RESUMO
Screening the whole genome of a cross between two inbred animal strains has proved to be a powerful method for detecting genetic loci underlying quantitative behavioural traits, but the level of resolution offered by quantitative trait loci (QTL) mapping is still too coarse to permit molecular cloning of the genetic determinants. To achieve high-resolution mapping, we used an outbred stock of mice for which the entire genealogy is known. The heterogeneous stock (HS) was established 30 years ago from an eight-way cross of C57BL/6, BALB/c, RIII, AKR, DBA/2, I, A/J and C3H inbred mouse strains. At the time of the experiment reported here, the HS mice were at generation 58, theoretically offering at least a 30-fold increase in resolution for QTL mapping compared with a backcross or an F2 intercross. Using the HS mice we have mapped a QTL influencing a psychological trait in mice to a 0.8-cM interval on chromosome 1. This method allows simultaneous fine mapping of multiple QTLs, as shown by our report of a second QTL on chromosome 12. The high resolution possible with this approach makes QTLs accessible to positional cloning.
Assuntos
Comportamento Animal/fisiologia , Mapeamento Cromossômico/métodos , Camundongos/genética , Animais , Cruzamento , Cromossomos Artificiais de Levedura , Marcadores Genéticos , Haplótipos , Desequilíbrio de Ligação , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos , Análise de RegressãoRESUMO
Psychological traits are commonly inferred from covariation in sets of behavioral measures that otherwise appear to have little in common. Emotionality in mice is such a trait, defined here by covariation in activity and defecation in a novel environment and emergence into the open arms of an elevated plus maze. Behavioral and quantitative trait analyses were conducted on four measures obtained from 879 mice from an F2 intercross. Three loci, on murine chromosomes 1, 12, and 15, were mapped that influence emotionality. This trait, inferred from studies of strain, sex, and individual differences in rodents, may be related to human susceptibility to anxiety or neuroticism.
Assuntos
Comportamento Animal , Mapeamento Cromossômico , Emoções , Ligação Genética , Animais , Defecação , Feminino , Genes , Variação Genética , Escore Lod , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fenótipo , Análise de RegressãoRESUMO
The ionotropic glutamate receptor subunit GluR6 undergoes developmentally and regionally regulated Q/R site RNA editing that reduces the calcium permeability of GluR6-containing kainate receptors. To investigate the functional significance of this editing in vivo, we engineered mice deficient in GluR6 Q/R site editing. In these mutant mice but not in wild types, NMDA receptor-independent long-term potentiation (LTP) could be induced at the medial perforant path-dentate gyrus synapse. This indicates that kainate receptors with unedited GluR6 subunits can mediate LTP. Behavioral analyses revealed no differences from wild types, but mutant mice were more vulnerable to kainate-induced seizures. Together, these results suggest that GluR6 Q/R site RNA editing may modulate synaptic plasticity and seizure vulnerability.
Assuntos
Plasticidade Neuronal/fisiologia , Edição de RNA/fisiologia , Receptores de Ácido Caínico/metabolismo , Convulsões/metabolismo , Sinapses/metabolismo , Animais , Sítios de Ligação/genética , Cálcio/metabolismo , Células Cultivadas , Giro Denteado/citologia , Giro Denteado/metabolismo , Feminino , Técnicas In Vitro , Ácido Caínico , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Camundongos Mutantes , Neurônios/metabolismo , Via Perfurante/citologia , Via Perfurante/metabolismo , Receptores de Ácido Caínico/genética , Convulsões/induzido quimicamente , Receptor de GluK2 CainatoRESUMO
The alpha7 subtype of nicotinic receptor is highly expressed in the hippocampus where it is purported to modulate release of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). The alpha7 receptor-mediated release of GABA is thought to contribute to hippocampal inhibition (gating) of response to repetitive auditory stimulation. This hypothesis is supported by observations of hippocampal auditory gating deficits in mouse strains with low levels of hippocampal alpha7 receptors compared to strains with high levels of hippocampal alpha7 receptors. The difficulty with comparisons between mouse strains, however, is that different strains have different genetic backgrounds. Thus, the observed interstrain differences in hippocampal auditory gating might result from factors other than interstrain variations in the density of hippocampal alpha7 receptors. To address this issue, hippocampal binding of the alpha7 receptor-selective antagonist alpha-bungarotoxin as well as hippocampal auditory gating characteristics were compared in C3H wild type and C3H alpha7 receptor null mutant heterozygous mice. The C3H alpha7 heterozygous mice exhibited significant reductions in hippocampal alpha7 receptor levels and abnormal hippocampal auditory gating compared to the C3H wild type mice. In addition, a general increase in CA3 pyramidal neuron responsivity was observed in the heterozygous mice compared to the wild type mice. These data suggest that decreasing hippocampal alpha7 receptor density results in a profound alteration in hippocampal circuit function.
Assuntos
Potenciais Evocados Auditivos/fisiologia , Hipocampo/fisiologia , Rede Nervosa/fisiologia , Receptores Nicotínicos/deficiência , Estimulação Acústica/métodos , Animais , Bungarotoxinas/farmacocinética , Eletroencefalografia/métodos , Potenciais Evocados Auditivos/efeitos dos fármacos , Feminino , Hipocampo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Rede Nervosa/efeitos dos fármacos , Antagonistas Nicotínicos/farmacocinética , Ligação Proteica/efeitos dos fármacos , Ensaio Radioligante/métodos , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Discovered in the 1940s by Selman Waksman, the aminoglycoside antibiotic streptomycin is clinically important in the treatment of tuberculosis worldwide. However, strains of Mycobacterium tuberculosis and other pathogenic bacteria have become resistant to streptomycin. One mechanism by which this can occur is through the action of phosphotransferases that attach a phosphate group to position 6 of the streptidine ring of streptomycin, thereby inactivating it. Two such phosphotransferases are APH(6)-Ia from producer strain Streptomyces griseus, and APH(6)-Id found in animal, plant and human pathogenic isolates. Here, we report the subcloning and expression in Escherichia coli of soluble recombinant APH(6)-Ia and Id enzymes. Sequencing of aph(6)-Ia revealed a one-nucleotide disagreement with the published sequence, such that the amino acid at position 262 is an alanine instead of a serine. The sequence of aph(6)-Id is identical to that of the gene found in transposon Tn5393 of plant pathogen Erwinia amylovora. The successful expression of soluble forms of these enzymes now paves the way for experiments to study their structure and function by using site-directed mutagenesis.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Streptomyces griseus/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Clonagem Molecular , Primers do DNA , Elementos de DNA Transponíveis/genética , Erwinia amylovora/genética , Escherichia coli , Dados de Sequência Molecular , Estrutura Molecular , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Presynaptic nicotinic acetylcholine receptors (nAChRs) on striatal synaptosomes stimulate dopamine release. Partial inhibition by the alpha3beta2-selective alpha-conotoxin-MII indicates heterogeneity of presynaptic nAChRs on dopamine terminals. We have used this alpha-conotoxin and UB-165, a novel hybrid of epibatidine and anatoxin-a, to address the hypothesis that the alpha-conotoxin-MII-insensitive subtype is composed of alpha4 and beta2 subunits. UB-165 shows intermediate potency, compared with the parent molecules, at alpha4beta2* and alpha3-containing binding sites, and resembles epibatidine in its high discrimination of these sites over alpha7-type and muscle binding sites. (+/-)-Epibatidine, (+/-)-anatoxin-a, and (+/-)-UB-165 stimulated [(3)H]-dopamine release from striatal synaptosomes with EC(50) values of 2.4, 134, and 88 nM, and relative efficacies of 1:0.4:0.2, respectively. alpha-Conotoxin-MII inhibited release evoked by these agonists by 48, 56, and 88%, respectively, suggesting that (+/-)-UB-165 is a very poor agonist at the alpha-conotoxin-MII-insensitive nAChR subtype. In assays of (86)Rb(+) efflux from thalamic synaptosomes, a model of an alpha4beta2* nAChR response, (+/-)-UB-165 was a very weak partial agonist; the low efficacy of (+/-)-UB-165 at alpha4beta2 nAChR was confirmed in Xenopus oocytes expressing various combinations of human nAChR subunits. In contrast, (+/-)-UB-165 and (+/-)-anatoxin-a were similarly efficacious and similarly sensitive to alpha-conotoxin-MII in increasing intracellular Ca(2+) in SH-SY5Y cells, a functional assay for native alpha3-containing nAChR. These data support the involvement of alpha4beta2* nAChR in the presynaptic modulation of striatal dopamine release and illustrate the utility of exploiting a novel partial agonist, together with a selective antagonist, to dissect the functional roles of nAChR subtypes in the brain.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Conotoxinas/metabolismo , Corpo Estriado/metabolismo , Dopamina/metabolismo , Agonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/metabolismo , Piridinas/metabolismo , Receptores Nicotínicos/metabolismo , Sinaptossomos/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacologia , Ligação Competitiva , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Células Cultivadas , Conotoxinas/farmacologia , Corpo Estriado/efeitos dos fármacos , Toxinas de Cianobactérias , Humanos , Toxinas Marinhas/metabolismo , Toxinas Marinhas/farmacologia , Microcistinas , Neurotoxinas/metabolismo , Neurotoxinas/farmacologia , Nicotina/metabolismo , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Piridinas/farmacologia , Ratos , Receptores Nicotínicos/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacos , Tropanos , XenopusRESUMO
We assessed the quality of overnight glycemic control and the frequency of the "dawn phenomenon" (nadir-0800 h glycemic increase) in 41 insulin-dependent diabetic patients treated by continuous subcutaneous insulin infusion (CSII). Mean plasma glucose levels were near-normal during the 24 h and, in particular, constant throughout the night. In a subset of six patients overnight plasma free insulin concentrations were also constant during CSII. The majority of profiles (88%) showed a glucose nadir from 2.0 to 5.9 mmol/L (most frequently at 0600 h) and had an 0800 h value from 2.0 to 6.9 mmol/L (92%). A large proportion (46%) of profiles showed a zero or negative nadir-0800 h glycemic increase. In 22 patients with three or more profiles recorded at the same basal insulin infusion rate, only one of 103 profiles had a fasting glycemic increase greater than an arbitrary value of 5.0 mmol/L (5.3), although many patients exhibited small dawn glycemic increases (e.g., 14 of 22 had a mean increase of from 0 to 2 mmol/L). In 12 subjects a 15% reduction in basal insulin infusion rate increased the mean +/- SEM dawn glycemic increase from 0.58 +/- 0.25 mmol/L to 2.7 +/- 0.76 mmol/L (P less than 0.025) as well as significantly increasing the nocturnal nadir and 0800 h plasma glucose concentrations. Thus, a marked dawn phenomenon is rare when a single but adequate basal infusion rate is used for CSII, and this questions the need in the majority of patients for preprogrammable pumps with nocturnal infusion rate changes.
Assuntos
Glicemia/análise , Ritmo Circadiano , Diabetes Mellitus Tipo 1/tratamento farmacológico , Sistemas de Infusão de Insulina , Adulto , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/fisiopatologia , Jejum , Humanos , Hiperglicemia/fisiopatologiaRESUMO
Eleven insulin-dependent diabetic patients were treated in random order by 2-mo continuous subcutaneous insulin infusion (CSII) or 2-mo conventional injection treatment (CIT) with crossover to the alternative regimen. Mean plasma glucose concentrations throughout the day were significantly lower during CSII than during CIT, but the percentage of plasma glucose values less than 2.5 mmol/L, obtained from outpatient self-collected diurnal profiles, was similar for both treatments (CSII vs. CIT: 5.9 and 4.8%, respectively). Reported symptomatic hypoglycemia at home was not significantly different in the whole group of patients treated by CSII or CIT but was reduced by a mean of 57% (P less than .02) in the five patients on CSII who experienced frequent symptomatic hypoglycemic episodes (greater than 4/2 mo) during CIT. Neither the plasma glucose concentration at which the patients recognized induced hypoglycemia nor the glycemic or counterregulatory hormone responses for 60 min thereafter were changed by CSII treatment.
Assuntos
Diabetes Mellitus Tipo 1/sangue , Hipoglicemia/etiologia , Insulina/administração & dosagem , Adulto , Glicemia/metabolismo , Diabetes Mellitus Tipo 1/tratamento farmacológico , Feminino , Hemodinâmica/efeitos dos fármacos , Humanos , Hipoglicemia/sangue , Hipoglicemia/fisiopatologia , Injeções , Sistemas de Infusão de Insulina/efeitos adversos , Masculino , Pessoa de Meia-Idade , Distribuição AleatóriaRESUMO
In a recent study, we reported that a restriction fragment length polymorphism associated with the alpha4 nicotinic receptor gene (Chrna4) may play a role in regulating differential sensitivity of LS and SS mouse lines to the seizure-inducing effects of nicotine. Since the alpha4 subunit (CHRNA4) is often found as a heteromer with the beta2 subunit (CHRNB2), alpha4 and beta2 cDNAs from the LS and SS mice were cloned and sequenced. A polymorphism in the coding portion of the alpha4 gene was found (1587A to G) which should result in a threonine/alanine substitution at position 529 (T529A). The LS and SS beta2 nicotinic receptor subunit cDNAs were identical. The potential consequences of the alpha4 polymorphism were evaluated using an ion (86Rb+) flux assay that likely measures the function of alpha4beta2-type receptors. LS-SS differences in maximal nicotine-stimulated ion flux were seen when bovine serum albumin (BSA) was not included but this difference was not seen when BSA was included in the perfusion buffer. Current evidence suggests that BSA may alter the ratio of nicotinic receptors that are in the ground state and desensitized forms. Thus, it may be that the Chrna4 T529A substitution leads to a difference in the ratio of the two receptor forms which then promotes differences in receptor function, as well as differential behavioural sensitivity to nicotine.
Assuntos
Nicotina/farmacologia , Polimorfismo Genético , Receptores Nicotínicos/genética , Rubídio/farmacocinética , Sono/genética , Sono/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico Ativo/efeitos dos fármacos , Bovinos , DNA Complementar/genética , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Nicotina/toxicidade , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Soroalbumina Bovina , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Tálamo/efeitos dos fármacos , Tálamo/metabolismoRESUMO
Evidence from human studies indicates that response to alcohol consumption and the probability of developing alcoholism have a heritable basis. The involvement of several genes encourages the application of classical genetics to elucidate the genetic basis of alcohol abuse. Quantitative genetics can provide evidence for modes of inheritance and estimates of gene number. Therefore, inbred strains and selective breeding programmes have been used to demonstrate a genetic basis for the physiological and behavioural effects of ethanol in rodents. Neurochemical studies have shown that ethanol has effects on membrane fluidity, and on membrane receptors, ion channels and enzymes (Fig. 1). Comparison of these in animal lines selected for a particular response (or lack of response) to ethanol has disclosed differences in membrane composition (notably in polysialogangliosides), and in the functioning of NaK-ATPase, Na+ channels and gamma-aminobutyric acid (GABAA) receptors. Not all of these effects were observed at relevant ethanol concentrations, but differences in the properties of GABAA receptors between lines are the most compelling. Thus classical genetic strategies, while labour intensive with respect to the generation and maintenance of animal lines, have proven useful in dissecting the molecular, cellular and behavioural effects of alcohol and their genetic variability.
Assuntos
Alcoolismo/genética , Alcoolismo/metabolismo , Química Encefálica/fisiologia , Animais , Modelos Animais de DoençasRESUMO
Previous studies have suggested that an abnormality in neuronal nicotinic acetylcholine receptor expression or function may be involved in the neuropathophysiology of schizophrenia. [(3)H]-nicotine and [(3)H]-epibatidine binding were compared in postmortem brain from control and schizophrenic subjects with varying smoking histories. In control subjects, increased receptor binding was seen in hippocampus, cortex, and caudate with increasing tobacco use. In contrast, schizophrenic smokers had reduced nicotinic receptor levels in these brain regions compared to control smokers. Chronic haloperidol and nicotine treatment, in the rat, was used to assess neuroleptic effects on receptor up-regulation by nicotine. A significant increase in cortical nicotinic receptors was seen in both nicotine treated as well as haloperidol and nicotine co-treated animals, suggesting that the abnormal regulation of high affinity neuronal nicotinic receptors in schizophrenics following nicotine use was not related to chronic neuroleptic treatment.
Assuntos
Encéfalo/metabolismo , Receptores Nicotínicos/metabolismo , Esquizofrenia/metabolismo , Fumar/metabolismo , Aconitina/análogos & derivados , Aconitina/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Animais , Antipsicóticos/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Criança , Feminino , Haloperidol/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Piridinas/metabolismo , Ratos , Receptores Nicotínicos/efeitos dos fármacos , Análise de RegressãoRESUMO
One function of the hippocampus is to ascertain the novelty of incoming sensations and encode significant new information into memory. The regulation of response to repeated stimuli may prevent overloading of this function by redundant sensory input. Recent pharmacological studies implicate the role of alpha-bungarotoxin-sensitive nicotinic cholinergic receptors in the inhibition of hippocampal response to repeated auditory stimuli. The number of hippocampal alpha-bungarotoxin-sensitive receptors has a major genetic determinant, as demonstrated by a significant variance between different inbred mouse strains. The purpose of the present study was to determine whether there was a related genetic correlation for the gating of auditory response. Nine inbred mouse strains, representing a continuum of hippocampal alpha-bungarotoxin binding, were tested for the electrophysiological response to repeated auditory stimulation, followed by whole hippocampus membrane alpha-bungarotoxin binding studies. Several parameters of the auditory evoked response showed significant genetic variance over the nine strains, and a significant correlation was found between hippocampal alpha-bungarotoxin binding and both the amplitude of the initial evoked response and its inhibition to repeated auditory stimuli. There was no correlation of the auditory evoked response with high-affinity nicotine binding. These data further support the hypothesis that alpha-bungarotoxin-sensitive nicotinic receptors are involved in the regulation of hippocampal response to repeated auditory stimuli and suggest that this function is genetically controlled.
Assuntos
Bungarotoxinas/metabolismo , Potenciais Evocados Auditivos/fisiologia , Hipocampo/fisiologia , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Estimulação Acústica , Animais , Autorradiografia , Eletrofisiologia , Hipocampo/anatomia & histologia , Hipocampo/metabolismo , Hibridização In Situ , Radioisótopos do Iodo , Masculino , Camundongos , Camundongos Endogâmicos , Nicotina/metabolismo , Agonistas Nicotínicos/metabolismo , RNA Mensageiro/biossíntese , Especificidade da EspécieRESUMO
Nicotinic acetylcholine receptor function and binding was measured in 12 brain regions from mice differing in beta2 subunit expression. Function was measured by on-line detection of (86)Rb(+) efflux stimulated under conditions that measure two pharmacologically distinct nicotinic responses: (1) stimulation with 10 microM nicotine, a response that is relatively sensitive to inhibition by the antagonist, dihydro-beta-erythroidine (DHbetaE); and (2) stimulation with 10 microM epibatidine in the presence of 2 microM DHbetaE, a response that is relatively resistant to inhibition by DHbetaE. Deletion of the beta2 subunit profoundly reduced both DHbetaE-sensitive and -resistant (86)Rb(+) efflux in each brain region and essentially eliminated activity in regions such as cerebral cortex and thalamus. However, residual activity was observed in regions such as olfactory bulbs and inferior colliculus. [(3)H]Epibatidine binding was measured under conditions that allow estimation of both high- and low-affinity sites. High-affinity sites sensitive to inhibition by the nicotinic agonist, cytisine, were virtually eliminated in every region by the beta2 null mutation. In contrast, only a subset of the high-affinity sites insensitive to inhibition by cytisine were eliminated in beta2 null mutants, suggesting receptor heterogeniety. Similarly, low affinity [(3)H]epibatidine binding was heterogeneous in that a fraction of the sites required the beta2 subunit. Many remaining sites were sensitive to inhibition by alpha-bungarotoxin indicating that a subset of the low affinity [(3)H]epibatidine binding are of the alpha7* subtype. Distinct regional variation was observed among the 12 brain regions. These studies confirm important roles for beta2-containing receptors in mediating pharmacologically distinct functions and as components of several identifiable binding sites.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Agonistas Nicotínicos/farmacologia , Piridinas/metabolismo , Receptores Nicotínicos/genética , Rubídio/metabolismo , Animais , Bungarotoxinas/farmacologia , Feminino , Genótipo , Cinética , Masculino , Camundongos , Potássio/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Radioisótopos de Rubídio , Estimulação Química , Veratridina/farmacologiaRESUMO
Previous studies have shown that adrenalectomy results in a small increase in hippocampal alpha-bungarotoxin binding, whereas seven days of chronic treatment with high doses of corticosterone results in decreases in alpha-bungarotoxin binding in several brain regions. The studies reported here examined the effects of different doses of corticosterone on brain alpha-bungarotoxin binding. C3H mice were adrenalectomized and treated with corticosterone-containing pellets (0.5-60%) for four days. Alpha-Bungarotoxin binding was measured in eight brain regions. Chronic treatment with corticosterone resulted in plasma corticosterone levels ranging from the low levels observed in an unstressed mouse during the daytime to levels significantly above those observed in mice during the night or as a result of stress. Adrenalectomy resulted in small increases in binding in hippocampus which was reversed by low dose corticosterone treatment. Chronic high-dose corticosterone treatment resulted in significant decreases in binding in four of the eight brain regions examined. Similar, but not identical, results were obtained in two other mouse strains (C57BL and DBA/2). These results argue that corticosterone levels play an important role in modulating the level of the brain nicotinic receptors that bind alpha-bungarotoxin with high affinity.
Assuntos
Encéfalo/efeitos dos fármacos , Bungarotoxinas/farmacologia , Corticosterona/farmacologia , Animais , Ligação Competitiva , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Radioimunoensaio , Fatores de TempoRESUMO
[(125)I]-Epibatidine binds to multiple nicotinic acetylcholine receptor (nAChR) subtypes with high affinity. In this study, [(125)I]-epibatidine was used to label and characterize a novel nAChR subtype found in mouse brain inferior colliculus, interpeduncular nucleus, and olfactory bulb homogenates. Binding of [(125)I]-epibatidine was saturable and apparently monophasic in each brain region (K:(D:)=71+/-12 pM mean+/-s.e.mean across regions) but inhibition of [(125)I]-epibatidine binding (200 pM) by A85380, cytisine and (-)-nicotine was biphasic, indicating the presence of multiple binding sites. The sites with lower agonist affinity comprised 30.0+/-2.2, 58.6+/-0.1 and 48.7+/-3.3% of specific [(125)I]-epibatidine (200 pM) binding in inferior colliculus, interpeduncular nucleus, and olfactory bulb homogenates, respectively. The affinity difference between A85380-sensitive and -resistant binding sites was particularly marked (approximately 1000 fold). Thus A85380 was used to differentiate agonist-sensitive and -resistant sites. The pharmacological profiles of the A85380-resistant sites in each region were assessed with inhibition binding experiments, using 14 agonists and five antagonists. The profiles were indistinguishable across regions, implying that A85380-resistant [(125)I]-epibatidine binding sites in inferior colliculus, interpeduncular nucleus, and olfactory bulb represent a single nAChR subtype. The pharmacological profile of the A85380-resistant sites is very different from that previously reported for high affinity (-)-[(3)H]-nicotine-, [(125)I]-alpha-bungarotoxin-, or [(125)I]-alpha-conotoxin MII-binding sites, suggesting that they represent a novel nAChR population in mouse brain.
Assuntos
Encéfalo/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Agonistas Nicotínicos/metabolismo , Piridinas/metabolismo , Receptores Nicotínicos/metabolismo , Alcaloides/farmacologia , Animais , Autorradiografia , Azetidinas/metabolismo , Azocinas , Sítios de Ligação , Radioisótopos do Iodo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nicotina/metabolismo , Quinolizinas , Receptores Nicotínicos/análiseRESUMO
Following a selective breeding program of heterogeneous mice for more than 30 generations, SS ("short sleep") and LS ("long sleep") lines have been developed on the basis of their sleep times when challenged with a single intraperitoneal dose of ethanol. The aromatic hydrocarbon responsiveness (Ah) locus encodes the Ah receptor, which regulates the induction of certain drug-metabolizing enzymes by polycyclic aromatic compounds such as 3-methylcholanthrene and tetrachlorodibenzo-p-dioxin. The C57BL/6 inbred mouse strain (B6; Ahb/Ahb) has a high-affinity Ah receptor, while the DBA/2 inbred mouse strain (D2; Ahd/Ahd) has a low-affinity Ah receptor. We show here that the SS inbred mouse line exhibits markedly elevated hepatic levels of the high-affinity Ah receptor, while the LS outbred mouse line contains the low-affinity Ah receptor. Among progeny of (B6D2)F1 X D2 backcross, the b/d heterozygote (having the high-affinity Ah receptor) was found to be several times more resistant than the d/d homozygote to a single dose of intraperitoneal ethanol. The D2.B6-Ahb congenic line is also several times more resistant to intraperitoneal ethanol than the B6.D2-Ahb congenic line is also several times more resistant to intraperitoneal ethanol than B6.D2-Ahd congenic line. We found that the waking blood ethanol levels are the same in b/d and d/d mice, suggesting that the relative ethanol resistance in b/d mice cannot be explained on the basis of a difference in central nervous system sensitivity. There are no differences between SS and LS mice or between b/d and d/d mice with regard to (i) blood acetaldehyde levels after a single intraperitoneal dose of ethanol, or (ii) hepatic alcohol dehydrogenase activities. There is a difference in the rate of ethanol elimination: SS more rapid than LS; b/d more rapid than d/d. Although SS mice have lower hepatic aldehyde dehydrogenase activities (cytosolic, mitochondrial low-Km: and mitochondrial high-Km forms) than LS mice, b/d and d/d do not show this difference. These data suggest that a selected mouse breeding program, based on resistance to a single intraperitoneal dose of ethanol, selects concurrently for the hepatic high-affinity Ah receptor. This selective advantage cannot be explained on the basis of changes in alcohol dehydrogenase or aldehyde dehydrogenase activities and might provide insight into the nature of the endogenous ligand for the Ah receptor.
Assuntos
Etanol/farmacologia , Hidrocarbonetos/metabolismo , Fígado/efeitos dos fármacos , Receptores de Droga/análise , Sono/efeitos dos fármacos , Acetaldeído/sangue , Álcool Desidrogenase/análise , Aldeído Desidrogenase/análise , Animais , Cruzamento , Resistência a Medicamentos , Etanol/metabolismo , Fígado/análise , Camundongos , Camundongos Endogâmicos , Receptores de Hidrocarboneto Arílico , Receptores de Droga/genéticaRESUMO
Inbred mouse strains have been shown to differ in their levels of brain alpha-bungarotoxin binding. These differences in alpha-bungarotoxin receptors have been shown to correlate with an animal's sensitivity to nicotine-induced seizures. Recent studies have shown that the alpha 7 nicotinic acetylcholine receptor subunit is the major alpha-bungarotoxin binding site in rodent brain. In this report, we examined whether mouse strains that differ in levels of alpha-bungarotoxin binding and sensitivity to nicotine-induced convulsions also differ for the alpha 7 subunit. A full-length murine alpha 7 cDNA was cloned and sequenced and found to be identical to that of a mouse alpha 7 cDNA recently reported. Subsequently, a comparison of alpha 7 cDNA sequences and RNA species was performed between two strains (C3H/2 and DBA/2) that differ in levels of brain alpha-bungarotoxin binding and sensitivity to nicotine-induced seizures. The only difference observed was a single nucleotide difference in the open reading frame of alpha 7 that does not affect the primary amino acid sequence. Inbred strains were also surveyed for restriction fragment length polymorphisms at the alpha 7 locus. Strain-specific polymorphisms were identified, and F2 and backcross animals from a classic genetic cross between C3H/2 and DBA/2 mice were compared for the inheritance of alpha 7 genotype and alpha-bungarotoxin receptor levels. A significant association between genotype and receptor levels was observed in both, the F2 and backcross generations. These results indicate that alpha 7 genotype is an important determinant of alpha-bungarotoxin receptor levels.
Assuntos
Encéfalo/metabolismo , Bungarotoxinas/metabolismo , Ligação Genética , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Polimorfismo de Fragmento de RestriçãoRESUMO
Inbred mouse strains vary in sensitivity to a number of behavioral and physiological effects produced by nicotine. Differences in sensitivity to nicotine are correlated with variance in the number of brain nicotinic receptors as measured in regionally dissected brain tissue. The studies reported here used quantitative autoradiography and in-situ hybridization methods to measure regional levels of alpha-bungarotoxin (alpha BTX) binding and alpha 7 mRNA levels. Two inbred mouse strains, ST/b and DBA/2, were compared because these strains differ maximally in sensitivity to nicotine-induced seizures and in alpha BTX binding measured in regional brain homogenates. The binding of alpha BTX was significantly greater in the St/b strain in 42 of 127 brain regions that were analyzed, and a trend towards increased binding was seen in many additional brain regions. The most consistent strain differences were found in hippocampal, thalamic and pontine nuclei. Strain differences in alpha 7 mRNA levels were also detected, but these were not as widespread as were the alpha BTX binding differences. The alpha 7 mRNA levels were significantly correlated with alpha BTX binding in both mouse strains which suggests that the strain differences in binding are related, in part, to the levels of alpha 7 mRNA.
Assuntos
Ligação Competitiva , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Bungarotoxinas/farmacologia , Receptores Nicotínicos/metabolismo , Animais , Autorradiografia , Expressão Gênica , Hipocampo/metabolismo , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos DBARESUMO
The startle response and adaptability of the startle response (prepulse inhibition and habituation) have been observed in animals. The studies reported here screened 8 inbred mouse strains to determine whether genetic factors influence these behaviors. Strain differences were found in both the sensitivity to acoustic startle and the magnitude of both the auditory and tactile startle as well as the magnitude of prepulse inhibition (PPI) of both tactile and acoustic startle. Neither the 2 startle responses nor the 2 forms of PPI were significantly correlated with one another, suggesting that different genes regulate these 2 forms of startle and PPI. Acoustic-acoustic PPI was significantly correlated, however, with hippocampal auditory gating (TC ratio) suggesting an overlap in the genes that regulate these 2 forms of sensory gating.
Assuntos
Estimulação Acústica , Camundongos Endogâmicos/fisiologia , Inibição Neural/genética , Reflexo de Sobressalto/genética , Animais , Habituação Psicofisiológica/genética , Habituação Psicofisiológica/fisiologia , Masculino , Camundongos , Inibição Neural/fisiologia , Ruído , Psicoacústica , Reflexo de Sobressalto/fisiologia , Tato/genética , Tato/fisiologiaRESUMO
The influences of genotype and sex on spontaneous motor activity in a Y-maze after nicotine administration and on nicotine concentrations in liver and brain were assessed in three inbred mouse strains. The rank order of liver nicotine elimination rates in these strains was found to be C57 > C3H = DBA for females and DBA > C3H = C57 for males. Within the C57 and C3H strains, females eliminated nicotine significantly faster than males, while DBA females and males eliminated nicotine at similar rates. The rank order of motor depression at early time points after nicotine administration was found to be DBA = C57 > C3H for both males and females. Females of all three strains demonstrated less sensitivity to nicotine's depressant effects than males. There did not appear to be any consistent association between rate of liver nicotine elimination or brain nicotine level and motor depression as measured in the Y-maze. Although variability in liver nicotine elimination and in brain nicotine content may account for some of the observed behavioral effects, these data suggest that strain and sex differences in tissue sensitivity to nicotine are of primary importance.