RESUMO
Activation of the dopaminergic (DA) neurons of the ventral tegmental area (VTA) by ethanol has been implicated in its rewarding and reinforcing effects. We previously demonstrated that ethanol enhances GABA release onto VTA-DA neurons via activation of 5-HT2C receptors and subsequent release of calcium from intracellular stores. Here we demonstrate that excitation of VTA-DA neurons by ethanol is limited by an ethanol-enhancement in GABA release. In this study, we performed whole-cell voltage clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs) and cell-attached recordings of action potential firing from VTA-DA neurons in midbrain slices from young Long Evans rats. Acute exposure to ethanol (75 mM) transiently enhanced the firing rate of VTA-DA neurons as well as the frequency of mIPSCs. Simultaneous blockade of both GABA(A) and GABA(B) receptors (Picrotoxin (75 µM) and SCH50911 (20 µM)) disinhibited VTA-DA firing rate whereas a GABA(A) agonist (muscimol, 1 µM) strongly inhibited firing rate. In the presence of picrotoxin, ethanol enhanced VTA-DA firing rate more than in the absence of picrotoxin. Additionally, a sub-maximal concentration of muscimol together with ethanol inhibited VTA-DA firing rate more than muscimol alone. DAMGO (3 µM) inhibited mIPSC frequency but did not block the ethanol-enhancement in mIPSC frequency. DAMGO (1 and 3 µM) had no effect on VTA-DA firing rate. Naltrexone (60 µM) had no effect on basal or ethanol-enhancement of mIPSC frequency. Additionally, naltrexone (20 and 60 µM) did not block the ethanol-enhancement in VTA-DA firing rate. Overall, the present results indicate that the ethanol enhancement in GABA release onto VTA-DA neurons limits the stimulatory effect of ethanol on VTA-DA neuron activity and may have implications for the rewarding properties of ethanol.
Assuntos
Dopamina/fisiologia , Etanol/farmacologia , Neurônios/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Área Tegmentar Ventral/efeitos dos fármacos , Ácido gama-Aminobutírico/fisiologia , Animais , Modelos Animais de Doenças , Masculino , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Ratos , Ratos Long-Evans , Transmissão Sináptica/fisiologia , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/fisiologiaRESUMO
Dopamine increases in the nucleus accumbens after ethanol administration in rats, but the contributions of the core and shell subregions to this response are unclear. The goal of this study was to determine the effect of various doses of i.v. ethanol infusions on dopamine in these two subregions of the nucleus accumbens. Male Long-Evans rats were infused with either acute i.v. ethanol (0.5, 1.0, 1.5 g/kg), repeated i.v. ethanol (four 1.0 g/kg infusions resulting in a cumulative dose of 4.0 g/kg), or saline as a control for each condition. Dopamine and ethanol were measured in dialysate samples from each experiment. The in vivo extraction fraction for ethanol of probes was determined using i.v. 4-methylpyrazole, and was used to estimate peak brain ethanol concentrations after the infusions. The peak brain ethanol concentrations after the 0.5, 1.0 and 1.5 g/kg ethanol infusions were estimated to be 20, 49 and 57 mM, respectively. A significant dopamine increase was observed for the 0.5 g/kg ethanol group when collapsed across subregions. However, both the 1.0 g/kg and 1.5 g/kg ethanol infusions produced significant increases in dopamine levels in the shell that were significantly higher than those in the core. An ethanol dose-response effect on dopamine in the shell was observed when saline controls, 0.5, 1.0, and 1.5 g/kg groups were compared. For the cumulative-dosing study, the first, second, and fourth infusions resulted in significant increases in dopamine in the shell. However, these responses were not significantly different from one another. The results of this study show that the shell has a stronger response than the core to i.v. ethanol, that dopamine in the shell increases in a dose-dependent manner between 0.5-1.0 g/kg doses, but that the response to higher ethanol doses reaches a plateau.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Dopamina/metabolismo , Etanol/farmacologia , Núcleo Accumbens/metabolismo , Álcool Desidrogenase/antagonistas & inibidores , Animais , Comportamento Animal/efeitos dos fármacos , Depressores do Sistema Nervoso Central/administração & dosagem , Depressores do Sistema Nervoso Central/farmacocinética , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Etanol/administração & dosagem , Etanol/farmacocinética , Fomepizol , Infusões Intravenosas , Masculino , Microdiálise , Núcleo Accumbens/anatomia & histologia , Núcleo Accumbens/efeitos dos fármacos , Pirazóis/farmacologia , Ratos , Ratos Long-EvansRESUMO
The medium spiny neurons of the nucleus accumbens receive both an excitatory glutamatergic input from forebrain and a dopaminergic input from the ventral tegmental area. This integration point may constitute a locus whereby the N-methyl-D-aspartate (NMDA)-subtype of glutamate receptors promotes drug reinforcement. Here we investigate how dopaminergic inputs alter the ethanol sensitivity of NMDA receptors in rats and mice and report that previous dopamine receptor-1 (D1) activation, culminating in dopamine and cAMP-regulated phosphoprotein-32 kD (DARPP-32) and NMDA receptor subunit-1 (NR1)-NMDA receptor phosphorylation, strongly decreases ethanol inhibition of NMDA responses. The regulation of ethanol sensitivity of NMDA receptors by D1 receptors was absent in DARPP-32 knockout mice. We propose that DARPP-32 mediated blunting of the response to ethanol subsequent to activation of ventral tegmental area dopaminergic neurons initiates molecular alterations that influence synaptic plasticity in this circuit, thereby promoting the development of ethanol reinforcement.
Assuntos
Etanol/farmacologia , Proteínas do Tecido Nervoso , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Fosfoproteínas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Fosfoproteína 32 Regulada por cAMP e Dopamina , Técnicas In Vitro , Camundongos , Camundongos Knockout , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Núcleo Accumbens/citologia , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Ratos , Receptores de Dopamina D1/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Área Tegmentar Ventral/fisiologiaRESUMO
BACKGROUND: The cellular mechanisms underlying acute tolerance to alcohol are unclear. This study aimed to determine whether hippocampal neurons have the ability to develop acute tolerance to alcohol in behaving rats. METHODS: Intrahippocampal microdialysis was performed in freely behaving rats, and the firing of single neurons in the dialysis area was recorded. The control microdialysis fluid, artificial cerebrospinal fluid (ACSF), was replaced with 1 M ethanol in ACSF for a 30 min period. One hour later, the ethanol perfusion was repeated. To test the functional integrity of the microdialysis probe in situ, each microdialysis session was completed with recording the effect of a 10-20 min perfusion of 500 microM N-methyl-D-aspartate (NMDA). The extracellular concentration profile of ethanol during intrahippocampal microdialysis with 1 M ethanol was estimated in a separate study in anesthetized rats. The ethanol content was measured in tissue slices surrounding the probe with gas chromatography (GC), and the generated data were analyzed with a mathematical model for microdialysis to estimate the concentration of ethanol at the recording site. RESULTS: The predominant effect of the first intrahippocampal microdialysis with ethanol was a decrease in firing rate in both pyramidal cells and interneurons. In contrast, such firing rate decrease did not develop during the second ethanol perfusion. Subsequent NMDA perfusion still induced robust changes in the electrical activity of the neurons. The estimated extracellular ethanol concentration at the recording site was 45-70 mM. CONCLUSION: This study revealed that hippocampal neurons have the ability to develop acute tolerance to a single exposure of clinically relevant concentrations of ethanol in behaving rats, without influences from the rest of the body.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Hipocampo/fisiologia , Neurônios/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Tolerância a Medicamentos , Hipocampo/citologia , Masculino , Microdiálise , Concentração Osmolar , Ratos , Ratos Long-EvansRESUMO
Amphetamine increases extracellular dopamine and induces locomotor and stereotypical behaviors in rats. This study examined the effect of the dopamine D2/D3 receptor antagonist sulpiride (50 mg/kg s.c.) on the dopaminergic response to amphetamine (0.5, 2.0, or 8.0 mg/kg i.p.) in male Sprague-Dawley rats. Extracellular dopamine in the striatum was monitored using in vivo microdialysis and high performance liquid chromatography with electrochemical detection. Dopamine concentration curves were analyzed using non-linear regression and residual F-testing. Amphetamine enhanced extracellular dopamine in a dose-dependent manner. Sulpiride augmented the increase in dopamine induced by 0.5 and 2 mg/kg amphetamine by decreasing the rate of dopamine concentration fall off in the extracellular space (P<0.05). Sulpiride also potentiated the amount of dopamine increased by 8 mg/kg amphetamine, but did so by affecting the maximum concentration achieved (P<0.05), not the onset or offset rates. We conclude that the primary effect of a dopamine D2/D3 receptor antagonist is a potentiation of the effect of amphetamine on extracellular striatal dopamine levels, which may contribute to the enhanced stereotypic effects observed when paired with amphetamine.
Assuntos
Anfetamina/farmacologia , Corpo Estriado/efeitos dos fármacos , Antagonistas de Dopamina/farmacologia , Dopamina/metabolismo , Sulpirida/farmacologia , Animais , Corpo Estriado/metabolismo , Antagonistas dos Receptores de Dopamina D2 , Relação Dose-Resposta a Droga , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Masculino , Microdiálise , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D3 , Fatores de TempoRESUMO
The Medicago Genome Initiative (MGI) is a database of EST sequences of the model legume MEDICAGO: truncatula. The database is available to the public and has resulted from a collaborative research effort between the Samuel Roberts Noble Foundation and the National Center for Genome Resources to investigate the genome of M.truncatula. MGI is part of the greater integrated MEDICAGO: functional genomics program at the Noble Foundation (http://www.noble.org ), which is taking a global approach in studying the genetic and biochemical events associated with the growth, development and environmental interactions of this model legume. Our approach will include: large-scale EST sequencing, gene expression profiling, the generation of M.truncatula activation-tagged and promoter trap insertion mutants, high-throughput metabolic profiling, and proteome studies. These multidisciplinary information pools will be interfaced with one another to provide scientists with an integrated, holistic set of tools to address fundamental questions pertaining to legume biology. The public interface to the MGI database can be accessed at http://www.ncgr.org/research/mgi.
Assuntos
Bases de Dados Factuais , Genoma de Planta , Medicago sativa/genética , Biologia Computacional , Etiquetas de Sequências Expressas , Fabaceae/genética , Internet , Plantas MedicinaisRESUMO
We performed a quantitative microdialysis study to determine whether the increase in dialysate dopamine from the nucleus accumbens caused by intraperitoneal administration of ethanol (1 g/kg) was due to enhanced dopamine release or inhibition of dopamine uptake. The Lönnroth method (no net flux), adapted for transient conditions, was used to follow the time course of true extracellular dopamine concentrations in the nucleus accumbens simultaneously with the in vivo recovery of dopamine across the microdialysis probe. Separate groups of rats were perfused with artificial cerebral spinal fluid containing 0, 4, 8, or 12 nM dopamine for the entire experiment. Samples were taken every 10 min. Each rat received a saline or an ethanol injection. The concentration of dopamine gained by or lost from the probe was plotted as a function of the concentration of dopamine perfused into the probe for each time point. Linear regression was used to determine the slope of the line (in vivo recovery) and the x-intercept (point of no net flux) for each plot. The in vivo recovery did not significantly change over time for the saline- or the ethanol-injected rats. However, the point of no net flux (true extracellular concentration of dopamine) significantly increased after the ethanol injection from 9.4+/-0.4 nM (mean of six basal samples) to 13.2+/-1.8 nM, at the maximum, but did not change after the saline injection. On the basis of these results, it is suggested that the primary mechanism by which ethanol increases dialysate dopamine levels in the nucleus accumbens after intraperitoneal administration is by increasing dopamine release from the terminals, rather than by inhibiting the dopamine transporter.
Assuntos
Dopamina/metabolismo , Etanol/farmacologia , Espaço Extracelular/metabolismo , Núcleo Accumbens/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Etanol/administração & dosagem , Cinética , Modelos Lineares , Masculino , Microdiálise , Núcleo Accumbens/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Subepithelial and intraepithelial lymphocytes of human adenoids and tonsils were characterized and directly compared to determine the potential contribution of these tissues to mucosal and systemic immune responses. The distribution of T and B cell subsets, cytokine patterns, and antibody (Ab) isotype profiles were similar for adenoids and tonsils. Both tissues contained predominantly B cells ( approximately 65%), approximately 5% macrophages, and 30% CD3(+) T cells. The T cells were primarily of the CD4(+) subset ( approximately 80%). Tonsillar intraepithelial lymphocytes were also enriched in B cells. The analysis of dispersed cells revealed a higher frequency of cells secreting IgG than IgA and the predominant Ig subclass profiles were IgG1 > IgG3 and IgA1 > IgA2, respectively. In situ analysis also revealed higher numbers of IgG- than IgA-positive cells. These IgG-positive cells were present in the epithelium and in the subepithelial zones of both tonsils and adenoids. Mitogen-triggered T cells from tonsils and adenoids produced both Th1- and Th2-type cytokines, clearly exhibiting their pluripotentiality for support of cell-mediated and Ab responses. Interestingly, antigen-specific T cells produced interferon-gamma and lower levels of interleukin-5. These results suggest that adenoids and tonsils of the nasopharyngeal-associated lymphoreticular tissues represent a distinct component of the mucosal-associated lymphoreticular tissues with features of both systemic and mucosal compartments.
Assuntos
Tonsila Faríngea/fisiologia , Linfócitos B/fisiologia , Nasofaringe/fisiologia , Tonsila Palatina/fisiologia , Linfócitos T/fisiologia , Tonsila Faríngea/citologia , Tonsila Faríngea/imunologia , Adolescente , Anticorpos/análise , Formação de Anticorpos , Antígenos/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Criança , Citocinas/biossíntese , Células Epiteliais/fisiologia , Humanos , Imunoglobulina A/análise , Imunoglobulina G/análise , Isotipos de Imunoglobulinas/análise , Mitógenos/farmacologia , Monócitos/citologia , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Fito-Hemaglutininas/farmacologia , Subpopulações de Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Traditional microdialysis techniques provide qualitative data, although quantitative data are often required for pharmacodynamic analyses. This study evaluated a potentially useful in vivo delivery technique to calibrate microdialysis probes for ethanol. We measured in vivo delivery extraction fractions within subjects across 2 days and found no change over time. We tested the effect of diffusion direction on extraction fraction and found that it was higher for ethanol diffusion out of the probe than for diffusion into the probe, both in vitro and in vivo. The in vivo extraction fraction ratio of diffusion(IN) versus diffusion(OUT) was 0.65+/-0.03. Finally, we predicted extracellular brain ethanol concentrations after 1 g/kg ethanol administration using in vivo delivery, "no net flux" dialysis, or in vivo delivery corrected for diffusion direction with the in vivo extraction fraction ratio. Both in vivo delivery and "no net flux" dialysis predicted brain concentrations that were approximately one-third lower than blood concentrations, whereas the corrected in vivo delivery predicted extracellular concentrations very similar to blood concentrations. We conclude that microdialysis calibration methods for ethanol require a measure of extraction fraction for diffusion into the probe. Further studies are needed to establish whether this effect is common to other alcohols.
Assuntos
Encéfalo/metabolismo , Calibragem/normas , Etanol/análise , Espaço Extracelular/química , Microdiálise/instrumentação , Animais , Química Encefálica , Difusão , Etanol/administração & dosagem , Estudos de Avaliação como Assunto , Espaço Extracelular/metabolismo , Feminino , Injeções Intravenosas , Modelos Lineares , Masculino , Perfusão , Valor Preditivo dos Testes , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
BACKGROUND: Dopamine release in the nucleus accumbens has been linked to the reinforcing effects of ethanol, but the time course or relationship of this response to ethanol concentrations in the brain has not been studied. METHODS: Various doses of ethanol (0-2.0 g/kg) were administered intraperitoneally to male Sprague Dawley rats, and dopamine and ethanol were simultaneously analyzed in dialysate samples from the nucleus accumbens. A separate study to compare the ethanol-induced dopamine response in male and female rats was carried out by using a 1 g/kg intraperitoneal dose of ethanol. RESULTS: In male rats, 1 and 2 g/kg ethanol significantly increased dialysate dopamine by 40% over basal, whereas 0.25 and 0.5 g/kg ethanol produced a nonsignificant 20% increase. Dialysate ethanol concentrations exhibited a curvilinear decline after reaching peak levels for the lower doses but showed a linear decrease after 1 and 2 g/kg. There was a dissociation between the time courses of extracellular dopamine and ethanol after 1 and 2 g/kg ethanol treatment. The dopamine response returned to basal within 90 min, whereas the ethanol concentrations remained elevated. In a separate study that compared male and female rats, the ratio of the dopamine response over basal to the dialysate ethanol concentrations was significantly decreased at 60 min after an injection of 1 g/kg. However, there were no differences between males and females. CONCLUSIONS: The dissociation between dopamine and ethanol levels may reflect the development of acute tolerance to ethanol-induced dopamine release in the nucleus accumbens within the time course of a single acute injection. Given the strong links between dopamine and ethanol reinforcement, our findings may be relevant for understanding the time course of ethanol's reinforcing effects in vivo.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Dopamina/metabolismo , Etanol/farmacocinética , Núcleo Accumbens/metabolismo , Animais , Depressores do Sistema Nervoso Central/administração & dosagem , Etanol/administração & dosagem , Feminino , Injeções Intraperitoneais , Masculino , Núcleo Accumbens/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Female rodents tend to drink more alcohol than males, a difference that emerges at puberty and appears to vary over the female estrous cycle. In addition, male and female rodents display different responses to alcohol; for example, female rats are reported to have faster elimination rates than males. We were interested in whether circulating ovarian hormones influence alcohol distribution to or elimination from the brain of rats, which might explain observed differences in drinking behavior. We administered 0.8 g/kg of ethanol via intraperitoneal injection to age-matched male and female Sprague-Dawley rats. Extracellular brain ethanol was sampled using microdialysis, and vascular ethanol concentrations were determined via tail blood collection, in two separate experiments. Ethanol pharmacokinetic parameters were calculated for both compartments. There were no differences in pharmacokinetic parameters due to gender or estrous cycle stage in brain ethanol concentration profiles. There were, however, differences in blood ethanol profiles: females showed faster elimination rates and a smaller area under the ethanol concentration versus time curve than males. In addition, the maximum concentration varied significantly across the estrous cycle. These results suggest that (1) circulating ovarian hormones do not influence alcohol distribution to the brain, but do influence distribution to more peripheral tissues such as the tail; and (2) apparent differences in tail blood alcohol levels may not reflect differences in brain levels.
Assuntos
Encéfalo/metabolismo , Etanol/farmacocinética , Animais , Área Sob a Curva , Estro/metabolismo , Etanol/sangue , Etanol/metabolismo , Feminino , Injeções Intraperitoneais , Masculino , Microdiálise , Ratos , Ratos Sprague-Dawley , Fatores SexuaisRESUMO
Changes in brain extracellular glutamate during movement stress were studied using in vivo microdialysis. Male Long-Evans rats were placed in a clear cylinder designed to elicit behavioral activation while undergoing microdialysis sampling from either the hippocampus, striatum or sensorimotor cortex. Glutamate levels were determined by high performance liquid chromatography with fluorescence detection in the dialysates taken before, during, and after exposure to the cylinder. Animals were in a behaviorally quiescent state before exposure to the cylinder, but they demonstrated increases in rearing, locomotion, and turning while in the cylinder. Dialysate glutamate levels were significantly enhanced in the samples taken while the rat was in the cylinder compared with samples taken before or after exposure to the cylinder. In a second study, rats were implanted with bilateral probes in the forelimb sensorimotor cortex, and one forelimb was immobilized by means of a plaster of paris cast. Glutamate, aspartate, serine, and taurine levels were quantified in casted animals. In casted animals, dialysate glutamate levels were lower on the side contralateral to the immobilized limb during both quiescence and movement stress. Aspartate and taurine, but not serine levels increased during movement stress in both the side contralateral and the side ipsilateral to the immobilized limb. These results suggest that there is extracellular overflow of glutamate and other neuroactive amino acids during spontaneous movement, and chronic disuse can suppress extracellular glutamate levels.
Assuntos
Corpo Estriado/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Atividade Motora/fisiologia , Córtex Motor/metabolismo , Aminoácidos/metabolismo , Animais , Masculino , Ratos , Ratos Long-EvansRESUMO
The opiate antagonist naltrexone suppresses ethanol-reinforced behavior in animals and decreases ethanol intake in humans. However, the mechanisms underlying these actions are not well understood. Experiments were designed to test the hypothesis that naltrexone attenuates the rewarding properties of ethanol by interfering with ethanol-induced stimulation of dopamine activity in the nucleus accumbens (NAcc). Simultaneous measures of the effects of naltrexone on dialysate dopamine levels in the NAcc and on operant responding for oral ethanol were used. Male Wistar rats were trained to self-administer ethanol (10-15%, w/v) in 0.2% (w/v) saccharin during daily 30 min sessions and were surgically prepared for intracranial microdialysis. Experiments began after reliable self-administration was established. Rats were injected with naltrexone (0.25 mg/kg, s.c.) or saline and 10 min later were placed inside the operant chamber for a 20 min waiting period with no ethanol available, followed by 30 min of access to ethanol. A transient rise in dialysate dopamine levels was observed during the waiting period, and this effect was not altered by naltrexone. Ethanol self-administration reliably increased dopamine levels in controls. Naltrexone significantly suppressed ethanol self-administration and prevented ethanol-induced increases in dialysate dopamine levels. Subsequent dose-effect analyses established that the latter effect was not merely a function of reduced ethanol intake but that naltrexone attenuated the efficacy of ethanol to elevate dialysate dopamine levels. These results suggest that suppression of ethanol self-administration by opiate antagonists is the result of interference with dopamine-dependent aspects of ethanol reinforcement, although possible additional effects via nondopaminergic mechanisms cannot be eliminated as a factor in opiate antagonist-induced reduction of ethanol intake.
Assuntos
Comportamento Animal/efeitos dos fármacos , Etanol/antagonistas & inibidores , Etanol/farmacologia , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Animais , Soluções para Diálise/química , Dopamina/análise , Relação Dose-Resposta a Droga , Etanol/administração & dosagem , Etanol/sangue , Masculino , Microdiálise , Núcleo Accumbens/metabolismo , Ratos , Ratos Wistar , AutoadministraçãoRESUMO
We have applied a steady-state theory of microdialysis to characterize the diffusion of ethanol through a microdialysis membrane and through rat striatum. Quantitative characterization required measurement of in vitro and in vivo extraction fractions for ethanol and determination of the clearance of ethanol from brain tissue during steady-state perfusion through a microdialysis probe. Extraction fraction of ethanol was determined in vitro by perfusing a known concentration of ethanol through probes immersed in water at 37 degrees C with stirring. The in vitro extraction fraction yielded a probe permeability value of 0.046 +/- 0.004 cm/min that is comparable with an estimate from published measurements for similar dialysis membranes. The in vivo extraction fraction was determined for probes placed in the striatum. Clearance of ethanol and a brain slice concentration profile of ethanol were determined by measurement of the amount of ethanol remaining in the brain tissue during steady-state perfusion of the probe. Steady state was achieved within 10 min after beginning the ethanol perfusion in vivo, and the extraction fraction was not altered by sedation of the rat with pentobarbital. The tissue concentration profile was symmetrical around the probe track, and ethanol was detected 1 mm from the probe. The experimental clearance rate constant value obtained for ethanol (2.0 +/- 0.3 min(-1)) was higher than that expected for removal solely by loss to the blood. The tissue diffusivity for ethanol, Dt, derived from the experimental measurements was 1.2 +/- 0.2 x 10(-5) cm2/sec. This value is greater than expected for interstitial diffusion, suggesting a substantial contribution by transcellular diffusion of ethanol as well. The predicted tissue concentration profile had a higher peak value and did not extend into the tissue (0.5 mm) as much as the experimental profile (1 mm), although there was reasonable agreement between experiment and theory. Our quantitative characterization of the microdialysis behavior of ethanol in brain provides a framework for interpretation of brain microdialysis experiments using ethanol by supplying, inter alia, a means for estimating the ethanol concentration achieved in the tissue volume being sampled by the probe.
Assuntos
Corpo Estriado/metabolismo , Etanol/farmacocinética , Animais , Corpo Estriado/efeitos dos fármacos , Relação Dose-Resposta a Droga , Etanol/farmacologia , Masculino , Taxa de Depuração Metabólica/fisiologia , Microdiálise , Modelos Teóricos , Ratos , Ratos Sprague-DawleyRESUMO
A cDNA and corresponding promoter region for a naturally occurring, feedback-insensitive anthranilate synthase (AS) alpha-subunit gene, ASA2, has been isolated from an unselected, but 5-methyl-tryptophan-resistant (5MTr), tobacco (Nicotiana tabacum) cell line (AB15-12-1). The ASA2 cDNA contains a putative transit peptide sequence, and Southern hybridization shows that more than one closely related sequence is present in the tobacco genome. The ASA2 cDNA complemented a trpE nonsense mutant Escherichia coli strain, allowing growth on 300 microm 5MT-containing minimal medium without tryptophan, and cell extracts contained feedback-insensitive AS activity. The 5MTr was lost when the E. coli strain was transformed with an ASA2 site-directed mutant (phenylalanine-107-arginine-108 --> serine-107-glutamine-108). Identical nucleotide sequences encoding the phenylalanine-107-arginine-108 region have been found in polymerase chain reaction-amplified 326-bp ASA2 genomic fragments of wild-type (5-methyl-tryptophan-sensitive [5MTs]) tobacco and a progenitor species. High-level ASA2 transcriptional expression was detected only in 5MTr-cultured cells, not in 5MTs cells or in plants. Promoter studies indicate that tissue specificity of ASA2 is controlled by the promoter region between -2252 and -607. Since the ASA2 promoter sequences are not substantially different in the 5MTr and 5MTs lines, the increased levels of ASA2 mRNA in the 5MTr lines are most likely due to changes in a regulatory gene affecting ASA2 expression.
Assuntos
Antranilato Sintase/metabolismo , Regulação da Expressão Gênica de Plantas , Nicotiana/enzimologia , Plantas Tóxicas , Sequência de Aminoácidos , Antranilato Sintase/química , Antranilato Sintase/genética , Sequência de Bases , Células Cultivadas , Primers do DNA , DNA Complementar , Escherichia coli/genética , Retroalimentação , Regulação Enzimológica da Expressão Gênica , Teste de Complementação Genética , Dados de Sequência Molecular , Plantas/enzimologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Nicotiana/citologia , Triptofano/farmacologiaRESUMO
To determine the site of action of systemic ethanol on dopaminergic function in the nucleus accumbens, we compared the effect of intraperitoneal (i.p.) and local administration of ethanol on interstitial dopamine concentration using microdialysis in freely moving rats. The i.p. administration of 1 g/kg of ethanol significantly increased the dialysate dopamine (DA) concentrations (approximately 40% above basal), compared with saline treatment. The concentration-time profile of DA and ethanol in dialysates was similar after two ethanol injections 4 hr apart. Local perfusion with several ethanol concentrations showed that 510 and 860 mM of ethanol caused a significant concentration-related increase in extracellular DA concentrations in the nucleus accumbens (510 mM, 28% increase; 860 mM, 62% increase). However, lower ethanol concentrations, 170 mM or below, failed to change basal DA concentrations. Stimulation with high potassium (50 mM) in artificial cerebrospinal fluid preceding local ethanol treatment increased dialysate DA concentrations to 523 +/- 83% of basal levels, confirming that the DA terminals were responsive to pharmacological manipulation. Basal DA levels in dialysates were approximately 70% calcium-dependent when tested at the end of the local perfusion experiments. Stereological examination of the nucleus accumbens revealed probe-induced damage, but did not detect additional damage by local perfusion of ethanol. When ethanol concentrations in the DA sampling area around the probe are taken into account in both systemic and local administration experiments, this study suggests that concentrations of ethanol associated with moderate intoxication do not directly affect the function of DA terminals in the nucleus accumbens. Therefore, the systemic effects of ethanol on nucleus accumbens DAergic function is more likely due to an interaction with sites other than the nucleus accumbens.
Assuntos
Intoxicação Alcoólica/fisiopatologia , Dopamina/metabolismo , Etanol/farmacologia , Espaço Extracelular/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Animais , Mapeamento Encefálico , Relação Dose-Resposta a Droga , Espaço Extracelular/fisiologia , Injeções Intraperitoneais , Masculino , Microdiálise , Núcleo Accumbens/fisiopatologia , Ratos , Ratos Sprague-Dawley , Receptores Dopaminérgicos/efeitos dos fármacos , Receptores Dopaminérgicos/fisiologiaRESUMO
Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secretory pathway, but molecular aspects about their involvement are poorly understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phaseolus vulgaris; Serpin from Manduca sexta) to direct a modified beta-glucuronidase (GUS) protein into the secretory pathway in transgenic tobacco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular fluid analysis (low- or high-ionic-strength conditions) but occurred in membranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equilibrated with the plasma membrane (PM) and the ER on linear sucrose gradients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed cultured cells prepared from GUS control and HRGP signal peptide (SP)-GUS-expressing plants suggested only cytosolic localization in GUS-expressing plants but substantial peripheral localization in HRGP SP-GUS plants, which is consistent with GUS being associated with the PM. Aqueous two-phase partitioning of microsomal membranes from HRGP SP-GUS and Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydrophobic moment plot analysis, suggest that properties of the SP-GUS protein result in its retention in the secretory pathway and PM.
Assuntos
Fusão Gênica Artificial , Glucuronidase/genética , Sinais Direcionadores de Proteínas/genética , Animais , Sequência de Bases , Western Blotting , Fracionamento Celular , Membrana Celular/enzimologia , Células Cultivadas , Glucuronidase/metabolismo , Glicoproteínas/genética , Manduca , Microscopia Confocal , Microssomos/enzimologia , Dados de Sequência Molecular , Proteínas de Plantas/genética , Plantas Tóxicas , Nicotiana/citologia , Nicotiana/genéticaRESUMO
Analysis of the genomic organization of tissue transglutaminase shows that the gene is 32.5 kilobases, contains 13 exons and 12 introns. Our results show that the sites for the two alternative splicing forms of tissue transglutaminase we reported earlier are located within exons 6 and 10 respectively. The 5'-upstream region of the gene has several potential regulatory promoter elements, and the 3'-exon contains about 50% of the total cDNA size and codes for the C-terminus of the protein. Alignment of deduced tissue transglutaminase amino acid sequence with other transglutaminases showed very similar intron splice positions.
Assuntos
Éxons , Íntrons , Transglutaminases/genética , Processamento Alternativo , Sequência de Aminoácidos , DNA , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Homologia de Sequência de AminoácidosRESUMO
We have isolated and characterized Tgmr, a copia-like retrotransposon, linked tightly to the Rps1-k allele that confers race-specific resistance of soybean to the the fungal pathogen Phytophthora sojae. Southern analysis followed by PCR and sequence analyses, using primers based on sequences flanking the insertion site confirmed that the element was inserted in the neighboring region of Rps1-k but not in that of the other four Rps1 alleles. This implies that Tgmr was transposed into the Rps1-k flanking site after the divergence of Rps1 alleles. Southern analysis of a series of diverse soybean cultivars revealed a high level of polymorphism of Tgmr-related sequences. These results indicate that this low copy retroelement family could have been active in the soybean genome in the recent past. Tgmr contains long terminal repeats (LTR) and four non-overlapping open reading frames (ORF), presumably originating from mutations leading to stop codons of a single ORF. The conserved domains for gag, protease, integrase, reverse transcriptase and RNaseH are present in the internal portion of the element. However, the protease, reverse transcriptase and RNaseH of this element are non-functional due to the presence of several stop codons. Possible transactivation of Tgmr and application of this element in insertional mutagenesis for soybean are discussed.
Assuntos
Alelos , Genes de Plantas , Ligação Genética , Glycine max/genética , Phytophthora/patogenicidade , Retroelementos , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cosmídeos/química , Marcadores Genéticos , Genoma de Planta , Imunidade Inata , Dados de Sequência Molecular , Phytophthora/genética , Polimorfismo de Fragmento de Restrição , Glycine max/química , Especificidade da EspécieRESUMO
The concentration-related effects of ethanol on extracellular dopamine (DA) in rat striatum were studied by direct perfusion through microdialysis probes in freely moving rats. Two sets of three ethanol concentrations were separately tested using a Latin square experimental design. Potassium stimulation with high potassium (50 mM) in artificial CSF (ACSF) preceding ethanol treatment confirmed the neuronal function of dopaminergic cells by increasing DA concentrations to 200-1,500% of basal levels. The perfusion with calcium-free ACSF applied at the end of each experiment confirmed the calcium dependency of the basal levels of extracellular DA by decreasing basal DA levels by 70%. The striatal volume measurement to examine the possible brain damage by direct ethanol perfusion suggested that ethanol did not increase the damage caused by the probe implantation at any ethanol concentration tested in this study. The 30-min direct perfusion of 510 and 860 mM ethanol resulted in a significant concentration-related stimulatory effect on the extracellular DA concentration in rat striatum (510 mM, 29% increase, p < 0.05; 860 mM, 66% increase, p < 0.05). However, there was no significant effect of ethanol at low concentrations, < or = 170 mM. Considering the effective ethanol concentration in tissue areas in which DA is sampled, the data suggest that concentrations of ethanol associated with moderate intoxication do not directly affect the extracellular concentration of DA in the striatum. Therefore, the systemic effects of ethanol on striatal DA found in previous studies may be caused by the interaction with sites other than the striatum.