RESUMO
The role of allopregnanolone on immature cerebellar granule cells (CGC) proliferation was studied. Allopregnanolone (0.1-1 microM) increased [(3)H]thymidine incorporation and cell number determined by neuronal counting and by an MTT colorimetric assay. The effect of the neurosteroid was completely prevented by preincubation with 10 mM MgCl(2), 10 microM nifedipine, 10 microM picrotoxin or by 50 microM bicuculine. We conclude that ALLO affects cerebellar neurogenesis by increasing calcium influx through voltage-gated calcium channels and GABA(A) receptors activation.
Assuntos
Cerebelo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Pregnanolona/farmacologia , Esteroides/farmacologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Canais de Cloreto/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/efeitos dos fármacosRESUMO
Many methods have been developed to quantify neuronal morphology: measurement of neurite length, neurite number, etc. However, none of these approaches provides a comprehensive view of the complexity of neuronal morphology. In this work we have analyzed the evaluation of fractal dimension (D) as a tool to represent and quantify changes in complexity of the dendritic arbor, in in vitro cultures grown under low-density conditions. Neurons grown in isolation developed a bipolar morphology corresponding to a fractal dimension close to the unit. The analysis showed that neuronal complexity increased when cells were incubated with a depolarizing potassium concentration and there was a correlation with an increase in fractal dimension (D5 mM KCl = 1.08 +/- 0.01, D25 mM KCl =1.25 +/- 0.01). We conclude that fractal dimension is a suitable parameter to quantify changes in neuronal morphological complexity.
Assuntos
Fractais , Neurônios/metabolismo , Neurônios/fisiologia , Animais , Cerebelo/citologia , Meios de Cultura Livres de Soro/farmacologia , Processamento de Imagem Assistida por Computador , Modelos Biológicos , Modelos Teóricos , Ratos , Ratos Sprague-Dawley , Toxina Tetânica/farmacologia , Fatores de TempoRESUMO
The presence of GABA and its receptors early in rodent nervous system development has lead to speculation on the role of this transmitter system in neuroblast proliferation, migration and differentiation. We studied the effect of GABA and GABA agonists on immature cerebellar granule cell proliferation and survival. Cerebellar granule cell suspensions were obtained from 6-8-day-old rats and grown in culture for up to 7 days in serum-containing or serum-free medium. The addition of GABA (0.1-100 microM) or muscimol (0.01-10 microM) 2 h after inoculation and harvested 22 h later, lead to an increase in 3H-thymidine incorporation over control samples with the correspondent increase in granule cells number assayed 48 h later. The effect on cell proliferation exerted by GABAA agonists was blocked by MgCl2 and nifedipine, as well as by the chloride channel blocker, picrotoxin (50 microM), and the GABAA receptor specific blocker, bicuculline (50 microM). The increase on cell proliferation induced by GABA also was blocked by PD98059 (75 microM), a specific inhibitor of the mitogen-activated protein kinase kinase (MAPKK). GABAA receptor-mediated proliferation was consistently seen in cells inoculated in serum-containing medium supplemented with 25 mM KCl but not seen in serum-free medium, with 5 mM or 25 mM KCl. The presence of serum did not enhance the survival of cerebellar granule cells grown for 7 days in either 5 mM or 25 mM KCl. Additionally, neither GABA nor muscimol applied from day 2 to day 7 in vitro affected cell survival in any culture condition. We conclude that GABA and GABAA receptor agonists influence granule cell proliferation but not survival and that this effect is mediated by a calcium influx via voltage-dependent calcium channel activation, with a subsequent activation of the MAPK cascade.
Assuntos
Senescência Celular/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Muscimol/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive disorder resulting from degeneration of motor neurons in the brain and spinal cord. Sporadic ALS (SALS) accounts for the majority of patients and the familial form (FALS) represents fewer than 10% of all cases. Since it was found that there are Cu/Zn superoxide dismutase (SODI) gene mutations in 20% of FALS patients and that FALS and SALS patients show similar clinical features, it has been postulated that both may share a common physiopathological mechanism. We studied Cu/Zn SOD1 activity in cytosolic extracts of erythrocytes from 125 normal individuals and 40 SALS patients. We found that enzyme activity does not change with age in control subjects and tends to decrease in most SALS patients older than 60 years. A subpopulation of five SALS patients had significantly increased SOD1 activity; four of these patients over 70 years old. There was no correlation between enzyme activity and time of onset of the disease, or clinical forms of the illness. The variation in SOD1 activity in ageing SALS patients compared with younger patients suggests that they may undergo an oxidative disbalance contributing to the development of the disease.
Assuntos
Envelhecimento/metabolismo , Esclerose Lateral Amiotrófica/enzimologia , Superóxido Dismutase/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Eritrócitos/enzimologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The present study examines the effect of depolarizing potassium concentrations on the proliferation of immature rat cerebellar neurons. Cells inoculated in serum free medium and 5 mM KCl (5 K) showed a high degree of 3H-thymidine incorporation that decreased 24-48 h after plating as differentiation began. During the first 24 h after inoculation, cells grown in high potassium (25 K), showed a 34 +/- 3% increase (mean +/- S.E.M., n = 12) in 3H-thymidine incorporation as compared with the values observed in 5 K. After 24 h in vitro, cells grown in 25 K showed 23 +/- 3% (mean +/- S.E.M., n = 3) less DNA synthesis than those inoculated in 5 K. The increase in DNA synthesis due to 25 K was blocked by MgCl2 and nifedipine, but not by omega-conotoxin GVIA, suggesting that it is mediated by a Ca2+ influx via voltage-gated calcium channels (VGCC) of the L-subtype. High potassium-induced cell proliferation was blocked by the mitogen-activated protein kinase kinase (MEK1) inhibitor (PD98059, 75 microM). The number of neurons counted after 48 h in vitro in 25 K was 35-100% above of the number obtained with 5 K and this increase also was blocked by MgCl2 and nifedipine. These data support the hypothesis that depolarizing activity during neurogenesis plays a role in the modulation of cerebellar granule cells proliferation.
Assuntos
Cerebelo/metabolismo , Espaço Extracelular/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno , Neurônios/metabolismo , Potássio/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Senescência Celular/fisiologia , Cerebelo/citologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , MAP Quinase Quinase 1 , Cloreto de Magnésio/farmacologia , Nifedipino/farmacologia , Concentração Osmolar , Peptídeos/farmacologia , Cloreto de Potássio/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Timidina/metabolismo , ômega-Conotoxina GVIARESUMO
The allosteric modulation of GABAA receptors in the rat brain cortex by neurosteroids was studied at different developmental stages. GABAA receptors were identified using [3H]muscimol binding to membrane preparations obtained from embryos and neonates (postnatal day 0-PN0; postnatal day 5-PN5). Data analysis disclosed a unique population of binding sites at all ages tested. An increase in the number of receptors was observed during development reaching almost adult levels at PN5. The neurosteroids pregnanolone and allopregnanolone failed to modulate [3H]muscimol specific binding in embryos and neonates, but a positive modulation was obtained in 5-day old animals. The addition of 1 microM pregnanolone induced a 3-6-fold increase [3H]muscimol affinity in PN5 (n = 3; P < 0.03), and a 2-fold increase in receptors number in adults (n = 3; P < 0.03). The differences observed in allosteric modulation during development suggest that a change occurred during the first week of life, and this change might affect GABAA receptor function.