RESUMO
Selective inhibition of the Na(+)/H(+) exchanger type 3 (NHE3) increases the firing rate of brainstem ventrolateral CO(2)/H(+) sensitive neurons, resembling the responses evoked by hypercapnic stimuli. In anesthetized animals, NHE3 inhibition has also been shown to stimulate the central chemosensitive drive. We aimed to analyze the respiratory-related brainstem regions affected by NHE3 inhibition in anaesthetized spontaneously-breathing rats with intact peripheral afferents. For that, c-Fos immunopositive cells were counted along the brainstem in rats intravenously infused with the selective NHE3 inhibitor AVE1599. A rostral extension of the ventral respiratory column which includes the pre-Bötzinger complex was activated by the NHE3 inhibitor. In addition, the number of c-Fos positive cells resulted significantly increased in the most rostral extension of the retrotrapezoid nucleus/parapyramidal region. In the pons, the intravenous infusion of AVE1599 activated the lateral parabrachial and Kölliker-Fuse nuclei. Thus, selective NHE3 inhibition in anaesthetized rats activates the respiratory network and evokes a pattern of c-Fos expressing cells similar to that induced by hypercapnia.
Assuntos
Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/fisiologia , Respiração , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Animais , Tronco Encefálico/citologia , Células Quimiorreceptoras/efeitos dos fármacos , Células Quimiorreceptoras/metabolismo , Relação Dose-Resposta a Droga , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Wistar , Trocador 3 de Sódio-HidrogênioRESUMO
The present study is concerned with the hypothetical toxicity of beta-N-methylamino-L-alanine (BMAA), a compound that has been hypothesized to produce amyotrophic lateral sclerosis/Parkinson-dementia complex. We have used the microdialysis technique to perfused different concentrations of BMAA in the rat's striatum 24h after the implantation of a microdialysis probe (day 1). BMAA perfusion produced a dose-response increase in the extracellular output of dopamine. Forty-eight hours after implantation of the probe (day 2), we have perfused MPP+ 1 mM to check the integrity of the dopaminergic terminals present around the cannula. Only the highest concentration of BMAA studied, 50mM, produced a clear decrease in the extracellular output of dopamine after MPP+ perfusion. However, this decrease was very similar, even smaller, to that obtained in a previous study carried out by us with MPP+ 1 mM, a dose much lower than that used for BMAA. Our model to study toxicity in the striatal dopaminergic terminal did not show that acute perfusion of BMAA at high doses produces a clear damage to the dopaminergic terminals.
Assuntos
Diamino Aminoácidos/toxicidade , Agonistas de Aminoácidos Excitatórios/toxicidade , Neostriado/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Diamino Aminoácidos/administração & dosagem , Animais , Morte Celular/efeitos dos fármacos , Circulação Cerebrovascular , Cromatografia Líquida de Alta Pressão , Toxinas de Cianobactérias , Dopamina/metabolismo , Eletroquímica , Agonistas de Aminoácidos Excitatórios/administração & dosagem , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Ácido Homovanílico/metabolismo , Microdiálise , Neostriado/irrigação sanguínea , Perfusão , Ratos , Ratos WistarRESUMO
We have previously described a neuroprotective action of (2S,2'R,3'R)-2-(2'3'-dicarboxycyclopropyl)glycine (DCG-IV), an agonist for group-II metabotropic receptors, on dopaminergic nerve terminals against the degeneration induced by 1-methyl-4-phenylpyridinium (MPP+). This effect was accompanied by an up-regulation of brain-derived neurotrophic factor (BDNF) mRNA expression in the rat striatum. We have now analyzed the phenotypic nature of the BDNF mRNA-expressing cells in response to intrastriatal injection of DCG-IV. Dual in situ hybridization and immunohistochemistry revealed that microglial cells but not astrocytes were responsible for this induction. Subsequent analysis demonstrated that this effect was accompanied by striking loss of striatal glutamic acid decarboxylase (GAD) mRNA and massive appearance of internucleosomal DNA fragmentation, a hallmark of apoptosis. A dose-response study demonstrated that doses of DCG-IV as low as 5 nmol was very toxic in terms GAD mRNA and apoptosis. 0.5 nmol of DCG-IV did not induce toxicity at all in terms of GAD mRNA and apoptosis. Activation of group-II metabotropic receptors in striatum with N-Acetyl-Asp-Glu (NAAG; a mGlu3 agonist) and (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (a mGlu2 and mGlu3 agonist) did not induce neither loss of GAD mRNA nor appearance of apoptosis (doses up to 20 nmol). In additional experiments, NAAG, in contrast to DCG-IV, failed to protect the striatal dopaminergic system against the degeneration induced by MPP+ as studied by microdialysis. Finally, we studied the mechanism by which DCG-IV is highly toxic. For that, selective antagonists of either metabotropic--(R,S)-alpha-methyl-4-carboxyphenylglycine and LY 341495--or ionotropic (N-methyl-D-aspartate, NMDA)--DL-2-amino-5-phosphonovaleric acid (AP-5) glutamate receptors --were co-administered with DCG-IV. Only AP-5 highly protected the striatum against the degeneration induced by DCG-IV. Since DCG-IV also activates the NMDA receptor at concentrations higher than 3 microM, it is conceivable that a intrastriatal concentration equal or higher than 3 microM after a single striatal injection of 5-20 nmol of DCG-IV. Our findings suggest that much caution must be exerted when testing the numerous neuroprotective effects ascribed to group-II metabotropic receptor activation, in particular when using DCG-IV. We conclude that the neuroprotectant capability of a given compound on a specific system does not exclude the possibility of inducing toxicity on a different one.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Corpo Estriado/efeitos dos fármacos , Ciclopropanos/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Microglia/efeitos dos fármacos , Degeneração Neural/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Animais , Corpo Estriado/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/fisiologia , Relação Dose-Resposta a Droga , Masculino , Microglia/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Receptores de Glutamato Metabotrópico/metabolismoRESUMO
Transplantation of embryonic dopaminergic neurons is an experimental therapy for Parkinson's disease, but limited tissue availability and suboptimal survival of grafted dopaminergic neurons impede more widespread clinical application. Glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-4/5 (NT-4/5) exert neurotrophic effects on dopaminergic neurons via different receptor systems. In this study, we investigated possible additive or synergistic effects of combined GDNF and NT-4/5 treatment on rat embryonic (embryonic day 14) nigral explant cultures grown for 8 days. Contrary to cultures treated with GDNF alone, cultures exposed to NT-4/5 and GDNF+NT-4/5 were significantly larger than controls (1.6- and 2.0-fold, respectively) and contained significantly more protein (1.6-fold). Treatment with GDNF, NT-4/5 and GDNF+NT-4/5 significantly increased dopamine levels in the culture medium by 1.5-, 2.5- and 4.7-fold, respectively, compared to control levels, and the numbers of surviving tyrosine hydroxylase-immunoreactive neurons increased by 1.7-, 2.1-, and 3.4-fold, respectively. Tyrosine hydroxylase enzyme activity was moderately increased in all treatment groups compared to controls. Counts of nigral neurons containing the calcium-binding protein, calbindin-D28k, revealed a marked increase in these cells by combined GDNF and NT-4/5 treatment. Western blots for neuron-specific enolase suggested an enhanced neuronal content in cultures after combination treatment, whereas the expression of glial markers was unaffected. The release of lactate dehydrogenase into the culture medium was significantly reduced for GDNF+NT-4/5-treated cultures only. These results indicate that combined treatment with GDNF and NT4/5 may be beneficial for embryonic nigral donor tissue either prior to, or in conjunction with, intrastriatal transplantation in Parkinson's disease.
Assuntos
Transplante de Tecido Encefálico/métodos , Sobrevivência de Enxerto/fisiologia , Neostriado/cirurgia , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Neurônios/efeitos dos fármacos , Doença de Parkinson/cirurgia , Substância Negra/efeitos dos fármacos , Animais , Calbindina 1 , Calbindinas , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Dopamina/metabolismo , Interações Medicamentosas/fisiologia , Feminino , Feto , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Sobrevivência de Enxerto/efeitos dos fármacos , Imuno-Histoquímica , L-Lactato Desidrogenase/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/transplante , Fosfopiruvato Hidratase/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Proteína G de Ligação ao Cálcio S100/metabolismo , Transplante de Células-Tronco , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Substância Negra/citologia , Substância Negra/transplante , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
We have studied the in vivo effect of the selective agonist for group II metabotropic glutamate receptors (2S, 2'R, 3'R)-2-(2'3'-dicarboxycyclopropyl)glycine (DCG-IV) against MPP+-induced toxicity on rat striatal dopaminergic nerve terminals by using both microdialysis and immunohistochemical techniques. Perfusion of 1 mM DCG-IV during 1 h protected dopaminergic nerve terminals against the degeneration induced by a 15-minute perfusion of 1 mM MPP+. In addition, the microglial cell population was markedly activated 24 h after DCG-IV perfusion. The astroglial cell population was only markedly activated around the microdialysis probe. This protective effect seems to be dependent on protein synthesis since 1 mM cycloheximide, an inhibitor of protein synthesis, abolished the neuroprotective effect of 1 mM DCG-IV against MPP+ toxicity. Perfusion of DCG-IV induced an upregulation of striatal brain-derived neurotrophic factor (BDNF) mRNA expressing cells which were confined precisely around the microdialysis probe. Taken together, our results suggest that the induction and release of brain-derived neurotrophic factor (BDNF) by activated glial cells induced by DCG-IV perfusion may account for its protective action against MPP+-induced dopaminergic terminal degeneration.
Assuntos
Corpo Estriado/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Glicina/análogos & derivados , Intoxicação por MPTP/prevenção & controle , Terminações Pré-Sinápticas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Benzoatos/farmacologia , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Corpo Estriado/citologia , Corpo Estriado/efeitos dos fármacos , Ciclopropanos/farmacologia , Modelos Animais de Doenças , Dopamina/análise , Dopamina/metabolismo , Dopaminérgicos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glicina/farmacologia , Imuno-Histoquímica , Hibridização In Situ , Intoxicação por MPTP/induzido quimicamente , Intoxicação por MPTP/patologia , Masculino , Microdiálise , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Glutamato Metabotrópico/agonistasRESUMO
The neurotoxic effect of dopamine (DA) and iron(III) on DAergic terminals in striatum has been studied by intracerebral microdialysis technique. Twenty-four hours after surgery (day 1), DA and/or iron(III) with and without DA reuptake inhibitor, nomifensine, were perfused for 1 h. Forty-eight hours after surgery (day 2), MPP(+) 1 mM was perfused for 15 min and the output of DA was measured, its amount being directly proportional to the remaining striatal DAergic terminals, supported by tyrosine hydroxylase immunohistochemistry technique. Perfusion of exogenous DA, as well as iron(III) 10 and 100 microM, did not produce any neurotoxic effect. However, perfusion of iron(III) (333 and 1000 microM) produced a concentration-dependent toxic effect. Co-perfusion of iron(III) at non-toxic concentration (100 microM) with DA (15 microM) produced a toxic effect. Elevation of the endogenous extracellular levels of DA by inhibiting its uptake with nomifensine increased the neurotoxic effect of iron(III) in a dose-dependent manner. The use of tetrodotoxin after elevation of DA with nomifensine partially prevented the neurotoxic effect of its co-perfusion with iron(III) (100 microM). These results suggest that DAergic system could be synergistically damaged by DA and iron(III). Thus, alterations in the clearance of DA from extracellular space along with an increase of iron may have significant consequences for DAergic system toxicity.
Assuntos
Corpo Estriado/efeitos dos fármacos , Dopamina/toxicidade , Ferro/toxicidade , Terminações Pré-Sinápticas/efeitos dos fármacos , 1-Metil-4-fenilpiridínio/farmacologia , Animais , Corpo Estriado/enzimologia , Corpo Estriado/patologia , Dopamina/administração & dosagem , Dopamina/análise , Dopamina/biossíntese , Inibidores da Captação de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Imuno-Histoquímica , Ferro/administração & dosagem , Masculino , Microdiálise , Nomifensina/farmacologia , Perfusão , Terminações Pré-Sinápticas/enzimologia , Terminações Pré-Sinápticas/patologia , Ratos , Ratos Wistar , Tetrodotoxina/farmacologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
In vivo microdialysis was used to investigate the mechanism behind the increase in extracellular dopamine (DA) induced by increase in extracellular serotonin (5-HT) level and 5-HT1 and 5-HT2 receptor activation. The following serotoninergic drugs were perfused in the absence or presence of nomifensine (5 microM) or tetrodotoxin (TTX; 2 microM): clomipramine (10, 500 and 1,000 microM), a selective 5-HT reuptake inhibitor; 8-OH-DPAT (50 and 500 microM), a 5-HT1A receptor agonist; and alpha-methyl-5-HT (1, 5 and 50 microM), a 5-HT2 receptor agonist. All the serotoninergic drugs studied increased DA extracellular output in a dose-dependent manner. The presence of nomifensine attenuated the effect of perfusion of clomipramine (500 microM) and completely abolished the effect of perfusion of 8-OH-DPAT (500 microM) and alpha-methyl-5-HT (5 microM) on DA extracellular output. Clomipramine (100-1,000 microM) perfusion produced a dose dependent increase in DOPAC extracellular output, which was stronger when clomipramine (500 microM) was co-perfused with nomifensine. 8-OH-DPAT and alpha-methyl-5-HT perfusion decreased DOPAC overflow. Addition of TTX to the perfusion fluid one hour before serotoninergic drugs perfusion, did not completely abolish the effect on dopamine extracellular output produced by the serotoninergic drugs. These data seem to indicate that increase in extracellular 5-HT level and 5-HT1 and 5-HT2 receptor activation increase in vivo DA extracellular output in the striatum mainly by a nonexocytotic mechanism involving DA uptake sites and, secondarily, by activation of 5-HT receptors.
Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Espaço Extracelular/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Clomipramina/farmacologia , Ácido Hidroxi-Indolacético/metabolismo , Masculino , Ratos , Ratos Wistar , Serotonina/análogos & derivados , Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologiaRESUMO
Desferrioxamine (DES) protective effect against 1-methyl-4-phenylpyridinium (MPP+) toxicity was evaluated by microdialysis in the substantia nigra. DES (1 microM to 10 mM) co-perfused with MPP+ (2.5 mM) on day 1, produced on day 2 a higher dopamine extracellular output after perfusion of MPP+ than in control-MPP+ perfusion experiments, in which no DES was administered on day 1. Both Ringer's perfusion alone (control-Ringer) and co-perfusion of DES (10 mM) with MPP+ (2.5 mM) on day 1 produced on day 2 similar increases in dopamine extracellular output after a second MPP+ perfusion. In the control-Ringer experiment, note that the MPP+ on day 2 is the first MPP+ perfusion. Perfusion of FeCl3 (200 microM) along with MPP+ (2.5 mM) and DES (100 microM) on day 1 completely abolished on day 2 the neuroprotective effect found with MPP+ (2.5 mM) and DES (100 microM). The ability of DES to protect against MPP+ toxicity may indicate a therapeutic strategy in the treatment of diseases when iron is implicated.
Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Desferroxamina/farmacologia , Dopamina/metabolismo , Compostos Férricos/farmacologia , Substância Negra/efeitos dos fármacos , 1-Metil-4-fenilpiridínio/antagonistas & inibidores , Animais , Cloretos , Masculino , Microdiálise , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Wistar , Substância Negra/metabolismo , Substância Negra/patologiaRESUMO
1. The present study concerns the possible relationship between glutamate excitotoxicity and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/1-methyl-4-phenylpyridinium (MPTP/MPP+) neurotoxicity on striatal dopaminergic terminals. 2. MPP+ neurotoxicity has been studied by means of two MPP+ perfusions separated by 24 h. After the second MPP+ 1 mM perfusion, dopamine extracellular output, measured by microdialysis, was considered to be an index of the dopaminergic neurone damage produced by the first MPP+ 1 mM perfusion. 3. High concentration (10 mM) of glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) stimulated basal release of dopamine and protected against the neurotoxic effect of MPP+. 4. PDC 10 mM perfusion produced an increase in the extracellular output of glutamate and aspartate, and a decrease in that of ascorbate. 5. The protective effect against MPP+ toxicity observed with PDC 10 mM was completely abolished when this glutamate uptake inhibitor was co-perfused with ascorbate 0.5 mM. 6. These results suggest that glutamate-induced neurotoxicity is not involved in MPP+ toxicity. The protective effect found with the glutamate uptake inhibitor could be due to a decrease in extracellular ascorbate levels.
Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Aminoácidos Essenciais/toxicidade , Ácido Ascórbico/fisiologia , Dopaminérgicos/toxicidade , Dopamina/fisiologia , Ácido Glutâmico/fisiologia , Neostriado/efeitos dos fármacos , Terminações Nervosas/efeitos dos fármacos , Animais , Ácido Ascórbico/metabolismo , Ácido Aspártico/metabolismo , Ácidos Dicarboxílicos/farmacologia , Dopamina/metabolismo , Espaço Extracelular/enzimologia , Espaço Extracelular/metabolismo , Imuno-Histoquímica , Intoxicação por MPTP , Masculino , Microdiálise , Neostriado/metabolismo , Inibidores da Captação de Neurotransmissores/farmacologia , Pirrolidinas/farmacologia , Ratos , Ratos Wistar , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Microdialysis was used to evaluate the effect of desferrioxamine (DES) against 1-methyl-4-phenylpyridinium (MPP+) toxicity. The presence of DES (40 fmol-40 nmol/15 min for a total of 90 min) in the Ringer solution, coperfused with MPP+ (40 nmol/15 min) on day 1, produced on day 2 a higher extracellular dopamine output after perfusion of MPP+ than in control MPP+ perfusion experiments, in which no DES was administered on day 1. Both Ringer perfusion alone (control Ringer) and coperfusion of 40 nmol DES with 40 nmol MPP+ on day 1 produced on day 2 similar increases in extracellular dopamine output after a second MPP+ perfusion. In the control Ringer experiment, note that the MPP+ on day 2 is the first MPP+ perfusion. Perfusion of 800 fmol FeCl3/15 min along with 40 nmol MPP+ and 400 fmol DES on day 1 completely abolished on day 2 the neuroprotective effect found with 40 nmol MPP+ and 400 fmol DES; 800 fmol FeCl3 did not increase the neurotoxic effect of 40 nmol MPP+ perfusion. The ability of DES to protect against MPP+ toxicity may indicate a therapeutic strategy in the treatment of diseases when iron is implicated.