RESUMO
The rationale of spinal administration of endothelin-1(ET-1) mediated anti-nociceptive effect has not been elucidated. ET-1 is reported to promote nuclear effluxion of histone deacetylase 5 (HDAC5) in myocytes, and spinal HDAC5 is implicated in modulation of pain processing. In this study, we aimed to investigate whether central ET-1 plays an anti-nociceptive role by facilitating spinal HDAC5 nuclear shuttling under neuropathic pain. Here, we demonstrate that upregulating spinal ET-1 attenuated the nociception induced by partial sciatic nerve ligation surgery and this analgesic effect mediated by ET-1 was attenuated by intrathecal injection of endothelin A receptor selective inhibitor (BQ123) or by blocking the exportation of nuclear HDAC5 by adeno-associated viruses targeting neuronal HDAC5 (AVV-HDAC5 S259/498A Mutant). Notably, ET-1 administration increased spinal glutamate acid decarboxylases (GAD65/67) expression via initiating HDAC5 nuclear exportation and increased the acetylation of histone 3 at lysine 9 (Acetyl-H3K9) in the promotor regions of spinal Gad1 and Gad2 genes. This was reversed by blocking endothelin A receptor function or by inhibiting the spinal neuronal nuclear exportation of HDAC5. Therefore, inducing spinal GABAergic neuronal HDAC5 nuclear exportation may be a novel therapeutic approach for managing neuropathic pain. PERSPECTIVE: Neuropathic pain is intractable in a clinical setting, and epigenetic regulation is considered to contribute to this processing. Characterizing the anti-nociceptive effect of ET-1 and investigating the associated epigenetic mechanisms in animal models may lead to the development of new therapeutic strategies and targets for treating neuropathic pain.
Assuntos
Analgesia , Antagonistas dos Receptores de Endotelina/farmacologia , Endotelina-1/metabolismo , Glutamato Descarboxilase/metabolismo , Histona Desacetilases/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Animais , Antagonistas dos Receptores de Endotelina/administração & dosagem , Endotelina-1/efeitos dos fármacos , Glutamato Descarboxilase/efeitos dos fármacos , Histona Desacetilases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeos Cíclicos/farmacologiaRESUMO
Mammalian taste buds are comprised of specialized neuroepithelial cells that act as sensors for molecules that provide nutrition (e.g., carbohydrates, amino acids, and salts) and those that are potentially harmful (e.g., certain plant compounds and strong acids). Type II and III taste bud cells (TBCs) detect molecules described by humans as "sweet," "bitter," "umami," and "sour." TBCs that detect metallic ions, described by humans as "salty," are undefined. Historically, type I glial-like TBCs have been thought to play a supportive role in the taste bud, but little research has been done to explore their role in taste transduction. Some evidence implies that type I cells may detect sodium (Na+) via an amiloride-sensitive mechanism, suggesting they play a role in Na+ taste transduction. We used an optogenetic approach to study type I TBCs by driving the expression of the light-sensitive channelrhodopsin-2 (ChR2) in type I GAD65+ TBCs of male and female mice. Optogenetic stimulation of GAD65+ TBCs increased chorda tympani nerve activity and activated gustatory neurons in the rostral nucleus tractus solitarius. "N neurons," whose NaCl responses were blocked by the amiloride analog benzamil, responded robustly to light stimulation of GAD65+ TBCs on the anterior tongue. Two-bottle preference tests were conducted under Na+-replete and Na+-deplete conditions to assess the behavioral impact of optogenetic stimulation of GAD65+ TBCs. Under Na+-deplete conditions GAD65-ChR2-EYFP mice displayed a robust preference for H2O illuminated with 470 nm light versus nonilluminated H2O, suggesting that type I glial-like TBCs are sufficient for driving a behavior that resembles Na+ appetite.SIGNIFICANCE STATEMENT This is the first investigation on the role of type I GAD65+ taste bud cells (TBCs) in taste-mediated physiology and behavior via optogenetics. It details the first definitive evidence that selective optogenetic stimulation of glial-like GAD65+ TBCs evokes neural activity and modulates behavior. Optogenetic stimulation of GAD65+ TBCs on the anterior tongue had the strongest effect on gustatory neurons that responded best to NaCl stimulation through a benzamil-sensitive mechanism. Na+-depleted mice showed robust preferences to "light taste" (H2O illuminated with 470 nm light vs nonilluminated H2O), suggesting that the activation of GAD65+ cells may generate a salt-taste sensation in the brain. Together, our results shed new light on the role of GAD65+ TBCs in gustatory transduction and taste-mediated behavior.
Assuntos
Apetite/fisiologia , Preferências Alimentares/fisiologia , Glutamato Descarboxilase/fisiologia , Optogenética/métodos , Células Receptoras Sensoriais/fisiologia , Sódio/deficiência , Papilas Gustativas/fisiologia , Amilorida/farmacologia , Animais , Apetite/efeitos dos fármacos , Channelrhodopsins , Nervos Cranianos/fisiologia , Diuréticos/farmacologia , Feminino , Preferências Alimentares/efeitos dos fármacos , Glutamato Descarboxilase/efeitos dos fármacos , Masculino , Camundongos , Células Receptoras Sensoriais/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Papilas Gustativas/efeitos dos fármacosRESUMO
BACKGROUND: Extracts from Boswellia serrata gum resin have anti-inflammatory effect and are used for treatment of a variety of chronic inflammatory diseases. It was previously demonstrated that the treatment with Boswellia serrata gum resin of LADA (Latent Autoimmune Diabetes in Adults) patients decreased blood levels of IA2 antibodies, one of the markers associated with LADA autoimmune diabetes. PRIMARY STUDY OBJECTIVE: The purpose of this study was to test whether Boswellia serrata gum resin also influences GAD65 autoantibodies as the other marker associated with LADA. METHODS/DESIGN: We report a case study of male patient diagnosed with LADA with positive GAD65 autoantibodies who was treated with extract from Boswellia serrata gum resin, during 9 months. Blood levels of GAD65 autoantibodies, fasting blood glucose levels and HbA1c were measured before the treatment and periodically during the treatment. RESULTS: Over the observed period, the blood levels of GAD65 autoantibodies linearly decreased about 25%. CONCLUSION: The study confirms that extract of Boswellia serrata gum resin seems to prevent insulitis in patients with LADA, as indicated by its action on both markers of autoimmune diabetes, i.e., GAD65 and IA2 autoantibodies. The possibility that the treatment with boswellic acids of LADA patients with positive autoantibodies could be beneficial on the course of the disease, calls for further investigation and a clinical study.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Autoanticorpos/sangue , Boswellia/química , Glutamato Descarboxilase/efeitos dos fármacos , Diabetes Autoimune Latente em Adultos/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Resinas Vegetais/uso terapêutico , Adulto , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/efeitos adversos , Diabetes Mellitus Tipo 1 , Glutamato Descarboxilase/imunologia , Humanos , Diabetes Autoimune Latente em Adultos/diagnóstico , Masculino , Extratos Vegetais/farmacologia , Resinas Vegetais/efeitos adversos , Resultado do TratamentoRESUMO
BACKGROUND: Ethanol (EtOH) has diverse effects on nervous system development, which includes development and survival of GABAergic neurons in a sonic hedgehog (Shh) and fibroblast growth factor (Fgf)-dependent mechanism. Cannabinoids also function as inhibitors of Shh signaling, raising the possibility that EtOH and cannabinoids may interact to broadly disrupt neuronal function during brain development. METHODS: Zebrafish embryos were exposed to a range of EtOH and/or cannabinoid receptor 1 (CB1R) agonist concentrations at specific developmental stages, in the absence or presence of morpholino oligonucleotides that disrupt shh expression. In situ hybridization was employed to analyze glutamic acid decarboxylase (gad1) gene expression as a marker of GABAergic neuron differentiation, and zebrafish behavior was analyzed using the novel tank diving test as a measure of risk-taking behavior. RESULTS: Combined acute subthreshold EtOH and CB1R agonist exposure results in a marked reduction in gad1 mRNA expression in zebrafish forebrain. Consistent with the EtOH and cannabinoid effects on Shh signaling, fgf8 mRNA overexpression rescues the EtOH- and cannabinoid-induced decrease in gad1 gene expression and also prevents the changes in behavior induced by EtOH and cannabinoids. CONCLUSIONS: These studies provide evidence that forebrain GABAergic neuron development and zebrafish risk-taking behavior are sensitive to both EtOH and cannabinoid exposure in a Shh- and Fgf-dependent mechanism, and provide additional evidence that a signaling pathway involving Shh and Fgf crosstalk is a critical target of EtOH and cannabinoids in FASD.
Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Fatores de Crescimento de Fibroblastos/genética , Neurônios GABAérgicos/efeitos dos fármacos , Proteínas Hedgehog/genética , Neurogênese/efeitos dos fármacos , Proteínas de Peixe-Zebra/genética , Animais , Comportamento Animal/efeitos dos fármacos , Embrião não Mamífero , Expressão Gênica , Glutamato Descarboxilase/efeitos dos fármacos , Glutamato Descarboxilase/genética , Proteínas Hedgehog/efeitos dos fármacos , Hibridização In Situ , Morfolinos , Neurogênese/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor CB1 de Canabinoide/agonistas , Assunção de Riscos , Peixe-Zebra , Proteínas de Peixe-Zebra/efeitos dos fármacosRESUMO
Maternal infection during pregnancy is considered a key risk factor for developing schizophrenia in offspring. There is evidence that maternal exposure to infectious agents is associated with fetal zinc deficiency. Due to the essential role of zinc in brain function and development, in the present study, we activated maternal immune system using lipopolysaccharide (LPS) as a model of schizophrenia to examine whether zinc supplementation throughout pregnancy can reverse LPS-induced deleterious effects. To test the hypothesis, pregnant rats were treated with intraperitoneal injection of either saline or LPS (0.5 mg/kg) at gestational day 15 and 16, and zinc supplementation (30 mg/kg) was administered throughout pregnancy by gavage. At postnatal day 60, Y-maze was used to evaluate working memory of offspring. Moreover, the expression levels of catechol O-methyltransferase (COMT) and glutamate decarboxylase 67 (GAD67) were measured in the frontal cortex of the brain samples. Only male offspring prenatally exposed to LPS showed a significant impairment in working memory. In addition, prenatal LPS exposure causes a moderate decrease in GAD67 expression level in the male pups, while COMT expression was found unchanged. Interestingly, zinc supplementation restored the alterations in working memory as well as GAD67 mRNA level in the male rats. No alteration was detected for neither working memory nor COMT/GAD67 genes expression in female offspring. This study demonstrates that zinc supplementation during pregnancy can attenuate LPS-induced impairments in male pups. These results support the idea to consume zinc supplementation during pregnancy to limit neurodevelopmental deficits induced by infections in offspring.
Assuntos
Suplementos Nutricionais , Glutamato Descarboxilase , Lipopolissacarídeos/farmacologia , Memória de Curto Prazo , Transtornos do Neurodesenvolvimento/prevenção & controle , Efeitos Tardios da Exposição Pré-Natal/prevenção & controle , Caracteres Sexuais , Oligoelementos/farmacologia , Zinco/farmacologia , Animais , Catecol O-Metiltransferase/metabolismo , Feminino , Glutamato Descarboxilase/efeitos dos fármacos , Glutamato Descarboxilase/metabolismo , Lipopolissacarídeos/administração & dosagem , Masculino , Memória de Curto Prazo/fisiologia , Transtornos do Neurodesenvolvimento/etiologia , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/fisiopatologia , Córtex Pré-Frontal/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , RNA Mensageiro , Ratos , Ratos Wistar , Oligoelementos/administração & dosagem , Zinco/administração & dosagemRESUMO
Bacterial L-aspartate α-decarboxylase (PanD) is a potential biocatalyst for the green production of ß-alanine, an important block chemical for manufacturing nitrogen-containing chemicals in bio-refinery field. It was reported that the poor catalytic stability caused by substrate inactivation limited the large-scale application. Here, we investigated the characters of inactivation by L-aspartate of PanD from Corynebacterium jeikeium (PDCjei), and found that L-aspartate induced a time-, and concentration-dependent inactivation of PDCjei with the values of KI and kinact being 288.4 mM and 0.235/min, respectively. To improve the catalytic stability of PDCjei, conserved amino acid residues essential to catalytic stability were analyzed by comparing the discrepancy in the observed inactivation rate of various sources. By an efficient colorimetric high-throughput screening method, four mutants with 3.18-24.69% higher activity were obtained from mutant libraries. Among them, the best mutation (R3K) also performed 66.38% higher catalytic stability than the wild type, showing great potential for industrial bio-production of ß-alanine.
Assuntos
Ácido Aspártico/metabolismo , Corynebacterium/enzimologia , Estabilidade Enzimática , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Mutagênese Sítio-Dirigida , Sequência de Aminoácidos , Ácido Aspártico/farmacologia , Bactérias/enzimologia , Carboxiliases/genética , Carboxiliases/metabolismo , Domínio Catalítico/genética , Estabilidade Enzimática/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glutamato Descarboxilase/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Microbiologia Industrial , Cinética , Alinhamento de Sequência , Especificidade por Substrato , Fatores de Tempo , beta-Alanina/biossínteseRESUMO
The cognitive symptoms of schizophrenia are poorly understood and difficult to treat. Estrogens may mitigate these symptoms via unknown mechanisms. To examine these mechanisms, we tested whether increasing estradiol (E) or decreasing luteinizing hormone (LH) could mitigate short-term episodic memory loss in a phencyclidine (PCP) model of schizophrenia. We then assessed whether changes in cortical or hippocampal GABA may underlie these effects. Female rats were ovariectomized and injected subchronically with PCP. To modulate E and LH, animals received estradiol capsules or Antide injections. Short-term episodic memory was assessed using the novel object recognition task (NORT). Brain expression of GAD67 was analyzed via western blot, and parvalbumin-containing cells were counted using immunohistochemistry. Some rats received hippocampal infusions of a GABAA agonist, GABAA antagonist, or GAD inhibitor before behavioral testing. We found that PCP reduced hippocampal GAD67 and abolished recognition memory. Antide restored hippocampal GAD67 and rescued recognition memory in PCP-treated animals. Estradiol prevented PCP's amnesic effect in NORT but failed to restore hippocampal GAD67. PCP did not cause significant differences in number of parvalbumin-expressing cells or cortical expression of GAD67. Hippocampal infusions of a GABAA agonist restored recognition memory in PCP-treated rats. Blocking hippocampal GAD or GABAA receptors in ovx animals reproduced recognition memory loss similar to PCP and inhibited estradiol's protection of recognition memory in PCP-treated animals. In summary, decreasing LH or increasing E can lessen short-term episodic memory loss, as measured by novel object recognition, in a PCP model of schizophrenia. Alterations in hippocampal GABA may contribute to both PCP's effects on recognition memory and the hormones' ability to prevent or reverse them.
Assuntos
Estradiol/fisiologia , Hormônio Luteinizante/fisiologia , Memória/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Glutamato Descarboxilase/efeitos dos fármacos , Hipocampo/metabolismo , Memória/fisiologia , Transtornos da Memória/metabolismo , Memória de Curto Prazo/efeitos dos fármacos , Fenciclidina/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/metabolismo , Reconhecimento Psicológico/efeitos dos fármacos , Esquizofrenia/metabolismoRESUMO
The citrus red mite, Panonychus citri, is one of the most economically and globally destructive mite pests of citrus. Acaricide resistance has been a growing problem in controlling this pest. As the main inhibitory neurotransmitter in organisms, γ-aminobutyric acid (GABA) is synthesized from the amino acid glutamate by the action of glutamate decarboxylases (GADs). In the present study, one novel GAD gene, PcGAD, was identified and characterized from P. citri. The opening reading frame of PcGAD contained 1548 nucleotides that encode 515 amino acids. The subsequent spatiotemporal expression pattern by RT-qPCR revealed that the expression levels of PcGAD were significantly higher in larvae than in adults. Challenging with various concentrations of abamectin resulted in the upregulation of PcGAD transcript levels. Furthermore, biochemical characterization indicated that changes in GAD activity coincided with its mRNA levels. High-performance liquid chromatography confirmed that the GABA contents of P. citri increased upon abamectin treatment. The application of abamectin induces PcGAD expression and activates GAD activity, thereby resulting in an increase in GABA content in P. citri, which contributes to the adaptability of the mite to abamectin challenge.
Assuntos
Glutamato Descarboxilase/metabolismo , Ivermectina/análogos & derivados , Tetranychidae , Ácido gama-Aminobutírico/metabolismo , Animais , Glutamato Descarboxilase/efeitos dos fármacos , Ivermectina/farmacologiaRESUMO
A 59-year-old Japanese woman developed diabetes mellitus without ketoacidosis in the presence of glutamic acid decarboxylase autoantibody (GADA) (24.7 U/mL). After the amelioration of her hyperglycemia, the patient had a relatively preserved serum C-peptide level. Her endogenous insulin secretion capacity remained almost unchanged during 5 years of insulin therapy. The patient's GADA titers normalized within 15 months. The islet-related autoantibodies, including GADA, are believed to be produced following the autoimmune destruction of pancreatic beta cells and are predictive markers of type 1 diabetes mellitus. Therefore, the transient appearance of GADA in our patient may have reflected pancreatic autoimmune processes that terminated without progression to insulin deficiency.
Assuntos
Autoanticorpos/sangue , Peptídeo C/sangue , Diabetes Mellitus Tipo 1/sangue , Glutamato Descarboxilase/sangue , Insulina/metabolismo , Pâncreas/metabolismo , Biomarcadores/sangue , Diabetes Mellitus Tipo 1/tratamento farmacológico , Progressão da Doença , Feminino , Glutamato Descarboxilase/efeitos dos fármacos , Humanos , Hiperglicemia/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Secreção de Insulina , Pessoa de Meia-Idade , Pâncreas/efeitos dos fármacos , Valor Preditivo dos Testes , Resultado do TratamentoRESUMO
Accumulating evidence supports the value of 5-HT1A receptor (5-HT1AR) agonists for dyskinesias that arise with long-term L-DOPA therapy in Parkinson's disease (PD). Yet, how 5-HT1AR stimulation directly influences the dyskinetogenic D1 receptor (D1R)-expressing striatonigral pathway remains largely unknown. To directly examine this, one cohort of hemiparkinsonian rats received systemic injections of Vehicle + Vehicle, Vehicle + the D1R agonist SKF81297 (0.8 mg/kg), or the 5-HT1AR agonist ±8-OH-DPAT (1.0 mg/kg) + SKF81297. Rats were examined for changes in abnormal involuntary movements (AIMs), rotations, striatal preprodynorphin (PPD), and glutamic acid decarboxylase (GAD; 65 and 67) mRNA via RT-PCR. In the second experiment, hemiparkinsonian rats received intrastriatal pretreatments of Vehicle (aCSF), ±8-OH-DPAT (7.5 mM), or ±8-OH-DPAT + the 5-HT1AR antagonist WAY100635 (4.6 mM), followed by systemic Vehicle or SKF81297 after which AIMs, rotations, and extracellular striatal glutamate and nigral GABA efflux were measured by in vivo microdialysis. Results revealed D1R agonist-induced AIMs were reduced by systemic and intrastriatal 5-HT1AR stimulation while rotations were enhanced. Although ±8-OH-DPAT did not modify D1R agonist-induced increases in striatal PPD mRNA, the D1R/5-HT1AR agonist combination enhanced GAD65 and GAD67 mRNA. When applied locally, ±8-OH-DPAT alone diminished striatal glutamate levels while the agonist combination increased nigral GABA efflux. Thus, presynaptic 5-HT1AR stimulation may attenuate striatal glutamate levels, resulting in diminished D1R-mediated dyskinetic behaviors, but maintain or enhance striatal postsynaptic factors ultimately increasing nigral GABA levels and rotational activity. The current findings offer a novel mechanistic explanation for previous results concerning 5-HT1AR agonists for the treatment of dyskinesia.
Assuntos
Agonistas de Dopamina/farmacologia , Atividade Motora/efeitos dos fármacos , Neostriado/efeitos dos fármacos , Receptor 5-HT1A de Serotonina , Receptores de Dopamina D1/agonistas , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Substância Negra/efeitos dos fármacos , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Benzazepinas/farmacologia , Dinorfinas/efeitos dos fármacos , Dinorfinas/metabolismo , Discinesia Induzida por Medicamentos , Glutamato Descarboxilase/efeitos dos fármacos , Glutamato Descarboxilase/metabolismo , Transtornos Parkinsonianos , Piperazinas/farmacologia , Precursores de Proteínas/efeitos dos fármacos , Precursores de Proteínas/metabolismo , Piridinas/farmacologia , Ratos , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologiaRESUMO
Brain injury during the last trimester to the first 1-4 years in humans is now thought to trigger an array of intellectual and emotional problems later in life, including disorders such as schizophrenia. In adult schizophrenic brains, there is a specific loss of neurons that co-express glutamic acid decarboxylase-parvalbumin (GAD67-PV). Loss of this phenotype is thought to occur in mature animals previously exposed to N-methyl-D: -aspartate receptor (NMDAR) antagonists during late gestation or at postnatal day 7 (P7). However, in similarly treated animals, we have previously shown that GAD67 and PV are unaltered in the first 24 h. To more precisely define when changes in these markers first occur, we exposed rat pups (P7 or P6-P10) to the NMDAR antagonist MK801 and at P11 co-stained brain sections for GAD67 or PV. In the cingulate cortex, we found evidence for a reduction in PV (GAD67 levels were very low to undetectable). In contrast, in the somatosensory cortex, we found that expression of GAD67 was reduced, but PV remained stable. Further, repeated but not single doses of MK801 were necessary to see such changes. Thus, depending on the region, NMDAR antagonism appears to influence expression of PV or GAD67, but not both. These observations could not have been predicted by previous studies and raise important questions as to how the GAD67-PV phenotype is lost once animals reach maturity. More importantly, such differential effects may be of great clinical importance, given that cognitive deficits are seen in children exposed to anesthetics that act by blocking the NMDAR.
Assuntos
Antagonistas de Aminoácidos Excitatórios/toxicidade , Glutamato Descarboxilase/metabolismo , Interneurônios/efeitos dos fármacos , Degeneração Neural/metabolismo , Parvalbuminas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Maleato de Dizocilpina/toxicidade , Glutamato Descarboxilase/efeitos dos fármacos , Giro do Cíngulo/efeitos dos fármacos , Giro do Cíngulo/metabolismo , Giro do Cíngulo/patologia , Imuno-Histoquímica , Interneurônios/metabolismo , Interneurônios/patologia , Degeneração Neural/induzido quimicamente , Degeneração Neural/fisiopatologia , Parvalbuminas/efeitos dos fármacos , Fenótipo , Ratos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Esquizofrenia/fisiopatologia , Córtex Somatossensorial/efeitos dos fármacos , Córtex Somatossensorial/metabolismo , Córtex Somatossensorial/patologia , Ácido gama-Aminobutírico/metabolismoRESUMO
Female rats are intensely affected by cocaine, with estrogen probably playing an important role in this effect. Progesterone modulates the GABA system and attenuates the effects of cocaine; however, there is no information about its relevance in changing GABA synthesis pathways after cocaine administration to female rats. Our objective was to investigate the influence of progesterone on the effects of repeated cocaine administration on the isoenzymes of glutamic acid decarboxylase (GAD65 and GAD67) mRNA in brain areas involved in the addiction circuitry. Ovariectomized, intact and progesterone replacement-treated female rats received saline or cocaine (30 mg/kg, ip) acutely or repeatedly. GAD isoenzyme mRNA levels were determined in the dorsolateral striatum (dSTR) and prefrontal cortex (PFC) by RT-PCR, showing that repeated, but not acute, cocaine decreased GADs/β-actin mRNA ratio in the dSTR irrespective of the hormonal condition (GAD65: P < 0.001; and GAD67: P = 0.004). In the PFC, repeated cocaine decreased GAD65 and increased GAD67 mRNA ratio (P < 0.05). Progesterone replacement decreased both GAD isoenzymes mRNA ratio after acute cocaine in the PFC (P < 0.001) and repeated cocaine treatment reversed this decrease (P < 0.001). These results suggest that cocaine does not immediately affect GAD mRNA expression, while repeated cocaine decreases both GAD65 and GAD67 mRNA in the dSTR of female rats, independently of their hormonal conditions. In the PFC, repeated cocaine increases the expression of GAD isoenzymes, which were decreased due to progesterone replacement.
Assuntos
Animais , Feminino , Ratos , Cocaína/farmacologia , Corpo Estriado/enzimologia , Glutamato Descarboxilase/efeitos dos fármacos , Córtex Pré-Frontal/enzimologia , Progesterona/farmacologia , Regulação da Expressão Gênica , Glutamato Descarboxilase/genética , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/metabolismoRESUMO
Female rats are intensely affected by cocaine, with estrogen probably playing an important role in this effect. Progesterone modulates the GABA system and attenuates the effects of cocaine; however, there is no information about its relevance in changing GABA synthesis pathways after cocaine administration to female rats. Our objective was to investigate the influence of progesterone on the effects of repeated cocaine administration on the isoenzymes of glutamic acid decarboxylase (GAD(65) and GAD(67)) mRNA in brain areas involved in the addiction circuitry. Ovariectomized, intact and progesterone replacement-treated female rats received saline or cocaine (30 mg/kg, ip) acutely or repeatedly. GAD isoenzyme mRNA levels were determined in the dorsolateral striatum (dSTR) and prefrontal cortex (PFC) by RT-PCR, showing that repeated, but not acute, cocaine decreased GADs/beta-actin mRNA ratio in the dSTR irrespective of the hormonal condition (GAD(65): P < 0.001; and GAD(67): P = 0.004). In the PFC, repeated cocaine decreased GAD(65) and increased GAD(67) mRNA ratio (P < 0.05). Progesterone replacement decreased both GAD isoenzymes mRNA ratio after acute cocaine in the PFC (P < 0.001) and repeated cocaine treatment reversed this decrease (P < 0.001). These results suggest that cocaine does not immediately affect GAD mRNA expression, while repeated cocaine decreases both GAD(65) and GAD(67) mRNA in the dSTR of female rats, independently of their hormonal conditions. In the PFC, repeated cocaine increases the expression of GAD isoenzymes, which were decreased due to progesterone replacement.
Assuntos
Cocaína/farmacologia , Corpo Estriado/enzimologia , Glutamato Descarboxilase/efeitos dos fármacos , Córtex Pré-Frontal/enzimologia , Progesterona/farmacologia , Animais , Feminino , Regulação da Expressão Gênica , Glutamato Descarboxilase/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Blockade of the N-methyl-d-aspartate receptor (NMDAR) in postnatal day 7 (P7) rats can promote rapid and robust induction of the pro-apoptotic marker activated caspase-3 (AC3) and loss of the GABAergic marker GAD67 at P56. Thus, we hypothesized that NMDAR blockade-induced AC3 occurs in GAD67 positive cells at P7. To test this idea, we injected P7 rat pups with vehicle or MK801 and after 8h (peak of AC3 induction) we examined brain sections for both AC3 and GAD67. Compared to vehicle, MK801 profoundly induced AC3 in all brain regions examined but co-expression of GAD67 in the same cells was not observed. However, in brain regions where punctate (synaptic) GAD67 was abundant (for example, layer IV of the somatosensory cortex), AC3 was robust. These data suggest that whereas somatic expression of AC3 and GAD67 may be non-overlapping, areas that exhibit punctate GAD67 (and are high in synaptic turnover) may be more vulnerable to MK801 exposure.
Assuntos
Encéfalo/metabolismo , Caspase 3/metabolismo , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glutamato Descarboxilase/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Encéfalo/efeitos dos fármacos , Caspase 3/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Imunofluorescência , Glutamato Descarboxilase/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , RatosRESUMO
Manganese is an essential nutrient, integral to proper metabolism of amino acids, proteins and lipids. Excessive environmental exposure to manganese can produce extrapyramidal symptoms similar to those observed in Parkinson's disease (PD). We used in vivo and in vitro models to examine cellular and circuitry alterations induced by manganese exposure. Primary mesencephalic cultures were treated with 10-800 microM manganese chloride which resulted in dramatic changes in the neuronal cytoskeleton even at subtoxic concentrations. Using cultures from mice with red fluorescent protein driven by the tyrosine hydroxylase (TH) promoter, we found that dopaminergic neurons were more susceptible to manganese toxicity. To understand the vulnerability of dopaminergic cells to chronic manganese exposure, mice were given i.p. injections of MnCl(2) for 30 days. We observed a 20% reduction in TH-positive neurons in the substantia nigra pars compacta (SNpc) following manganese treatment. Quantification of Nissl bodies revealed a widespread reduction in SNpc cell numbers. Other areas of the basal ganglia were also altered by manganese as evidenced by the loss of glutamic acid decarboxylase 67 in the striatum. These studies suggest that acute manganese exposure induces cytoskeletal dysfunction prior to degeneration and that chronic manganese exposure results in neurochemical dysfunction with overlapping features to PD.
Assuntos
Dopamina/metabolismo , Intoxicação por Manganês/metabolismo , Manganês/toxicidade , Neurônios/metabolismo , Substância Negra/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Gânglios da Base/efeitos dos fármacos , Gânglios da Base/metabolismo , Gânglios da Base/fisiopatologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Glutamato Descarboxilase/efeitos dos fármacos , Glutamato Descarboxilase/metabolismo , Cloreto de Magnésio/toxicidade , Intoxicação por Manganês/fisiopatologia , Camundongos , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Ratos , Substância Negra/efeitos dos fármacos , Substância Negra/fisiopatologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Dopamine (DA) affects GABA neuronal function in the striatum and together these neurotransmitters play a large role in locomotor function. We recently reported that unilateral striatal administration of GDNF, a growth factor that has neurotrophic effects on DA neurons and enhances DA release, bilaterally increased striatal neuron activity related to locomotion in aged rats. We hypothesized that the GDNF enhancement of DA function and resulting bilateral enhancement of striatal neuronal activity was due to prolonged bilateral changes in DA- and GABA-regulating proteins. Therefore in these studies we assessed dopamine- and GABA-regulating proteins in the striatum and substantia nigra (SN) of 24 month old Fischer 344 rats, 30 days after a single unilateral striatal delivery of GDNF. The nigrostriatal proteins investigated were the DA transporter (DAT), tyrosine hydroxylase (TH), and TH phosphorylation and were examined by blot-immunolabeling. The striatal GABA neuron-related proteins were examined by assay of the DA D1 receptor, DARPP-32, DARPP-32 Thr34 phosphorylation, and glutamic acid decarboxylase (GAD). Bilateral effects of GDNF on TH and DAT occurred only in the SN, as 30 microg GDNF increased ser19 phosphorylation, and 100 microg GDNF decreased DAT and TH protein levels. GDNF also produced bilateral changes in GAD protein in the striatum. A decrease in DARPP-32 occurred in the ipsilateral striatum, while increased D1 receptor and DARPP-32 phosphorylation occurred in the contralateral striatum. The 30 microg GDNF infusion into the lateral striatum was confined to the ipsilateral striatum and substantia nigra. Thus, long-lasting bilateral effects of GDNF on proteins regulating DA and GABA neuronal function likely alter physiological properties in neurons, some with bilateral projections, associated with locomotion. Enhanced nigrostriatal excitability and DA release by GDNF may trigger these bilateral effects.
Assuntos
Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Substância Negra/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Fosfoproteína 32 Regulada por cAMP e Dopamina/efeitos dos fármacos , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Relação Dose-Resposta a Droga , Lateralidade Funcional/efeitos dos fármacos , Lateralidade Funcional/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Glutamato Descarboxilase/efeitos dos fármacos , Glutamato Descarboxilase/metabolismo , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Receptores de Dopamina D1/efeitos dos fármacos , Receptores de Dopamina D1/metabolismo , Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The amygdala, the dorsal periaqueductal gray (dPAG), and the medial hypothalamus have long been recognized to be a neural system responsible for the generation and elaboration of unconditioned fear in the brain. It is also well known that this neural substrate is under a tonic inhibitory control exerted by GABA mechanisms. However, whereas there is a growing body of evidence to suggest that the amygdala and dPAG are also able to integrate conditioned fear, it is still unclear, however, how the distinct hypothalamic nuclei participate in fear conditioning. In this work we aimed to examine the extent to which the gabaergic mechanisms of this brain region are involved in conditioned fear using the fear-potentiated startle (FPS). Muscimol, a GABA-A receptor agonist, and semicarbazide, an inhibitor of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD), were used as an enhancer and inhibitor of the GABA mechanisms, respectively. Muscimol and semicarbazide were injected into the anterior hypothalamus (AHN), the dorsomedial part of the ventromedial nucleus (VMHDM), the dorsomedial (DMH) or the dorsal premammillary (PMD) nuclei of male Wistar rats before test sessions of the fear conditioning paradigm. The injections into the DMH and PMD did not produce any significant effects on FPS. On the other hand, muscimol injections into the AHN and VMHDM caused significant reduction in FPS. These results indicate that injections of muscimol and semicarbazide into the DMH and PMD fail to change the FPS, whereas the enhancement of the GABA transmission in the AHN and VMHDM produces a reduction of the conditioned fear responses. On the other hand, the inhibition of this transmission led to an increase of this conditioned response in the AHN. Thus, whereas DMH and PMD are known to be part of the caudal-most region of the medial hypothalamic defensive system, which integrates unconditioned fear, systems mediating conditioned fear select the AHN and VMHDM nuclei that belong to the rostral-most portion of the hypothalamic defense area. Thus, distinct subsets of neurons in the hypothalamus could mediate different aspects of the defensive responses.
Assuntos
Aprendizagem por Associação/fisiologia , Condicionamento Operante/fisiologia , Medo/fisiologia , Hipotálamo/metabolismo , Ácido gama-Aminobutírico/metabolismo , Análise de Variância , Animais , Aprendizagem por Associação/efeitos dos fármacos , Condicionamento Operante/efeitos dos fármacos , Núcleo Hipotalâmico Dorsomedial/efeitos dos fármacos , Núcleo Hipotalâmico Dorsomedial/metabolismo , Inibidores Enzimáticos/farmacologia , Agonistas GABAérgicos/farmacologia , Glutamato Descarboxilase/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Hipotálamo Anterior/efeitos dos fármacos , Hipotálamo Anterior/metabolismo , Masculino , Muscimol/farmacologia , Ratos , Ratos Wistar , Reflexo de Sobressalto/fisiologia , Semicarbazidas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Núcleo Hipotalâmico Ventromedial/efeitos dos fármacos , Núcleo Hipotalâmico Ventromedial/metabolismoRESUMO
Brain cell vulnerability to neurologic insults varies greatly, depending on their neuronal subpopulation. Among cells that survive a pathological insult such as ischemia or brain trauma, some may undergo morphological and/or biochemical changes that could compromise brain function. We previously reported that surviving cortical GABAergic neurons exposed to glutamate in vitro displayed an NMDA receptor (NMDAR)-mediated alteration in the levels of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) [Monnerie, H., Le Roux, P., 2007. Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro. Exp. Neurol. 205, 367-382]. In this study, we examined the mechanisms by which glutamate excitotoxicity caused a change in cortical GABAergic neurons' GAD protein levels. Removing extracellular calcium prevented the NMDAR-mediated decrease in GAD protein levels, measured using Western blot techniques, whereas inhibiting calcium entry through voltage-gated calcium channels had no effect. Glutamate's effect on GAD protein isoforms was significantly attenuated by preincubation with the cysteine protease inhibitor N-Acetyl-L-Leucyl-L-Leucyl-L-norleucinal (ALLN). Using class-specific protease inhibitors, we observed that ALLN's effect resulted from the blockade of calpain and cathepsin protease activities. Cell-free proteolysis assay confirmed that both proteases were involved in glutamate-induced alteration in GAD protein levels. Together these results suggest that glutamate-induced excitotoxic stimulation of NMDAR in cultured cortical neurons leads to altered GAD protein levels from GABAergic neurons through intracellular calcium increase and protease activation including calpain and cathepsin. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation and inhibition that is often observed after injury.
Assuntos
Córtex Cerebral/enzimologia , Cisteína Endopeptidases/metabolismo , Glutamato Descarboxilase/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/enzimologia , Ácido gama-Aminobutírico/metabolismo , Animais , Dano Encefálico Crônico/enzimologia , Dano Encefálico Crônico/fisiopatologia , Sinalização do Cálcio/efeitos dos fármacos , Calpaína/metabolismo , Catepsinas/metabolismo , Células Cultivadas , Córtex Cerebral/fisiopatologia , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática/efeitos dos fármacos , Epilepsia/enzimologia , Epilepsia/fisiopatologia , Glutamato Descarboxilase/efeitos dos fármacos , Ácido Glutâmico/toxicidade , Hipóxia-Isquemia Encefálica/enzimologia , Hipóxia-Isquemia Encefálica/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/efeitos dos fármacos , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme for gamma-aminobutyric acid (GABA) biosynthesis. Previously, we reported the presence of truncated forms of GAD in vivo and in vitro. In addition, an unidentified endogenous protease responsible for proteolytic cleavage of full-length GAD (fGAD) to its truncated form (tGAD) was also observed. In this communication, we report that mu-calpain is a good candidate for conversion of fGAD(67) to tGAD(67). This conclusion is based on the following observations: 1. purified recombinant GAD(67) is cleaved by mu-calpain at specific sites; 2. in brain synaptosomal preparation, GAD(67) is cleaved to its truncated form by an endogenous protease which is inhibited by specific calpain inhibitors; 3. in mu-calpain knockout mice, the level of tGAD in the brain is greatly reduced compared with the wild type; 4. when mu-calpain gene is silenced by siRNA, the level of tGAD is also markedly reduced compared to the control group; and 5. mu-calpain is activated by neuronal stimulation and Ca(2+)-influx. The physiological significance of calpain in regulation of GABA synthesis and GABAergic neurotransmission is also discussed.
Assuntos
Encéfalo/metabolismo , Calpaína/fisiologia , Glutamato Descarboxilase/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/ultraestrutura , Cálcio/metabolismo , Calpaína/farmacologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Deleção de Genes , Glutamato Descarboxilase/efeitos dos fármacos , Camundongos , Camundongos Knockout/genética , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Frações Subcelulares , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Transfecção/métodosRESUMO
Prolonged treatment with L-DOPA induces highly disabling dyskinesia in Parkinson's disease (PD) patients. In contrast, dopaminergic agonists display variably dyskinetic outcome, depending on pharmacokinetic/pharmacodynamic profile. The present study was aimed at assessing behavioral and biochemical correlates of intense or mild dyskinesia displayed by the different dopamine (DA) receptors stimulation in a rat model of PD. The effect of subchronic stimulation of the D(1) receptor by SKF38393, and the D(2)/D(3) receptor by ropinirole was evaluated in unilaterally 6-hydroxyDA-lesioned rats. Sensitization of contralateral turning (SCT) behavior and abnormal involuntary movements (AIMs) were assessed as behavioral correlates of dyskinetic responses. Opioid peptides mRNA in the dorsolateral striatum (dlStr) and glutamic acid decarboxylase (GAD67) mRNA content in globus pallidus (GP), were evaluated as an index of neuroadaptive changes occurring in the direct and indirect basal ganglia pathways. Subchronic SKF38393 caused AIMs and SCT whereas ropinirole elicited SCT only, indicating that both drugs induced some dyskinetic response, albeit of different type. Peptides mRNA evaluation in dlStr, showed that SKF38393 subchronic treatment was associated to an overexpression of both dynorphin (DYN) and enkephalin (ENK) mRNAs, in the direct and indirect striatal pathway respectively. In contrast, a decrease in DYN mRNA levels only was observed after treatment with ropinirole. Analysis of GAD67 mRNA levels in the GP showed an increase after both D(1) and D(2)/D(3) agonist treatments. Results suggest that presence of SCT alone or SCT plus AIMs might represent correlates of the differential severity of dyskinetic movements induced by treatment with low (ropinirole) or high (SKF38393) dyskinetic potential. Neuroadaptive increases in opioid peptide expression in both direct and indirect striatal pathways were associated to the appearance of AIMs alone. In contrast, increase of GAD67 mRNA in the GP was associated to both behavioral responses (SCT and AIMs), suggesting that neuroadaptive changes in this area were unrelated to the difference in dyskinetic potential of drugs.