RESUMO
An instability of the mature cell phenotype is thought to participate to the formation of gliomas, primary brain tumors deriving from astrocytes and/or neural stem cells. Transforming growth factor alpha (TGFalpha) is an erbB1 ligand overexpressed in the earliest stages of gliomas, and exerts trophic effects on gliomal cells and astrocytes. Here, we questioned whether prolonged TGFalpha exposure affects the stability of the normal mature astrocyte phenotype. We first developed astrocyte cultures devoid of residual neural stem cells or progenitors. We demonstrate that days of TGFalpha treatment result in the functional conversion of a population of mature astrocytes into radial glial cells, a population of neural progenitors. TGFalpha-generated radial glial cells support embryonic neurons migration, and give birth to cells of the neuronal lineage, expressing neuronal markers and the electrophysiological properties of neuroblasts. Lengthening TGFalpha treatment to months results in the delayed appearance of cells with neural stem cells properties: they form floating cellular spheres that are self-renewing, can be clonally derived from a single cell and differentiated into cells of the neuronal lineage. This study uncovers a novel population of mature astrocytes capable, in response to a single epigenetic factor, to regress progressively into a neural stem-like cell stage via an intermediate progenitor stage.
Assuntos
Astrócitos/citologia , Diferenciação Celular/efeitos dos fármacos , Neurônios/metabolismo , Células-Tronco/citologia , Fator de Crescimento Transformador alfa/farmacologia , Animais , Astrócitos/metabolismo , Linhagem da Célula , Movimento Celular , Células Cultivadas , Eletrofisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Receptores ErbB/metabolismo , Feminino , Feto/citologia , Feto/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/citologia , Neurônios/citologia , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/farmacologia , Células-Tronco/metabolismoRESUMO
Astrocyte death has been implicated in several neuropathological diseases, but the identification of molecules susceptible of promoting astrocyte survival has been elusive. We investigated whether transforming growth factor alpha (TGFalpha), an erbB1/EGFR ligand, which promotes glioma progression and affects astrocyte metabolism at embryonic and adult stages, regulates astrocyte survival. Primary serum-free astrocyte cultures from post-natal mouse and fetal human cortices were used. Transforming growth factor alpha protected both species of astrocytes from staurosporine-induced apoptosis. In serum-free medium, mouse astrocytes did not survive beyond 2 months while TGFalpha-treated astrocytes survived up to 12 months. Transforming growth factor alpha also promoted long-term survival of human astrocytes. We additionally extended TGFalpha proliferative effects to human astrocytes. After 3 days of permanent application, TGFalpha induced a major downregulation of both erbB1 and erbB2. This downregulation did not impair the functional activation of the receptors, as ascertained by their tyrosine phosphorylation and the continuous stimulation of both ERK/MAPK and PI3K/Akt pathways up to 7 days, the longest time examined. The full cellular effects of TGFalpha required activation of both transduction pathways. Enhanced proliferation and survival thus define TGFalpha as a gliatrophin for mammalian astrocytes. These results demonstrate that in normal, non-transformed astrocytes, sustained and functional erbBs activation is achieved without bypassing ligand-induced receptors downregulation.
Assuntos
Astrócitos/metabolismo , Regulação para Baixo/efeitos dos fármacos , Receptores ErbB/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Fator de Crescimento Transformador alfa/farmacologia , Envelhecimento/metabolismo , Animais , Astrócitos/citologia , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebelar/citologia , Córtex Cerebelar/embriologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glioma/metabolismo , Humanos , Camundongos , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador alfa/metabolismoRESUMO
A major aim of neurobiology today is to improve understanding of the signaling pathways that couple rapid events, such as the action potential and neurotransmitter release, to long-lasting changes in synaptic strength and increased neuronal survival. These adaptations involve interactions of neurons with other cells and with the extracellular matrix. They use, in part, the same molecular machinery that controls adhesion, motility or survival in non-neuronal cells. This machinery includes two homologous non-receptor tyrosine kinases, FAK and PYK2/CAKbeta, and the associated SRC-family tyrosine kinases. Specific brain isoforms of FAK with distinct properties are regulated by neurotransmitters, whereas PYK2/CAKbeta is highly sensitive to depolarization. The multiplicity of the pathways that can be activated by these tyrosine kinases indicates their importance in signal transduction in the adult brain.
Assuntos
Encéfalo/enzimologia , Moléculas de Adesão Celular/metabolismo , Plasticidade Neuronal/fisiologia , Proteínas Tirosina Quinases/metabolismo , Adulto , Animais , Sobrevivência Celular/fisiologia , Quinase 1 de Adesão Focal , Quinase 2 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Técnicas In Vitro , Vias Neurais/fisiologia , Neurotransmissores/biossíntese , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
In the striatum, dopamine D(1) and adenosine A(2A) receptors stimulate the production of cAMP, which is involved in neuromodulation and long-lasting changes in gene expression and synaptic function. Positive coupling of receptors to adenylyl cyclase can be mediated through the ubiquitous GTP-binding protein Galpha(S) subunit or through the olfactory isoform, Galpha(olf), which predominates in the striatum. In this study, using double in situ hybridization, we show that virtually all striatal efferent neurons, identified by the expression of preproenkephalin A, substance P, or D(1) receptor mRNA, contained high amounts of Galpha(olf) mRNA and undetectable levels of Galpha(s) mRNA. In contrast, the large cholinergic interneurons contained both Galpha(olf) and Galpha(s) transcripts. To assess the functional relationship between dopamine or adenosine receptors and G-proteins, we examined G-protein levels in the striatum of D(1) and A(2A) receptor knock-out mice. A selective increase in Galpha(olf) protein was observed in these animals, without change in mRNA levels. Conversely, Galpha(olf) levels were decreased in animals lacking a functional dopamine transporter. These results indicate that Galpha(olf) protein levels are regulated through D(1) and A(2A) receptor usage. To determine the functional consequences of changes in Galpha(olf) levels, we used heterozygous Galpha(olf) knock-out mice, which possess half of the normal Galpha(olf) levels. In these animals, the locomotor effects of amphetamine and caffeine, two psychostimulant drugs that affect dopamine and adenosine signaling, respectively, were markedly reduced. Together, these results identify Galpha(olf) as a critical and regulated component of both dopamine and adenosine signaling.
Assuntos
Adenosina/metabolismo , Corpo Estriado/metabolismo , Dopamina/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Receptores de Dopamina D1/metabolismo , Receptores Purinérgicos P1/metabolismo , Anfetamina/farmacologia , Animais , Cafeína/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina , Proteínas Heterotriméricas de Ligação ao GTP/genética , Heterozigoto , Hibridização In Situ , Masculino , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Neurônios/classificação , Neurônios/metabolismo , Especificidade de Órgãos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor A2A de Adenosina , Receptores de Dopamina D1/deficiência , Receptores de Dopamina D1/genética , Receptores Purinérgicos P1/deficiência , Receptores Purinérgicos P1/genéticaRESUMO
Halothane-anaesthetized cats were implanted with push-pull cannulae to demonstrate the in vivo release of cholecystokinin-like immunoreactivity (CCK-LI) in the substantia nigra and the ipsilateral caudate nucleus. The spontaneous and the calcium-dependent potassium-evoked release of CCK-LI were observed in both structures. In addition, the local application of tetrodotoxin (10-6 M) reduced the spontaneous release of the peptide. 6-OHDA lesions made in the substantia nigra pars compacta led to a complete destruction of nigrostriatal dopaminergic neurons. CCK-LI levels were not affected in the caudate nucleus but were reduced substantially in the substantia nigra. The activation of dopaminergic cells induced by the nigral application of alpha-methyl-para-tyrosine (10-4 M) stimulated the release of CCK-LI and dopamine in the ipsilateral caudate nucleus, whilst opposite effects were seen in the substantia nigra. Similar results were obtained when dopaminergic transmission was blocked in the caudate nucleus suggesting that the evoked release of CCK-LI by the alpha-methyl-para-tyrosine treatment originates from dopaminergic nerve terminals and not from other CCK-LI containing fibres in response to released dopamine. Dopamine (10-7 M) as well as the D1 agonist SKF 38393 (10-5 M) stimulated CCK-LI release when applied into the caudate nucleus while the D2 agonist, LY 171555 (10-6 M) slightly reduced peptide release. The local application of cholecystokinin-8 sulfate (CCK-8S) (10-8 M, for 30 min) into the substantia nigra pars compacta increased the firing rate of dopaminergic cells and stimulated the release of newly synthesized 3H-dopamine from dendrites and nerve terminals. These results suggest, but do not definitively prove, that, in the cat, CCK-LI and dopamine are coreleased from nigrostriatal mixed dopaminergic/CCK-LI neurons and that CCK-LI released from dendrites is, like dopamine, involved in the regulation of the activity of these cells.
RESUMO
We assayed the content of neurotransmitters (or their synthesizing enzymes) and neuropeptides in the hypothalamus of control and parkinsonian brains post mortem. Only dopamine concentrations were lower than normal in Parkinson's disease, suggesting that deficiency in hypothalamic dopamine transmission may play a role in the autonomic and endocrine abnormalities of this disorder.
Assuntos
Hipotálamo/metabolismo , Neurotransmissores/metabolismo , Doença de Parkinson/metabolismo , Idoso , Dopamina/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Norepinefrina/metabolismoRESUMO
[(3)H]Boc[Nle(28,31)]CCK(27)-(33) ([(3)H]BDNL-CCK(7)) is a new ligand for cholecystokinin (CCK) receptors, endowed with a high specific activity (100 Ci/mmol). Binding sites for this ligand were visualized in the rat brain by autoradiography [(3)H]BDNL-CCK(7) binds specifically to an apparent single class of CCK receptors on rat striatum sections with a K(d) of 1.76 nM and a B(max) of 57 fmol/mg protein. Unsulfated CCK(8) was two times less potent than sulfated CCK(8) to displace binding of [(3)H]BDNL-CCK(7). Binding sites for [(3)H]BDNL-CCK(7) were present in many brain regions, the highest concentrations occurring in cortex, olfactory bulbs, nucleus accumbens, and medium to high concentrations in striatum, hippocampus, and several nuclei of thalamus, hypothalamus and amygdala. In the same experimental conditions, the binding sites for [(125)I]BH-CCK(8) showed similar specificity and localization. We thus used both ligands to investigate the subregional distributions of CCK receptors in nucleus accumbens and hippocampus, where a highly organized topography of action of CCK has been reported. In nucleus accumbens, the CCK binding sites were concentrated in the anterior portion of the nucleus, whereas very low densities were observed within medial posterior nucleus accumbens, where injection of CCK has been shown to potentiate dopamine-induced hyperlocomotion. p]In hippocampus, CCK receptors were concentrated in the polymorphic zone of the hilus of the dentate gyrus and in stratum lacunosum moleculare of Ammon's horn. Very few receptors were observed in other regions of hippocampus, including stratum pyramidale and stratum moleculare. This is in contrast with the presence of numerous CCK terminals and the potent effect of CCK in these areas. The distributions of CCK receptors reported here in both nucleus accumbens and hippocampus were discussed in correlation with the distribution of CCK neurons and terminals, the related anatomical pathways, and the pharmacological profiles of the effects of CCK in these regions.
RESUMO
In mammalian brain, dopaminergic (DA) cell bodies located in the ventral mesencephalon give rise to meso-cortical, meso-limbic and meso-striatal systems. Among these, the meso-cortical DA pathway is particularly involved in the processing of emotional and cognitive responses. We demonstrate that the rat meso-cortical neurons specifically contain, in addition to DA, another transmitter, Neurotensin. If this co-localization exists in man, it may provide an anatomical substratum for the biological theory of schizophrenia as well as an indication that potential anti-psychotic drugs which act differentially on the DA ascending transmissions can be developed.
Assuntos
Encéfalo/metabolismo , Dopamina/metabolismo , Neurônios/metabolismo , Neurotensina/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Hidroxidopaminas/farmacologia , Masculino , Neurônios/efeitos dos fármacos , Norepinefrina/metabolismo , Núcleo Accumbens/metabolismo , Especificidade de Órgãos , Oxidopamina , Ratos , Ratos EndogâmicosRESUMO
As shown in the rat by estimation of dopamine (DA) and noradrenaline (NA) levels, bilateral 6-hydroxydopamine (6-OHDA, 4 micrograms/microliter) lesions made in the ventral mesencephalic tegmentum (VMT) destroy both ascending DA and NA neurones. Pretreatment of rats with desmethylimipramine (DMI, 30 mg/kg, i.p.), 30 min before microinjection of 6-OHDA into the VMT partially prevented the destruction of the DA neurones innervating the prefrontal and cingulate cortices but not those innervating subcortical structures (nucleus accumbens, olfactory tubercles, septum). Results obtained from the prefrontal cortex of rats with extensive lesions of the ascending NA neurones performed 15 days prior to the 6-OHDA lesions of the VMT in the presence of DMI, imply that NA innervation of the VMT seems to be required for DMI to protect the cortical DA neurons from the neurotoxic effect of 6-OHDA.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Desipramina/farmacologia , Hidroxidopaminas/antagonistas & inibidores , Sistema Límbico/efeitos dos fármacos , Tegmento Mesencefálico/efeitos dos fármacos , Animais , Dopamina/análise , Giro do Cíngulo/efeitos dos fármacos , Masculino , Vias Neurais/efeitos dos fármacos , Norepinefrina/análise , Núcleo Accumbens/efeitos dos fármacos , Bulbo Olfatório/efeitos dos fármacos , Oxidopamina , Ratos , Septo Pelúcido/efeitos dos fármacosRESUMO
The regional distribution of immunoassayable CCK-8 was determined in 12 regions of control and parkinsonian human brain, with specific attention to the possible regional coexistence of CCK-8 with dopamine. In Parkinson's disease, CCK-8-I levels were only decreased in the substantia nigra where dopamine cell bodies lie, and not in striatal and corticolimbic dopamine projecting areas. Our results suggest that the major proportion of dopaminergic neurones degenerated in Parkinson's disease may not contain the CCK-8 peptide.
Assuntos
Depressores do Apetite/análise , Química Encefálica , Colecistocinina/análise , Doença de Parkinson/patologia , Fragmentos de Peptídeos/análise , Substância Negra/análise , Autopsia , Encéfalo/patologia , Humanos , Sincalida , Distribuição TecidualRESUMO
The mixed CCK8/DA meso-nucleus accumbens pathway was used as a model to study the effects of some pharmacological treatments on the two coexisting transmitters. Reserpine (7 mg/kg i.p.), which depletes monoamine vesicles, induced as early as 1 h following its injection a selective decrease (36%) of CCK8 levels in the posterior part of the nucleus accumbens, an area innervated by the mixed CCK8/DA projection. In contrast, this treatment was without effect on CCK8 levels in the anterior nucleus accumbens and the ventral striatum, two areas which contain distinct CCK8 and DA innervations. Apomorphine (5 mg/kg i.p.), which is known to inhibit the firing rate of DA cells, did not block the reserpine- induced decrease in CCK8 levels suggesting that reserpine is acting on CCK8 storage. This mechanism of action was further substantiated by results obtained with alpha-methyl-p-tyrosine (alpha-MpT, 200 mg/kg i.p.) since no change in CCK8 levels was observed 4 h after this treatment. However, a selective decrease (35%) in CCK8 levels was found in the posterior part of the nucleus accumbens 20 h after two successive alpha-MpT injections. This suggested that long-term interruption of DA transmission resulted in an activation of CCK8/DA cells leading to a release of CCK8. The partial effect of reserpine on total CCK8 stores in CCK8/DA fibers suggests that the peptide is distributed in two types of storage compartments, one of them being sensitive to reserpine and possibly corresponding to mixed CCK8/DA vesicles.
Assuntos
Dopamina/análise , Mesencéfalo/análise , Núcleo Accumbens/análise , Núcleos Septais/análise , Sincalida/análise , Animais , Apomorfina/farmacologia , Corpo Estriado/análise , Masculino , Metiltirosinas/farmacologia , Vias Neurais/análise , Radioimunoensaio/métodos , Ratos , Ratos Endogâmicos , Reserpina/farmacologia , alfa-MetiltirosinaRESUMO
Bilateral injections of 6-OHDA in the ventral mesencephalic tegmentum (VMT) destroy the DA afferents of the nucleus accumbens but do not induce any denervation supersensitivity of the D1 receptors in the nucleus accumbens. Because other non-DA afferent fibers might also regulate D1 receptors in that structure, bilateral ablations of the prefrontal cortex were performed. This lesion induced a 55% decrease of [3H]glutamic acid high-affinity uptake activity in the nucleus accumbens. When the prefronto-cortical ablation was performed simultaneously with the bilateral injection of 6-OHDA in the VMT, a marked hypersensitivity of DA-sensitive adenylate cyclase activity was observed in the nucleus accumbens (+52% increase of Vmax and a two-fold decrease of the Kapp for DA) while the ablation of the prefrontal cortex alone induced only a +14% (P less than 0.01) increase of D1 receptors in that structure. These results indicate that the regulation of D1 receptors in the nucleus accumbens is not solely dependent on the presynaptic DA innervation and that other non-DA fibers, such as those of the cortico-nucleus accumbens pathway, might contribute to it.
Assuntos
Lobo Frontal/fisiologia , Núcleo Accumbens/fisiologia , Receptores Dopaminérgicos/fisiologia , Núcleos Septais/fisiologia , Adenilil Ciclases/metabolismo , Animais , Vias Eferentes/fisiologia , Glutamatos/metabolismo , Ácido Glutâmico , Masculino , Ratos , Ratos EndogâmicosRESUMO
The bilateral electrolytic lesion of the ventral mesencephalic tegmentum (VMT) induces, in the rat, behavioural deficits such as locomotor hyperactivity and disappearance of spontaneous alternation ('VMT syndrome'). When a specific 6-hydroxy dopamine (6-OHDA) destruction of the dorsal noradrenergic (NA) ascending pathway was superimposed to an electrolytic lesion of the VMT, animals recovered a normal locomotor activity and the possibility to alternate. Since many studies indicate that the development of the 'VMT syndrome' is linked to the disruption of the dopaminergic (DA) meso-cortico-limbic transmission, it is proposed that the recovery observed is due to an interaction between NA and DA ascending systems in cortical and/or subcortical structures; noradrenergic innervation would have a permissive role on the expression of the 'VMT syndrome', possibly via a mechanism of heteroregulation of DA receptors by NA fibers.
Assuntos
Córtex Cerebral/fisiologia , Comportamento Exploratório , Atividade Motora , Núcleo Accumbens/fisiologia , Núcleos Septais/fisiologia , Tegmento Mesencefálico/fisiologia , Animais , Dopamina/análise , Lateralidade Funcional , Hidroxidopaminas , Masculino , Norepinefrina/análise , Oxidopamina , Ratos , Ratos EndogâmicosRESUMO
In a previous study using differential pulse voltammetry we demonstrated an interaction between dopaminergic activity in the amygdala and the nucleus accumbens. In the present study, by post-mortem biochemical measurements, we showed that bilateral 6-OHDA lesions of DA innervation of the amygdala leads to an increase in DA activity in the nucleus accumbens (DOPAC/DA ratio +24%) and a reduction (DOPAC/DA ratio -40%) in the prefrontal cortex. In addition, after these lesions in the amygdala, there was an increased behavioral sensitivity to D-amphetamine, demonstrated by enhanced locomotor activity. Increased understanding of the interregulations between dopaminergic activity in forebrain structures may help explain forebrain functions and/or dysfunctions.
Assuntos
Tonsila do Cerebelo/fisiologia , Córtex Cerebral/fisiologia , Dextroanfetamina/farmacologia , Dopamina/fisiologia , Locomoção/efeitos dos fármacos , Núcleo Accumbens/fisiologia , Núcleos Septais/fisiologia , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Hidroxidopaminas/farmacologia , Masculino , Oxidopamina , Ratos , Ratos EndogâmicosRESUMO
The nucleus accumbens of the rat receives a mixed DA/CCK8 innervation in its posterior part while its anterior part is innervated by distinct DA and CCK8 fibres. In vitro, the addition of CCK8 (0.3-1 microM) potentiated the activating effect of DA (10-30 microM) on adenylate cyclase in tissue homogenates obtained from the posterior part of the nucleus accumbens, whilst this activating effect was reduced by CCK8 in the anterior part. These results suggest the existence of two types of regulation of the D1 receptor by CCK8 depending on the identity (mixed or not mixed) of their innervating fibres.
Assuntos
Adenilil Ciclases/metabolismo , Núcleo Accumbens/enzimologia , Receptores Dopaminérgicos/efeitos dos fármacos , Núcleos Septais/enzimologia , Sincalida/farmacologia , Animais , Masculino , Ratos , Ratos EndogâmicosRESUMO
The four cases of dyskinesia at the "beginning and end of dose" caused by L-Dopa presented in this series were characterized by four essential features: 1) their onset at the beginning and end of the period of effectiveness of a dose of L-Dopa + IDC (benserazide); 2) their ballic and dystonic appearance associated with a reinforcement of Parkinsonian signs; 3) the possibility of their reduction by an increase and fractionning of the daily dose of L-Dopa; 4) the particular nature of the underlying Parkinsonian problem in which they were seen, i.e. the young age at onset of the disease, the severity of akinesia, and the quality of the clinical response to L-Dopa. Thus on the basis of the circumstances of their development, their appearance, and their treatment, such forms of dyskinesia at the "beginning and end of dose" appear to be different from classical "mid-dose" dyskinesia. In addition, they pose a new physiolopathological problem.
Assuntos
Benserazida/uso terapêutico , Discinesia Induzida por Medicamentos/etiologia , Hidrazinas/uso terapêutico , Levodopa/efeitos adversos , Doença de Parkinson/tratamento farmacológico , Quimioterapia Combinada , Feminino , Humanos , Levodopa/administração & dosagem , Levodopa/uso terapêutico , Masculino , Pessoa de Meia-IdadeRESUMO
This study involved twleve parkinsonian patients exposed to abnormal movements provoked by L-DOPA. Including six patients with "mid-dose" dyskinesias and six others with "onset and end of dose" dyskinesias, Correlation between the circumstances of onset of abnormal movements and plasma concentrations of DOPA and O-methyl-DOPA, after administration of a dose of L-DOPA + IDC, gave the following results: 1) mid-dose dyskinesias appeared with the highest plasma concentrations of DOPA, at the maximum therapeutic effect; 2) onset and end of dose occurred during rise and fall in plasma levels of DOPA, coinciding with the relief and the reappearance of parkinsonian symptoms respectively; 3) no correlation could be established between plasma concentrations of O-methyl-DOPA and the duration of the period of clinical remission or of abnormal movements. These biochemical data, completed by the neuropharmacological study of one patient with onset and end of dose abnormal movements suggest the predominant role of a disturbance in central dopaminergic mechanisms in the genesis of abnormal movements, whatever their nature.