RESUMEN
The involvement of metabotropic glutamate receptors type 5 (mGluR5) in drug-induced behaviours is well-established but limited information is available on their functional roles in addiction-relevant brain areas like the nucleus accumbens (NAc). This study demonstrates that pharmacological and synaptic activation of mGluR5 increases the spike discharge of medium spiny neurons (MSNs) in the NAc. This effect was associated with the appearance of a slow afterdepolarization (ADP) which, in voltage-clamp experiments, was recorded as a slowly inactivating inward current. Pharmacological studies showed that ADP was elicited by mGluR5 stimulation via G-protein-dependent activation of phospholipase C and elevation of intracellular Ca(2+) levels. Both ADP and spike aftercurrents were significantly inhibited by the Na(+) channel-blocker, tetrodotoxin (TTX). Moreover, the selective blockade of persistent Na(+) currents (I(NaP)), achieved by NAc slice pre-incubation with 20 nm TTX or 10 \#956;m riluzole, significantly reduced the ADP amplitude, indicating that this type of Na(+) current is responsible for the mGluR5-dependent ADP. mGluR5 activation also produced significant increases in I(NaP), and the pharmacological blockade of this current prevented the mGluR5-induced enhancement of spike discharge. Collectively, these data suggest that mGluR5 activation upregulates I(NaP) in MSNs of the NAc, thereby inducing an ADP that results in enhanced MSN excitability. Activation of mGluR5 will significantly alter spike firing in MSNs in vivo, and this effect could be an important mechanism by which these receptors mediate certain aspects of drug-induced behaviours.
Asunto(s)
Núcleo Accumbens/citología , Núcleo Accumbens/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sodio/metabolismo , Potenciales de Acción , Animales , Catecoles/farmacología , Fenómenos Electrofisiológicos , Glicina/análogos & derivados , Glicina/farmacología , Técnicas In Vitro , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Núcleo Accumbens/efectos de los fármacos , Técnicas de Placa-Clamp , Receptor del Glutamato Metabotropico 5 , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
Although cyclic nucleotide-gated (CNG) channels are expressed in numerous brain areas, little information is available on their functions in CNS neurons. The aim of the present study was to define the distribution of CNG channels in the rat medial vestibular nucleus (MVN) and their possible involvement in regulating MVN neuron (MVNn) excitability. The majority of MVNn expressed both CNG1 and CNG2 A subunits. In whole-cell current-clamp experiments carried out on brainstem slices containing the MVNn, the membrane-permeant analogues of cyclic nucleotides, 8-Br-cGMP and 8-Br-cAMP (1 mM), induced membrane depolarizations (8.9 +/- 0.8 and 9.2 +/- 1.0 mV, respectively) that were protein kinase independent. The cGMP-induced depolarization was associated with a significant decrease in the membrane input resistance. The effects of cGMP on membrane potential were almost completely abolished by the CNG channel blockers, Cd(2+) and L-cis-diltiazem, but they were unaffected by blockade of hyperpolarization-activated cyclic nucleotide-gated channels. In voltage-clamp experiments, 8-Br-cGMP induced non-inactivating inward currents (-22.2 +/- 3.9 pA) with an estimated reversal potential near 0 mV, which were markedly inhibited by reduction of extracellular Na(+) and Ca(2+) concentrations. Membrane depolarization induced by CNG channel activation increased the firing rate of MVNn without changing the action potential shape. Collectively, these findings provide novel evidence that CNG channels affect membrane potential and excitability of MVNn. Such action should have a significant impact on the function of these neurons in sensory-motor integration processes. More generally, it might represent a broad mechanism for regulating the excitability of different CNS neurons.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Neuronas/metabolismo , Núcleos Vestibulares/metabolismo , Potenciales de Acción , Animales , AMP Cíclico/fisiología , GMP Cíclico/fisiología , Masculino , Potenciales de la Membrana , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Subunidades de Proteína/metabolismo , Ratas , Ratas WistarRESUMEN
We previously reported that exposure to extremely low-frequency electromagnetic fields (ELFEFs) increases the expression and function of voltage-gated Ca2+)channels and that Ca2+ influx through Ca(v)1 channels plays a key role in promoting the neuronal differentiation of neural stem/progenitor cells (NSCs). The present study was conducted to determine whether ELFEFs influence the neuronal differentiation of NSCs isolated from the brain cortices of newborn mice by modulating Ca(v)1-channel function. In cultures of differentiating NSCs exposed to ELFEFs (1 mT, 50 Hz), the percentage of cells displaying immunoreactivity for neuronal markers (beta-III-tubulin, MAP2) and for Ca(v)1.2 and Ca(v)1.3 channels was markedly increased. NSC-differentiated neurons in ELFEF-exposed cultures also exhibited significant increases in spontaneous firing, in the percentage of cells exhibiting Ca2+ transients in response to KCl stimulation, in the amplitude of these transients and of Ca2+ currents generated by the activation of Ca(v)1 channels. When the Ca(v)1-channel blocker nifedipine (5 microM) was added to the culture medium, the neuronal yield of NSC differentiation dropped significantly, and ELFEF exposure no longer produced significant increases in beta-III-tubulin- and MAP2-immunoreactivity rates. In contrast, the effects of ELFEFs were preserved when NSCs were cultured in the presence of either glutamate receptor antagonists or Ca(v)2.1- and Ca(v)2.2-channel blockers. ELFEF stimulation during the first 24 h of differentiation caused Ca(v)1-dependent increases in the number of cells displaying CREB phosphorylation. Our data suggest that ELFEF exposure promotes neuronal differentiation of NSCs by upregulating Ca(v)1-channel expression and function.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Diferenciación Celular , Campos Electromagnéticos , Neuronas/citología , Regulación hacia Arriba , Animales , Proliferación Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Fenotipo , Fosforilación , Células Madre/citología , Factores de Tiempo , Tubulina (Proteína)/metabolismoRESUMEN
Trimethyltin (TMT) intoxication is considered a suitable experimental model to study the molecular basis of selective hippocampal neurodegeneration as that occurring in several neurodegenerative diseases. We have previously shown that rat hippocampal neurons expressing the Ca(2+)-binding protein calretinin (CR) are spared by the neurotoxic action of TMT hypothetically owing to their ability to buffer intracellular Ca(2+) overload. The present study was aimed at determining whether intracellular Ca(2+) homeostasis dysregulation is involved in the TMT-induced neurodegeneration and if intracellular Ca(2+)-buffering mechanisms may exert a protective action in this experimental model of neurodegeneration. In cultured rat hippocampal neurons, TMT produced time- and concentration-dependent [Ca(2+)](i) increases that were primarily due to Ca(2+) release from intracellular stores although Ca(2+) entry through Ca(v)1 channels also contributed to [Ca(2+)](i) increases in the early phase of TMT action. Cell pre-treatment with the Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (2 muM) significantly reduced the TMT-induced neuronal death. Moreover, CR(+) neurons responded to TMT with smaller [Ca(2+)](i) increases. Collectively, these data suggest that the neurotoxic action of TMT is mediated by Ca(2+) homeostasis dysregulation, and the resistance of hippocampal neurons to TMT (including CR(+) neurons) is not homogeneous among different neuron populations and is related to their ability to buffer intracellular Ca(2+) overload.
Asunto(s)
Señalización del Calcio/fisiología , Hipocampo/fisiología , Homeostasis/fisiología , Líquido Intracelular/fisiología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/metabolismo , Compuestos de Trimetilestaño/toxicidad , Animales , Señalización del Calcio/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Hipocampo/efectos de los fármacos , Homeostasis/efectos de los fármacos , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Degeneración Nerviosa/inducido químicamente , Neuronas/efectos de los fármacos , Neuronas/fisiología , Ratas , Ratas WistarRESUMEN
Amyloid beta-peptide (Abeta) plays a fundamental role in the pathogenesis of Alzheimer's disease. We recently reported that the redox state of the methionine residue in position 35 of amyloid beta-peptide (Abeta) 1-42 (Met35) strongly affects the peptide's ability to trigger apoptosis and is thus a major determinant of its neurotoxicity. Dysregulation of intracellular Ca(2+) homeostasis resulting in the activation of pro-apoptotic pathways has been proposed as a mechanism underlying Abeta toxicity. Therefore, we investigated correlations between the Met35 redox state, Abeta toxicity, and altered intracellular Ca(2+) signaling in human neuroblastoma IMR32 cells. Cells incubated for 6-24 h with 10 microM Abeta1-42 exhibited significantly increased KCl-induced Ca(2+) transient amplitudes and resting free Ca(2+) concentrations. Nifedipine-sensitive Ca(2+) current densities and Ca(v)1 channel expression were markedly enhanced by Abeta1-42. None of these effects were observed when cells were exposed to Abeta containing oxidized Met35 (Abeta1-42(Met35-Ox)). Cell pre-treatment with the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (1 microM) or the Ca(v)1 channel blocker nifedipine (5 microM) significantly attenuated Abeta1-42-induced apoptosis but had no effect on Abeta1-42(Met35-Ox) toxicity. Collectively, these data suggest that reduced Met35 plays a critical role in Abeta1-42 toxicity by rendering the peptide capable of disrupting intracellular Ca(2+) homeostasis and thereby provoking apoptotic cell death.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Líquido Extracelular/efectos de los fármacos , Homeostasis/efectos de los fármacos , Metionina/metabolismo , Fragmentos de Péptidos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/genética , Canales de Calcio/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quelantes/farmacología , Relación Dosis-Respuesta a Droga , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Etiquetado Corte-Fin in Situ/métodos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Neuroblastoma/metabolismo , Nifedipino/farmacología , Oxidación-Reducción , Técnicas de Placa-Clamp , Cloruro de Potasio/farmacologíaRESUMEN
HL-60 leukemia cells, Rat-1 fibroblasts and WI-38 diploid fibroblasts were exposed for 24-72 h to 0.5-1.0-mT 50-Hz extremely low frequency electromagnetic field (ELF-EMF). This treatment induced a dose-dependent increase in the proliferation rate of all cell types, namely about 30% increase of cell proliferation after 72-h exposure to 1.0 mT. This was accompanied by increased percentage of cells in the S-phase after 12- and 48-h exposure. The ability of ELF-EMF to induce DNA damage was also investigated by measuring DNA strand breaks. A dose-dependent increase in DNA damage was observed in all cell lines, with two peaks occurring at 24 and 72 h. A similar pattern of DNA damage was observed by measuring formation of 8-OHdG adducts. The effects of ELF-EMF on cell proliferation and DNA damage were prevented by pretreatment of cells with an antioxidant like alpha-tocopherol, suggesting that redox reactions were involved. Accordingly, Rat-1 fibroblasts that had been exposed to ELF-EMF for 3 or 24 h exhibited a significant increase in dichlorofluorescein-detectable reactive oxygen species, which was blunted by alpha-tocopherol pretreatment. Cells exposed to ELF-EMF and examined as early as 6 h after treatment initiation also exhibited modifications of NF kappa B-related proteins (p65-p50 and I kappa B alpha), which were suggestive of increased formation of p65-p50 or p65-p65 active forms, a process usually attributed to redox reactions. These results suggest that ELF-EMF influence proliferation and DNA damage in both normal and tumor cells through the action of free radical species. This information may be of value for appraising the pathophysiologic consequences of an exposure to ELF-EMF.
Asunto(s)
Proliferación Celular/efectos de la radiación , Daño del ADN/efectos de la radiación , Desoxiguanosina/análogos & derivados , Oxidación-Reducción , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Western Blotting , Línea Celular Tumoral , Aductos de ADN , Desoxiguanosina/metabolismo , Relación Dosis-Respuesta en la Radiación , Campos Electromagnéticos , Fibroblastos/metabolismo , Citometría de Flujo , Fluoresceínas/farmacología , Radicales Libres , Células HL-60 , Humanos , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno , Factores de Tiempo , alfa-Tocoferol/farmacologíaRESUMEN
Sinusoidal vestibular stimulation induces in the intact rabbit in prone position a periodic alternating drift (PAD), evident in the earth horizontal plane when the animal is rotated about the vertical axis but weak in the vertical one when the animal is rotated about the longitudinal axis. It has been hypothesized that these oscillations are related to an intrinsic instability of the velocity storage, due to the length of its time constant. The velocity storage has the longest time constant aligned with the vertical axis, and it changes its orientation with the gravity vector. The present research examined the spatial orientation of PAD in relation to changes of the animal position with respect to gravity. Normal pigmented rabbits were sinusoidally oscillated about their longitudinal axes to evoke vertical eye responses. The stimulation was carried out with the animal in prone position and with the animal in nose-up condition. With the animal in prone position, PAD had a weak vertical component, but an evident horizontal component was visible. When the animal was in nose-up position, the horizontal component of PAD was clearly visible, while the vertical component was negligible. In both stimulation conditions PAD period and peak velocity were not modulated by the stimulus characteristics. These results are consistent with a model of PAD based on an interaction between velocity storage and the cerebellar adaptation-habituation circuit.
Asunto(s)
Movimientos Oculares/fisiología , Fijación Ocular/fisiología , Sensación de Gravedad/fisiología , Reflejo Vestibuloocular/fisiología , Percepción Espacial/fisiología , Vestíbulo del Laberinto/fisiología , Análisis de Varianza , Animales , Orientación/fisiología , ConejosRESUMEN
Although data from our laboratory and others suggest that nitric oxide (NO) exerts an overall inhibitory action on high-voltage-activated Ca2+ channels, conflicting observations have been reported regarding its effects on N-type channels. We performed whole-cell and cell-attached patch-clamp recordings in IMR32 cells to clarify the functional role of NO in the modulation of N channels of human neuronal cells. During depolarizing steps to +10 mV from V(h) = -90 mV, the NO donor, sodium nitroprusside (SNP; 200 microm), reduced macroscopic N currents by 34% (p < 0.01). The magnitude of inhibition was similar at all voltages tested (range, -40 to +50 mV). No significant inhibition was observed when SNP was applied together with the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium salt (300 microm), or after cell treatment with the guanylate cyclase inhibitor, 1H-[1,2,4] oxadiazole [4,3-a] quinoxalin-1-one (10 microm). 8-bromoguanosine-cGMP (8-Br-cGMP) (400 microm) mimicked the effects of SNP, reducing Ba2+ currents by 37% (p < 0.001). Cell treatment with the protein kinase G (PKG) inhibitor KT5823 (1 microm) or guanosine 3',5'-cyclic monophosphorothioate, 8-(4-chloro-phenylthio)-Rp-isomer, triethylammonium salt (20 microm) virtually abolished the effects of 8-Br-cGMP. At the single-channel level, 8-Br-cGMP reduced the channel open probability by 59% and increased both the mean shut time and the null sweep probability, but it had no significant effects on channel conductance, mean open time, or latency of first openings. These data suggest that NO inhibits N-channel gating through cGMP and PKG. The consequent decrease in Ca2+ influx through these channels may affect different neuronal functions, including neurotransmitter release.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Neuroblastoma/metabolismo , Óxido Nítrico/farmacología , Calcio/metabolismo , Canales de Calcio Tipo N/efectos de los fármacos , GMP Cíclico/antagonistas & inhibidores , GMP Cíclico/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Humanos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Neuroblastoma/tratamiento farmacológico , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Óxido Nítrico/biosíntesis , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Técnicas de Placa-Clamp , Tiempo de Reacción/efectos de los fármacos , Sistemas de Mensajero Secundario/fisiología , Células Tumorales CultivadasRESUMEN
The role of cyclic nucleotide-gated (CNG) channels in sensory signal transduction in retinal and olfactory cells is widely recognized, but there is increasing evidence that they also play more general functions in the central nervous system as downstream effectors of cyclic nucleotides. Here, we demonstrate the expression of the alpha-subunit of rod- and olfactory-type CNG channels (CNG1 and CNG2, respectively) in the rat medial vestibular nucleus (MVN). Nested polymerase chain reaction revealed CNG channel mRNA in the MVN, and CNG1 and CNG2 proteins were also detected by Western blotting and immunohistochemistry. Finally, electrophysiological evidence is provided suggesting that CNG channels play a functional role in the MVN.
Asunto(s)
Canales Iónicos/genética , Núcleos Vestibulares/fisiología , Animales , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Expresión Génica , Canales Iónicos/metabolismo , Masculino , Técnicas de Cultivo de Órganos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Ratas , Ratas Wistar , Núcleos Vestibulares/metabolismoRESUMEN
Pyrrolidine-dithiocarbamate (PDTC), a synthetic compound widely used in cell biological investigations, recently attracted considerable interest as a putative anticancer agent. However, different dithiocarbamates have previously shown to cause neurological symptoms and morphological alterations in peripheral nerves. The purpose of the present study was to determine whether a 15-day oral administration with low doses of PDTC may produce adverse effects in peripheral nerves of rats. Female Wistar rats were assigned to receive PDTC [0.1, 0.5 or 1.0mmol/(kg body weight/day)] by gavage for 15 days. Reduced conduction velocity was observed by electrophysiological analysis in tibial nerves of treated animals, accompanied by a marked decrease in Shwann cell S100-protein expression determined by immunohistochemistry. Electron microscopy evaluation revealed marked myelin degeneration in the fibers of treated animals. In particular, both morphological and electrophysiological data suggested an impairment of large, fast conducting fibers, whereas the smallest and slowest ones remained intact. However, the activity of plasma and liver alkaline-phosphatase, an enzymic marker of hepatic dithiocarbamate toxicity, was not altered by the treatment. The total contents of the redox-active metal copper increased in tibial nerves of treated rats and was accompanied by raised levels of lipid peroxidation products. This finding suggests a role for oxidative stress in the development of PDTC-induced pathological and functional alterations of tibial nerves. The observation that a 15-day treatment with low doses of PDTC causes functional and morphological derangement of peripheral nerves advices against the possible use of this compound as a chemopreventive agent against cancer.
Asunto(s)
Antioxidantes/toxicidad , Vaina de Mielina/efectos de los fármacos , Pirrolidinas/toxicidad , Tiocarbamatos/toxicidad , Nervio Tibial/efectos de los fármacos , Degeneración Walleriana/inducido químicamente , Fosfatasa Alcalina/metabolismo , Animales , Biomarcadores/metabolismo , Cobre/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Técnicas para Inmunoenzimas , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/enzimología , Vaina de Mielina/ultraestructura , Conducción Nerviosa/efectos de los fármacos , Conducción Nerviosa/fisiología , Ratas , Ratas Wistar , Proteínas S100/metabolismo , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Nervio Tibial/patología , Nervio Tibial/fisiopatología , Degeneración Walleriana/patologíaRESUMEN
Possible correlation between the effects of electromagnetic fields (EFs) on voltage-gated Ca(2+) channels, cell proliferation and apoptosis was investigated in neural and neuroendocrine cells. Exposure to 50 Hz EFs significantly enhanced proliferation in human neuroblastoma IMR32 (+40%) and rat pituitary GH3 cells (+38%). In IMR32 cells EF stimulation also inhibited puromycin- and H(2)O(2)-induced apoptosis (-22 and -33%, respectively). EF effects on proliferation and apoptosis were counteracted by Ca(2+) channel blockade. In whole-cell patch-clamp experiments 24-72 h exposure to EFs increased macroscopic Ba(2+)-current density in both GH3 (+67%) and IMR32 cells (+40%). Single-channel recordings showed that gating of L and N channels was instead unaffected, thus suggesting that the observed enhancement of current density was due to increased number of voltage-gated Ca(2+) channels. Western blot analysis of plasma membrane-enriched microsomal fractions of GH3 and IMR32 cells confirmed enhanced expression of Ca(2+) channel subunit alpha(1) following exposure to EFs. These data provide the first direct evidence that EFs enhance the expression of voltage-gated Ca(2+) channels on plasma membrane of the exposed cells. The consequent increase in Ca(2+) influx is likely responsible for the EF-induced modulation of neuronal cell proliferation and apoptosis.
Asunto(s)
Apoptosis/efectos de la radiación , Canales de Calcio Tipo L/efectos de la radiación , Canales de Calcio Tipo N/efectos de la radiación , Campos Electromagnéticos , Neuroblastoma/metabolismo , Hipófisis/metabolismo , Animales , Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , División Celular/efectos de los fármacos , División Celular/efectos de la radiación , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Electrofisiología , Humanos , Peróxido de Hidrógeno/farmacología , Neuroblastoma/patología , Oxidantes/farmacología , Técnicas de Placa-Clamp , Hipófisis/citología , Puromicina/farmacología , RatasRESUMEN
The unconventional gaseous transmitter nitric oxide (NO) markedly influences most of mechanisms involved in the regulation of intracellular Ca2+ homeostasis. In excitable cells, Ca2+ signaling mainly depends on the activity of voltage-gated Ca2+ channels (VGCCs). In the present paper, we will review data from our laboratory and others characterizing NO-induced modulation of Ca(v)1 (L-type) and Ca(v)2.2 (N-type) channels. In particular, we will explore experimental evidence indicating that NO's inhibition of channel gating is produced via cGMP-dependent protein kinase and examine some of the numerous cell functions that are potentially influenced by the action of NO on Ca2+ channels.
Asunto(s)
Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo N/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Óxido Nítrico/fisiología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , HumanosRESUMEN
Epidemiological and experimental findings suggest that chronic infection with Herpes simplex virus type 1 (HSV-1) may be a risk factor for Alzheimer's disease (AD), but the molecular mechanisms underlying this association have not been fully identified. We investigated the effects of HSV-1 on excitability and intracellular calcium signaling in rat cortical neurons and the impact of these effects on amyloid precursor protein (APP) processing and the production of amyloid-ß peptide (Aß). Membrane depolarization triggering firing rate increases was observed shortly after neurons were challenged with HSV-1 and was still evident 12 hours postinfection. These effects depended on persistent sodium current activation and potassium current inhibition. The virally induced hyperexcitability triggered intracellular Ca(2+) signals that significantly increased intraneuronal Ca(2+) levels. It also enhanced activity- and Ca(2+)-dependent APP phosphorylation and intracellular accumulation of Aß42. These findings indicate that HSV-1 causes functional changes in cortical neurons that promote APP processing and Aß production, and they are compatible with the co-factorial role for HSV-1 in the pathogenesis of AD suggested by previous findings.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Calcio/fisiología , Corteza Cerebral/virología , Herpesvirus Humano 1/fisiología , Neuronas/virología , Fragmentos de Péptidos/metabolismo , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Potenciales de la Membrana/genética , Neuronas/metabolismo , Fosforilación/genética , Ratas , Regulación hacia Arriba/genética , Replicación Viral/genéticaRESUMEN
Throughout life, new neurons are continuously generated in the hippocampus, which is therefore a major site of structural plasticity in the adult brain. We recently demonstrated that extremely low-frequency electromagnetic fields (ELFEFs) promote the neuronal differentiation of neural stem cells in vitro by up-regulating Ca(v)1-channel activity. The aim of the present study was to determine whether 50-Hz/1 mT ELFEF stimulation also affects adult hippocampal neurogenesis in vivo, and if so, to identify the molecular mechanisms underlying this action and its functional impact on synaptic plasticity. ELFEF exposure (1 to 7 h/day for 7 days) significantly enhanced neurogenesis in the dentate gyrus (DG) of adult mice, as documented by increased numbers of cells double-labeled for 5-bromo-deoxyuridine (BrdU) and doublecortin. Quantitative RT-PCR analysis of hippocampal extracts revealed significant ELFEF exposure-induced increases in the transcription of pro-neuronal genes (Mash1, NeuroD2, Hes1) and genes encoding Ca(v)1.2 channel α(1C) subunits. Increased expression of NeuroD1, NeuroD2 and Ca(v)1 channels was also documented by Western blot analysis. Immunofluorescence experiments showed that, 30 days after ELFEF stimulation, roughly half of the newly generated immature neurons had survived and become mature dentate granule cells (as shown by their immunoreactivity for both BrdU and NeuN) and were integrated into the granule cell layer of the DG. Electrophysiological experiments demonstrated that the new mature neurons influenced hippocampal synaptic plasticity, as reflected by increased long-term potentiation. Our findings show that ELFEF exposure can be an effective tool for increasing in vivo neurogenesis, and they could lead to the development of novel therapeutic approaches in regenerative medicine.
Asunto(s)
Hipocampo/efectos de la radiación , Neurogénesis/efectos de la radiación , Animales , Antimetabolitos , Western Blotting , Bromodesoxiuridina , Canales de Calcio/efectos de la radiación , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Campos Electromagnéticos , Técnica del Anticuerpo Fluorescente , Hipocampo/citología , Potenciación a Largo Plazo , Masculino , Ratones , Ratones Endogámicos C57BL , Red Nerviosa/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Ca(2+) influx through voltage-gated Ca(2+) channels, especially the L-type (Ca(v)1), activates downstream signaling to the nucleus that affects gene expression and, consequently, cell fate. We hypothesized that these Ca(2+) signals may also influence the neuronal differentiation of neural stem/progenitor cells (NSCs) derived from the brain cortex of postnatal mice. We first studied Ca(2+) transients induced by membrane depolarization in Fluo 4-AM-loaded NSCs using confocal microscopy. Undifferentiated cells (nestin(+)) exhibited no detectable Ca(2+) signals whereas, during 12 days of fetal bovine serum-induced differentiation, neurons (beta-III-tubulin(+)/MAP2(+)) displayed time-dependent increases in intracellular Ca(2+) transients, with DeltaF/F ratios ranging from 0.4 on day 3 to 3.3 on day 12. Patch-clamp experiments revealed similar correlation between NSC differentiation and macroscopic Ba(2+) current density. These currents were markedly reduced (-77%) by Ca(v)1 channel blockade with 5 microm nifedipine. To determine the influence of Ca(v)1-mediated Ca(2+) influx on NSC differentiation, cells were cultured in differentiative medium with either nifedipine (5 microm) or the L-channel activator Bay K 8644 (10 microm). The latter treatment significantly increased the percentage of beta-III-tubulin(+)/MAP2(+) cells whereas nifedipine produced opposite effects. Pretreatment with nifedipine also inhibited the functional maturation of neurons, which responded to membrane depolarization with weak Ca(2+) signals. Conversely, Bay K 8644 pretreatment significantly enhanced the percentage of responsive cells and the amplitudes of Ca(2+) transients. These data suggest that NSC differentiation is strongly correlated with the expression of voltage-gated Ca(2+) channels, especially the Ca(v)1, and that Ca(2+) influx through these channels plays a key role in promoting neuronal differentiation.
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Canales de Calcio Tipo L/fisiología , Diferenciación Celular/fisiología , Corteza Cerebral/citología , Neuronas/citología , Células Madre/fisiología , Compuestos de Anilina/metabolismo , Animales , Animales Recién Nacidos , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/fisiología , Recuento de Células/métodos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica/métodos , Proteínas de Filamentos Intermediarios/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Ratones , Microscopía Confocal/métodos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nestina , Nifedipino/farmacología , Técnicas de Placa-Clamp/métodos , Cloruro de Potasio/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Factores de Tiempo , Tubulina (Proteína)/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Xantenos/metabolismoRESUMEN
The possibility that mechanical stimulation of selected muscles can act directly on the nervous system inducing persistent changes of motor performances was explored. On the basis of literature, stimulating parameters were chosen to stimulate the central nervous system and to avoid muscle fibre injuries. A sinusoidal mechanical vibration was applied, for three consecutive days, on the quadriceps muscle in seven subjects that performed a muscular contraction (VC). The same stimulation paradigm was applied on seven subjects in relaxed muscle condition (VR) and seven subjects were not treated at all (NV). Two sessions (PRE and POST) of isometric and isotonic tests were performed separated for 21 days, in all studied groups 7 days before and 15 days after stimulation, whilst an isokinetic test was performed on VC only. In the isometric test, the time of force development showed a significant decrease only in VC (POST vs PRE mean 27.8%, P < 0.05). In the isotonic test, the subjects' had to perform a fatiguing leg extension against a load. In this condition, the fatigue resistance increased greatly in VC (mean 40.3%, P < 0.001), increased slightly in VR and there was no difference in NV. In Isokinetic test, at several angular velocities, significantly less time was required to reach the force peak (mean 20.2% P < 0.05). The findings could be ascribed to plastic changes in proprioceptive processing, leading to an improvement in knee joint control. Such action delineates a new tool in sports training and in motor rehabilitation.
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Destreza Motora/fisiología , Contracción Muscular/fisiología , Fatiga Muscular/fisiología , Músculo Esquelético/fisiología , Esfuerzo Físico/fisiología , Adaptación Fisiológica/fisiología , Adulto , Femenino , Humanos , Masculino , Músculo Esquelético/inervación , Estimulación Física/métodos , VibraciónRESUMEN
Changes in intracellular Ca2+ levels are an important signal underlying neuron-glia cross-talk, but little is known about the possible role of voltage-gated Ca2+ channels (VGCCs) in controlling glial cell Ca2+ influx. We investigated the pharmacological and biophysical features of VGCCs in cultured rat cortical astrocytes. In whole-cell patch-clamp experiments, L-channel blockade (5 microM nifedipine) reduced Ba2+ current amplitude by 28% of controls, and further decrease (32%) was produced by N-channel blockade (3 microM omega-conotoxin-GVIA). No significant additional changes were observed after P/Q channel blockade (3 microM omega-conotoxin-MVIIC). Residual current (36% of controls) amounted to roughly the same percentage (34%) that was abolished by R-channel blockade (100 nM SNX-482). Electrophysiological evidence of L-, N-, and R-channels was associated with RT-PCR detection of mRNA transcripts for VGCC subunits alpha1C (L-type), alpha1B (N-type), and alpha1E (R-type). In cell-attached recordings, single-channel properties (L-currents: amplitude, -1.21 +/- 0.02 pA at 10 mV; slope conductance, 22.0 +/- 1.1 pS; mean open time, 5.95 +/- 0.24 ms; N-currents: amplitude, -1.09 +/- 0.02 pA at 10 mV; slope conductance, 18.0 +/- 1.1 pS; mean open time, 1.14 +/- 0.02 ms; R-currents: amplitude, -0.81 +/- 0.01 pA at 20 mV; slope conductance, 10.5 +/- 0.3 pS; mean open time, 0.88 +/- 0.02 ms) resembled those of corresponding VGCCs in neurons. These novel findings indicate that VGCC expression by cortical astrocytes may be more varied than previously thought, suggesting that these channels may indeed play substantial roles in the regulation of astrocyte Ca2+ influx, which influences neuron-glia cross-talk and numerous other calcium-mediated glial-cell functions.
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Astrocitos/fisiología , Canales de Calcio Tipo L/fisiología , Canales de Calcio Tipo N/fisiología , Canales de Calcio Tipo R/fisiología , Corteza Cerebral/fisiología , Animales , Astrocitos/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/biosíntesis , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo N/biosíntesis , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo R/biosíntesis , Canales de Calcio Tipo R/genética , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Electrofisiología , Ratas , Ratas WistarRESUMEN
The inducible isoform of nitric oxide (NO) synthase (iNOS), expressed in endothelium, epithelium, and inflammatory cells, produces a large amount of NO. Previous studies on mouse intestine indicate that a muscular iNOS may have a role in the storage of intraluminal content. In this study we investigated the presence and function of iNOS in the colonic smooth muscle cells of 2- and 12-month-old dystrophic (mdx) mice. By using an in vitro isovolumic technique, and immunohistochemical and Western blot analysis, we demonstrated that iNOS is expressed and active in the smooth muscle cells of normal mouse and defective in young adult (2-month-old) mdx mice. Therefore, an altered activity of the muscle iNOS might explain the motility disorders observed in the colon of mdx mice and, from a clinical point of view, the impairment of intestinal function in dystrophic patients.
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Colon/enzimología , Músculo Liso/enzimología , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/fisiopatología , Óxido Nítrico Sintasa/metabolismo , Animales , Colon/citología , Colon/fisiopatología , Regulación Enzimológica de la Expresión Génica , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Músculo Liso/citología , Músculo Liso/fisiopatología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatología , Miocitos del Músculo Liso/enzimología , Óxido Nítrico Sintasa de Tipo II , Peristaltismo/fisiologíaRESUMEN
The effects of nitric oxide (NO) on the discharge rate of medial vestibular nucleus neurons (MVNn) were investigated in rat brainstem slices. The NO-donor sodium nitroprusside (SNP, 200 microM) caused a marked enhancement (+36.7%) of MVNn spontaneous firing rate, which was prevented by the NO-scavenger, carboxy-PTIO (300 microM). The SNP effects were not modified (+37.4%) by synaptic uncoupling, suggesting that NO influences intrinsic membrane properties of MVNn rather than the synaptic input they receive. The excitatory action of SNP was virtually abolished by slice pretreatment with the soluble guanylyl cyclase inhibitor, ODQ (10 microM), and it was mimicked (+33.1%) by the cGMP analogue 8-Br-cGMP (400 microM). Protein kinase G (PKG) and cAMP/protein kinase A (PKA) were both excluded as downstream effectors of the NO/cGMP-induced excitation. However, the cyclic nucleotide-gated (CNG) channel blockers, L-cis-diltiazem (LCD, 100 microM) and Sp-8-Br-PET-cGMPS (100 microM), significantly reduced the firing rate increase produced by 8-Br-cGMP. Moreover, LCD alone decreased spontaneous MVNn firing (-19.7%), suggesting that putative CNG channels may contribute to the tonic control of resting MVNn discharge. 8-Br-cAMP (1 mM) also elicited excitatory effects in MVNn (+40.8%), which occluded those induced by 8-Br-cGMP, indicating that the two nucleotides share a common target. Finally, nested-polymerase chain reaction assay revealed the expression of CNG channel alpha subunit transcript in MVNn. Our data provide the first demonstration that NO/cGMP signalling modulates MVNn spontaneous firing through a mechanism that is independent of PKG or PKA and probably involves activation of CNG channels.