RESUMO
OBJECTIVE: To determine glutamate and aspartate levels in the cerebrospinal fluid (CSF) in patients with sporadic amyotrophic lateral sclerosis (SALS) grouped according to El Escorial diagnostic criteria, and to perform an in vitro assessment of the neurotoxicity of the CSF in murine cortical neurons. METHODS: SALS patients were sorted according to El Escorial diagnostic criteria. Glutamate and aspartate were measured in the CSF using high performance liquid chromatography. Cultured cortical neuron viability was determined after exposure to CSF for 24 h. RESULTS: Glutamate levels were elevated in 28 out of the 29 patients with definite, probable or possible SALS. There were no differences in glutamate concentrations when the three clinical forms of the disease were compared; neither there were significant variation across disease duration and clinical presentation. In agreement with previous reports, we concluded that CSF-SALS-induced in vitro neurotoxicity is mediated by ionotropic glutamate receptors. We found no relationship between the degree of in vitro neurotoxicity and glutamate concentration in the CSF. CONCLUSIONS: Glutamate but not aspartate CSF levels may contribute to ALS pathogenesis. However, glutamate levels may not influence the degree of diagnosis certainty or lesion extension.
Assuntos
Esclerose Lateral Amiotrófica/líquido cefalorraquidiano , Esclerose Lateral Amiotrófica/diagnóstico , Ácido Aspártico/líquido cefalorraquidiano , Líquido Cefalorraquidiano/metabolismo , Ácido Glutâmico/líquido cefalorraquidiano , Neurônios/fisiologia , Adulto , Idoso , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebral/fisiologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Fatores de TempoRESUMO
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
To compare different culture conditions for neuroprotection assays in cultured cortic neurons, we evaluated cell viability after H2O2 exposure in cells cultured with standard N2 and with the enriched B-27 developed by GIBCO, both serum-free supplements. The following additives/associations were compared: N2 (+N2), B-27 (+B-27), 10% FBS (+FBS), 1% FBS in combination with N2 (FBS/N2) or N2 supplement preceded by an 1 hour precoating with 10% FBS (N2 + precoated). Our data demonstrated that B-27 is as efficient as 10% FBS to support neuronal growth for more than a week. As shown by phase-contrast optics cells grown in N2 started degenerating within 24-48 hours although measurable absorbance was seen with MTT. The pre-coating procedure failed to modify substantially cell viability as compared with N2 alone. Dose-response curves for H2O2 to induce neuronal apoptosis were almost identical for B-27 and serum supplemented samples. Catalase (100 U/ml) or vitamin E (200 microM) prevented cell death in both culture conditions. Our results indicate that DMEM/B-27 provides a serum-free cell culture environment that allows neurons to grow with optimal cell viability, comparable to that obtained with serum. We conclude that this culture condition reveals as a useful tool to test the efficacy of neuroprotectants when a serum free medium is required.
Assuntos
Sangue , Córtex Cerebral/efeitos dos fármacos , Meios de Cultura/farmacologia , Peróxido de Hidrogênio/farmacologia , Neurotoxinas/farmacologia , Oxidantes/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Meios de Cultura Livres de Soro/farmacologia , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Estresse OxidativoRESUMO
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 aim of the present study was to determine the developmental pattern of progesterone metabolism in rat brain and spinal cord from embryonic day 13 (E13) to the perinatal period. A marked decrease in the 5alpha-reduction of progesterone in brain cortex was observed between E13 and postnatal day 5 (P5). Isopregnanolone was the predominant isomer in E13 in both cortex and spinal cord and its synthesis diminished gradually, while the concentration of allopregnanolone did not change significantly during development. The placental tissue was able to synthesize the 3alpha and 3beta isomers in E13, E16 and E19 embryos with allopregnanolone being the major metabolite in all the samples. We conclude that embryonic central nervous system tissues are able to synthesize neurosteroids at least from stage E13 and that they are developmentally regulated.
Assuntos
Sistema Nervoso Central/embriologia , Sistema Nervoso Central/enzimologia , Placenta/enzimologia , Pregnanolona/biossíntese , Progesterona/biossíntese , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Animais , Radioisótopos de Carbono , Cromatografia em Camada Fina , Feminino , Masculino , Gravidez , Progesterona/análise , Progesterona/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Envelhecimento/metabolismo , Córtex Cerebral/metabolismo , Pregnanolona/farmacologia , Receptores de GABA-A/metabolismo , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Animais Recém-Nascidos/metabolismo , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário e Fetal , Muscimol/metabolismo , Ratos , Ratos Wistar , Receptores de GABA-A/efeitos dos fármacos , Esteroides/farmacologiaRESUMO
In this paper we describe the effects of X-radiation on the viability of cerebellar granule cells grown in culture. Cell cultures were exposed to X-rays 2 h after plating and then grown for 1-7 days. Two days after X-ray exposure with a dose-range of 0.1-2 Gy (acute effect), a significant decrease in neuronal number was observed. The magnitude of the lethal effect was directly correlated to the dose of X-ray applied. When the interval between plating and irradiation was increased, the acute lethal effect of X-rays decreased. 3H-thymidine incorporation was maximal during the first 24 h in vitro and decreased to nearly blank levels, after 72 h. In some experiments, cells present in each culture dish were counted at day 2 and at day 7. We observed that the number of cells present in sham-irradiated cultures decreased from day 2 to day 7, reflecting cell death after several days in vitro. The cell loss observed in X-irradiated cultures was significantly greater as compared with sham-irradiated cultures, confirming the deleterious effect of X-ray on cell survival. This effect was completely prevented by GM1 (6.5, 10 and 30 microM) added 48 h after X-ray exposure, but not 1 h after plating. We conclude that X-rays induce two different effects: an acute effect related to impaired DNA synthesis which is very active during the first 24 h in vitro, and a long-term effect owing to a sublethal damage in the surviving neuronal population.
Assuntos
Cerebelo/efeitos dos fármacos , Gangliosídeo G(M1)/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos da radiação , Radicais Livres , Neurônios/citologia , Neurônios/efeitos da radiação , Ratos , Ratos WistarRESUMO
1. The in vitro effects of N-(2-chloroethyl)-N-ethyl-bromobenzylamine (DSP4) were studied in the rat vas deferens. 2. DSP4 inhibited the biphasic motor response induced by field stimulation in a concentration-dependent manner. The concentration of DSP4 that elicited 50% of the maximal inhibition of the twitch response induced by 3 Hz was 10 microM. 3. DSP4 10 microM abolished the motor response induced by exogenously applied noradrenaline and 0.1 mM ATP. Phentolamine (an alpha-adrenoceptor blocker) prevented DSP4 inhibitory effect. 4. DSP4 inhibitory effect was no due to the activation of alpha 2-presynaptic adrenoceptor mechanisms. 5. DSP4 impairs neurotransmission in the rat vas deferens by a postsynaptic alpha 1-adrenoceptor blockade and by an inhibition of the purinergic response.
Assuntos
Benzilaminas/farmacologia , Inibidores da Captação de Neurotransmissores/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Ducto Deferente/inervação , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/farmacologia , Animais , Estimulação Elétrica , Técnicas In Vitro , Contração Isométrica/efeitos dos fármacos , Masculino , Contração Muscular/efeitos dos fármacos , Norepinefrina/antagonistas & inibidores , Norepinefrina/farmacologia , Fentolamina/farmacologia , Ratos , Ratos Wistar , Reserpina/farmacologia , Ducto Deferente/efeitos dos fármacos , Ioimbina/farmacologiaRESUMO
1. In the whole rat vas deferens 20 microM noradrenaline (NA) and 0.011 microM clonidine decreased (36 +/- 7.4% and 80 +/- 6.0% respectively) the motor response induced by hypogastric nerve stimulation. These effects were reverted by 1 microM yohimbine. Amphetamine 5.4 microM failed to antagonize the inhibitory effect of NA and attenuated clonidine effect. 2. The effect of amphetamine was not altered by preincubation with either cocaine 1 microM, (-)-propranolol 0.3 microM or cocaine plus prazosin 0.028 microM. 3. In reserpine pretreated animals amphetamine 5.4 microM shifted to the right the concentration-response curve (CRC) to clonidine 0.62 +/- 0.05 log units with a KB value of 1.83 +/- 0.30 microM. 4. Binding of [3H]clonidine and [3H]prazosin were inhibited by amphetamine. Amphetamine was 90 times more potent to inhibit [3H]clonidine binding. 5. The results obtained suggest a possible direct interaction between clonidine and amphetamine on alpha-adrenoceptor.
Assuntos
Anfetamina/farmacologia , Clonidina/antagonistas & inibidores , Receptores Adrenérgicos alfa/efeitos dos fármacos , Animais , Ligação Competitiva/efeitos dos fármacos , Clonidina/farmacologia , Estimulação Elétrica , Técnicas In Vitro , Contração Isométrica/efeitos dos fármacos , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Norepinefrina/farmacologia , Prazosina/farmacologia , Próstata/fisiologia , Ratos , Ratos Endogâmicos , Reserpina/farmacologia , Ducto Deferente/efeitos dos fármacosRESUMO
1 The interaction between amphetamine and the alpha 2-adrenoreceptor agonists, clonidine and guanabenz, was studied in the submaxillary gland of anaesthetised rats. 2 Low doses of clonidine (10 micrograms/kg) and guanabenz (10 micrograms/kg) inhibited the secretory responses induced by methacholine and substance P, respectively. 3 Amphetamine (300 micrograms/kg) antagonized the inhibitory effects of both alpha 2-agonists. This dose of amphetamine alone did not show sialagogic effects. 4 Atropine (1 micrograms/kg) diminished the secretory responses to methacholine as much as clonidine (10 micrograms/kg). Amphetamine did not modify the blockade by atropine. 5 Guanabenz (10 micrograms/kg) markedly decreased the secretory responses to substance P, an effect that was also prevented by amphetamine. 6 Reserpine pretreatment (5 mg/kg, i.p., 18 h) did not alter the effect of amphetamine. 7 These results indicate that the interaction between amphetamine and alpha 2-adrenoreceptor agonists is unrelated to the indirect effect of this amine and suggest a direct interaction between the drug and postsynaptic inhibitory alpha 2-adrenoreceptors.
Assuntos
Anfetamina/farmacologia , Receptores Adrenérgicos alfa/efeitos dos fármacos , Glândula Submandibular/efeitos dos fármacos , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Relação Dose-Resposta a Droga , Interações Medicamentosas , Feminino , Ratos , Ratos Endogâmicos , Glândula Submandibular/metabolismoRESUMO
The effects of alpha-adrenoceptor and dopaminoceptor agonists and antagonists were investigated on presynaptic receptors in the prostatic portion of rat vas deferens. The variable studied was the early component (250 msec) of the motor response elicited by field stimulation (single pulses). All the experiments were carried out in the presence of cocaine 30 mumol/l and hydrocortisone 28 mumol/l so as to block the sites of amines loss and 1-propranolol 0.3 mumol/l to block beta-adrenoceptors. Clonidine, noradrenaline (NA) and dopamine (DA) inhibited the motor response in a concentration-dependent manner. DA was 10 and 10(4) times less potent than NA and clonidine respectively. The selective D2 agonist, LY 141865, failed to inhibit the motor response even at a high concentration (30 mumol/l). Yohimbine (0.1, 0.3 and 1 mumol/l) antagonized competitively the effect of clonidine, NA and DA showing similar - log KB values (7.57; 7.68 and 7.09 respectively). Likewise, idaxozan (0.03 mumol/l) blocked the inhibitory effect of DA in the same order of potency (- log KB = 7.81). On the other hand, pimozide 0.21 mumol/l and Schering 23390 3 mumol/l antagonized the inhibitory effect of DA, showing a lower potency than the other antagonists. Taken together, these findings do not support the hypothesis that DA activates a specific population of prejunctional dopaminoceptors to inhibit the motor response elicited by field stimulation in the presence of cocaine, hydrocortisone and 1-propranolol in the prostatic portion of the rat vas deferens. Instead, the population of prejunctional alpha 2-adrenoceptor may be involved.
Assuntos
Clonidina/farmacologia , Dopamina/farmacologia , Neurônios Motores/efeitos dos fármacos , Receptores de Neurotransmissores/efeitos dos fármacos , Sódio/farmacologia , Ducto Deferente/efeitos dos fármacos , Animais , Estimulação Elétrica , Masculino , Neurônios Motores/fisiologia , Condução Nervosa/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Receptores Dopaminérgicos/fisiologia , Ducto Deferente/fisiologiaRESUMO
The effects of alpha-adrenoceptor and dopaminoceptor agonists and antagonists were investigated on presynaptic receptors in the prostatic portion of rat vas deferens. The variable studied was the early component (250 msec) of the motor response elicited by field stimulation (single pulses). All the experiments were carried out in the presence of cocaine 30 mumol/l and hydrocortisone 28 mumol/l so as to block the sites of amines loss and 1-propranolol 0.3 mumol/l to block beta-adrenoceptors. Clonidine, noradrenaline (NA) and dopamine (DA) inhibited the motor response in a concentration-dependent manner. DA was 10 and 10(4) times less potent than NA and clonidine respectively. The selective D2 agonist, LY 141865, failed to inhibit the motor response even at a high concentration (30 mumol/l). Yohimbine (0.1, 0.3 and 1 mumol/l) antagonized competitively the effect of clonidine, NA and DA showing similar - log KB values (7.57; 7.68 and 7.09 respectively). Likewise, idaxozan (0.03 mumol/l) blocked the inhibitory effect of DA in the same order of potency (- log KB = 7.81). On the other hand, pimozide 0.21 mumol/l and Schering 23390 3 mumol/l antagonized the inhibitory effect of DA, showing a lower potency than the other antagonists. Taken together, these findings do not support the hypothesis that DA activates a specific population of prejunctional dopaminoceptors to inhibit the motor response elicited by field stimulation in the presence of cocaine, hydrocortisone and 1-propranolol in the prostatic portion of the rat vas deferens. Instead, the population of prejunctional alpha 2-adrenoceptor may be involved.