RESUMO
There is evidence that suggests that increased corticotropin-releasing hormone (CRH) release in the central nucleus of the amygdala underlies the anxiogenic and stress-like consequences of withdrawal that are common in phenomenology to all drugs of abuse. The present studies were undertaken to determine levels of CRH mRNA in the amygdala, and also in the hypothalamus, frontal cortex and brainstem after short-term (2 days) and intermediate-term (10 days) cocaine withdrawal (with continued saline injections) from chronic (14 days) 'binge' pattern cocaine administration (3 x 15 mg/kg per day at hourly intervals). Confirming our recent finding of an activation of stress responsive hypothalamic-pituitary-adrenal activity during early cocaine withdrawal, there was a significant elevation of plasma corticosterone level after 2-day cocaine withdrawal. There was also a significant elevation of CRH mRNA levels in the amygdala, but not in the hypothalamus, frontal cortex or brainstem after 2-day cocaine withdrawal. A negative correlation between amygdalar CRH mRNA and plasma corticosterone levels was found in the 2-day cocaine withdrawn rats but not in control rats, suggesting that CRH neurons in the amygdala may be differentially responsive to glucocorticoids after chronic cocaine exposure and withdrawal. There were no changes in either plasma corticosterone or amygdalar CRH mRNA levels after 10-day cocaine withdrawal. Our findings of an increase in amygdalar CRH gene expression during early cocaine withdrawal support a potentially important role for amygdalar CRH activity in the anxiogenic and aversive consequences of withdrawal from cocaine during a time when humans are most subject to relapse.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/fisiologia , Transtornos Relacionados ao Uso de Cocaína/fisiopatologia , Hormônio Liberador da Corticotropina/genética , Síndrome de Abstinência a Substâncias/fisiopatologia , Animais , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/fisiologia , Doença Crônica , Cocaína/farmacologia , Corticosterona/sangue , Inibidores da Captação de Dopamina/farmacologia , Encefalinas/genética , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/fisiologia , Expressão Gênica/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/fisiologia , Masculino , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/fisiologia , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344RESUMO
We have previously demonstrated that there are stimulatory effects of acute (1 day) "binge" cocaine on corticotropin-releasing hormone (CRH) gene expression in the rat hypothalamus and on the stress responsive hypothalamic-pituitary-adrenal (HPA) activity. The first aim of the present study was to investigate the possible role of dopamine (DA) D1- or D2-like receptors (D1R or D2R) in modulating these acute effects. Administration of acute "binge" cocaine (3x15 mg/kg, i.p.) was preceded by injections of either the selective D1R antagonist (SCH23390, 2 mg/kg) or D2R antagonist (sulpiride, 50 mg/kg). The D1R or D2R blockade by SCH23390 or sulpiride, respectively, did not alter the mRNA levels of CRH in the hypothalamus, CRH-R1 or proopiomelanocortin (POMC) in the anterior pituitary. However, the acute "binge" cocaine-induced increase in hypothalamic CRH mRNA levels was not found in the rats that received either D1R or D2R antagonist pretreatment. In the anterior pituitary, acute "binge" cocaine or its combinations with either DA antagonist did not alter CRH-R1 receptor or POMC mRNA levels. Both the D1R and D2R antagonists attenuated the elevation of plasma corticosterone levels induced by acute "binge" cocaine. These results suggest that both D1R and D2R mediate acute cocaine's stimulatory effect on HPA axis at the hypothalamic CRH level. Neurobiological evidence has demonstrated functional interactions between dopaminergic and opioidergic systems that regulate preproenkephalin and preprodynorphin gene expression in the striatum. The second aim of our study was to investigate the roles that D1R or D2R could play in regulation of POMC mRNA levels in the hypothalamus in response to acute "binge" cocaine. The D2R blockade by sulpiride increased POMC mRNA levels in the hypothalamus, indicating that D2R exerts a tonic inhibitory effect on hypothalamic POMC gene expression. The POMC mRNA increases induced by the D2R blockade were attenuated by acute "binge" cocaine. Neither the D2R blockade nor acute "binge" cocaine altered POMC mRNA levels in the amygdala, anterior pituitary or neurointermediate lobe of the pituitary. In contrast to the D2R, the D1R blockade by SCH23390, acute "binge" cocaine or their combination had no effect on hypothalamic POMC mRNA levels. These results support a specific role for D2R in acute cocaine's effects on hypothalamic POMC gene expression.
Assuntos
Cocaína/farmacologia , Hormônio Liberador da Corticotropina/metabolismo , Antagonistas de Dopamina/farmacologia , Inibidores da Captação de Dopamina/administração & dosagem , Inibidores da Captação de Dopamina/farmacologia , Hipotálamo/efeitos dos fármacos , Pró-Opiomelanocortina/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Benzazepinas/farmacologia , Corticosterona/sangue , Hormônio Liberador da Corticotropina/genética , Interações Medicamentosas , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Hipotálamo/anatomia & histologia , Masculino , Hipófise/efeitos dos fármacos , Pró-Opiomelanocortina/genética , RNA Mensageiro/metabolismo , Radioimunoensaio/métodos , Ratos , Ratos Endogâmicos F344 , Sulpirida/farmacologiaRESUMO
As opiates increase dopamine transmission, we measured the effects of morphine on dopamine-related genes using a real-time optic PCR assay that reliably detects small differences in mRNA in discrete brain regions. Tissue from dopaminoceptive and dopaminergic brain regions was collected from rats injected twice daily for 7 days with saline or increasing doses of morphine. Tissues were assayed for D1, D2 and D3 dopamine receptor mRNAs (D1R, D2R and D3R), as well as for mRNAs for tyrosine hydroxylase (TH) and the dopamine transporter (DAT). The neuron-associated mRNAs for SNAP-25 and synaptophysin, as well as the glial-associated mRNA for S100-beta and three 'housekeeping' mRNAs, were also measured. As reported previously by others, there was no alteration in D1R mRNA and a 25% decrease in D2R mRNA in the caudate-putamen, 2 h after the final morphine injection. Importantly, in the same RNA extracts, D3R mRNA showed significant increases of 85% in the caudate-putamen and 165% in the ventral midbrain, including the substantia nigra and ventral tegmental area. There were no other significant morphine effects. Mapping of brain regions in saline control rats agreed with previous studies, including showing the presence of low abundance TH mRNA and the absence of DAT mRNA in the caudate-putamen. The finding that chronic, intermittent injections of morphine caused an increase in D3R mRNA extends our understanding of the ability of D3R agonists to reduce the effects of morphine.
Assuntos
Encéfalo/efeitos dos fármacos , Dopamina/metabolismo , Glicoproteínas de Membrana , Morfina/farmacologia , Neurônios/efeitos dos fármacos , Receptores de Dopamina D2/genética , Regulação para Cima/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Dependência de Morfina/genética , Dependência de Morfina/metabolismo , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D3 , Proteínas S100/genética , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Sinaptofisina/genética , Proteína 25 Associada a Sinaptossoma , Tirosina 3-Mono-Oxigenase/genética , Regulação para Cima/genética , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismoRESUMO
Drugs abused by humans are thought to activate areas in the ventral striatum of the brain that engage the organism in important adaptive behaviors, such as eating. In support of this, we report here that striatal regions of sugar-dependent rats show alterations in dopamine and opioid mRNA levels similar to morphine-dependent rats. Specifically, after a chronic schedule of intermittent bingeing on a sucrose solution, mRNA levels for the D2 dopamine receptor, and the preproenkephalin and preprotachykinin genes were decreased in dopamine-receptive regions of the forebrain, while D3 dopamine receptor mRNA was increased. While morphine affects gene expression across the entire dopamine-receptive striatum, significant differences were detected in the effects of sugar on the nucleus accumbens and adjacent caudate-putamen. The effects of sugar on mRNA levels were of greater magnitude in the nucleus accumbens than in the caudate-putamen. These areas also showed clear differences in the interactions among the genes, especially between D3R and the other genes. This was revealed by a novel multivariate analysis method that identified cooperative interactions among genes, specifically in the nucleus accumbens but not the caudate-putamen. Finally, a role for these cooperative interactions in a load-sharing response to perturbations caused by sugar was supported by the finding of a different pattern of correlations between the genes in the two striatal regions. These findings support a major role for the nucleus accumbens in mediating the effects of naturally rewarding substances and extend an animal model for studying the common substrates of drug addiction and eating disorders.
Assuntos
Encéfalo/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Morfina/farmacologia , Vias Neurais/metabolismo , Recompensa , Sacarose/farmacologia , Animais , Encéfalo/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Encefalinas/genética , Transtornos da Alimentação e da Ingestão de Alimentos/genética , Transtornos da Alimentação e da Ingestão de Alimentos/metabolismo , Expressão Gênica/genética , Perfilação da Expressão Gênica , Masculino , Vias Neurais/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Peptídeos Opioides/genética , Precursores de Proteínas/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D2/genética , Receptores de Dopamina D3 , Transtornos Relacionados ao Uso de Substâncias/genética , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Taquicininas/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Tolerance to the stimulatory effects of cocaine on the hypothalamic-pituitary-adrenal (HPA) axis develops after chronic 'binge' cocaine exposure in the rat. This blunting of HPA axis activity in response to cocaine is associated with a cocaine-induced reduction of corticotropin-releasing hormone (CRH) mRNA level in the hypothalamus. There is limited information about the effects of withdrawal from chronic cocaine on HPA activity. The present studies were undertaken to determine levels of the HPA hormones adrenocorticotropic hormone (ACTH) and corticosterone across 10 days of withdrawal following chronic 'binge' pattern cocaine administration (3 x 15 mg/kg/day at hourly intervals) for 14 days. Male Fischer rats showed a significantly attenuated HPA axis response to chronic 'binge' pattern cocaine administration 30 min after the last injection on the 14th day, as measured by both plasma ACTH and corticosterone levels at the nadir time point. Twenty-four hours following the final administration of 'binge' cocaine (the 1st day of withdrawal), a significant elevation of plasma ACTH levels and a modest, but significant, elevation of plasma corticosterone levels were found at the nadir time point. This acute withdrawal-related activation of the hormones of the HPA axis was no longer found on the 10th day of withdrawal. In the anterior pituitary, levels of both proopiomelanocortin (POMC) and CRH-receptor 1 (R1) mRNAs were significantly higher than saline controls on the 14th day of chronic 'binge' cocaine and were at control levels on the 4th day of withdrawal. In the neurointermediate lobe of the pituitary, a sustained reduction in POMC mRNA levels was observed on the 3rd, 7th and 14th day of chronic 'binge' cocaine, but POMC mRNA was at control levels by the 4th day of withdrawal. In the hypothalamus, POMC mRNA levels showed a transient decrease on the 1st day of 'binge' cocaine with no change during chronic 'binge' cocaine or its withdrawal. CRH mRNA levels in the hypothalamus were not different from saline controls on the 1st and 4th days of withdrawal. Taken together, the present results show that after development of adaptation or tolerance to chronic 'binge' cocaine there is an increase in HPA activity during acute cocaine withdrawal. In addition to being associated with CRH input from the hypothalamus, the activation of the HPA axis by cocaine withdrawal may be, at least in part, due to the increased POMC and/or CRH-R1 gene expression observed in the anterior pituitary after chronic 'binge' cocaine.
Assuntos
Glândulas Suprarrenais/efeitos dos fármacos , Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Hipófise/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Pró-Opiomelanocortina/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Síndrome de Abstinência a Substâncias/metabolismo , Glândulas Suprarrenais/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Animais , Northern Blotting , Cocaína/administração & dosagem , Corticosterona/metabolismo , Hormônio Liberador da Corticotropina/efeitos dos fármacos , Hormônio Liberador da Corticotropina/metabolismo , Inibidores da Captação de Dopamina/administração & dosagem , Encefalinas/metabolismo , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Masculino , Hipófise/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Pró-Opiomelanocortina/efeitos dos fármacos , Precursores de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Radioimunoensaio , Ratos , Ratos Endogâmicos F344 , Receptores de Hormônio Liberador da Corticotropina/efeitos dos fármacos , Receptores de Hormônio Liberador da Corticotropina/genética , Fatores de TempoRESUMO
BACKGROUND: PCR in principle can detect a single target molecule in a reaction mixture. Contaminating bacterial DNA in reagents creates a practical limit on the use of PCR to detect dilute bacterial DNA in environmental or public health samples. The most pernicious source of contamination is microbial DNA in DNA polymerase preparations. Importantly, all commercial Taq polymerase preparations inevitably contain contaminating microbial DNA. Removal of DNA from an enzyme preparation is problematical. METHODOLOGY/PRINCIPAL FINDINGS: This report demonstrates that the background of contaminating DNA detected by quantitative PCR with broad host range primers can be decreased greater than 10-fold through the simple expedient of Taq enzyme dilution, without altering detection of target microbes in samples. The general method is: For any thermostable polymerase used for high-sensitivity detection, do a dilution series of the polymerase crossed with a dilution series of DNA or bacteria that work well with the test primers. For further work use the concentration of polymerase that gave the least signal in its negative control (H(2)O) while also not changing the threshold cycle for dilutions of spiked DNA or bacteria compared to higher concentrations of Taq polymerase. CONCLUSIONS/SIGNIFICANCE: It is clear from the studies shown in this report that a straightforward procedure of optimizing the Taq polymerase concentration achieved "treatment-free" attenuation of interference by contaminating bacterial DNA in Taq polymerase preparations. This procedure should facilitate detection and quantification with broad host range primers of a small number of bona fide bacteria (as few as one) in a sample.
Assuntos
Taq Polimerase/química , Técnicas de Tipagem Bacteriana/métodos , Sequência de Bases , Clonagem Molecular , DNA/análise , DNA/isolamento & purificação , DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , DNA Bacteriano/genética , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Pseudomonas fluorescens/genética , RNA Ribossômico 16S/química , Homologia de Sequência do Ácido Nucleico , Taq Polimerase/metabolismo , beta-Lactamases/metabolismoRESUMO
The algebra of target theory for damage by radiation was laid out by Atwood and Norman in 1949. Their equations provide a widely embraced framework for distinguishing single-hit and multi-hit mechanisms of damage. The present work asks whether in vitro damage to DNA duplexes by different agents affects amplification by the polymerase chain reaction (PCR) in a single-hit manner. Real-time monitoring of fluorescent PCR product (qPCR) was used to measure the fraction of DNA (S) surviving doses (D) of three damaging agents: gamma irradiation, DNase I, and UV radiation. The log fraction surviving was compared to the best-fit straight line predicted for a random single-hit model (lnS=kD). Human DNA targets for analysis were segments of multiple (nested) DNA lengths from the nuclear and the mitochondrial genomes within 10% of 150, 250, 350, 450, 650, 1000 and 2000 bases. For gamma irradiation, the results were consistent with a single-hit model for all segment sizes. In the case of DNase I, the shortest segment (150 bp), for both genomic and mitochondrial DNA, experienced more damage at low concentrations of DNase than the random single-hit model predicted. Conversely, in the case of UV, all segments of the nuclear target gene were less damaged at low doses and more damaged at high doses than predicted by the one hit model. These deviations from the predictions of a random single-hit model were interpreted as evidence for concerted activity in the case of DNase and of a multi-hit, sequence-dependent mechanism in the case of UV, perhaps due to the accumulation of lesions that slowed but did not entirely block Taq polymerase elongation.
Assuntos
Dano ao DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , DNA/efeitos da radiação , Desoxirribonuclease I/metabolismo , Raios gama , Humanos , Modelos Estatísticos , Reação em Cadeia da Polimerase/métodos , Raios UltravioletaRESUMO
Cocaine administration increases activity at dopamine receptors, increases preprodynorphin (ppDyn) gene expression in the caudate-putamen (CPu), and activates the stress responsive hypothalamic-pituitary-adrenal (HPA) axis. To examine the hypothesis that mu-opioid receptors (MOR) may play roles in these cocaine effects, we tested the effects of acute "binge" pattern cocaine administration in mice with targeted disruption of the MOR gene. Wild-type (+/+) and homozygous MOR-deficient (-/-) mice received three injections of 15 mg/kg cocaine at 1-h intervals. Mice were sacrificed 30 min after the last injection and mRNAs for ppDyn and preproenkephalin (ppEnk) in the CPu and nucleus accumbens (NAc), and for type I corticotropin-releasing hormone receptor (CRH(1) receptor) and pro-opiomelanocortin (POMC) in the hypothalamus and pituitary, were measured by solution hybridization RNase protection assays. Cocaine elevated ppDyn mRNA in the CPu, but not NAc, of both the MOR -/- and wild-type mice. ppEnk mRNA in the CPu, but not NAc, was lower in MOR -/- mice than in wild-type mice following cocaine administration. Hypothalamic CRH(1) receptor and POMC mRNAs were expressed at similar levels in untreated and in cocaine-treated mice of each genotype. However, there were lower basal levels of CRH(1) receptor mRNA in the anterior pituitary of the MOR -/- mice than in wild-type mice and the MOR -/- mice failed to show the cocaine-induced decreases in CRH(1) receptor mRNA found in the wild-type mice. Cocaine activated the HPA axis similarly in MOR -/- and wild-type mice, as reflected in similar increases in plasma corticosterone levels in both genotypes. These results support a specific role for MORs in acute cocaine effects on striatal ppEnk gene expression and fail to support critical roles for these receptors in acute cocaine's effects on either ppDyn gene expression or HPA activation. MOR -/- mice are useful models for studying cocaine effects on ppEnk gene expression that could aid interpretation of the similar postmortem phenomena found in human cocaine addicts.