RESUMO
Astrocytes release glutamate (Glu) by the mobilisation of intracellular concentrations of Ca++. The rationale of the present work was to test whether Glu and its agonists, known to affect intracellular Ca++ content via the activation of metabotropic and ionotropic receptors, could modulate the astrocytic release of excitatory aminoacids. NMR experiments showed that Glu released uniformly labelled [13C] Glu in the incubation medium of rat astrocytes in primary cultures. Further experiments confirmed this finding and showed that the incubation of these cells with agonists and antagonists of Glu ionotropic and metabotropic receptors, produced a different modulation of Glu and aspartate release. The observed activations of the various receptors suggest a complex modulation of the release of the excitatory aminoacids. Such a release of is interpreted in terms of metabolic microzonation.
Assuntos
Ácido Aspártico/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/metabolismo , Animais , Células Cultivadas , Espectroscopia de Ressonância Magnética , Ratos , Ratos Sprague-DawleyRESUMO
After injection of 6-hydroxydopamine into the lateral part of the rat substantia nigra, tissue dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were reduced in the corresponding lateral part of the ipsilateral caudate/putamen (CP) complex (13, 40 and 56% of controls, respectively). In this region, tyrosine hydroxylase (TH, the rate limiting enzyme of the DA synthesis) immunoautoradiography decreased by more than 80% as was the case for the binding of tritiated GBR12935 (a specific marker of the DA-carrier protein). In the medial region of the CP, only very moderate reductions of DA, DOPAC and HVA (77, 76 and 84% of controls, respectively) were observed. In this region, TH immunoautoradiography and GBR12935 binding were only reduced by about 20% reflecting weak DA denervation. However, using in vivo voltammetry, extracellular basal DA levels were found to be particularly high in the medial region of CP complex when compared to unoperated animals (up to 235%). In the medial region, TH activity was also significantly increased (161%) but the electrical stimulation of DA fibers produced the same DA overflow in control and lesioned animals. From these results, it may be concluded that elevated basal DA levels in this region cannot be attributed to the reduced DA uptake and/or to an increased ability of DA neurons to release DA in response to impulse flow.
Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Espaço Extracelular/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Substância Negra/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Autorradiografia , Proteínas de Transporte/metabolismo , Núcleo Caudado/metabolismo , Contagem de Células , Proteínas da Membrana Plasmática de Transporte de Dopamina , Estimulação Elétrica , Eletrodos Implantados , Ácido Homovanílico/metabolismo , Masculino , Feixe Prosencefálico Mediano/fisiologia , Oxidopamina/administração & dosagem , Putamen/metabolismo , Ratos , Ratos Sprague-Dawley , Substância Negra/efeitos dos fármacos , Substância Negra/enzimologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
It is well established that midbrain dopamine neurons innervating the striatum, release their neurotransmitter through an exocytotic process triggered by the neural firing and involving a transient calcium entry in the terminals. Long ago, it had been proposed, however, that another mechanism of release could co-exist with classical exocytosis, involving the reverse-transport of the cytosolic amine by the carrier, ordinarily responsible for uptake function. This atypical mode of release could be evoked directly at the preterminal level by multiple environmental endogenous factors involving transient alterations of the sodium gradient. It cannot be excluded that this mode of release participates in the firing-induced release. In contrast with the classical exocytosis of a preformed DA pool, the reverse-transport of DA requires simultaneous alterations of intraterminal amine metabolism including synthesis and displacement from storage compartment. The concept of a reverse-transport of dopamine is coming from the observations that releasing substances, such as amphetamine-related molecules, actually induce this type of transport. A large set of arguments advocates that reverse-transport plays a role in the maintenance of basal extracellular DA concentration in striatum. It was also often evoked in physiopathological situations including ischemia, neurodegenerative processes, etc. The most recent studies suggest that this release could occur mainly outside the synapses, and thus could constitute a major feature in the paracrine transmission, sometimes evoked for DA.
Assuntos
Dopamina/metabolismo , Anfetamina/farmacologia , Animais , Transporte Biológico Ativo/fisiologia , Encefalopatias/metabolismo , Isquemia Encefálica/metabolismo , Corpo Estriado/metabolismo , Dopamina/fisiologia , Inibidores da Captação de Dopamina/farmacologia , Camundongos Mutantes Neurológicos/metabolismo , Terminações Nervosas/metabolismo , Substância NegraRESUMO
The in vivo and ex vivo distributions and the pharmacological profile of the fluorinated phenylpiperazine derivative p-[(18)F]MPPF (4-(2'-methoxyphenyl)-1-[2'-(N-2"-pyridinyl)-p-fluorobenzamido]-et hyl piperazine) were evaluated in the cat brain as a potential selective antagonist for 5-HT(1A) receptors using PET. After intravenous injection of p-[(18)F]MPPF in cats, there was a rapid accumulation of radioactivity in the brain, with 4% of the total radioactivity injected present in the brain at 4 minutes postinjection. The highest uptakes of radioactivity were observed in the hippocampus and cingulate cortex, regions known to be rich in 5-HT(1A) receptors, whereas lower levels of radioactivity were observed in the cerebellum. The mean ratio of radioactivity in the hippocampus to the cerebellum was 4.29 (SD = 0.21; n = 5) from 40 to 90 minutes postinjection of p-[(18)F]MPPF. The corresponding ratio for the cingulate cortex was 3.01 (SD = 0.16; n = 5). Specific binding in the hippocampus and the cingulate cortex was markedly reduced following injection of unlabeled WAY-100635 and pindolol but was unaffected by treatment with alpha1, 5-HT(2), or reuptake inhibitor agents indicating reversibility and selectivity of p-[(18)F]MPPF binding to 5-HT(1A) receptors. Ex vivo autoradiographic study with p-[(18)F]MPPF in cat brain sections showed labeling of areas rich in 5-HT(1A) receptors with a regional brain distribution that closely matched that observed using PET. These results indicate that p-[(18)F]MPPF may be a useful candidate for noninvasive PET imaging of 5-HT(1A) receptors in the living human brain.
Assuntos
Aminopiridinas/farmacocinética , Encéfalo/metabolismo , Piperazinas/farmacocinética , Piridinas/farmacocinética , Receptores de Serotonina/metabolismo , Antagonistas da Serotonina/farmacocinética , Animais , Encéfalo/diagnóstico por imagem , Gatos , Masculino , Receptores 5-HT1 de Serotonina , Distribuição Tecidual , Tomografia Computadorizada de EmissãoRESUMO
To investigate the contribution of the dopamine (DA) synthesis to both the calcium-dependent and the carrier-mediated, mechanisms of DA release in the striatum, anaesthetized rats were locally superfused in the striatum with a push pull cannula supplied with an artificial CSF containing tritiated tyrosine. DA, dihydroxyphenylacetic acid (DOPAC) and their respective specific activity were measured in effluent and used to evaluate changes in the DA synthesizing rate. Excluding calcium ions from the CSF only partially reduced spontaneous DA release (70%) still leaving a possible carrier-mediated DA release. This effect was not additive with a local superfusion with 0.1 mM a-methyl-p-tyrosine, a blocker of DA synthesis, suggesting that synthesis could already be reduced by calcium-free superfusion. Local superfusion with 100 microM cadmium in the presence or not of calcium ions, increased the DA release (220 and 350%, respectively), simultaneously reducing DA synthesis. Local application of 1 microM calcium ionophore (A23187) was without effect on the basal release of DA but enhanced DA synthesis and increased the amphetamine-evoked and carrier-mediated amine release. We conclude that DA synthesis can be a modulatory process of the firing-independent and carrier-mediated amine release while it weakly affects the classical calcium-dependent release.
Assuntos
Sinalização do Cálcio , Cálcio/farmacologia , Corpo Estriado/metabolismo , Dopamina/biossíntese , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Anfetamina/farmacologia , Animais , Cádmio/farmacologia , Calcimicina/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Ionóforos/farmacologia , Masculino , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Ratos , Ratos Wistar , Tirosina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Metiltirosina/farmacologiaRESUMO
Polyamine contents were determined in human temporal lobe epilepsy. In the seven patients studied, stereoelectroencephalography (SEEG) located the epileptogenic focus in Ammon's horn and neuropathological findings were limited to hippocampal gliosis and sclerosis. Each polyamine exhibited a specific regional distribution. The most important variations were observed for spermidine and spermine while putrescine levels varied less. The regional variation was predominant in middle > posterior > anterior parts of the temporal lobe. Spermine contents and the spermidine/spermine (SPD/SPM) index varied especially in the middle and posterior parts of the hippocampus. Metabolic SPD/SPM index and spermidine levels were found to be drastically increased in almost all limbic parts when compared to neocortical regions. The opposite was observed for spermine. The heterogeneous distribution of polyamines was compared to abnormal electrical activities recorded by SEEG: SPD/SPM index and spermidine levels were sharply increased in seizure onset areas and high levels of spermine were detected in temporal cortex propagation areas. The presently reported heterogeneity of polyamine contents might contribute to modulate differentially the local control of excitability in human temporal epilepsy.
Assuntos
Epilepsia do Lobo Temporal/metabolismo , Poliaminas/análise , Adulto , Cromatografia Líquida de Alta Pressão , Eletroencefalografia/métodos , Epilepsia do Lobo Temporal/fisiopatologia , Feminino , Humanos , MasculinoRESUMO
The effects of moderate changes in extracellular dopamine concentrations on the in vivo binding of specific dopaminergic D2 radioligands with different affinities and kinetics were investigated in rats. Either [125I]NCQ298 (Kd = 19 pM), or [25I]iodolisuride (Kd = 0.27 nM) or [3H]raclopride (Kd = 1.5 nM) were administered intravenously (IV) to animals 1 h after the intraperitoneal (IP) injection of either alpha-methyl-p-tyrosine (AMPT) (250 mg/kg) or nomifensine (15 mg/kg), or saline. The kinetics of radioactivity concentration in the striatum, cerebellum, and plasma were measured for up to 4 h after [125I]NCQ298 or [125I]iodolisuride injection and up to 1.5 h after [3H]raclopride injection. For each tracer, the striatum-to-cerebellum radioactivity concentration ratios (S/C) and the binding potential (BP), calculated as the association to dissociation binding rate constant ratios (k3/k4), were assessed and related to the changes in extracellular dopamine concentration induced by drug treatments. Results show that S/C and BP of [3H]raclopride were significantly diminished by pretreatment with nomifensine, a drug that increases extracellular dopamine concentration. Nomifensine pretreatment induced no changes in the in vivo binding indexes of the high affinity [125I]NCQ298 and a slight but not significant decrease of the binding indexes of 125I]iodolisuride. Treatment with AMPT, which induced a 40% reduction in dopamine concentration, did not change [125I]NCQ298 binding indexes but slightly increased those of [3H]raclopride and [125I]iodolisuride. In conclusion, the change of dopamine concentration induces modification of radiotracer kinetics. Thus, the combined use of tracers with high and low affinities could allow us to obtain information both on receptor density and neurotransmitter release in vivo. However, as indicated by the [3H]raclopride study with AMPT, small changes in the concentration of intrasynaptic dopamine cannot be easily detected.
Assuntos
Cerebelo/metabolismo , Dopamina/farmacologia , Receptores de Dopamina D2/metabolismo , Córtex Visual/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Cerebelo/efeitos dos fármacos , Dopamina/metabolismo , Radioisótopos do Iodo , Lisurida/análogos & derivados , Lisurida/metabolismo , Masculino , Nomifensina/farmacologia , Racloprida , Compostos Radiofarmacêuticos/metabolismo , Ratos , Ratos Wistar , Salicilamidas/metabolismo , Tomografia Computadorizada de Emissão , Trítio , Córtex Visual/efeitos dos fármacos , alfa-Metiltirosina/farmacologiaRESUMO
Colchicine, an axonal transport blocking agent, was unilaterally injected in the medial forebrain bundle of rats. As early as 18 h after the injection a rapid decrease in TH-mRNA level was observed in the substantia nigra and the ventral tegmental area (SN/VTA) on the injected side. In contrast, TH protein levels remained stable for 48 h, and decreased later in both cells bodies and terminals (caudate/putamen). The number of TH-immunopositive cells in SN/VTA increased after colchicine equally in both sides, excluding a neurotoxic effect. These results suggest that TH gene expression is controlled by a retrogradely transported activating factor rather than by feedback inhibition by the end product, i.e. TH protein.
Assuntos
Transporte Axonal/fisiologia , Axônios/fisiologia , Colchicina/farmacologia , Mesencéfalo/enzimologia , Neurônios/enzimologia , Transcrição Gênica , Tirosina 3-Mono-Oxigenase/biossíntese , Animais , Transporte Axonal/efeitos dos fármacos , Axônios/efeitos dos fármacos , Núcleo Caudado/enzimologia , Lateralidade Funcional , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , Mesencéfalo/fisiologia , Terminações Nervosas/enzimologia , Neurônios/efeitos dos fármacos , Putamen/enzimologia , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Substância Negra/efeitos dos fármacos , Substância Negra/enzimologia , Tegmento Mesencefálico/efeitos dos fármacos , Tegmento Mesencefálico/enzimologia , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
No-carrier-added 4-[18F]fluoro-N-[2-[1-(2-methoxyphenyl)-1 piperazinyl]ethyl-N-2-pyridinyl-benzamide (p-[18F]MPPF) was synthesized by nucleophilic substitution of the corresponding nitro compound in the presence of Kryptofix 222 and K2CO3 by microwave heating (3 min, 500 W) using a remotely controlled radiosynthesis. Baseline separation of p-[18F]MPPF from the nitro derivative was performed on a semipreparative HPLC C18 column. After Sep-Pak formulation, the radiopharmaceutical was obtained with a radiochemical yield of 25% (EOS) in about 70 min. Specific radioactivity averaged between 1-5 Ci/micromol EOS. Labelling of the ortho and meta derivatives was also attempted. Brain uptake of p-[18F]MPPF was studied with PET on fluothane-anesthetized cats. Following intravenous injection of p-[18F]MPPF, high accumulation of radioactivity was observed in the hippocampus and cerebral cortex. Low levels of radioactivity were observed in cerebellum. At 30 min, the mean hippocampus/cerebellum and cortex/cerebellum ratios were 5 and 3.8, respectively. The accumulation of the tracer was blocked by prior administration of reference WAY-100635, demonstrating the specificity of the ligand.
Assuntos
Encéfalo/metabolismo , Piperazinas/síntese química , Piperazinas/farmacocinética , Piridinas/síntese química , Piridinas/farmacocinética , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Antagonistas da Serotonina/síntese química , Antagonistas da Serotonina/farmacocinética , Tomografia Computadorizada de Emissão , Animais , Gatos , Cromatografia Líquida de Alta Pressão , Feminino , Radioisótopos de Flúor/farmacocinética , Estrutura Molecular , Controle de Qualidade , Ensaio RadioliganteRESUMO
The expression of enkephalin in neurons of the rat forebrain was studied by in situ hybridization and immunohistochemistry after unilateral injections of ibotenic acid into the bed nucleus of the stria terminalis. Initially, we observed that the destruction of nerve cell bodies in this nucleus resulted in a prominent bilateral increase in the number of neuronal perikarya immunoreactive for [Met]enkephalin in the lateral/basolateral amygdaloid complex-especially in the anterior division of the latter nucleus-as compared with NaCl-injected rats. In a separate set of experiments, this effect was associated with a significant (two times) enhancement of the number of nerve cell bodies containing preproenkephalin A messenger RNAs in the same amygdaloid nucleus ipsilateral to the injection, as compared with controls. In the hypothalamus of both experimental and control rats, the nerve cell bodies immunoreactive for [Met]enkephalin were few since the animals were not pretreated with colchicine, and the effects of the lesion were difficult to appreciate. However, using in situ hybridization, numerous nerve cell bodies containing preproenkephalin A messenger RNAs were detected bilaterally in the perifornical area, the paraventricular (parvocellular division) and the ventromedial nuclei of the hypothalamus. In the latter nucleus, the lesion of the bed nucleus of the stria terminalis resulted in a strong decrease (about two times) in the number of labelled cell bodies as compared with the controls, whereas no significant changes were found bilaterally in the paraventricular nucleus. In agreement with some data of the literature, our results indicate that the bed nucleus of the stria terminalis plays an important role in the regulation of neuropeptide genes expression in certain regions of the limbic system. Such a role is often exerted by nerve fibres afferents to the nerve cell bodies considered. However, from numerous neuroanatomical data of the literature, it appears more probable that the induction or inhibition of the expression of enkephalin in presynaptic neurons is due to the disappearance of their postsynaptic target in the bed nucleus of the stria terminalis.
Assuntos
Tonsila do Cerebelo/metabolismo , Encefalinas/metabolismo , Hipotálamo/metabolismo , Sistema Límbico/fisiologia , Prosencéfalo/fisiologia , Animais , Ácido Ibotênico/farmacologia , Imuno-Histoquímica , Hibridização In Situ , Sistema Límbico/efeitos dos fármacos , Masculino , Prosencéfalo/efeitos dos fármacos , Ratos , Ratos WistarAssuntos
Corpo Estriado/efeitos dos fármacos , Dopamina/biossíntese , Estradiol/farmacologia , Ácido 3,4-Di-Hidroxifenilacético/análise , Animais , Inibidores das Descarboxilases de Aminoácidos Aromáticos , Corpo Estriado/metabolismo , Dopamina/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Hidrazinas/farmacologia , Levodopa/análise , Ovariectomia , RatosRESUMO
The acute effect of physiological doses of estradiol (E2) on the dopaminergic activity in the striatum was studied. In a first series of experiments, ovariectomized rats were injected with 17 alpha or 17 beta E2 (125, 250, or 500 ng/kg of body weight, s.c.), and in situ tyrosine hydroxylase (TH) activity (determined by DOPA accumulation in the striatum after intraperitoneal administration of NSD 1015) was quantified. A dose-dependent increase in striatal TH activity was observed within minutes after 17 beta (but not 17 alpha) E2 treatment. To examine whether E2 acts directly on the striatum, in a second series of experiments, anesthetized rats were implanted in the striatum with a push-pull cannula supplied with an artificial CSF containing [3H]tyrosine. The extracellular concentrations of total and tritiated dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured at 20-min intervals. Addition of 10(-9) M 17 beta (but not 17 alpha) E2 to the superfusing fluid immediately evoked an approximately 50% increase in [3H]DA and [3H]DOPAC extracellular concentrations, but total DA and DOPAC concentrations remained constant. This selective increase in the newly synthesized DA and DOPAC release suggested that E2 affects DA synthesis rather than DA release. Finally, to determine whether this rapid E2-induced stimulation of DA synthesis was a consequence of an increase in TH level of phosphorylation, the enzyme constant of inhibition by DA (Ki(DA)) was calculated. Incubation of striatal slices in the presence of 10(-9) M 17 beta (but not 17 alpha) E2 indeed evoked an approximate twofold increase in the Ki(DA) of one form of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopamina/biossíntese , Estradiol/farmacologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Hidrazinas/farmacologia , Ratos , Ratos Wistar , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
A push-pull cannula supplied with artificial CSF was implanted in the striatum of anaesthetized rats, and the basal extracellular DA and DOPAC was assayed in the superfusates using HPLC and electrochemical detection. Simultaneously, a carbon fibre electrode was implanted in close proximity of the cannula and the evoked DA release was detected by differential pulse amperometry during stimulation of the DA axons. Local treatments with cadmium (100 microM) blocked the evoked DA release (-90%), but substantially increased the basal extracellular DA (+125%). The effects of glutamate agonists NMDA (1 mM) and kainate (0.1 mM), known to increase basal extracellular DA were confirmed (+150% and +60% respectively). It was, however, simultaneously observed that the evoked DA release was inhibited (-80% and -50%, respectively). Amphetamine (1 microM) released DA (+150%) and produced also an increase (+100%) of the evoked DA release. These results, apparently conflicting, show that the two mechanisms releasing dopamine (firing-dependent and not) can be directly and simultaneously observed. These two releasing processes appear to be not strictly antagonist. They are also differently and independently modulated by calcium and by local influences such those conveyed by glutamate.
Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Anfetamina/farmacologia , Animais , Cádmio/farmacologia , Ácido Caínico/farmacologia , Masculino , N-Metilaspartato/farmacologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The effects of a chronic imipramine treatment on the mesoamygdaloid pathway of rats were examined. Using semiquantitative immunocytochemical techniques, it was observed that the level of TH mRNA was decreased in the ventral tegmental area (VTA). In contrast, the TH protein was increased in both the VTA and amygdala. The TH activity was decreased in the amygdala when assessed under normal conditions but increased after a preincubation to phosphorylate the enzyme, suggesting a lowering of the protein-specific activity in the terminals. These results show that TH protein turnover in the mesoamygdaloid neurons can be reduced by chronic imipramine treatments, thereby producing an accumulation of inactive TH protein in the neurons while also decreasing TH gene activity in the cell bodies.
Assuntos
Dopamina/metabolismo , Imipramina/farmacologia , Mesencéfalo/fisiologia , Neurônios/fisiologia , Tirosina 3-Mono-Oxigenase/genética , Tonsila do Cerebelo , Animais , Expressão Gênica , Masculino , Fosforilação , Ratos , Ratos WistarRESUMO
Twenty weeks after ibotenic acid lesions of the striatum, the amount of tyrosine hydroxylase (TH) in this structure was markedly increased. This was accompanied by a 3-fold increase in TH mRNA levels in the ipsilateral subtantia nigra (SN). Striatal levels of dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) were markedly reduced. In the nucleus accumbens, spared by the lesion, DA neurotransmission was also altered, as evidenced by a reduction of DA and DOPAC, but no increase in TH could be detected. TH mRNA levels were moderately enhanced in the ventral tegmental area (VTA). Thus, lesioning in the striatum induces TH gene activation in both SN and VTA neurones, not strictly related to DA function at the terminal level.
Assuntos
Corpo Estriado/metabolismo , RNA Mensageiro/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Encéfalo/metabolismo , Encefalopatias/induzido quimicamente , Núcleo Caudado/efeitos dos fármacos , Núcleo Caudado/patologia , Doença Crônica , Corpo Estriado/patologia , Dopamina/metabolismo , Ácido Ibotênico/farmacologia , Masculino , Putamen/efeitos dos fármacos , Putamen/patologia , Ratos , Ratos Wistar , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The mechanism of the short-term activation by prolactin (PRL) of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by 3,4-dihydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a dose-dependent increase within 2 h of incubation of the hypothalamic slices with PRL. To determine whether a phosphorylation process was involved in this increase in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition. In control median eminences, two kinetically different forms of TH coexisted, one exhibiting a Ki(DA) value of 29.92 +/- 0.49 microM, the other being approximately 15-fold more sensitive to DA inhibition with a Ki(DA) of 1.96 +/- 0.09 microM, likely corresponding to a phosphorylated and active form and to a nonphosphorylated and less active form, respectively. After PRL treatment, the TH form of low Ki(DA) remained unaffected, whereas the Ki(DA) of the purported active form of TH increased to 62.6 +/- 0.8 microM, suggesting an increase in the enzyme phosphorylation. This increase in the Ki(DA) of TH was selectively prevented by GF 109203X, a potent and selective inhibitor of protein kinase C, but not by a specific inhibitor of protein kinase A or calmodulin. Finally, this action of PRL could be mimicked by 12-O-tetradecanoylphorbol 13-acetate (a direct activator of protein kinase C). These results suggest that PRL, at the median eminence level, activates TH by increasing the enzyme phosphorylation and that this action may involve an activation of protein kinase C.
Assuntos
Dopamina/fisiologia , Hipotálamo/metabolismo , Neurônios/metabolismo , Prolactina/farmacologia , Proteína Quinase C/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Dopamina/farmacologia , Ativação Enzimática , Feminino , Hipotálamo/citologia , Técnicas In Vitro , Eminência Mediana/enzimologia , Ovariectomia , Inibidores de Proteínas Quinases , Ratos , Ratos Wistar , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/antagonistas & inibidoresRESUMO
In the present study, we describe the neurochemical effects of intranigral injections of colchicine in the rat forebrain using immunohistochemistry and in situ hybridization. The observations on the injected side are compared to the contralateral one and to the sham-operated rats. We demonstrate that such injections are able to strongly enhance the immunoreactivity for Met-enkephalin (ME), substance P (SP) and neuropeptide Y (NPY) in numerous nerve cell bodies of the limbic system (injected side), whereas the levels of the corresponding mRNAs are differently modified according to the region examined. A clear correlation between the enhancement of the immunostaining for ME and SP and that of the preproenkephalin (PPA) and preprotachychinin gene transcripts was observed in neuronal perikarya of the medial amygdaloid nucleus (SP), of the dorsolateral hypothalamus (ME) and of the ventromedial hypothalamic nucleus (SP). These observations are interpreted as an induction--or increased expression--of neuropeptide genes in neuronal perikarya postsynaptic to nerve fibers originating in the midbrain and brain stem. In this case, colchicine is thought to block the electrophysiological activity of ascending nerve fibers (anterograde and postsynaptic effect). In the case where the enhancement of the immunoreactivity for the studied neuropeptides was associated with no change or a decreased expression of the corresponding genes in the same brain areas, colchicine may have blocked the axoplasmic transport of peptides in nerve fibers projecting to the midbrain and/or brain stem (6). This may result in a retrograde accumulation of peptides in the nerve cell bodies of origin and, eventually, in a negative feedback regulation of the corresponding encoding genes in these perikarya (retrograde and presynaptic effect of colchicine). The drastic behavioral effects of bilateral intranigral injections of colchicine, on ingestive behavior in particular, have been studied in a following paper.
Assuntos
Colchicina/farmacologia , Neuropeptídeos/biossíntese , Prosencéfalo/metabolismo , Substância Negra/fisiologia , Animais , Colchicina/administração & dosagem , Encefalina Metionina/biossíntese , Encefalinas/biossíntese , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Hibridização In Situ , Injeções , Neuropeptídeo Y/biossíntese , Prosencéfalo/anatomia & histologia , Prosencéfalo/efeitos dos fármacos , Precursores de Proteínas/biossíntese , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Substância P/biossíntese , Tirosina 3-Mono-Oxigenase/imunologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
In the present study, we demonstrate the existence of numerous peptidergic afferents to the bed nucleus of the stria terminalis (BNST) using the retrograde transport of gold-labeled wheat germ agglutinin-apo-peroxidase (G-WGA-HRP) combined with the indirect immunoperoxidase method after intraparenchymatous injections of colchicine. At first, we show that local injections of colchicine alone into the BNST are able to induce the retrograde accumulation of peptides until the nerve cell bodies of origin, probably because of the blockade of axonal transport in nerve terminal arborizations innervating this nucleus. The actual existence of putative peptidergic afferents to the BNST indicated by the local injections of colchicine was established using: a) the retrograde transport of G-WGA-HRP from the BNST combined with immunocytochemistry after administration of colchicine at the same place, b) the anterograde "transport" of the fluorescent tracer DiI from selected nuclei of the forebrain. We demonstrate that the neurons immunoreactive for enkephalins, neurotensin, or substance P that innervate the BNST are localized mainly in the central amygdaloid nucleus, the paraventricular thalamic nucleus, and the ventromedial hypothalamic nucleus ipsilateral to the injection, as well as bilaterally in the magnocellular paraventricular and perifornical regions of the hypothalamus. From these results it may be concluded that intracerebral injections of colchicine constitute a powerful tool to search for multiple peptidergic afferents to a given brain nucleus using only immunohistochemistry. The existence of these pathways, however, must be verified by other neuroanatomical methods because of the problem of nerve fibers of passage.
Assuntos
Colchicina , Neurônios Aferentes/fisiologia , Neuropeptídeos/fisiologia , Núcleos Talâmicos/fisiologia , Animais , Transporte Axonal/fisiologia , Carbocianinas , Encefalina Metionina/imunologia , Encefalina Metionina/metabolismo , Encefalinas/imunologia , Encefalinas/metabolismo , Peroxidase do Rábano Silvestre , Ácido Ibotênico/farmacologia , Imuno-Histoquímica , Masculino , Neurotensina/imunologia , Neurotensina/metabolismo , Ratos , Ratos Wistar , Substância P/imunologia , Substância P/metabolismo , Núcleos Talâmicos/anatomia & histologia , Núcleos Talâmicos/citologia , Tirosina 3-Mono-Oxigenase/imunologia , Tirosina 3-Mono-Oxigenase/metabolismo , Conjugado Aglutinina do Germe de Trigo-Peroxidase do Rábano Silvestre , Aglutininas do Germe de TrigoRESUMO
The short-term inhibition by estradiol of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by L-3,4-dihydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a 30-40% decrease within 1 h of incubation with estradiol. To determine whether a dephosphorylation process was involved in this decline in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition: In controls, we observed that two kinetically different forms of TH coexisted, with one exhibiting a Ki(DA) of 26.4 +/- 2 microM and the other being approximately 10-fold more sensitive to DA inhibition, with a Ki(DA) of 2.56 +/- 0.17 microM, likely corresponding to a phosphorylated and active form and to a nonphosphorylated and poorly active form, respectively. Conversely, after estradiol treatment all TH molecules exhibited the same Ki(DA) of 2.5 +/- 0.3 microM. This effect was stereospecific, because 17 alpha-estradiol could not promote it, whereas with 17 beta-estradiol, it could be observed at only 10(-11) M and after a short delay (30 min). Finally, this decrease in the Ki(DA) of the purported active form of TH could be prevented by okadaic acid (an inhibitor of protein phosphatases). These results suggest that estradiol can act directly on the mediobasal hypothalamus to trigger a rapid decline in TH activity and that this action may involve a decrease in TH phosphorylation.
Assuntos
Estradiol/farmacologia , Hipotálamo/enzimologia , Neurônios/enzimologia , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Catálise , Dactinomicina/farmacologia , Dopamina/farmacologia , Éteres Cíclicos/farmacologia , Feminino , Hipotálamo/citologia , Técnicas In Vitro , Eminência Mediana/enzimologia , Neurônios/citologia , Ácido Okadáico , Fosfoproteínas Fosfatases/antagonistas & inibidores , Proteínas/antagonistas & inibidores , Ratos , Ratos Wistar , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/antagonistas & inibidoresRESUMO
Polyamine (tissue) concentrations have been studied in hippocampus and temporal neocortex from patients with temporal lobe epilepsy. Depth electrode recordings demonstrated hippocampal origin of the seizures, the temporal neocortex being involved during the discharge propagation. Neuropathological examination of excised tissues showed glial proliferation or glioma in Ammon's horn (CA), whereas the temporal neocortex did not exhibit any histological abnormality. Polyamine (putrescine or PUT, spermidine or SPD, spermine or SPM) concentrations were determined on surgical samples from the hippocampus and various areas of temporal neocortex. Human post-mortem tissue from temporal lobe regions was used for controls. In post-mortem controls and temporal neocortex specimens from epileptic patients, polyamine levels were similar (in nmol/g wet weight: PUT = 40-100; SPD = 200-350; SPM = 100-200). In CA, polyamine levels exhibited striking changes: SPD content was significantly increased (350-700 nmol/g) while SPM was lowered (50-100). PUT was only increased in CA invaded by the tumoral process (100-180). Accordingly, a very high SPD/SPM molar ratio in the abnormal CA region was observed, indicating an acceleration of polyamine neosynthesis which is usually related to ornithine decarboxylase induction. Metabolic changes in polyamines appear to be selective of human epileptic hippocampus. A relationship between glial proliferation (gliosis or neoplasia), epileptic firing and polyamines is discussed.