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1.
Brain Res ; 1677: 26-32, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-28951233

RESUMO

Mutations in the SCN1A gene causing either loss or gain of function have been frequently found in patients affected by genetic epilepsy with febrile seizures plus (GEFS+) or Dravet syndrome (also named severe myoclonic epilepsy in infancy SMEI). By mutation screening of the SCN1A gene, we identified for the first time a case of two missense mutations in cis (p.[Arg1525Gln;Thr297Ile]) in all affected individuals of an Italian family showing GEFS+ and idiopathic generalized epilepsy (IGE). The p.Arg1525Gln mutation was not previously reported yet and was predicted to be pathological by prediction tools, whereas the p.Thr297Ile was already identified in patients showing SMEI. Functional studies revealed that the Nav1.1 channels harboring both mutations were characterized by a significant shift in the activation curve towards more positive potentials. Our data demonstrate that the p.Arg1525Gln represents a novel mutation in the SCN1A gene altering the channel properties in the co-presence of the p.Thr297Ile.


Assuntos
Epilepsia Generalizada/genética , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Convulsões Febris/genética , Epilepsia Generalizada/fisiopatologia , Família , Feminino , Células HEK293 , Humanos , Masculino , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Convulsões Febris/fisiopatologia
3.
Mol Psychiatry ; 22(5): 689-702, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27021819

RESUMO

SHANK3 (also called PROSAP2) genetic haploinsufficiency is thought to be the major cause of neuropsychiatric symptoms in Phelan-McDermid syndrome (PMS). PMS is a rare genetic disorder that causes a severe form of intellectual disability (ID), expressive language delays and other autistic features. Furthermore, a significant number of SHANK3 mutations have been identified in patients with autism spectrum disorders (ASD), and SHANK3 truncating mutations are associated with moderate to profound ID. The Shank3 protein is a scaffold protein that is located in the postsynaptic density (PSD) of excitatory synapses and is crucial for synapse development and plasticity. In this study, we investigated the molecular mechanisms associated with the ASD-like behaviors observed in Shank3Δ11-/- mice, in which exon 11 has been deleted. Our results indicate that Shank3 is essential to mediating metabotropic glutamate receptor 5 (mGlu5)-receptor signaling by recruiting Homer1b/c to the PSD, specifically in the striatum and cortex. Moreover, augmenting mGlu5-receptor activity by administering 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide ameliorated the functional and behavioral defects that were observed in Shank3Δ11-/- mice, suggesting that pharmaceutical treatments that increase mGlu5 activity may represent a new approach for treating patients that are affected by PMS and SHANK3 mutations.


Assuntos
Transtorno do Espectro Autista/tratamento farmacológico , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Benzamidas/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Pirazóis/farmacologia , Receptor de Glutamato Metabotrópico 5/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Deleção Cromossômica , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/metabolismo , Cromossomos Humanos Par 22/genética , Cromossomos Humanos Par 22/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Éxons , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Proteínas de Arcabouço Homer/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Proteínas dos Microfilamentos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Densidade Pós-Sináptica/metabolismo , Transdução de Sinais , Transmissão Sináptica
5.
Cell Death Dis ; 5: e1342, 2014 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-25032865

RESUMO

Loss of ataxia telangiectasia mutated (ATM) kinase, a key factor of the DNA damage response (DDR) pathway, causes the cancer predisposing and neurodegenerative syndrome ataxia-telangiectasia (A-T). To investigate the mechanisms of neurodegeneration, we have reprogrammed fibroblasts from ATM-null A-T patients and normal controls to pluripotency (human-induced pluripotent stem cells), and derived from these neural precursor cells able to terminally differentiate into post-mitotic neurons positive to >90% for ß-tubulin III+/microtubule-associated protein 2+. We show that A-T neurons display similar voltage-gated potassium and sodium currents and discharges of action potentials as control neurons, but defective expression of the maturation and synaptic markers SCG10, SYP and PSD95 (postsynaptic density protein 95). A-T neurons exhibited defective repair of DNA double-strand breaks (DSBs) and repressed phosphorylation of ATM substrates (e.g., γH2AX, Smc1-S966, Kap1-S824, Chk2-T68, p53-S15), but normal repair of single-strand breaks, and normal short- and long-patch base excision repair activities. Moreover, A-T neurons were resistant to apoptosis induced by the genotoxic agents camptothecin and trabectedin, but as sensitive as controls to the oxidative agents. Most notably, A-T neurons exhibited abnormal accumulation of topoisomerase 1-DNA covalent complexes (Top1-ccs). These findings reveal that ATM deficiency impairs neuronal maturation, suppresses the response and repair of DNA DSBs, and enhances Top1-cc accumulation. Top1-cc could be a risk factor for neurodegeneration as they may interfere with transcription elongation and promote transcriptional decline.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Ataxia Telangiectasia/enzimologia , Células-Tronco Pluripotentes Induzidas/enzimologia , Neurônios/enzimologia , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/fisiopatologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Topoisomerases Tipo I/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas de Membrana , Mitose , Neurônios/citologia , Fosforilação , Estatmina
6.
Br J Pharmacol ; 150(6): 792-7, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17279091

RESUMO

BACKGROUND AND PURPOSE: Topiramate is a novel anticonvulsant known to modulate the activity of several ligand- and voltage-gated ion channels in neurons. The mechanism of action of topiramate, at a molecular level, is still unclear, but the phosphorylation state of the channel/receptor seems to be a factor that is able to influence its activity. We investigated the consequences of phosphorylation of the sodium channel on the effect of topiramate on tetrodotoxin (TTX)-sensitive transient Na(+) current (I(NaT)). EXPERIMENTAL APPROACH: I(NaT) was recorded in dissociated neurons of rat sensorimotor cortex using whole-cell patch-clamp configuration. KEY RESULTS: We found that topiramate (100 microM) significantly shifted the steady-state I(NaT) inactivation curve in a hyperpolarized direction. In neurons pre-treated with a PKC-activator, 1-oleoyl-2-acetyl-sn-glycerol (OAG; 2 microM), the net effect of topiramate on steady-state I(NaT) inactivation was significantly decreased. In addition, OAG also slightly shifted the I(NaT) activation curve in a hyperpolarized direction, while perfusion with topiramate had no effect on the parameters of I(NaT) activation. CONCLUSIONS AND IMPLICATIONS: These data show that PKC-activation can modulate the effect of topiramate on I(NaT). This suggests that channel phosphorylation in physiological or pathological conditions (such as epiliepsy), can alter the action of topiramate on sodium currents.


Assuntos
Frutose/análogos & derivados , Proteína Quinase C/metabolismo , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/metabolismo , Animais , Anticonvulsivantes/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Diglicerídeos/farmacologia , Frutose/farmacologia , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Córtex Motor/efeitos dos fármacos , Córtex Motor/metabolismo , Técnicas de Patch-Clamp , Fosforilação , Ratos , Ratos Sprague-Dawley , Tetrodotoxina/toxicidade , Topiramato
7.
J Neurophysiol ; 95(6): 3460-8, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16467432

RESUMO

We evaluated the characteristics of the persistent sodium current (I(NaP)) in pyramidal neurons of layers II/III and V in slices of rat sensorimotor cortex using whole cell patch-clamp recordings. In both layers, I(NaP) began activating around -60 mV and was half-activated at -43 mV. The I(NaP) peak amplitude and density were significantly higher in layer V. The voltage-dependent I(NaP) steady-state inactivation occurred at potentials that were significantly more positive in layer V (V(1/2): -42.3 +/- 1.1 mV) than in layer II/III (V(1/2): -46.8 +/- 1.6 mV). In both layers, a current fraction corresponding to about 25% of the maximal peak amplitude did not inactivate. The time course of I(NaP) inactivation and recovery from inactivation could be fitted with a biexponential function. In layer V pyramidal neurons the faster time constant of development of inactivation had variable values, ranging from 158.0 to 1,133.8 ms, but it was on average significantly slower than that in layer II/III (425.9 +/- 80.5 vs. 145.8 +/- 18.2 ms). In both layers, I(NaP) did not completely inactivate even with very long conditioning depolarizations (40 s at -10 mV). Recovery from inactivation was similar in the two layers. Layer V intrinsically bursting and regular spiking nonadapting neurons showed particularly prolonged depolarized plateau potentials when Ca2+ and K+ currents were blocked and slower early phase of I(NaP) development of inactivation. The biexponential kinetics characterizing the time-dependent inactivation of I(NaP) in layers II/III and V indicates a complex inactivating process that is incomplete, allowing a residual "persistent" current fraction that does not inactivate. Moreover, our data indicate that I(NaP) has uneven inactivation properties in pyramidal neurons of different layers of rat sensorimotor cortex. The higher current density, the rightward shifted voltage dependency of inactivation as well the slower kinetics of inactivation characterizing I(NaP) in layer V with respect to layer II/III pyramidal neurons may play a significant role in their ability to fire recurrent action potential bursts, as well in the high susceptibility to generate epileptic events.


Assuntos
Potenciais de Ação/fisiologia , Ativação do Canal Iônico/fisiologia , Córtex Motor/fisiologia , Rede Nervosa/fisiologia , Canais de Sódio/fisiologia , Sódio/metabolismo , Córtex Somatossensorial/fisiologia , Animais , Células Cultivadas , Potenciais da Membrana , Ratos , Ratos Sprague-Dawley
8.
Neuroscience ; 127(1): 63-8, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15219669

RESUMO

We investigated the interference of protein-kinase C (PKC)-dependent Na(+) channel phosphorylation on the inhibitory effect that the antiepileptic drug topiramate (TPM) has on persistent Na(+) currents (I(NaP)) by making whole cell patch-clamp and intracellular recordings of rat sensorimotor cortex neurons. The voltage-dependent activation of I(NaP) was significantly shifted in the hyperpolarizing direction when PKC was activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG). TPM reduced the peak amplitude of I(NaP), but it did not counteract the OAG-induced shift in I(NaP) activation. Firing property experiments showed that the firing threshold was lowered by OAG. TPM was unable to counteract this effect, which may be due to OAG-dependent enhancement of the contribution of subthreshold I(NaP). These data suggest that PKC activation may limit the effect of the anticonvulsant TPM on the persistent fraction of Na(+) currents. The channel phosphorylation that may occur in cortical neurons as a result of physiological or pathological (e.g. epileptic) events can modulate the action of TPM on Na(+) currents.


Assuntos
Membrana Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Frutose/análogos & derivados , Frutose/farmacologia , Neurônios/efeitos dos fármacos , Proteína Quinase C/metabolismo , Canais de Sódio/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Anticonvulsivantes/farmacologia , Membrana Celular/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Diglicerídeos/farmacologia , Ativadores de Enzimas/farmacologia , Neurônios/metabolismo , Técnicas de Patch-Clamp , Fosforilação/efeitos dos fármacos , Proteína Quinase C/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Canais de Sódio/metabolismo , Topiramato
9.
Proc Natl Acad Sci U S A ; 98(26): 15348-53, 2001 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-11742069

RESUMO

Inactivation is a fundamental characteristic of Na(+) channels, and small changes cause skeletal muscle paralysis and myotonia, epilepsy, and cardiac arrhythmia. Brain Na(v)1.2a channels have faster inactivation than cardiac Na(v)1.5 channels, but minor differences in inactivation gate structure are not responsible. We constructed chimeras in which the C termini beyond the fourth homologous domains of Na(v)1.2a and Na(v)1.5 were exchanged. Replacing the C-terminal domain (CT) of Na(v)1.2a with that of Na(v)1.5 (Na(v)1.2/1.5CT) slowed inactivation at +40 mV approximately 2-fold, making it similar to Na(v)1.5. Conversely, replacing the CT of Na(v)1.5 with that of Na(v)1.2a (Nav1.5/1.2CT) accelerated inactivation, making it similar to Na(v)1.2a. Activation properties were unaffected. The voltage dependence of steady-state inactivation of Na(v)1.5 is 16 mV more negative than that of Na(v)1.2a. The steady-state inactivation curve of Na(v)1.2a was shifted +12 mV in Na(v)1.2/1.5CT, consistent with destabilization of the inactivated state. Conversely, Na(v)1.5/1.2CT was shifted -14 mV relative to Na(v)1.5, consistent with stabilization of the inactivated state. Although these effects of exchanging C termini were consistent with their effects on inactivation kinetics, they magnified the differences in the voltage dependence of inactivation between brain and cardiac channels rather than transferring them. Thus, other parts of these channels determine the basal difference in steady-state inactivation. Deletion of the distal half of either the Na(v)1.2 or Na(v)1.5 CTs accelerated open-state inactivation and negatively shifted steady-state inactivation. Thus, the C terminus has a strong influence on kinetics and voltage dependence of inactivation in brain Na(v)1.2 and cardiac Na(v)1.5 channels and is primarily responsible for their differing rates of channel inactivation.


Assuntos
Encéfalo/metabolismo , Miocárdio/metabolismo , Canais de Sódio/fisiologia , Linhagem Celular , Humanos , Cinética , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Deleção de Sequência , Canais de Sódio/química , Canais de Sódio/genética , Canais de Sódio/metabolismo
10.
J Gen Physiol ; 118(3): 291-302, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11524459

RESUMO

beta-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3-S4 loop at the extracellular end of the S4 voltage sensor in domain II of the alpha subunit. Here, we probe the role of gating charges in the IIS4 segment in beta-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances beta-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the beta-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from -80 to -140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor-trapping model in which the beta-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances toxin-induced channel activation.


Assuntos
Ativação do Canal Iônico/fisiologia , Venenos de Escorpião/farmacologia , Canais de Sódio/fisiologia , Arginina/fisiologia , Linhagem Celular , Eletrofisiologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Membranas/efeitos dos fármacos , Membranas/metabolismo , Mutagênese Sítio-Dirigida/genética , Mutagênese Sítio-Dirigida/fisiologia , Técnicas de Patch-Clamp , Canais de Sódio/efeitos dos fármacos
11.
J Pharmacol Exp Ther ; 288(3): 960-8, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10027832

RESUMO

The actions of the antiepileptic drug topiramate (TPM) on Na+ currents were assessed using whole-cell patch-clamp recordings in dissociated neocortical neurons and intracellular recordings in neocortical slices. Relatively low TPM concentrations (25-30 microM) slightly inhibited the persistent fraction of Na+ current in dissociated neurons and reduced the Na+-dependent long-lasting action potential shoulders, which can be evoked in layer V pyramidal neurons after Ca++ and K+ current blockade. Conversely, the same drug concentrations were ineffective in reducing the amplitude of the fast Na+-dependent action potentials evoked in slices or the peak of transient Na+ (INaf) current evoked in isolated neurons from a physiological holding potential. Consistent INaf inhibition became, however, evident only when the neuronal membrane was kept depolarized to enhance resting Na+ channel inactivation. TPM (100 microM) was ineffective on the voltage dependence of activation but induced a leftward shift of the steady-state INaf inactivation curve. The drug-induced inhibitory effect increased with the duration of membrane depolarization, and the recovery of INaf after long membrane depolarizations was slightly delayed in comparison with that observed under control conditions. The obtained evidence suggests that the anticonvulsant action of TPM may operate by stabilizing channel inactivation, which can be induced by depolarizing events similar to those occurring in chronic epileptic conditions. Concurrently, the slight but significant inhibition of the persistent fraction of the Na+ current, obtained with the application of relatively low TPM concentrations, may contribute toward its anticonvulsant effectiveness by modulating the near-threshold depolarizing events that are sustained by this small current fraction.


Assuntos
Anticonvulsivantes/farmacologia , Frutose/análogos & derivados , Neocórtex/efeitos dos fármacos , Sódio/metabolismo , Animais , Frutose/farmacologia , Técnicas In Vitro , Neocórtex/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Canais de Sódio/efeitos dos fármacos , Topiramato
12.
Epilepsy Res ; 32(1-2): 304-8, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9761329

RESUMO

The effect of valproate (VPA) on Na+ currents (INa), was studied by means of voltage clamp recordings using whole-cell patch clamp configuration in 21 acutely dissociated neocortical neurons. Concentrations of VPA up to 200 microM failed to induce any detectable decrease in fast INa (I(Naf)), but the persistent fraction (I(NaP)) was significantly reduced by low VPA concentrations (10-30 microM), corresponding to the lower values of the 'therapeutic' range in epileptic patients. Since it is known that I(NaP) critically regulates the firing properties of pyramidal neurons, it is suggested that the anticonvulsant effectiveness of VPA is mainly due to its effect on I(NaP).


Assuntos
Neocórtex/fisiologia , Neurônios/fisiologia , Canais de Sódio/efeitos dos fármacos , Ácido Valproico/farmacologia , Animais , Anticonvulsivantes/uso terapêutico , Células Cultivadas , Epilepsia/tratamento farmacológico , Humanos , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Canais de Sódio/fisiologia , Ácido Valproico/uso terapêutico
13.
J Physiol ; 507 ( Pt 1): 105-16, 1998 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-9490824

RESUMO

1. The experiments were performed on sensorimotor cortex using current-clamp intracellular recordings in layer V pyramidal neurones and whole-cell voltage-clamp recordings in dissociated pyramidal neurones. The intracellularly recorded neurones were classified on the basis of their firing characteristics as intrinsically bursting (IB) and regular spiking (RS). The RS neurones were further subdivided into adapting (RSAD) or non-adapting (RSNA), depending on the presence or absence of spike frequency adaptation. Since burst firing in neocortical pyramidal neurones has previously been suggested to depend on the persistent fraction of Na+ current (INa, p), pharmacological manipulations with drugs affecting INa inactivation have been employed. 2. ATX II, a toxin derived from Anemonia sulcata, selectively inhibited INa fast inactivation in dissociated neurones. In current-clamp experiments on neocortical slices, ATX II enhanced the naturally occurring burst firing in IB neurones and revealed the ability of RSNA neurones to discharge in bursts, whereas in RSAD neurones it increased firing frequency, without inducing burst discharges. During the ATX II effect, in all the three neuronal subclasses, episodes of a metastable condition occurred, characterized by long-lasting depolarizing shifts, triggered by action potentials, which were attributed to a peak in the toxin-induced inhibition of INa inactivation. The ATX II effect on IB and RSNA neurones was compared with that induced by veratridine and iodoacetamide. Veratridine induced a small increase in the INa and a large shift to the left in the voltage dependence of INa activation. Accordingly, its major effect on firing characteristics was the induction of prolonged tonic discharges, associated with burst facilitation less pronounced than that induced by ATX II. The alkylating agent iodoacetamide was able to induce a selective small increase in the INa,p, with a similar but less pronounced effect than ATX II on firing behaviour. 3. The present results show that pharmacological manipulations capable of slowing down INa inactivation significantly enhance burst behaviour in IB neurones and promote burst firing in otherwise non-bursting RSNA neurones. We suggest that IB and, to a lesser extent, RSNA neurones are endowed with a relatively large fraction of INa,p which, in physiological conditions, is sufficient to sustain bursting in IB but not in RSNA neurones.


Assuntos
Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiologia , Venenos de Cnidários/farmacologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Sódio/fisiologia , Alquilantes/farmacologia , Animais , Córtex Cerebral/citologia , Condutividade Elétrica , Técnicas In Vitro , Iodoacetamida/farmacologia , Íons , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Veratridina/farmacologia
14.
Biophys J ; 74(2 Pt 1): 831-42, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9533695

RESUMO

An extensive evaluation of the possible alterations affecting physiological voltage signals recorded with patch-clamp amplifiers (PCAs) working in the current-clamp (CC) mode was carried out by following a modeling approach. The PCA output voltage and current signals obtained during CC recordings performed under simplified experimental conditions were exploited to determine the equations describing the generation of error currents and voltage distortions by PCAs. The functions thus obtained were used to construct models of PCAs working in the CC mode, which were coupled to numerical simulations of neuronal bioelectrical behavior; this allowed us to evaluate the effects of the same PCAs on different physiological membrane-voltage events. The models revealed that rapid signals such as fast action potentials are preferentially affected, whereas slower events, such as low-threshold spikes, are less altered. Prominent effects of model PCAs on fast action potentials were alterations of their amplitude, duration, depolarization and repolarization speeds, and, most notably, the generation of spurious afterhyperpolarizations. Processes like regular firing and burst firing could also be altered, under particular conditions, by the model PCAs. When a cell consisting of more than one single intracellular compartment was considered, the model PCAs distorted fast equalization transients. Furthermore, the effects of different experimental and cellular parameters (series resistance, cell capacitance, temperature) on PCA-generated artifacts were analyzed. Finally, the simulations indicated that no off-line correction based on manipulations of the error-current signals returned by the PCAs can be successfully performed in the attempt to recover unperturbed voltage signals, because of alterations of the overall current flowing through the cell-PCA system.


Assuntos
Potenciais de Ação/fisiologia , Membrana Celular/fisiologia , Modelos Biológicos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Animais , Gânglios Espinais/fisiologia , Matemática , Potenciais da Membrana/fisiologia
15.
Trends Neurosci ; 19(12): 530-4, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8961481

RESUMO

A growing number of experimental studies have used patch-clamp amplifiers (PCAs) in the current-clamp (CC) mode to investigate classical excitability. In this paper we show that the measurements obtained in this way are affected by errors due to the electronic design of the PCA input section. We present experimental evidence of such errors, and demonstrate that they derive from PCA current absorption. Moreover, we propose a new PCA input-circuit configuration for the CC mode, which is suitable for accurately recording physiological voltage signals and is perfectly compatible with the standard voltage-clamp mode.


Assuntos
Técnicas de Patch-Clamp/normas , Potenciais de Ação/fisiologia , Artefatos , Técnicas de Patch-Clamp/instrumentação , Técnicas de Patch-Clamp/métodos
16.
Pediatr Med Chir ; 17(4): 299-302, 1995.
Artigo em Italiano | MEDLINE | ID: mdl-7491322

RESUMO

Early-onset infection findings caused by Group B Streptococcus occur within 24 hours of birth (60 per cent of cases) but they may appear anytime during the first 5 days of life. In our experience early-onset infection affects both preterm and term neonates. The Authors report the usual clinical signs described for bacterial infections. Unusual findings are also reported: among 34 infants with early-onset infection, the congenital diaphragmatic hernia was associated with GBS septicemia in two neonates; beads of perspiration were the first only clinical finding in one neonate too. Two cases of late-onset infection are also reported.


Assuntos
Infecções Estreptocócicas/diagnóstico , Streptococcus agalactiae , Fatores Etários , Anticorpos Antibacterianos/análise , Peso ao Nascer , Feminino , Humanos , Recém-Nascido , Masculino , Gravidez , Complicações Infecciosas na Gravidez/diagnóstico , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/imunologia , Streptococcus agalactiae/isolamento & purificação
17.
EMBO J ; 14(6): 1075-83, 1995 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-7720698

RESUMO

Hormonal stimulation of voltage-dependent Ca2+ channels in pituitary cells is thought to contribute to the sustained phase of Ca2+ entry and secretion induced by secretion stimulating hormones and has been suggested as a mechanism for refilling the Ca2+ stores. Using the cell-attached patch-clamp technique, we studied the stimulation of single Ca2+ channels by thyrotropin-releasing hormone (TRH) in rat GH3 cells. We show that TRH applied from the bath switched the activity of single L-type Ca2+ channels from a gating mode with very low open probability (po) to a gating mode with slightly smaller conductance but 10 times higher po. Interconversions between these two gating modes were also observed under basal conditions, where the equilibrium was shifted towards the low po mode. TRH applied from the pipette had no effect, indicating the involvement of a cytosolic compound in the stimulatory pathway. We show that TRH does not potentiate all the L-type Ca2+ channels in a given membrane patch and report evidence for co-expression of two functionally different L-type Ca2+ channels. Our results uncover the biophysical mechanism of hormonal stimulation of voltage-dependent Ca2+ channels in GH3 cells and are consistent with differential modulation of different subtypes of dihydropyridine-sensitive Ca2+ channels.


Assuntos
Canais de Cálcio/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Hipófise/fisiologia , Hormônio Liberador de Tireotropina/farmacologia , Animais , Canais de Cálcio/fisiologia , Potenciais da Membrana , Técnicas de Patch-Clamp , Hipófise/citologia , Ratos
18.
Eur J Neurosci ; 6(3): 381-91, 1994 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-8019675

RESUMO

We studied, in rat sensory neurons, the modulation of high voltage-activated Ca2+ currents (ICa) mediated by the pertussis toxin-sensitive activation of muscarinic receptors, which were found to be of subtypes M2 or M4. Muscarine reversibly blocked somatic Ca2+ spikes but strong predepolarizations only partially relieved the inhibited Ca2+ current. On the other hand, the putative coupling messenger could not rapidly diffuse towards channels whose activity was recorded from a macro-patch. The perforated patch technique virtually prevented the response rundown present during whole-cell experiments. Both omega-conotoxin GVIA (omega-CgTx)-sensitive channels and omega-CgTx- and dihydropyridine-resistant channels are coupled to the muscarinic receptor, but not the L-channel. When measured in the same neuron, dose-response relationships for the first and subsequent agonist applications differed; maximal inhibition, the reciprocal of half-maximal concentration and the Hill coefficient were always highest in the first trial. Muscarine and oxotremorine exhibited monotone dose-response curves, but oxotremorine-M showed non-linear relationships which became monotonic when cells were intracellularly perfused with inhibitors of protein kinase A (PKA) and C (PKC), suggesting that either PKA or receptor-induced PKC could phosphorylate and thus inactive G-proteins or other unknown proteins involved in inhibitory muscarinic actions on ICa. In summary, these data provide a preliminary pharmacological characterization of the muscarinic inhibition of the Ca2+ channels in sensory neurons, with implications about agonist specificity and the interplay between signalling pathways.


Assuntos
Canais de Cálcio/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Receptores Muscarínicos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , ômega-Conotoxinas , Potenciais de Ação/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Condutividade Elétrica , Técnicas In Vitro , Cinética , Potenciais da Membrana/efeitos dos fármacos , Venenos de Moluscos/farmacologia , Muscarina/farmacologia , Peptídeos/farmacologia , Inibidores de Proteínas Quinases , Ratos , Ratos Sprague-Dawley
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