RESUMO
Amyotrophic lateral sclerosis (ALS) is a lethal disorder characterized by the gradual degeneration of motor neurons in the cerebrospinal axis. Whether upper motor neuron hyperexcitability, which is a feature of ALS, provokes dysfunction of glutamate metabolism and degeneration of lower motor neurons via an anterograde process is undetermined. To examine whether early changes in upper motor neuron activity occur in association with glutamatergic alterations, we performed whole-cell patch-clamp recordings to analyze excitatory properties of Layer V cortical motor neurons and excitatory postsynaptic currents (EPSCs) in presymptomatic G93A mice modeling familial ALS (fALS). We found that G93A Layer V pyramidal neurons exhibited altered EPSC frequency and rheobase values indicative of their hyperexcitability status. Biocytin loading of these hyperexcitable neurons revealed an expansion of their basal dendrite arborization. Moreover, we detected increased expression levels of the vesicular glutamate transporter 2 in cortical Layer V of G93A mice. Altogether our data show that functional and structural neuronal alterations associate with abnormal glutamatergic activity in motor cortex of presymptomatic G93A mice. These abnormalities, expected to enhance glutamate release and to favor its accumulation in the motor cortex, provide strong support for the view that upper motor neurons are involved early on in the pathogenesis of ALS.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Córtex Motor/patologia , Córtex Motor/fisiopatologia , Neurônios Motores/patologia , Neurônios Motores/fisiologia , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Potenciais de Ação , Esclerose Lateral Amiotrófica/metabolismo , Animais , Dendritos/patologia , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores , Histona-Lisina N-Metiltransferase/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Córtex Motor/metabolismo , Neurônios Motores/metabolismo , Superóxido Dismutase-1/genética , Sinapses/metabolismoRESUMO
miR-34a is involved in the regulation of the fate of different cell types. However, the mechanism by which it controls the differentiation programme of neural cells remains largely unknown. Here, we investigated the role of miR-34a in neurogenesis and maturation of developing neurons and identified Doublecortin as a new miR-34a target. We found that the overexpression of miR-34a in vitro significantly increases precursor proliferation and influences morphology and function of developing neurons. Indeed, miR-34a overexpressing neurons showed a decreased expression of several synaptic proteins and receptor subunits, a decrement of NMDA-evoked current density and, interestingly, a more efficient response to synaptic stimulus. In vivo, miR-34a overexpression showed stage-specific effects. In neural progenitors, miR-34a overexpression promoted cell proliferation, in migratory neuroblasts reduced the migration and in differentiating newborn neurons modulated process outgrowth and complexity. Importantly, we found that rats overexpressing miR-34a in the brain have better learning abilities and reduced emotionality.
Assuntos
Comportamento Animal , Forma Celular , MicroRNAs/metabolismo , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Animais , Sequência de Bases , Bromodesoxiuridina/metabolismo , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Córtex Cerebral/citologia , Cognição , Dependovirus/metabolismo , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Emoções , Feminino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Dados de Sequência Molecular , Neurite (Inflamação)/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fenótipo , Ratos Wistar , Células-Tronco/citologiaRESUMO
The progressive production and subsequent accumulation of ß-amyloid (Aß), a proteolytic fragment of the membrane-associated amyloid precursor protein (APP), plays a central role in Alzheimer's Disease (AD). Aß is released in a soluble form that may be responsible for cognitive dysfunction in the early stages of the disease, then progressively forms oligomeric, multimeric and fibrillar aggregates, triggering neurodegeneration. Eventually, the aggregation and accumulation of Aß culminates with the formation of extracellular plaques, one of the morphological hallmarks of the disease, detectable post-mortem in AD brains. In this review we report the known structural features of amyloid peptides and fibrils, and we give an overview of all small molecules that have been found to interact with Aß aggregation. Deeper knowledge of the mechanism leading to amyloid fibrils along with their molecular structure and the molecular interactions responsible for activity of small molecules could supply useful information for the design of new AD therapeutic agents.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Animais , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
The tachykinin endecapeptide substance P (SP) has been demonstrated to exert a functional role in neurodegenerative disorders, including Alzheimer's disease (AD). Aim of the present study was to evaluate the SP neuroprotective potential against apoptosis induced by the neurotoxic beta-amyloid peptide (A beta) in cultured rat cerebellar granule cells (CGCs). We found that SP protects CGCs against both A beta(25-35)- and A beta(1-42)-induced apoptotic CGCs death as revealed by live/dead cell assay, Hoechst staining and caspase(s)-induced PARP-1 cleavage, through an Akt-dependent mechanism. Since in CGCs the fast inactivating or A-type K(+) current (I(KA)) was potentiated by A beta treatment through up-regulation of Kv4 subunits, we investigated whether I(KA) and the related potassium channel subunits could be involved in the SP anti-apoptotic activity. Patch-clamp experiments showed that the A beta-induced increase of I(KA) current amplitude was reversed by SP treatment. In addition, as revealed by Western blot analysis and immunofluorescence studies, SP prevented the up-regulation of Kv4.2 and Kv4.3 channel subunits expression. These results indicate that SP plays a role in the regulation of voltage-gated potassium channels in A beta-mediated neuronal death and may represent a new approach in the understanding and treatment of AD.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Cerebelo/citologia , Neurônios/efeitos dos fármacos , Canais de Potássio Shal/metabolismo , Substância P/farmacologia , Animais , Animais Recém-Nascidos , Biofísica , Caspase 3/metabolismo , Células Cultivadas , Estimulação Elétrica , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Proteína Oncogênica v-akt/metabolismo , Técnicas de Patch-Clamp/métodos , Fragmentos de Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Canais de Potássio Shal/efeitos dos fármacosRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal progressive neuropathy associated with the degeneration of spinal and brainstem motor neurons. Although ALS is essentially considered as a lower motor neuron disease, prefrontal cortex atrophy underlying executive function deficits have been extensively reported in ALS patients. Here, we examine whether prefrontal cortex neuronal abnormalities and related cognitive impairments are present in presymptomatic G93A Cu/Zn superoxide dismutase mice, a mouse model for familial ALS. Structural characteristics of prelimbic/infralimbic (PL/IL) medial prefrontal cortex (mPFC) neurons were studied in 3-month-old G93A and wild-type mice with the Golgi-Cox method, while mPFC-related cognitive operations were assessed using the conditioned fear extinction paradigm. Sholl analysis performed on the dendritic material showed a reduction in dendrite length and branch nodes on basal dendrites of PL/IL neurons in G93A mice. Spine density was also decreased on basal dendrite segments of branch order five. Consistent with the altered morphology of PL/IL cortical regions, G93A mice showed impaired extinction of conditioned fear. Our findings indicate that abnormal prefrontal cortex connectivity and function are appreciable before the onset of motor disturbances in this model.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/psicologia , Transtornos Cognitivos/genética , Extinção Psicológica/fisiologia , Córtex Pré-Frontal/anormalidades , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/complicações , Animais , Forma Celular/genética , Transtornos Cognitivos/enzimologia , Transtornos Cognitivos/fisiopatologia , Condicionamento Psicológico/fisiologia , Espinhas Dendríticas/patologia , Modelos Animais de Doenças , Progressão da Doença , Medo/fisiologia , Predisposição Genética para Doença , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Malformações do Sistema Nervoso/enzimologia , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/fisiopatologia , Vias Neurais/anormalidades , Vias Neurais/enzimologia , Vias Neurais/fisiopatologia , Córtex Pré-Frontal/enzimologia , Córtex Pré-Frontal/fisiopatologia , Células Piramidais/patologia , Coloração pela PrataRESUMO
In the current study, we have evaluated the ability of substance P (SP) and other neurokinin 1 receptor (NK1) agonists to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death (S-K5). We also established the presence of SP high affinity NK1 transcripts and the NK1 protein localization in the membrane of a sub-population of CGCs. Moreover, SP significantly and dose-dependently reduced the Akt 1/2 and Erk1/2 dephosphorylation induced by S-K5 conditions, as demonstrated by Western blot analysis. Surprisingly, in SP-treated CGCs caspase-3 activity was not inhibited, while the calpain-1 activity was moderately reduced. Corroborating this result, SP blocked calpain-mediated cleavage of tau protein, as demonstrated by the reduced appearance of a diagnostic fragment of 17 kDa by Western blot analysis. In addition, SP induced a significant reduction of the delayed rectifier K+ currents (Ik) in about 42% of the patched neurons, when these were evoked with depolarizing potential steps. Taken together, the present results demonstrate that the activation of NK1 receptors expressed in CGCs promote the neuronal survival via pathways involving Akt and Erk activation and by inhibition of Ik which can contribute to the neuroprotective effect of the peptide.
Assuntos
Cerebelo/efeitos dos fármacos , Canais de Potássio de Retificação Tardia/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fármacos Neuroprotetores , Substância P/farmacologia , Animais , Western Blotting , Calpaína/antagonistas & inibidores , Caspases/metabolismo , Cerebelo/citologia , Grânulos Citoplasmáticos/fisiologia , Canais de Potássio de Retificação Tardia/efeitos dos fármacos , Eletrofisiologia , Ativação Enzimática/efeitos dos fármacos , Imuno-Histoquímica , Microscopia de Contraste de Fase , Proteína Oncogênica v-akt/fisiologia , Técnicas de Patch-Clamp , Potássio/fisiologia , Ratos , Ratos Wistar , Receptores da Neurocinina-1/efeitos dos fármacos , Receptores da Neurocinina-1/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Substância P/análogos & derivados , Taquicininas/agonistasRESUMO
The production of no-carrier-added (NCA) alpha-emitter (211)At/(211g)Po radionuclides for high-LET targeted radiotherapy and immunoradiotherapy, through the (209)Bi(alpha,2n) reaction, together with the required wet radiochemistry and radioanalytical quality controls carried out at LASA is described, through dedicated irradiation experiments at the MC-40 cyclotron of JRC-Ispra. The amount of both the gamma-emitter (210)At and its long half-lived alpha-emitting daughter (210)Po is optimised and minimised by appropriate choice of energy and energy loss of alpha particle beam. The measured excitation functions for production of the main radioisotopic impurity (210)At-->(210)Po are compared with theoretical predictions from model calculations performed at ENEA.
Assuntos
Astato/química , Ciclotrons , Polônio/química , Astato/isolamento & purificação , Polônio/isolamento & purificação , Radioterapia , Espectrometria gamaRESUMO
The wish of this work is the study of the effect of electromagnetic (EMF) radiations at a frequency of 50 Hz on the development of cerebellar granule neurons (CGN). Granule neurons, prepared from newborn rat cerebellum (8 days after birth), were cultured after plate-seeding in the presence of EMF radiations, with the plan of characterizing their cellular and molecular biochemistry, after exposure to the electromagnetic stimulus. Five days challenge to EMF radiations showed, by the cytotoxic glutamate (Glu) pulse test, a 30% decrease of cells survival, while only 5% of mortality was reported for unexposed sample. Moreover, blocking the glutamate receptor (GluR) with the Glu competitor MK-801, no toxicity effect after CGN challenge to EMF radiations and Glu was detected. By patch-clamp recording technique, the Kainate-induced currents from 6 days old exposed CGN exhibited a significant increase with respect to control cells. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses show that EMF exposure of rats CGN, induces a change in both GluRs proteins and mRNAs expression with respect to control. In addition, the use of monoclonal antibody raised against neurofilament protein (NF-200) reveals an increase in NF-200 synthesis in the exposed CGN. All these results indicate that exposure to non-ionizing radiations contribute to a premature expression of GluRs reducing the life span of CGN, leading to a more rapid cell maturation.
Assuntos
Diferenciação Celular/efeitos da radiação , Senescência Celular/efeitos da radiação , Cerebelo/citologia , Neurônios/citologia , Neurônios/fisiologia , Radiação , Animais , Animais Recém-Nascidos , Western Blotting , Células Cultivadas , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Neurofilamentos/metabolismo , Neurônios/metabolismo , Ratos , Ratos Wistar , Distribuição TecidualRESUMO
There are many evidences implicating glutamatergic toxicity as a contributory factor in the selective neuronal injury occurring in amyotrophic lateral sclerosis (ALS). This neurodegenerative disorder is characterized by the progressive loss of motor neurons, whose pathogenesis is thought to involve Ca(2+) influx mediated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate receptors (AMPARs). In the present study we report alterations in the AMPARs function in a transgenic mouse-model of the human SOD1(G93A) familial ALS. Compared with those expressed in motor neurons carrying the human wild type gene, AMPAR-gated channels expressed in motor neurons carrying the human mutant gene exhibited modified permeability, altered agonist cooperativity between the sites involved in the process of channel opening and were responsible for slower spontaneous synaptic events. These observations demonstrate that the SOD1(G93A) mutation induces changes in AMPAR functions which may underlie the increased vulnerability of motor neurons to glutamatergic excitotoxicity in ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Neurônios Motores/metabolismo , Mutação , Receptores de AMPA/metabolismo , Medula Espinal/metabolismo , Superóxido Dismutase/metabolismo , Alanina/genética , Esclerose Lateral Amiotrófica/enzimologia , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Eletrofisiologia , Glicina/genética , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Neurônios Motores/enzimologia , Técnicas de Patch-Clamp , Medula Espinal/enzimologia , Superóxido Dismutase/genética , Regulação para CimaRESUMO
Apart from the extensive loss of motor neurons, degeneration of midbrain dopaminergic cells has been described in both familial and sporadic forms of amyotrophic lateral sclerosis (ALS). Mice overexpressing the mutant human Cu/Zn superoxide dismutase (SOD1) show an ALS-like phenotype in that they show a progressive death of motor neurons accompanied by degeneration of dopaminergic cells. To describe the functional alterations specifically associated with this dopaminergic dysfunction, we have investigated the corticostriatal synaptic plasticity in mice overexpressing the human SOD1 (SOD1+) and the mutated (Gly(93)-->Ala) form (G93A+) of the same enzyme. We show that repetitive stimulation of the corticostriatal pathway generates long-term depression (LTD) in SOD1+ mice and in control (G93A-/SOD1-) animals, whereas in G93A+ mice the same stimulation generates an N-methyl-D-aspartic acid receptor-dependent long-term potentiation. No significant alterations were found in the intrinsic membrane properties of striatal medium spiny neurons and basal corticostriatal synaptic transmission of G93A+ mice. Bath perfusion of dopamine or the D(2) dopamine receptor agonist quinpirole restored LTD in G93A+ mice. Consistent with these in vitro results, habituation of locomotor activity and striatal-dependent active avoidance learning were impaired in G93A+ mice. Thus, degeneration of dopaminergic neurons in the substantia nigra of G93A+ mice causes substantial modifications in striatal synaptic plasticity and related behaviors, and may be a cellular substrate of the extrapyramidal motor and cognitive disorders observed in familial and sporadic ALS.
Assuntos
Potenciação de Longa Duração/fisiologia , Mutação , Plasticidade Neuronal/fisiologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Valina/análogos & derivados , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Alanina/genética , Animais , Aprendizagem da Esquiva/fisiologia , Cálcio/metabolismo , Membrana Celular/fisiologia , Corpo Estriado/fisiologia , Modelos Animais de Doenças , Agonistas de Dopamina/farmacologia , Estimulação Elétrica , Genótipo , Glicina/genética , Giro do Cíngulo/anatomia & histologia , Giro do Cíngulo/fisiologia , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Vias Neurais , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Quimpirol/farmacologia , Valina/farmacologiaRESUMO
This study investigated whether the mechanism of action of levetiracetam (LEV) is related to effects on neuronal voltage-gated Na+ or T-type Ca2+currents. Rat neocortical neurones in culture were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study voltage-gated Na+ current. Additionally, visually identified pyramidal neurones in the CA1 area of rat hippocampal slices were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study low-voltage-gated (T-type) Ca2+ current. LEV (10 microM-1 mM) did not modify the Na+ current amplitude and did not change (200 microM) the steady-state activation and inactivation, the time to peak, the fast kinetics of the inactivation and the recovery from the steady-state inactivation of the Na+ current. Likewise, LEV (32-100 microM) did not modify the amplitude and did not change the steady-state activation and inactivation, the time to peak, the fast kinetics of the inactivation and the recovery from the steady-state inactivation of the T-type Ca2+current. In conclusion, neuronal voltage-gated Na+ channels do not appear directly involved in the antiepileptic mechanism of action of LEV, and LEV was devoid of effect on the low-voltage-gated (T-type) Ca2+ current in hippocampal neurones.
Assuntos
Canais de Cálcio Tipo T/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Nootrópicos/farmacologia , Piracetam/farmacologia , Canais de Sódio/efeitos dos fármacos , Animais , Corpo Estriado/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Levetiracetam , Piracetam/análogos & derivados , Ratos , Ratos Wistar/embriologia , Substância Negra/efeitos dos fármacos , Tálamo/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismoRESUMO
The action of purinergic antagonists on kainate-induced currents was studied in rat cortical neurons in primary culture using the whole-cell configuration of the patch-clamp technique. The amplitude of the currents induced by kainate in cortical neurons was concentration-dependent (EC(50)=106 microM). Pyridoxal-phosphate-6-azophenyll-2',4'-disulphonic acid 4-sodium (PPADS), a P2X antagonist, was ineffective in the reduction of the kainate-induced current in cortical neurons, while 2, 2'-pyridylisatogen (PIT), basilen blue (BB) and suramin, respectively two selective P2Y and a non-selective P2 receptor antagonist, caused a reduction in the amplitude of the current induced by kainate. BB decreased the inward current induced by kainate at all holding potentials and the reduction was dose-dependent (EC(50)=34 microM). The total conductance of the neurons for the kainate-induced current was significantly reduced (P<0.01) and the effect was completely reversible. BB furthermore reduced the kainate-induced current in granule and hippocampal neurons and decreased the amplitude of the alpha-amino-3-hydroxy-5-methyl-4-isoxalepropionic acid (AMPA)-evoked current in cortical neurons. Cholera toxin (ChTx) did not affect the action of BB on the kainate-induced currents in cortical neurons and moreover, when guanosine 5'-o-(3-thiotriphosphate) (GTPgammaS) was added to the electrode solution, the kainate-induced currents were still reduced by 100 microM BB. The maximal response to kainate decreased in the presence of 20 microM BB without changing its EC(50), indicating a non-competitive mechanism of inhibition. These results demonstrate that preferential P2Y receptor antagonists are able to modulate the kainate and AMPA-induced currents in central neurons, suggesting a potential use of these compounds as neuroprotective agents.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Neurônios/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2 , Potenciais de Ação/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral , Embrião de Mamíferos , Hipocampo , Neurônios/fisiologia , Ratos , Ratos WistarRESUMO
The protective effects of riluzole against the neuronal damage caused by O2 and glucose deprivation (ischemia) was investigated in rat cortical slices by recording electrophysiologically the cortico-cortical field potential and by evaluating histologically the severity of neuronal death. Five minutes of ischemia determined an irreversible depression of the amplitude of the field potential. In addition, this insult caused a clear enhancement of the number of death cells that were specifically colored with trypan blue (a vital colorant which stains altered cells). We found that riluzole, which by itself depressed the synaptic transmission, neuroprotected when perfused 15-20 min before and during ischemia. In fact, due to the treatment with riluzole, the ischemia-induced irreversible depression of the field potential recovered and less cells were stained with trypan blue. These findings demonstrate that riluzole prevents neuronal death in an in vitro model of ischemia and suggest a therapeutic use of this drug in order to reduce the pathophysiological outcomes of stroke.
Assuntos
Isquemia Encefálica/patologia , Lobo Frontal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Riluzol/farmacologia , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/fisiopatologia , Contagem de Células/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Lobo Frontal/patologia , Lobo Frontal/fisiopatologia , Proteína Glial Fibrilar Ácida/análise , Glucose/metabolismo , Técnicas In Vitro , Neurônios/patologia , Neurônios/fisiologia , Fármacos Neuroprotetores/uso terapêutico , Oxigênio/metabolismo , Ratos , Ratos Wistar , Riluzol/uso terapêutico , Fatores de Tempo , Azul TripanoRESUMO
The whole-cell configuration of the patch-clamp recording was used to study the voltage-dependent Na+ currents in a model system for the familial form of amyotrophic lateral sclerosis (ALS) associated with mutations in Cu,Zn superoxide dismutase. Here we report that the amplitude of voltage-gated Na+ currents is significantly reduced in cell lines expressing mutant Cu,Zn superoxide dismutase G93A when compared with the parental, untransfected cell line and to a cell line expressing the wild-type enzyme. This effect is associated with a shift toward positive values of the steady-state inactivation curve of the Na+ currents. These results indicate that expression of a Cu,Zn superoxide dismutase typical of patients affect with familial ALS influence the functionality of the voltage-dependent Na+ channels; this effect may contribute to the pathogenesis of the disease.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Canais de Sódio/fisiologia , Sódio/metabolismo , Superóxido Dismutase/genética , 4-Aminopiridina/farmacologia , Cádmio/farmacologia , Cálcio/metabolismo , Estimulação Elétrica , Humanos , Ativação do Canal Iônico/fisiologia , Mutação/fisiologia , Degeneração Neural/metabolismo , Neuroblastoma , Neurônios/química , Neurônios/enzimologia , Estresse Oxidativo/fisiologia , Técnicas de Patch-Clamp , Potássio/metabolismo , Superóxido Dismutase/metabolismo , Células Tumorais CultivadasRESUMO
We used field potential recording techniques to examine whether felbamate (FBM), lamotrigine (LTG), and lidocaine (LID) protect against the irreversible functional damage induced by transient ischemia. Five minutes of ischemia caused a depression of the field potential in rat cortical slices, which did not recover even after more than 1 h of washout. The N-methyl-D-aspartate (NMDA) antagonist ketamine (50 microM) protected against depression of the field caused by ischemia. On the other hand, the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2.3-dione (CNQX) (10 microM) had protective effects only if co-applied with ketamine. We found that either FBM (30-300 microM), which did not modify the amplitude of the field EPSP, or LTG (10-300 microM), which reversibly depressed the excitatory synaptic transmission, had a marked protective effect when superfused before and during the ischemic insult. After FBM (100 microM) and LTG (100 microM), the field EPSP recovered by 84 +/- 1% and 73 +/- 2.7% of control, respectively. Furthermore, LID (30-300 microM) was less effective than FBM and LTG in inducing a functional recovery from the damage caused by ischemia (58 +/- 1.8%). The rank order of potency, based on the maximal protection caused by the three drugs, was FBM > LTG > LID. Our results suggest that a noticeable neuroprotection can be obtained during glucose and O2 deprivation by preventive therapeutic regimens which use the two recently marketed anticonvulsant drugs, FBM and LTG.
Assuntos
Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/efeitos dos fármacos , Isquemia/fisiopatologia , Lidocaína/farmacologia , Fármacos Neuroprotetores/farmacologia , Propilenoglicóis/farmacologia , Triazinas/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Eletrofisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Felbamato , Técnicas In Vitro , Lamotrigina , Masculino , Fenilcarbamatos , Ratos , Ratos WistarRESUMO
The actions of the neuroprotective and anticonvulsant agent riluzole on voltage-activated currents were studied in primary cultures of rat cortical neurons by using whole-cell patch-clamp recording techniques. Isolated Na+, Ca2+ and K+ currents were generated in these cells by depolarizing commands from a holding potential of - 80 mV. Riluzole (10-300 microM) reversibly reduced in a dose-dependent manner the inward Na+ currents with an IC50 of 51 microM in all the tested neurons (n=29). This drug also shifted the steady-state inactivation curve of the sodium current towards more negative values (about 20mV, n=15) while it did not change significantly the decay phase of the Na+ current. Furthermore, riluzole (100 and 300 microM; n=5 and n=3, respectively) did not modulate the inward Ca2+ currents evoked by depolarizing steps on cortical cells. An additional concentration-dependent effect of riluzole was observed on the outward potassium currents. In fact, while the amplitude of the peak of the outward current (IA) was not changed significantly, the amplitude of the late component of the outward K+ current (Iss) was markedly decreased during the perfusion of riluzole (IC50=88 microM; n=16). It is concluded that riluzole modulates the Na+- and the late K+-dependent currents in cortical neurons. Both phenomena may explain, at least in part, the anticonvulsant and neuroprotective properties of this compound.
Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Neurônios/química , Fármacos Neuroprotetores/farmacologia , Canais de Potássio/fisiologia , Riluzol/farmacologia , Canais de Sódio/fisiologia , 4-Aminopiridina/farmacologia , Animais , Cádmio/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Estimulação Elétrica , Neurônios/fisiologia , Técnicas de Patch-Clamp , Potássio/metabolismo , Ratos , Ratos Wistar , Sódio/metabolismo , Tetraetilamônio/farmacologiaRESUMO
Whole-cell, voltage-clamp recordings were made from rat cerebellar granule cells in culture under experimental conditions designed to study voltage-gated Na+ currents that were elicited by depolarizing commands from a holding potential of -60 mV up to +20 mV. These tetrodotoxin-sensitive inward currents were reduced in a dose-related manner by bath application of the structurally novel, anticonvulsant drug topiramate (10-1000 microM; n = 16). Dose-response analysis of this effect revealed an IC50 of 48.9 microM. Topiramate also made the steady-state inactivation curve of this current shift toward more negative values (midpoint of the inactivation curve -46.9 mV under control conditions and -56.5 mV during topiramate application; n = 5). We propose that these effects may contribute to control the sustained depolarizations with repetitive firing of action potentials that occur within neuronal networks during seizure activity. Therefore they may represent a mechanism of action for this novel anticonvulsant drug.
Assuntos
Anticonvulsivantes/farmacologia , Cerebelo/fisiologia , Frutose/análogos & derivados , Ativação do Canal Iônico/efeitos dos fármacos , Canais de Sódio/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Cerebelo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Frutose/farmacologia , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Sódio/metabolismo , Tetrodotoxina/farmacologia , TopiramatoRESUMO
PURPOSE: To study the mechanism or mechanisms of action of lamotrigine (LTG) and, in particular, to establish its effects on the function of NA+ channels in mammalian central neurons. METHODS: Rat cerebellar granule cells in culture were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study voltage-gated Na+ currents. RESULTS: Extracellular application of LTG (10-500 microM, n = 21) decreased in a dose-related manner a tetrodotoxin-sensitive inward current that was elicited by depolarizing commands (from -80 to +20 mV). The peak amplitude of this Na(+)-mediated current was diminished by 38.8 +/- 12.2% (mean +/- SD, n = 6) during application of 100 microM LTG, and the dose-response curve of this effect indicated an IC50 of 145 microM. The reduction in the inward currents produced by LTG was not associate with any significant change in the current decay, whereas the voltage dependency of the steady-state inactivation shifted toward more negative values (midpoint of the inactivation curve: -47.5 and -59.0 mV under control conditions and during application of 100 microM LTG, respectively, n = 4). CONCLUSIONS: Our findings indicate that LTG reduces the amplitude of voltage-gated Na+ inward current in rat cerebellar granule cells and induces a negative shift of the steady-state inactivation curve. Both mechanisms may be instrumental in controlling the repetitive firing of action potentials (AP) that occurs in neuronal networks during seizure activity.
Assuntos
Anticonvulsivantes/farmacologia , Canais de Cálcio/efeitos dos fármacos , Córtex Cerebelar/citologia , Neurônios/efeitos dos fármacos , Triazinas/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Células Cultivadas , Córtex Cerebelar/efeitos dos fármacos , Lamotrigina , Potenciais da Membrana/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos WistarRESUMO
Current induced in cultured cerebellar granule cells by the bath application of kainate (500 microM) was measured using the conventional patch-clamp technique. Two different kinds of responses were observed after the agonist perfusion. Some cells exhibited small inward whole-cell currents: 116 +/- 40 pA (7 cells) at a clamp potential of -60 mV; in other cells, the agonist induced significantly larger currents: 420 +/- 35 pA (6 cells) at a clamp potential of -60 mV. The current flowing in the agonist-activated ionic channels was indirectly estimated by processing the fluctuations of whole-cell current by means of an original parametric method. Mean conductance of the underlying channels was then determined from the single-channel current estimated at different clamp potentials. In the cells exhibiting small inward currents, the mean conductance was equal to 0.5 +/- 0.2 pS (7 cells), whereas in the cells with large inward currents it was 3 +/- 0.4 pS (6 cells). This result gives a coherent explanation of the different kinds of responses observed at macroscopic level in the whole-cell current and confirms that kainate-activated channels can exhibit different levels of conductance.
Assuntos
Cerebelo/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Ácido Caínico/farmacologia , Processamento de Sinais Assistido por Computador , Animais , Células Cultivadas , Cerebelo/citologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Ácido Caínico/metabolismo , Modelos Biológicos , Técnicas de Patch-Clamp , Perfusão , Ratos , Ratos WistarRESUMO
Our previous studies have shown that the response to the excitotoxic action of glutamate by cultured cerebellar granule cells depends upon the cell density or the volume of medium in which they have been grown: the higher the cell density or the lower the volume, the higher the response to glutamate. We have hypothesized that this variable response is due to the formation in culture of a glutamate-sensitizing activity GSA more abundantly in conditioned medium derived from high-density or low-volume cultures than that present in low-density or high volume cultures and capable of restoring sensitivity in previously resistant granule cells. In order to elucidate the mechanism of action of glutamate-sensitizing activity, we measured the extent and function of NMDA receptors in low- and high-volume cultures and assessed the effect of glutamate-sensitizing activity on the same receptors. We found that under high-volume conditions the extent of MK-801 binding, the amount of NMDA receptor type 1, the currents evoked in whole cells after an NMDA pulse and the response of cultured cells to this ligand were markedly reduced compared with low-volume cultures. Addition of glutamate-sensitizing activity to high-volume cultures increased their glutamate sensitivity, the NMDA-evoked currents, the extent of MK-801 binding and the amount of NMDA receptor type 1 protein present. The corresponding mRNA transcripts, on the contrary, were unchanged in high-volume, low-volume and high-volume GSA-treated cultures.