA/efectos de los fármacos , Animales , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
Alcohol and nicotine are the two most widely used and misused drugs around the world, and co-consumption of both substances is highly prevalent. Multiple lines of evidence show a profound effect of sex in many aspects of alcohol and nicotine reward, with women having more difficulty quitting smoking and showing a faster progression toward developing alcohol use disorder compared with men. Both alcohol and nicotine require neuronal nicotinic acetylcholine receptors (nAChRs) to elicit rewarding effects within the mesolimbic system, representing a shared molecular pathway that likely contributes to the frequent comorbidity of alcohol and nicotine dependence. However, the majority of preclinical studies on the mechanisms of alcohol and nicotine reward behaviors utilize only male rodents, and thus our understanding of alcohol and nicotine neuropharmacology relies heavily on male data. As preclinical research informs the development and refinement of therapies to help patients reduce drug consumption, it is critical to understand the way biological sex and sex hormones influence the rewarding properties of alcohol and nicotine. In this review, we summarize what is known about sex differences in rodent models of alcohol and nicotine reward behaviors with a focus on neuronal nAChRs, highlighting exciting areas for future research. Additionally, we discuss the way circulating sex hormones may interact with neuronal nAChRs to influence reward-related behavior.
RESUMEN
Drugs of abuse engage overlapping but distinct molecular and cellular mechanisms to enhance dopamine (DA) signaling in the mesocorticolimbic circuitry. DA neurons of the ventral tegmental area (VTA) are key substrates of drugs of abuse and have been implicated in addiction-related behaviors. Enhanced VTA DA neurotransmission evoked by drugs of abuse can engage inhibitory G-protein-dependent feedback pathways, mediated by GABAB receptors (GABABRs) and D2 DA receptors (D2Rs). Chemogenetic inhibition of VTA DA neurons potently suppressed baseline motor activity, as well as the motor-stimulatory effect of cocaine and morphine, confirming the critical influence of VTA DA neurons and inhibitory G-protein signaling in these neurons on this addiction-related behavior. To resolve the relative influence of GABABR-dependent and D2R-dependent signaling pathways in VTA DA neurons on behavioral sensitivity to drugs of abuse, we developed a neuron-specific viral CRISPR/Cas9 approach to ablate D2R and GABABR in VTA DA neurons. Ablation of GABABR or D2R did not impact baseline physiological properties or excitability of VTA DA neurons, but it did preclude the direct somatodendritic inhibitory influence of GABABR or D2R activation. D2R ablation potentiated the motor-stimulatory effect of cocaine in male and female mice, whereas GABABR ablation selectively potentiated cocaine-induced activity in male subjects only. Neither D2R nor GABABR ablation impacted morphine-induced motor activity. Collectively, our data show that cocaine and morphine differ in the extent to which they engage inhibitory G-protein-dependent feedback pathways in VTA DA neurons and highlight key sex differences that may impact susceptibility to various facets of addiction.
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Cocaína , Área Tegmental Ventral , Animales , Cocaína/farmacología , Neuronas Dopaminérgicas , Femenino , Proteínas de Unión al GTP , Masculino , Ratones , Morfina/farmacologíaRESUMEN
The prevalence of alcohol use disorders (AUDs) has steadily increased in the United States over the last 30 years. Alcohol acts on multiple receptor systems including the nicotinic acetylcholine receptors (nAChRs), which are known to mediate alcohol consumption and reward. We previously reported that the preclinical drug sazetidine-A, a nAChR agonist and desensitizer, reduces alcohol consumption without affecting nicotine consumption in C57BL/6J mice. Here, we found that sazetidine-A enhances the expression of alcohol aversion without affecting the expression or acquisition of conditioned alcohol reward in C57BL/6J mice. Microinjection of sazetidine-A into the ventral midbrain targeting the ventral tegmental area (VTA) reduced binge alcohol consumption, implicating this region in mediating the effects of sazetidine-A. Furthermore, the sazetidine-A-induced reduction in alcohol consumption was mediated by non-α4 containing nAChRs, as sazetidine-A reduced binge alcohol consumption in both α4 knock-out and wild-type mice. Finally, we found that in mice pretreated with sazetidine-A, alcohol induced Fos transcript in Th-, but not Gad2-expressing neurons in the VTA as measured by increased Fos transcript expression. In summary, we find that sazetidine-A enhances the expression of alcohol aversion, which may underlie the reduction in alcohol consumption induced by sazetidine-A. Elucidating the identity of non-α4 nAChRs in alcohol aversion mechanisms will provide a better understanding the complex role of nAChRs in alcohol addiction and potentially reveal novel drug targets to treat AUDs.
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Disuasivos de Alcohol/farmacología , Alcoholismo/tratamiento farmacológico , Azetidinas/farmacología , Agonistas Nicotínicos/farmacología , Piridinas/farmacología , Animales , Consumo Excesivo de Bebidas Alcohólicas/tratamiento farmacológico , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-fos/efectos de los fármacos , Receptores Nicotínicos , Recompensa , Área Tegmental Ventral/efectos de los fármacosRESUMEN
Nicotine and alcohol are the most commonly abused substances worldwide and are frequently coabused. Nicotinic acetylcholine receptors (nAChRs) containing the α6 and ß3 subunits are expressed in neural reward circuits and are critical for nicotine and alcohol reward. nAChRs are dynamically regulated by signaling molecules such as protein kinase C epsilon (PKCε), which impact transcription of α6 and ß3 subunit mRNA (Chrna6 and Chrnb3, respectively). Previous work found decreased expression of Chrna6 and Chrnb3 transcripts in the ventral midbrain of male PKCε-/- mice, who also consume less nicotine and alcohol compared with wild-type (WT) littermates. Using RT-qPCR, we show that female PKCε-/- mice have higher expression of Chrna6 and Chrnb3 transcripts in the ventral midbrain, which functionally impacts nAChR-dependent behavior as female but not male PKCε-/- mice exhibit locomotor hypersensitivity to low-dose (0.25 mg/kg i.p.) nicotine. Female PKCε-/- mice show no differences in alcohol-induced sedation in the loss-of-righting reflex assay (4.0 g/kg i.p.) compared with WT littermates, whereas male PKCε-/- mice have enhanced sedation compared with WT mice. Female PKCε-/- mice also show reduced immobility time in response to varenicline (1.0 mg/kg i.p.) compared with WT littermates in the tail suspension test, and this effect was absent in male mice. Additionally, we found that female PKCε-/- mice show altered alcohol and nicotine consumption patterns in chronic voluntary two-bottle choice assays. Our data reveal a bidirectional effect of sex in the transcriptional regulation of nicotinic receptors by PKCε, highlighting the importance of studying both sexes in preclinical animal models.
Asunto(s)
Etanol/farmacología , Proteína Quinasa C-epsilon/metabolismo , Receptores Nicotínicos/metabolismo , Vareniclina/farmacología , Animales , Femenino , Masculino , Mesencéfalo/efectos de los fármacos , Mesencéfalo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Agonistas Nicotínicos/farmacología , Proteína Quinasa C-epsilon/genética , Subunidades de Proteína/metabolismo , Receptores Nicotínicos/genética , Reflejo de Enderezamiento/efectos de los fármacos , Recompensa , Factores Sexuales , Área Tegmental Ventral/efectos de los fármacosRESUMEN
RATIONALE: Alcohol and nicotine addiction are prevalent conditions that co-occur. Despite the prevalence of co-use, factors that influence the suppression and enhancement of concurrent alcohol and nicotine intake are largely unknown. OBJECTIVES: Our goals were to assess how nicotine abstinence and availability influenced concurrent alcohol consumption and to determine the impact of quinine adulteration of alcohol on aversion-resistant alcohol consumption and concurrent nicotine consumption. METHODS: Male and female C57BL/6J mice voluntarily consumed unsweetened alcohol, nicotine, and water in a chronic 3-bottle choice procedure. In experiment 1, nicotine access was removed for 1 week and re-introduced the following week, while the alcohol and water bottles remained available at all times. In experiment 2, quinine (100-1000 µM) was added to the 20% alcohol bottle, while the nicotine and water bottles remained unaltered. RESULTS: In experiment 1, we found that alcohol consumption and preference were unaffected by the presence or absence of nicotine access in both male and female mice. In experiment 2a, we found that quinine temporarily suppressed alcohol intake and enhanced concurrent nicotine, but not water, preference in both male and female mice. In experiment 2b, chronic quinine suppression of alcohol intake increased nicotine consumption and preference in female mice without affecting water preference, whereas it increased water and nicotine preference in male mice. CONCLUSIONS: Quinine suppression of alcohol consumption enhanced the preference for concurrent nicotine preference in male and female mice, suggesting that mice compensate for the quinine adulteration of alcohol by increasing their nicotine preference.
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Consumo de Bebidas Alcohólicas/psicología , Conducta de Elección/efectos de los fármacos , Etanol/administración & dosificación , Nicotina/administración & dosificación , Uso de Tabaco/psicología , Animales , Conducta de Elección/fisiología , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Quinina/administración & dosificación , AutoadministraciónRESUMEN
Late adolescence and young adulthood, corresponding to the high school and college years, are vulnerable periods for increased alcohol and nicotine use. The dramatic increase in the prevalence of electronic cigarette use is particularly concerning in these age groups. Late adolescents and young adults are more likely to engage in cycles of binge drug consumption, and alcohol and nicotine are frequently used together. However, there are few data examining the combination of alcohol and nicotine in binge models in animal models. In this study, our objectives were to determine how voluntary nicotine consumption beginning in late adolescence influenced subsequent binge alcohol consumption in young adulthood, how a combination of alcohol and nicotine binge consumption differed from alcohol-only binge consumption, and whether nicotine would be consumed when presented in a binge procedure. Male C57BL/6J mice voluntarily consumed unsweetened alcohol and nicotine in continuous-access bottle-choice procedures in combination with cycles of drinking-in-the-dark. Our results show that experience with voluntary nicotine consumption in late adolescence did not affect subsequent binge alcohol consumption in early adulthood. However, mice that consumed nicotine in adolescence showed an initial decrease in alcohol preference, and consequently increase in nicotine preference, on the first session of combined ethanol and nicotine binge consumption in adulthood compared with mice that drank only water during late adolescence. Lastly, we found that mice readily consumed unsweetened nicotine when presented in a binge procedure, and the level of consumption exceeded the nicotine consumption observed in the combination alcohol and nicotine binge. Our data show that expansion of the patterns of alcohol and nicotine co-consumption in a mouse models is possible, which will enable us to dissect relevant molecular targets underlying these consumption patterns and better inform drug development efforts.
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Consumo Excesivo de Bebidas Alcohólicas/psicología , Etanol/administración & dosificación , Nicotina/administración & dosificación , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , AutoadministraciónRESUMEN
Alcohol and nicotine addiction are frequently co-morbid. The nicotinic acetylcholine receptors (nAChRs) are critical for both alcohol and nicotine addiction mechanisms, since nAChR drugs that reduce nicotine consumption have been shown to also reduce alcohol consumption. Sazetidine-A, a pre-clinical nAChR drug with agonist and desensitizing effects at α4ß2 and α7 nAChRs, has been reported to reduce alcohol consumption and nicotine self-administration in rats when administered at high doses. However, this effect has not been replicated in mice. In this study, we examined the effect of sazetidine-A on alcohol and nicotine consumption in male and female mice utilizing voluntary oral consumption procedures previously developed in our lab. We found that sazetidine-A (1â¯mg/kg, i.p) reduced overnight alcohol consumption, but did not affect nicotine consumption when presented either alone or concurrently with alcohol. Sazetidine-A did not reduce water or saccharin consumption at any dose tested. In a chronic co-consumption experiment in which either alcohol or nicotine was re-introduced after one week of forced abstinence, sazetidine-A attenuated post-abstinence consumption of alcohol but not nicotine. Sazetidine-A also significantly reduced alcohol consumption in an acute, binge drinking-in-the-dark procedure. Finally, we tested the effect of sazetidine-A on alcohol withdrawal, and found that sazetidine-A significantly reduced handling-induced convulsions during alcohol withdrawal. Collectively, these data suggest a novel role for the nAChR targets of sazetidine-A in specifically mediating alcohol consumption, separate from the involvement of nAChRs in mediating nicotine consumption. Delineation of this pathway may provide insight into novel therapies for the treatment of alcohol use disorders.
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Consumo de Bebidas Alcohólicas/tratamiento farmacológico , Azetidinas/uso terapéutico , Agonistas Nicotínicos/uso terapéutico , Piridinas/uso terapéutico , Tabaquismo/tratamiento farmacológico , Animales , Conducta de Elección/efectos de los fármacos , Condicionamiento Operante/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Etanol/farmacología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Nicotina/farmacología , Autoadministración , Síndrome de Abstinencia a Sustancias/tratamiento farmacológico , Factores de TiempoRESUMEN
BACKGROUND: Despite the high cost and widespread prevalence of alcohol use disorders, treatment options are limited, underscoring the need for new, effective medications. Previous results using protein kinase C epsilon (PKCε) knockout mice, RNA interference against PKCε, and peptide inhibitors of PKCε predict that small-molecule inhibitors of PKCε should reduce alcohol consumption in humans. METHODS: We designed a new class of PKCε inhibitors based on the Rho-associated protein kinase (ROCK) inhibitor Y-27632. In vitro kinase and binding assays were used to identify the most potent compounds. Their effects on ethanol-stimulated synaptic transmission; ethanol, sucrose, and quinine consumption; ethanol-induced loss of righting; and ethanol clearance were studied in mice. RESULTS: We identified two compounds that inhibited PKCε with Ki <20 nM, showed selectivity for PKCε over other kinases, crossed the blood-brain barrier, achieved effective concentrations in mouse brain, prevented ethanol-stimulated gamma-aminobutyric acid release in the central amygdala, and reduced ethanol consumption when administered intraperitoneally at 40 mg/kg in wild-type but not in Prkce-/- mice. One compound also reduced sucrose and saccharin consumption, while the other was selective for ethanol. Both transiently impaired locomotion through an off-target effect that did not interfere with their ability to reduce ethanol intake. One compound prolonged recovery from ethanol-induced loss of righting but this was also due to an off-target effect since it was present in Prkce-/- mice. Neither altered ethanol clearance. CONCLUSIONS: These results identify lead compounds for development of PKCε inhibitors that reduce alcohol consumption.
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Consumo de Bebidas Alcohólicas/tratamiento farmacológico , Núcleo Amigdalino Central/enzimología , Proteína Quinasa C-epsilon/genética , Inhibidores de Proteínas Quinasas/farmacología , Transmisión Sináptica/efectos de los fármacos , Alcoholismo/enzimología , Alcoholismo/fisiopatología , Amidas/farmacocinética , Amidas/farmacología , Animales , Núcleo Amigdalino Central/efectos de los fármacos , Núcleo Amigdalino Central/fisiopatología , Depresores del Sistema Nervioso Central/farmacología , Condicionamiento Psicológico , Modelos Animales de Enfermedad , Etanol , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibidores de Proteínas Quinasas/farmacocinética , Piridinas/farmacocinética , Piridinas/farmacologíaRESUMEN
Alcohol and nicotine are often used together, and there is a high rate of co-occurrence between alcohol and nicotine addiction. Most animal models studying alcohol and nicotine interactions have utilized passive drug administration, which may not be relevant to human co-addiction. In addition, the interactions between alcohol and nicotine in female animals have been understudied, as most studies have used male animals. To address these issues, we developed models of alcohol and nicotine co-consumption in male and female mice that utilized voluntary, oral consumption of unsweetened alcohol, nicotine and water. We first examined drug consumption and preference in single-drug, sequential alcohol and nicotine consumption tests in male and female C57BL/6 and DBA/2J mice. We then tested chronic continuous and intermittent access alcohol and nicotine co-consumption procedures. We found that male and female C57BL/6 mice readily co-consumed unsweetened alcohol and nicotine. In our continuous co-consumption procedures, we found that varying the available nicotine concentration during an alcohol abstinence period affected compensatory nicotine consumption during alcohol abstinence, and affected rebound alcohol consumption when alcohol was re-introduced. Consumption of alcohol and nicotine in an intermittent co-consumption procedure produced higher alcohol consumption levels, but not nicotine consumption levels, compared with the continuous co-consumption procedures. Finally, we found that intermittent alcohol and nicotine co-consumption resulted in physical dependence. Our data show that these voluntary co-consumption procedures can be easily performed in mice and can be used to study behavioral interactions between alcohol and nicotine consumption, which may better model human alcohol and nicotine co-addiction.
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Abstinencia de Alcohol/psicología , Consumo de Bebidas Alcohólicas/psicología , Etanol/administración & dosificación , Nicotina/administración & dosificación , Síndrome de Abstinencia a Sustancias/psicología , Trastornos Relacionados con Sustancias/psicología , Consumo de Bebidas Alcohólicas/genética , Consumo de Bebidas Alcohólicas/tendencias , Animales , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Especificidad de la Especie , Síndrome de Abstinencia a Sustancias/genética , Trastornos Relacionados con Sustancias/genéticaRESUMEN
BACKGROUND: Prkcz has been identified as a gene whose expression is positively correlated with ethanol (EtOH) consumption in mice and is also induced by EtOH. Two proteins are produced from Prkcz: protein kinase M zeta (PKMζ), which is expressed in the nervous system and protein kinase C zeta (PKCζ), which is expressed in other tissues. We examined Prkcz(-/-) mice that lack PKCζ and PKMζ to investigate the role of this gene in behavioral responses to EtOH. METHODS: Male Prkcz(-/-) and wild-type littermates were tested for EtOH consumption using 4 procedures: 24-hour intermittent access, 4-hour limited intermittent access, 4-day drinking-in-the-dark, and 24-hour continuous access. We also assessed the acute hypnotic effect of EtOH, EtOH reward, and taste preference for sweet-, bitter-, salty-, and umami-flavored solutions. Finally, we determined whether EtOH could increase PKMζ and PKCζ transcripts and protein expression in wild-type mice using quantitative PCR and Western blot analysis. RESULTS: Prkcz(-/-) mice consumed more EtOH than their wild-type littermates in both intermittent access procedures, but not in the drinking-in-the-dark or 24-hour continuous access procedures. EtOH exposure increased Prkcz transcripts in cultured PC12 cells, and intermittent EtOH consumption increased PKMζ protein in the ventral striatum of wild-type mice. CONCLUSIONS: Absence of PKMζ in the brain is associated with increased EtOH intake during procedures that incorporate intermittent consumption sessions every other day. Our data suggest that EtOH induces PKMζ, which acts in a negative feedback loop to limit binge-like EtOH consumption.
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Consumo de Bebidas Alcohólicas/metabolismo , Etanol/administración & dosificación , Eliminación de Gen , Proteína Quinasa C/deficiencia , Regulación hacia Arriba/fisiología , Consumo de Bebidas Alcohólicas/genética , Consumo de Bebidas Alcohólicas/prevención & control , Animales , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células PC12 , Proteína Quinasa C/genética , Ratas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Protein kinase M-ζ (PKM-ζ) is a constitutively active form of atypical protein kinase C that is exclusively expressed in the brain and implicated in the maintenance of long-term memory. Most studies that support a role for PKM-ζ in memory maintenance have used pharmacological PKM-ζ inhibitors such as the myristoylated zeta inhibitory peptide (ZIP) or chelerythrine. Here we use a genetic approach and target exon 9 of the Prkcz gene to generate mice that lack both protein kinase C-ζ (PKC-ζ) and PKM-ζ (Prkcz(-/-) mice). Prkcz(-/-) mice showed normal behaviour in a cage environment and in baseline tests of motor function and sensory perception, but displayed reduced anxiety-like behaviour. Notably, Prkcz(-/-) mice did not show deficits in learning or memory in tests of cued fear conditioning, novel object recognition, object location recognition, conditioned place preference for cocaine, or motor learning, when compared with wild-type littermates. ZIP injection into the nucleus accumbens reduced expression of cocaine-conditioned place preference in Prkcz(-/-) mice. In vitro, ZIP and scrambled ZIP inhibited PKM-ζ, PKC-ι and PKC-ζ with similar inhibition constant (K(i)) values. Chelerythrine was a weak inhibitor of PKM-ζ (K(i) = 76 µM). Our findings show that absence of PKM-ζ does not impair learning and memory in mice, and that ZIP can erase reward memory even when PKM-ζ is not present.
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Eliminación de Gen , Memoria/fisiología , Proteína Quinasa C/deficiencia , Proteína Quinasa C/genética , Animales , Ansiedad/genética , Conducta Animal , Benzofenantridinas/farmacología , Cocaína , Condicionamiento Clásico , Señales (Psicología) , Exones/genética , Miedo , Femenino , Masculino , Ratones , Proteína Quinasa C/análisis , Proteína Quinasa C/inmunologíaRESUMEN
Nicotine addiction and alcohol use disorders are very widespread and often occur together. Currently, there is no single drug approved for the simultaneous treatment of both conditions. Although these conditions share common genetic factors, the molecular mechanisms underlying their comorbidity are unknown. We have previously shown that mice lacking protein kinase C epsilon (PKCε) show decreased ethanol self-administration and reward as well as increased aversion to ethanol. Here we find that Prkce(-/-) mice self-administer less nicotine and show decreased conditioned place preference for nicotine compared with wild-type mice. In Prkce(-/-) mice, these behaviors are associated with reduced levels of α(6) and ß(3) nicotinic receptor subunit mRNA in the ventral midbrain and striatum as well as a functional deficit in cholinergic modulation of dopamine release in nucleus accumbens. Our results indicate that PKCε regulates reward signaling through α(6)-containing nicotinic receptors and suggest that PKCε could be a target for the treatment of comorbid nicotine and alcohol addictions.
Asunto(s)
Dopamina/metabolismo , Nicotina/metabolismo , Núcleo Accumbens/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Animales , Condicionamiento Clásico/fisiología , Dopamina/administración & dosificación , Dopaminérgicos/administración & dosificación , Dopaminérgicos/metabolismo , Femenino , Estimulantes Ganglionares/administración & dosificación , Estimulantes Ganglionares/metabolismo , Expresión Génica , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Nicotina/administración & dosificación , Proteína Quinasa C-epsilon/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Nicotínicos/genética , Recompensa , Autoadministración , Transducción de Señal , Percepción Espacial/fisiologíaRESUMEN
Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharmacotherapies to treat drug addiction.
Asunto(s)
Proteínas del Tejido Nervioso/fisiología , Proteínas Quinasas/fisiología , Trastornos Relacionados con Sustancias/enzimología , Animales , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Estimulantes del Sistema Nervioso Central/farmacología , Condicionamiento Clásico/efectos de los fármacos , Tolerancia a Medicamentos , Etanol/farmacología , Humanos , Drogas Ilícitas/farmacología , Memoria/efectos de los fármacos , Ratones , Ratones Noqueados , Plasticidad Neuronal/fisiología , Neurotransmisores/fisiología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/fisiología , Recompensa , Transducción de Señal/fisiología , Síndrome de Abstinencia a Sustancias/enzimología , Síndrome de Abstinencia a Sustancias/fisiopatología , Trastornos Relacionados con Sustancias/tratamiento farmacológicoAsunto(s)
Hidrocarburo de Aril Hidroxilasas/genética , Nicotina/administración & dosificación , Nicotina/metabolismo , Agonistas Nicotínicos/sangre , Agonistas Nicotínicos/metabolismo , Oxidorreductasas N-Desmetilantes/genética , Cese del Hábito de Fumar , Administración Cutánea , Administración por Inhalación , Cotinina/análogos & derivados , Cotinina/sangre , Citocromo P-450 CYP2B6 , Genotipo , Humanos , Desequilibrio de Ligamiento , Nicotina/sangre , Cese del Hábito de Fumar/métodosRESUMEN
BACKGROUND: CYP2B6 is the primary enzyme involved in bupropion (Zyban; GlaxoSmithKline, Research Triangle Park, North Carolina) metabolism. Genetic polymorphisms in CYP2B6, such as CYP2B6*6, can alter bupropion metabolism and may affect bupropion treatment outcome. METHODS: Subjects participated in a smoking cessation clinical trial of bupropion versus placebo. The main outcome was a 7-day point prevalence abstinence rate measured 10 weeks after the start of treatment (i.e., end of treatment) and at the 6-month follow-up; secondary outcomes were severity of adverse effects, withdrawal, and urge to smoke. Subjects were haplotyped for the CYP2B6*6 variants. RESULTS: Among smokers in the CYP2B6*6 group (CYP2B6*1/*6 or CYP2B6*6/*6 genotype, n = 147, 45% of the population), bupropion produced significantly higher abstinence rates than placebo at the end of treatment (32.5% vs. 14.3%, p = .01) and at the 6-month follow-up (31.2% vs. 12.9%, p = .008). In contrast, bupropion was no more effective than placebo for smokers in the CYP2B6*1 group (CYP2B6*1/*1, n = 179) at the end of treatment (31.0% vs. 31.6%, p = .93) or at the 6-month follow-up (22.0% vs. 21.5%, p = .94). There was a significant genotype by treatment interaction at the end of treatment (odds ratio [OR] = 2.97, confidence interval [CI] = 1.05-8.40, p = .04), which was similar at 6-month follow-up (OR = 2.98, CI = .98-9.06, p = .05). CONCLUSIONS: These data suggest that smokers with the CYP2B6*6 genotype have a higher liability to relapse on placebo and that they may be good candidates for bupropion treatment for smoking cessation.