RESUMO
Chronic alcohol exposure results in widespread dysregulation of gene expression that contributes to the pathogenesis of Alcohol Use Disorder (AUD). Long noncoding RNAs are key regulators of the transcriptome that we hypothesize coordinate alcohol-induced transcriptome dysregulation and contribute to AUD. Based on RNA-Sequencing data of human prefrontal cortex, basolateral amygdala and nucleus accumbens of AUD versus non-AUD brain, the human LINC01265 and its predicted murine homolog Gm41261 (i.e., TX2) were selected for functional interrogation. We tested the hypothesis that TX2 contributes to ethanol drinking and behavioral responses to ethanol. CRISPR/Cas9 mutagenesis was used to create a TX2 mutant mouse line in which 306 base-pairs were deleted from the locus. RNA analysis revealed that an abnormal TX2 transcript was produced at an unchanged level in mutant animals. Behaviorally, mutant mice had reduced ethanol, gaboxadol and zolpidem-induced loss of the righting response and reduced tolerance to ethanol in both sexes. In addition, a male-specific reduction in two-bottle choice every-other-day ethanol drinking was observed. Male TX2 mutants exhibited evidence of enhanced GABA release and altered GABAA receptor subunit composition in neurons of the nucleus accumbens shell. In C57BL6/J mice, TX2 within the cortex was cytoplasmic and largely present in Rbfox3+ neurons and IBA1+ microglia, but not in Olig2+ oligodendrocytes or in the majority of GFAP+ astrocytes. These data support the hypothesis that TX2 mutagenesis and dysregulation impacts ethanol drinking behavior and ethanol-induced behavioral responses in mice, likely through alterations in the GABAergic system.
Assuntos
Alcoolismo , RNA Longo não Codificante , Humanos , Feminino , Camundongos , Masculino , Animais , Etanol/toxicidade , RNA Longo não Codificante/genética , Alcoolismo/genética , Consumo de Bebidas Alcoólicas/genética , Receptores de GABA-A/genética , Mutação , Camundongos Endogâmicos C57BLRESUMO
Neuroimmune pathways regulate brain function to influence complex behavior and play a role in several neuropsychiatric diseases, including alcohol use disorder (AUD). In particular, the interleukin-1 (IL-1) system has emerged as a key regulator of the brain's response to ethanol (alcohol). Here we investigated the mechanisms underlying ethanol-induced neuroadaptation of IL-1ß signaling at GABAergic synapses in the prelimbic region of the medial prefrontal cortex (mPFC), an area responsible for integrating contextual information to mediate conflicting motivational drives. We exposed C57BL/6J male mice to the chronic intermittent ethanol vapor-2 bottle choice paradigm (CIE-2BC) to induce ethanol dependence, and conducted ex vivo electrophysiology and molecular analyses. We found that the IL-1 system regulates basal mPFC function through its actions at inhibitory synapses on prelimbic layer 2/3 pyramidal neurons. IL-1ß can selectively recruit either neuroprotective (PI3K/Akt) or pro-inflammatory (MyD88/p38 MAPK) mechanisms to produce opposing synaptic effects. In ethanol naïve conditions, there was a strong PI3K/Akt bias leading to a disinhibition of pyramidal neurons. Ethanol dependence produced opposite IL-1 effects - enhanced local inhibition via a switch in IL-1ß signaling to the canonical pro-inflammatory MyD88 pathway. Ethanol dependence also increased cellular IL-1ß in the mPFC, while decreasing expression of downstream effectors (Akt, p38 MAPK). Thus, IL-1ß may represent a key neural substrate in ethanol-induced cortical dysfunction. As the IL-1 receptor antagonist (kineret) is already FDA-approved for other diseases, this work underscores the high therapeutic potential of IL-1 signaling/neuroimmune-based treatments for AUD.
Assuntos
Alcoolismo , Etanol , Camundongos , Masculino , Animais , Etanol/farmacologia , Interleucina-1beta/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Camundongos Endogâmicos C57BL , Córtex Pré-Frontal/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Astrocytes are fundamental building blocks of the central nervous system. Their dysfunction has been implicated in many psychiatric disorders, including alcohol use disorder, yet our understanding of their functional role in ethanol intoxication and consumption is very limited. Astrocytes regulate behavior through multiple intracellular signaling pathways, including G-protein coupled-receptor (GPCR)-mediated calcium signals. To test the hypothesis that GPCR-induced calcium signaling is also involved in the behavioral effects of ethanol, we expressed astrocyte-specific excitatory DREADDs in the prefrontal cortex (PFC) of mice. Activating Gq-GPCR signaling in PFC astrocytes increased drinking in ethanol-naïve mice, but not in mice with a history of ethanol drinking. In contrast, reducing calcium signaling with an astrocyte-specific calcium extruder reduced ethanol intake. Cortical astrocyte calcium signaling also altered the acute stimulatory and sedative-hypnotic effects of ethanol. Astrocyte-specific Gq-DREADD activation increased both the locomotor-activating effects of low dose ethanol and the sedative-hypnotic effects of a high dose, while reduced astrocyte calcium signaling diminished sensitivity to the hypnotic effects. In addition, we found that adenosine A1 receptors were required for astrocyte calcium activation to increase ethanol sedation. These results support integral roles for PFC astrocytes in the behavioral actions of ethanol that are due, at least in part, to adenosine receptor activation.
Assuntos
Alcoolismo , Astrócitos , Consumo de Bebidas Alcoólicas , Animais , Sinalização do Cálcio , Etanol/toxicidade , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Overactivation of neuroimmune signaling has been linked to excessive ethanol consumption. Toll-like receptors (TLRs) are a major component of innate immune signaling and initiate anti- and pro-inflammatory responses via intracellular signal transduction cascades. TLR7 is upregulated in post-mortem brain tissue from humans with alcohol use disorder (AUD) and animals with prior exposure to ethanol. Despite this evidence, the role of TLR7 in the regulation of voluntary ethanol consumption has not been studied. We test the hypothesis that TLR7 activation regulates voluntary ethanol drinking behavior by administering a TLR7 agonist (R848) during an intermittent access drinking procedure in mice. Acute activation of TLR7 reduced ethanol intake, preference, and total fluid intake due, at least in part, to an acute sickness response. However, chronic pre-treatment with R848 resulted in tolerance to the adverse effects of the drug and a subsequent increase in ethanol consumption. To determine the molecular machinery that mediates these behavioral changes, we evaluated gene expression after acute and chronic TLR7 activation. We found that acute TLR7 activation produces brain region specific changes in expression of immune pathway genes, whereas chronic TLR7 activation causes downregulation of TLRs and blunted cytokine induction, suggesting molecular tolerance. Our results demonstrate a novel role for TLR7 signaling in regulating voluntary ethanol consumption. Taken together, our findings suggest TLR7 may be a viable target for development of therapies to treat AUD.
Assuntos
Alcoolismo , Receptor 7 Toll-Like , Consumo de Bebidas Alcoólicas , Animais , Etanol , Camundongos , Camundongos Endogâmicos C57BL , Receptores Toll-LikeRESUMO
In this chapter, we review the effects of global null mutant and overexpressing transgenic mouse lines on voluntary self-administration of alcohol. We examine approximately 200 publications pertaining to the effects of 155 mouse genes on alcohol consumption in different drinking models. The targeted genes vary in function and include neurotransmitter, ion channel, neuroimmune, and neuropeptide signaling systems. The alcohol self-administration models include operant conditioning, two- and four-bottle choice continuous and intermittent access, drinking in the dark limited access, chronic intermittent ethanol, and scheduled high alcohol consumption tests. Comparisons of different drinking models using the same mutant mice are potentially the most informative, and we will highlight those examples. More mutants have been tested for continuous two-bottle choice consumption than any other test; of the 137 mouse genes examined using this model, 97 (72%) altered drinking in at least one sex. Overall, the effects of genetic manipulations on alcohol drinking often depend on the sex of the mice, alcohol concentration and time of access, genetic background, as well as the drinking test.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Alcoolismo/genética , Modelos Animais de Doenças , Etanol/administração & dosagem , Animais , Comportamento de Escolha/efeitos dos fármacos , Comportamento de Escolha/fisiologia , Condicionamento Operante/efeitos dos fármacos , Condicionamento Operante/fisiologia , Camundongos , Camundongos Transgênicos , Autoadministração , Caracteres SexuaisRESUMO
Alcoholism is associated with dysregulation in the neural circuitry that mediates motivated and goal-directed behaviors. The dopaminergic (DA) connection between the ventral tegmental area (VTA) and the nucleus accumbens is viewed as a critical component of the neurocircuitry mediating alcohol's rewarding and behavioral effects. We sought to determine the effects of binge alcohol drinking on global gene expression in VTA DA neurons. Alcohol-preferring C57BL/6J × FVB/NJ F1 hybrid female mice were exposed to a modified drinking in the dark (DID) procedure for 3 weeks, while control animals had access to water only. Global gene expression of laser-captured tyrosine hydroxylase (TH)-positive VTA DA neurons was measured using microarrays. A total of 644 transcripts were differentially expressed between the drinking and nondrinking mice, and 930 transcripts correlated with alcohol intake during the last 2 days of drinking in the alcohol group. Bioinformatics analysis of alcohol-responsive genes identified molecular pathways and networks perturbed in DA neurons by alcohol consumption, which included neuroimmune and epigenetic functions, alcohol metabolism and brain disorders. The majority of genes with high and specific expression in DA neurons were downregulated by or negatively correlated with alcohol consumption, suggesting a decreased activity of DA neurons in high drinking animals. These changes in the DA transcriptome provide a foundation for alcohol-induced neuroadaptations that may play a crucial role in the transition to addiction.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Neurônios Dopaminérgicos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Área Tegmentar Ventral/metabolismo , Consumo de Bebidas Alcoólicas/efeitos adversos , Animais , Comportamento Aditivo/genética , Dopamina/metabolismo , Etanol/farmacologia , Feminino , Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Núcleo Accumbens/efeitos dos fármacosRESUMO
Chronic alcohol consumption changes gene expression, likely causing persistent remodeling of synaptic structures via altered translation of mRNAs within synaptic compartments of the cell. We profiled the transcriptome from synaptoneurosomes (SNs) and paired total homogenates (THs) from mouse amygdala following chronic voluntary alcohol consumption. In SN, both the number of alcohol-responsive mRNAs and the magnitude of fold-change were greater than in THs, including many GABA-related mRNAs upregulated in SNs. Furthermore, SN gene co-expression analysis revealed a highly connected network, demonstrating coordinated patterns of gene expression and highlighting alcohol-responsive biological pathways, such as long-term potentiation, long-term depression, glutamate signaling, RNA processing and upregulation of alcohol-responsive genes within neuroimmune modules. Alterations in these pathways have also been observed in the amygdala of human alcoholics. SNs offer an ideal model for detecting intricate networks of coordinated synaptic gene expression and may provide a unique system for investigating therapeutic targets for the treatment of alcoholism.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Etanol/efeitos adversos , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Transcriptoma/genética , Alcoolismo/genética , Alcoolismo/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Animais , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcriptoma/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismoRESUMO
The IL-1 receptor antagonist (IL-1ra), encoded by the Il1rn gene, is an endogenous antagonist of the IL-1 receptor. Studies of Il1rn knockout (KO) and wild type (WT) mice identified differences in several ethanol-related behaviors, some of which may be mediated by GABAergic transmission in the central nucleus of the amygdala (CeA). In this study we examined phasic (both evoked and spontaneous) and tonic GABAergic transmission in the CeA of Il1rn KO and WT mice and the ethanol sensitivity of these GABAergic synapses. The mean amplitude of baseline evoked GABAA-inhibitory postsynaptic potentials (IPSPs), and the baseline frequency of spontaneous GABAA-inhibitory postsynaptic currents (sIPSCs), but not the frequency of miniature GABAA-IPSCs (mIPSCs), were significantly increased in KO compared to WT mice, indicating enhanced presynaptic action potential-dependent GABA release in the CeA of KO mice. In KO mice, we also found a cell-type specific switch in the ongoing tonic GABAA receptor conductance such that the tonic conductance in low threshold bursting (LTB) neurons is lost and a tonic conductance in late spiking (LS) neurons appears. Notably, the ethanol-induced facilitation of evoked and spontaneous GABA release was lost in most of the CeA neurons from KO compared to WT mice. Ethanol superfusion increased the sIPSC rise and decay times in both KO and WT mice, suggesting ethanol-induced postsynaptic effects. The pretreatment of CeA slices with exogenous IL-1ra (Kineret; 100ng/ml) returned sIPSC frequency in KO mice to the levels found in WT. Importantly, Kineret also restored ethanol-induced potentiation of the sIPSC frequency in the KO mice. These results show that IL-1ra regulates baseline GABAergic transmission in the CeA and is critical for the ethanol effects at these synapses.
Assuntos
Tonsila do Cerebelo/metabolismo , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Neurônios GABAérgicos/metabolismo , Potenciais Pós-Sinápticos Inibidores/genética , Proteína Antagonista do Receptor de Interleucina 1/genética , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Neurônios GABAérgicos/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Camundongos , Camundongos Knockout , Técnicas de Patch-ClampRESUMO
GABA type A receptors (GABA(A)-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABA(A)-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011). All together, they indicate that aversive property of ethanol is dependent on ethanol action on α2-containing GABA(A)-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABA(A)-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 (-/-) and α3 (-/Y) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism.
Assuntos
Intoxicação Alcoólica/genética , Aprendizagem da Esquiva/fisiologia , Receptores de GABA-A/genética , Recuperação de Função Fisiológica/efeitos dos fármacos , Paladar/genética , Doença Aguda , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Etanol/administração & dosagem , Etanol/toxicidade , Ligação Genética/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Recuperação de Função Fisiológica/genética , Paladar/efeitos dos fármacosRESUMO
Behavioral studies on genetically diverse mice have proven powerful for determining relationships between phenotypes and have been widely used in alcohol research. Most of these studies rely on naturally occurring genetic polymorphisms among inbred strains and selected lines. Another approach is to introduce variation by engineering single-gene mutations in mice. We have tested 37 different mutant mice and their wild-type controls for a variety (31) of behaviors and have mined this data set by K-means clustering and analysis of correlations. We found a correlation between a stress-related response (activity in a novel environment) and alcohol consumption and preference for saccharin. We confirmed several relationships detected in earlier genetic studies, including positive correlation of alcohol consumption with saccharin consumption and negative correlations with conditioned taste aversion and alcohol withdrawal severity. Introduction of single-gene mutations either eliminated or greatly diminished these correlations. The three tests of alcohol consumption used (continuous two-bottle choice and two limited access tests: drinking in the dark and sustained high alcohol consumption) share a relationship with saccharin consumption, but differ from each other in their correlation networks. We suggest that alcohol consumption is controlled by multiple physiological systems where single-gene mutations can disrupt the networks of such systems.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Comportamento Animal/fisiologia , Comportamento de Escolha/fisiologia , Etanol/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento de Escolha/efeitos dos fármacos , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Paladar/genéticaRESUMO
Previous studies showed that mice with genetic predisposition for high alcohol consumption as well as human alcoholics show changes in brain expression of genes related to immune signaling. In addition, mutant mice lacking genes related to immune function show decreased alcohol consumption (Blednov et al., 2011), suggesting that immune signaling promotes alcohol consumption. To test the possibility that activation of immune signaling will increase alcohol consumption, we treated mice with lipopolysaccaride (LPS; 1mg/kg, i.p.) and tested alcohol consumption in the continuous two-bottle choice test. To take advantage of the long-lasting activation of brain immune signaling by LPS, we measured drinking beginning one week or one month after LPS treatment and continued the studies for several months. LPS produced persistent increases in alcohol consumption in C57BL/6J (B6) inbred mice, FVBxB6F1 and B6xNZBF1 hybrid mice, but not in FVB inbred mice. To determine if this effect of LPS is mediated through binding to TLR4, we tested mice lacking CD14, a key component of TLR4 signaling. These null mutants showed no increase of alcohol intake after treatment with LPS. LPS treatment decreased ethanol-conditioned taste aversion but did not alter ethanol-conditioned place preference (B6xNZBF1 mice). Electrophysiological studies of dopamine neurons in the ventral tegmental area showed that pretreatment of mice with LPS decreased the neuronal firing rate. These results suggest that activation of immune signaling promotes alcohol consumption and alters certain aspects of alcohol reward/aversion.
Assuntos
Consumo de Bebidas Alcoólicas/imunologia , Comportamento de Escolha/efeitos dos fármacos , Condicionamento Psicológico/efeitos dos fármacos , Etanol/administração & dosagem , Lipopolissacarídeos/farmacologia , Neurônios/efeitos dos fármacos , Análise de Variância , Animais , Comportamento de Escolha/fisiologia , Condicionamento Psicológico/fisiologia , Eletrofisiologia , Camundongos , Neurônios/imunologia , Autoadministração , Especificidade da EspécieRESUMO
Knock-in mice were constructed with mutations in the α1 (H(270), A(277)) and α2 (H(270), A(277)) subunits of the GABAA receptor, which resulted in receptors that lacked modulation by ethanol but retained normal responses to GABA in vitro. A key question is whether these mutant receptors also function normally in vivo. Perturbation of brain function was evaluated by gene expression profiling in the cerebral cortex and by behavioral pharmacology experiments with GABAergic drugs. Analysis of individual transcripts found only six transcripts that were changed in α1 knock-in mice and three in the α2 mutants (p<0.05, corrected for multiple comparisons). Two transcripts that are sensitive to neuronal activity, Arc and Fos, increased about 250% in the α2 mutants, and about 50% in the α1 mutants. Behavioral effects (loss of righting reflex, rotarod) of flurazepam and pentobarbital were not different between α2 mutants and wild-type, but they were enhanced for α1 knock-in mice. These results indicate that introduction of these mutations in the α2 subunit of the GABAA receptor does not produce marked perturbation of brain function, as measured by gene expression and GABAergic behavioral responses, but the same mutations in the α1 subunit produce more pronounced changes, especially in GABAergic function.
Assuntos
Comportamento Animal/fisiologia , Regulação da Expressão Gênica/genética , Mutação/genética , Receptores de GABA-A , Animais , Comportamento Animal/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Flurazepam/farmacologia , Flurazepam/uso terapêutico , Moduladores GABAérgicos/farmacologia , Moduladores GABAérgicos/uso terapêutico , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Transtornos dos Movimentos/tratamento farmacológico , Transtornos dos Movimentos/genética , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Pentobarbital/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismoRESUMO
GABA type A receptors (GABA(A)-Rs) are potential targets of ethanol. However, there are multiple subtypes of this receptor, and, thus far, individual subunits have not been definitively linked with specific ethanol behavioral actions. Interestingly, though, a chromosomal cluster of four GABA(A)-R subunit genes, including α2 (Gabra2), was associated with human alcoholism (Am J Hum Genet 74:705-714, 2004; Pharmacol Biochem Behav 90:95-104, 2008; J Psychiatr Res 42:184-191, 2008). The goal of our study was to determine the role of receptors containing this subunit in alcohol action. We designed an α2 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity in a recombinant expression system. Knockin mice containing this mutant subunit were tested in a range of ethanol behavioral tests. These mutant mice did not develop the typical conditioned taste aversion in response to ethanol and showed complete loss of the motor stimulant effects of ethanol. Conversely, they also demonstrated changes in ethanol intake and preference in multiple tests. The knockin mice showed increased ethanol-induced hypnosis but no difference in anxiolytic effects or recovery from acute ethanol-induced motor incoordination. Overall, these studies demonstrate that the effects of ethanol at GABAergic synapses containing the α2 subunit are important for specific behavioral effects of ethanol that may be relevant to the genetic linkage of this subunit with human alcoholism.
Assuntos
Aprendizagem da Esquiva/fisiologia , Condicionamento Psicológico/fisiologia , Etanol/administração & dosagem , Atividade Motora/genética , Receptores de GABA-A/genética , Paladar/genética , Consumo de Bebidas Alcoólicas/genética , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Condicionamento Psicológico/efeitos dos fármacos , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Receptores de GABA-A/fisiologia , Paladar/efeitos dos fármacos , Xenopus laevisRESUMO
C57BL/6J x FVB/NJ F1 (B6 x FVB) mice consume more alcohol than C57BL/6J x NZB/B1NJ F1 (B6 x NZB) mice and this high alcohol consumption is stable after abstinence whereas B6 x NZB show reduced consumption, thus providing models of Sustained Alcohol Preference (SAP) and Reduced Alcohol Preference (RAP). In female hybrids, we assessed several behavioral responses to define behaviors which might predict SAP and RAP. B6 x FVB exhibited less severe ethanol-induced conditioned taste aversion and were less sensitive to ethanol-induced loss of righting reflex than B6 x NZB. Both hybrids demonstrated ethanol-induced place preference and a low ethanol withdrawal severity. We found that these hybrids differ in their sensitivity to the aversive and sedative, but not rewarding, effects of ethanol. Results of elevated plus maze, mirror chamber, and locomotor tests reveal B6 x FVB mice are less anxious and more active than B6 x NZB mice. Results obtained offer insights about factors that determine SAP and RAP in these new genetic models of alcohol consumption.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Comportamento Animal/efeitos dos fármacos , Condicionamento Operante/efeitos dos fármacos , Reflexo/efeitos dos fármacos , Animais , Cruzamentos Genéticos , Etanol/administração & dosagem , Feminino , Heterozigoto , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NZB , Camundongos Endogâmicos , Atividade Motora , Autoadministração , Especificidade da EspécieRESUMO
We showed that F1 hybrid genotypes may provide a broader variety of ethanol drinking phenotypes than the inbred progenitor strains used to create the hybrids (Blednov et al. in Alcohol Clin Exp Res 29:1949-1958, 2005). To extend this work, we characterized alcohol consumption as well as intake of other tastants (saccharin, quinine and sodium chloride) in five inbred strains of mice (FVB, SJL, B6, BUB, NZB) and in their reciprocal F1 hybrids with B6 (FVBxB6; B6xFVB; NZBxB6; B6xNZB; BUBxB6; B6xBUB; SJLxB6; B6xSJL). We also compared ethanol intake in these mice for several concentrations before and after two periods of abstinence. F1 hybrid mice derived from the crosses of B6 and FVB and also B6 and SJL drank higher levels of ethanol than their progenitor strains, demonstrating overdominance for two-bottle choice drinking test. The B6 and NZB hybrid showed additivity in two-bottle choice drinking, whereas the hybrid of B6 and BUB demonstrated full or complete dominance. Genealogical origin, as well as non-alcohol taste preferences (sodium chloride), predicted ethanol consumption. Mice derived from the crosses of B6 and FVB showed high sustained alcohol preference and the B6 and NZB hybrids showed reduced alcohol preference after periods of abstinence. These new genetic models offer some advantages over inbred strains because they provide high, sustained, alcohol intake, and should allow mapping of loci important for the genetic architecture of these traits.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Alcoolismo/genética , Etanol/farmacologia , Animais , Cruzamentos Genéticos , Genes Dominantes , Variação Genética , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Modelos Genéticos , Fenótipo , Quinina/farmacologia , Cloreto de Sódio/farmacologiaRESUMO
The vanilloid receptor TRPV1 is activated by ethanol and this may be important for some of the central and peripheral actions of ethanol. To determine if this receptor has a role in ethanol-mediated behaviors, we studied null mutant mice in which the Trpv1 gene was deleted. Mice lacking this gene showed significantly higher preference for ethanol and consumed more ethanol in a two-bottle choice test as compared with wild type littermates. Null mutant mice showed shorter duration of loss of righting reflex induced by low doses of ethanol (3.2 and 3.4 g/kg) and faster recovery from motor incoordination induced by ethanol (2 g/kg). However, there were no differences between null mutant and wild type mice in severity of ethanol-induced acute withdrawal (4 g/kg) or conditioned taste aversion to ethanol (2.5 g/kg). Two behavioral phenotypes (decreased sensitivity to ethanol-induced sedation and faster recovery from ethanol-induced motor incoordination) seen in null mutant mice were reproduced in wild type mice by injection of a TRPV1 antagonist, capsazepine (10 mg/kg). These two ethanol behaviors were changed in the opposite direction after injection of capsaicin, a selective TRPV1 agonist, in wild type mice. The studies provide the first evidence that TRPV1 is important for specific behavioral actions of ethanol.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Consumo de Bebidas Alcoólicas/psicologia , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Canais de Cátion TRPV/genética , Animais , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Depressores do Sistema Nervoso Central/efeitos adversos , Depressores do Sistema Nervoso Central/farmacocinética , Comportamento de Escolha , Etanol/efeitos adversos , Etanol/farmacocinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Equilíbrio Postural/efeitos dos fármacos , Síndrome de Abstinência a Substâncias/psicologia , Canais de Cátion TRPV/agonistas , Paladar/efeitos dos fármacosRESUMO
C57BL/6 inbred mice have been widely used as research models; however, widespread demand has led to the creation of several B6 substrains with markedly different phenotypes. In this study, we report that two substrains of C57BL/6 mice, C57BL/6J (B6J) and C57BL/6NCrl (B6C), separated over 50 years ago at two different breeding facilities differ significantly in alcohol consumption and alcohol preference. The genomes of these two substrains are estimated to differ by only 1-2% of all gene loci, providing a unique opportunity to extract particular expression signatures between these substrains that are associated with quantifiable behavioral differences. Expression profiling of the cortex and striatum, hippocampus, cerebellum and the ventral brain region from alcohol-naïve B6C and B6J mice showed intervals on three chromosomes that are enriched in clusters of coregulated transcripts significantly divergent between the substrains. Additional analysis identified two genomic regions containing putative copy number differences between the substrains. One such region on chromosome 14 contained an estimated 3n copy number in the B6J genome compared with B6C. Within this interval, a gene of unknown function, D14Ertd449e, was found to be both associated with alcohol preference and vary in copy number across several inbred strain lineages. H2afz, Psen1, Wdfy1 and Clu were also identified as candidate genes that may be involved in influencing alcohol consumption.
Assuntos
Transtornos do Sistema Nervoso Induzidos por Álcool/genética , Alcoolismo/genética , Química Encefálica/genética , Predisposição Genética para Doença/genética , Genoma/genética , Transcrição Gênica/genética , Consumo de Bebidas Alcoólicas/genética , Consumo de Bebidas Alcoólicas/metabolismo , Transtornos do Sistema Nervoso Induzidos por Álcool/metabolismo , Transtornos do Sistema Nervoso Induzidos por Álcool/fisiopatologia , Alcoolismo/metabolismo , Alcoolismo/fisiopatologia , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Mapeamento Cromossômico , Análise Mutacional de DNA , Modelos Animais de Doenças , Feminino , Dosagem de Genes/genética , Perfilação da Expressão Gênica , Testes Genéticos , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Especificidade da EspécieRESUMO
To directly evaluate the association between taste perception and alcohol intake, we used three different mutant mice, each lacking a gene expressed in taste buds and critical to taste transduction: alpha-gustducin (Gnat3), Tas1r3 or Trpm5. Null mutant mice lacking any of these three genes showed lower preference score for alcohol and consumed less alcohol in a two-bottle choice test, as compared with wild-type littermates. These null mice also showed lower preference score for saccharin solutions than did wild-type littermates. In contrast, avoidance of quinine solutions was less in Gnat3 or Trpm5 knockout mice than in wild-type mice, whereas Tas1r3 null mice were not different from wild type in their response to quinine solutions. There were no differences in null vs. wild-type mice in their consumption of sodium chloride solutions. To determine the cause for reduction of ethanol intake, we studied other ethanol-induced behaviors known to be related to alcohol consumption. There were no differences between null and wild-type mice in ethanol-induced loss of righting reflex, severity of acute ethanol withdrawal or conditioned place preference for ethanol. Weaker conditioned taste aversion (CTA) to alcohol in null mice may have been caused by weaker rewarding value of the conditioned stimulus (saccharin). When saccharin was replaced by sodium chloride, no differences in CTA to alcohol between knockout and wild-type mice were seen. Thus, deletion of any one of three different genes involved in detection of sweet taste leads to a substantial reduction of alcohol intake without any changes in pharmacological actions of ethanol.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Proteínas Heterotriméricas de Ligação ao GTP/genética , Receptores Acoplados a Proteínas G/genética , Canais de Cátion TRPM/genética , Paladar/genética , Consumo de Bebidas Alcoólicas/psicologia , Animais , Aprendizagem da Esquiva , Condicionamento Clássico , Cruzamentos Genéticos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Quinina , SacarinaRESUMO
Volatile anesthetics and alcohols enhance transmission mediated by gamma-aminobutyric acid type A receptors (GABA(A)Rs) in the central nervous system, an effect that may underlie some of the behavioral actions of these agents. Substituting a critical serine residue within the GABA(A)R alpha(1) subunit at position 270 with the larger residue histidine eliminated receptor modulation by isoflurane, but it also affected receptor gating (increased GABA sensitivity). To correct the shift in GABA sensitivity of this mutant, we mutated a second residue, leucine at position 277 to alanine. The double mutant alpha(1)(S270H,L277A)beta(2)gamma(2S) GABA(A)R was expressed in Xenopus laevis oocytes and human embryonic kidney (HEK)293 cells, and it had near-normal GABA sensitivity. However, rapid application of a brief GABA pulse to receptors expressed in HEK293 cells revealed that the deactivation was faster in double mutant than in wild-type receptors. In all heterologous systems, the enhancing effect of isoflurane and ethanol was greatly decreased in the double mutant receptor. Homozygous knockin mice harboring the double mutation were viable and presented no overt abnormality, except hyperactivity. This knockin mouse line should be useful in determining which behavioral actions of volatile anesthetics and ethanol are mediated by the GABA(A)Rs containing the alpha(1) subunit.
Assuntos
Etanol/farmacologia , Moduladores GABAérgicos/farmacologia , Isoflurano/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Mutação , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Relação Estrutura-Atividade , Xenopus , Zinco/farmacologia , Ácido gama-Aminobutírico/farmacologiaRESUMO
Glycine receptors (GlyR) are ligand-gated ion channels that inhibit neurotransmission in the spinal cord and brainstem, and mutations in GlyR can cause the human disease hyperekplexia, which is characterized by elevated startle responses. Recently, the GlyR alpha1S267Q mutation was shown to disrupt normal GlyR function, and knock-in mice harboring this mutation displayed profoundly increased acoustic startle responses and reduced glycine-stimulated the chloride flux [Findlay, G.S., Phelan, R., Roberts, M.T., Homanics, G.E., Bergeson, S.E., Lopreato, G.F., Mihic, S.J., Blednov, Y.A., Harris, R.A. 2003. Glycine receptor knock-in mice and hyperekplexia: comparisons with the null mutant. J Neurosci 23, 8051-8059.]. In this study, a transgenic mouse model expressing this S267Q mutation was evaluated using similar techniques to determine if these mice are similarly affected. Male transgenic mice displayed increased acoustic startle responses. However, decreases in glycine-stimulated strychnine-sensitive radioactive chloride (36Cl-) uptake were not observed in spinal cord and brainstem synaptoneurosomes from transgenic mice. No changes in habituation or prepulse inhibition of startle responses or spontaneous locomotion in response to taurine were observed as a result of presence of the transgene. Consistent with previous studies using immunoblotting and strychnine binding [Findlay, G.S., Wick, M.J., Mascia, M.P., Wallace, D., Miller, G.W., Harris, R.A., Blednov, Y.A. 2002. Transgenic expression of a mutant glycine receptor decreases alcohol sensitivity of mice. J Pharmacol Exp Ther 300, 526-534.], the glycine-stimulated strychnine-sensitive chloride flux of cortical microsacs in transgenic mice confirmed the ectopic expression of transgenic GlyR. These results support both the idea that transgenic expression of the S267Q mutation produces a less dramatic phenotype as compared to the knock-in mouse model as well as the idea that the in vivo acoustic startle test (as compared to the in vitro chloride flux assay) is particularly sensitive to disruptions in GlyR function.