Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 19.819
Filtrar
1.
Adv Exp Med Biol ; 1131: 73-91, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646507

RESUMO

Imaging techniques may overcome the limitations of electrode techniques to measure locally not only membrane potential changes, but also ionic currents. Here, we review a recently developed approach to image native neuronal Ca2+ currents from brain slices. The technique is based on combined fluorescence recordings using low-affinity Ca2+ indicators possibly in combination with voltage sensitive dyes. We illustrate how the kinetics of a Ca2+ current can be estimated from the Ca2+ fluorescence change and locally correlated with the change of membrane potential, calibrated on an absolute scale, from the voltage fluorescence change. We show some representative measurements from the dendrites of CA1 hippocampal pyramidal neurons, from olfactory bulb mitral cells and from cerebellar Purkinje neurons. We discuss the striking difference in data analysis and interpretation between Ca2+ current measurements obtained using classical electrode techniques and the physiological currents obtained using this novel approach. Finally, we show how important is the kinetic information on the native Ca2+ current to explore the potential molecular targets of the Ca2+ flux from each individual Ca2+ channel.


Assuntos
Canais de Cálcio , Neuroimagem , Animais , Cálcio/metabolismo , Canais de Cálcio/fisiologia , Dendritos/fisiologia , Humanos , Potenciais da Membrana/fisiologia , Imagem Óptica , Células Piramidais/fisiologia
2.
Nat Commun ; 10(1): 2659, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201323

RESUMO

In contrast to the plasma membrane, the vacuole membrane has not yet been associated with electrical excitation of plants. Here, we show that mesophyll vacuoles from Arabidopsis sense and control the membrane potential essentially via the K+-permeable TPC1 and TPK channels. Electrical stimuli elicit transient depolarization of the vacuole membrane that can last for seconds. Electrical excitability is suppressed by increased vacuolar Ca2+ levels. In comparison to wild type, vacuoles from the fou2 mutant, harboring TPC1 channels insensitive to luminal Ca2+, can be excited fully by even weak electrical stimuli. The TPC1-loss-of-function mutant tpc1-2 does not respond to electrical stimulation at all, and the loss of TPK1/TPK3-mediated K+ transport affects the duration of TPC1-dependent membrane depolarization. In combination with mathematical modeling, these results show that the vacuolar K+-conducting TPC1 and TPK1/TPK3 channels act in concert to provide for Ca2+- and voltage-induced electrical excitability to the central organelle of plant cells.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Canais de Cálcio/metabolismo , Potenciais da Membrana/fisiologia , Vacúolos/fisiologia , Proteínas de Arabidopsis/genética , Canais de Cálcio/genética , Sinalização do Cálcio/fisiologia , Membranas Intracelulares/fisiologia , Mutação com Perda de Função , Células do Mesofilo/citologia , Células do Mesofilo/fisiologia , Plantas Geneticamente Modificadas , Potássio/metabolismo , Canais de Potássio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo
3.
Invest Ophthalmol Vis Sci ; 60(6): 2294-2303, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31117121

RESUMO

Purpose: The concentration of protons in the aqueous humor (AH) of the vertebrate eye is maintained close to blood pH; however, pathologic conditions and surgery may shift it by orders of magnitude. We investigated whether and how changes in extra- and intracellular pH affect the physiology and function of trabecular meshwork (TM) cells that regulate AH outflow. Methods: Electrophysiology, in conjunction with pharmacology, gene knockdown, and optical recording, was used to track the pH dependence of transmembrane currents and mechanotransduction in primary and immortalized human TM cells. Results: Extracellular acidification depolarized the resting membrane potential by inhibiting an outward K+-mediated current, whereas alkalinization hyperpolarized the cells and augmented the outward conductance. Intracellular acidification with sodium bicarbonate hyperpolarized TM cells, whereas removal of intracellular protons with ammonium chloride depolarized the membrane potential. The effects of extra- and intracellular acid and alkaline loading were abolished by quinine, a pan-selective inhibitor of two-pore domain potassium (K2P) channels, and suppressed by shRNA-mediated downregulation of the mechanosensitive K2P channel TREK-1. Extracellular acidosis suppressed, whereas alkalosis facilitated, the amplitude of the pressure-evoked TREK-1-mediated outward current. Conclusions: These results demonstrate that TM mechanotransduction mediated by TREK-1 channels is profoundly sensitive to extra- and intracellular pH shifts. Intracellular acidification might modulate aqueous outflow and IOP by stimulating TREK-1 channels.


Assuntos
Concentração de Íons de Hidrogênio , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Pressão , Malha Trabecular/fisiologia , Células Cultivadas , Humanos , Mecanotransdução Celular/fisiologia , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp
4.
Phys Rev E ; 99(3-1): 032407, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30999441

RESUMO

A 15-state kinetic model for Na^{+} channel gating that describes the coupling between three activation sensors, a two-stage fast inactivation process, and slow inactivated states may be reduced to equations for a 6-state system by application of the method of multiple scales. By expressing the occupation probabilities for closed states and the open state in terms of activation and fast inactivation variables, and assuming that activation has a faster relaxation than inactivation and that the activation sensors are mutually independent, the kinetic equations may be further reduced to rate equations for activation, and coupled fast and slow inactivation that describe spike frequency adaptation, a repetitive bursting oscillation in the neural membrane, and a cardiac action potential with a plateau oscillation. The fast inactivation rate function is, in general, dependent on the activation variable m(t) but may be approximated by a voltage-dependent function, and the rate function for entry into the slow inactivated state is dependent on the fast inactivation variable.


Assuntos
Potenciais da Membrana/fisiologia , Modelos Biológicos , Miocárdio/metabolismo , Neurônios/metabolismo , Canais de Sódio/metabolismo , Algoritmos , Animais , Cátions Bivalentes/metabolismo , Simulação por Computador , Cinética , Probabilidade , Sódio/metabolismo , Fatores de Tempo
5.
Vis Neurosci ; 36: E003, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30915923

RESUMO

Variability in the electrophysiological properties of homotypic photoreceptors is widespread and is thought to facilitate functioning under disparate illumination conditions. Compound eyes of insects have three sources of variability: inter-individual, intra-individual, and intra-ommatidial, the latter two overlapping. Here, I explored the causes of variability in Periplaneta americana, a nocturnal insect characterized by highly variable photoreceptor responses. By recording from photoreceptors in dissociated ommatidia, including consecutive recordings from photoreceptors in the same ommatidium (SO), I studied the variability of six properties: whole-cell membrane capacitance (Cm), phototransduction latency, maximal conductance (Gmax) and the slope factor of the sustained Kv current, absolute sensitivity in dim light, and sustained light-induced current (LIC) amplitude in bright light. Coefficient of variation (CV) metrics were used to compare variances in four experimental groups: SO, same animal (SA), all data combined "full sample" (FS), and full sample of all SO recordings (FSSO). For the normally distributed parameters Cm, Gmax, slope factor, and latency, the highest CV values were found in FS and FSSO, intermediate in SA, and the lowest in SO. On average, SO variance accounted for 47% of the full-sample variance in these four parameters. Absolute sensitivity and LIC values were not normally distributed, and the differences in variability between SO and FS/FSSO groups were smaller than for the other four parameters. These results indicate two main sources of variability, intra-ommatidial and inter-individual. Inter-individual variability was investigated by exposing adult cockroaches to constant light or dark for several months. In both groups, the majority of CV measures for the six parameters decreased compared to control, indicating substantial contribution of phenotypic plasticity to inter-individual differences. Analysis of variability of resting potential and elementary voltage responses revealed that resting potential is mainly determined by the sustained Kv conductance, whereas voltage bump amplitude is mainly determined by current bump amplitude and Cm.


Assuntos
Olho Composto de Artrópodes/fisiologia , Adaptação à Escuridão/fisiologia , Periplaneta/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Potenciais de Ação/fisiologia , Animais , Eletrofisiologia , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Estimulação Luminosa , Visão Ocular/fisiologia
6.
Nat Commun ; 10(1): 1302, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30899018

RESUMO

Humans and vocal animals use vocalizations to communicate with members of their species. A necessary function of auditory perception is to generalize across the high variability inherent in vocalization production and classify them into behaviorally distinct categories ('words' or 'call types'). Here, we demonstrate that detecting mid-level features in calls achieves production-invariant classification. Starting from randomly chosen marmoset call features, we use a greedy search algorithm to determine the most informative and least redundant features necessary for call classification. High classification performance is achieved using only 10-20 features per call type. Predictions of tuning properties of putative feature-selective neurons accurately match some observed auditory cortical responses. This feature-based approach also succeeds for call categorization in other species, and for other complex classification tasks such as caller identification. Our results suggest that high-level neural representations of sounds are based on task-dependent features optimized for specific computational goals.


Assuntos
Córtex Auditivo/fisiologia , Percepção Auditiva/fisiologia , Callithrix/fisiologia , Neurônios/fisiologia , Vocalização Animal/fisiologia , Estimulação Acústica , Animais , Córtex Auditivo/anatomia & histologia , Eletrodos Implantados , Feminino , Cobaias , Humanos , Masculino , Potenciais da Membrana/fisiologia , Neurônios/citologia , Som , Espectrografia do Som/métodos , Técnicas Estereotáxicas
7.
Molecules ; 24(4): 775, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30795543

RESUMO

Individuals with end-stage diabetic peripheral neuropathy present with decreased pain sensation. Transient receptor potential vanilloid type 1 (TRPV1) is implicated in pain signaling and resides on sensory dorsal root ganglion (DRG) neurons. We investigated the expression and functional activity of TRPV1 in DRG neurons of the Ins2+/Akita mouse at 9 months of diabetes using immunohistochemistry, live single cell calcium imaging, and whole-cell patch-clamp electrophysiology. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence assay was used to determine the level of Reactive Oxygen Species (ROS) in DRGs. Although TRPV1 expressing neuron percentage was increased in Ins2+/Akita DRGs at 9 months of diabetes compared to control, capsaicin-induced Ca2+ influx was smaller in isolated Ins2+/Akita DRG neurons, indicating impaired TRPV1 function. Consistently, capsaicin-induced Ca2+ influx was decreased in control DRG neurons cultured in the presence of 25 mM glucose for seven days versus those cultured with 5.5 mM glucose. The high glucose environment increased cytoplasmic ROS accumulation in cultured DRG neurons. Patch-clamp recordings revealed that capsaicin-activated currents decayed faster in isolated Ins2+/Akita DRG neurons as compared to those in control neurons. We propose that in poorly controlled diabetes, the accelerated rate of capsaicin-sensitive TRPV1 current decay in DRG neurons decreases overall TRPV1 activity and contributes to peripheral neuropathy.


Assuntos
Cálcio/metabolismo , Capsaicina/farmacologia , Neuropatias Diabéticas/metabolismo , Gânglios Espinais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Dor/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Neuropatias Diabéticas/genética , Neuropatias Diabéticas/patologia , Modelos Animais de Doenças , Fluoresceínas/química , Corantes Fluorescentes/química , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Regulação da Expressão Gênica , Glucose/farmacologia , Transporte de Íons/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Imagem Óptica , Dor/genética , Dor/fisiopatologia , Técnicas de Patch-Clamp , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Análise de Célula Única , Canais de Cátion TRPV/genética
8.
Nat Commun ; 10(1): 787, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770809

RESUMO

Mutations that modulate the activity of ion channels are essential tools to understand the biophysical determinants that control their gating. Here, we reveal the conserved role played by a single amino acid position (TM2.6) located in the second transmembrane domain of two-pore domain potassium (K2P) channels. Mutations of TM2.6 to aspartate or asparagine increase channel activity for all vertebrate K2P channels. Using two-electrode voltage-clamp and single-channel recording techniques, we find that mutation of TM2.6 promotes channel gating via the selectivity filter gate and increases single channel open probability. Furthermore, channel gating can be progressively tuned by using different amino acid substitutions. Finally, we show that the role of TM2.6 was conserved during evolution by rationally designing gain-of-function mutations in four Caenorhabditis elegans K2P channels using CRISPR/Cas9 gene editing. This study thus describes a simple and powerful strategy to systematically manipulate the activity of an entire family of potassium channels.


Assuntos
Potenciais da Membrana/fisiologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Drosophila , Evolução Molecular , Humanos , Invertebrados , Potenciais da Membrana/genética , Mutação/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Vertebrados
9.
Mol Pharmacol ; 95(4): 376-385, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30739093

RESUMO

The neurohypophyseal hormone oxytocin (OT) regulates biologic functions in both peripheral tissues and the central nervous system. In the central nervous system, OT influences social processes, including peer relationships, maternal-infant bonding, and affiliative social relationships. In mammals, the nonapeptide OT structure is highly conserved with leucine in the eighth position (Leu8-OT). In marmosets (Callithrix), a nonsynonymous nucleotide substitution in the OXT gene codes for proline in the eighth residue position (Pro8-OT). OT binds to its cognate G protein-coupled receptor (OTR) and exerts diverse effects, including stimulation (Gs) or inhibition (Gi/o) of adenylyl cyclase, stimulation of potassium channel currents (Gi), and activation of phospholipase C (Gq). Chinese hamster ovary cells expressing marmoset or human oxytocin receptors (mOTRs or hOTRs, respectively) were used to characterize OT signaling. At the mOTR, Pro8-OT was more efficacious than Leu8-OT in measures of Gq activation, with both peptides displaying subnanomolar potencies. At the hOTR, neither the potency nor efficacy of Pro8-OT and Leu8-OT differed with respect to Gq signaling. In both mOTR- and hOTR-expressing cells, Leu8-OT was more potent and modestly more efficacious than Pro8-OT in inducing hyperpolarization. In mOTR cells, Leu8-OT-induced hyperpolarization was modestly inhibited by pretreatment with pertussis toxin (PTX), consistent with a minor role for Gi/o activation; however, the Pro8-OT response in mOTR and hOTR cells was PTX insensitive. These findings are consistent with membrane hyperpolarization being largely mediated by a Gq signaling mechanism leading to Ca2+-dependent activation of K+ channels. Evaluation of the influence of apamin, charybdotoxin, paxilline, and TRAM-34 demonstrated involvement of both intermediate and large conductance Ca2+-activated K+ channels.


Assuntos
Cálcio/metabolismo , Leucina/metabolismo , Ocitocina/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Potássio/metabolismo , Prolina/metabolismo , Receptores de Ocitocina/metabolismo , Animais , Células CHO , Cricetulus , Humanos , Potenciais da Membrana/fisiologia , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/metabolismo
10.
eNeuro ; 6(1)2019 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30783614

RESUMO

The biological mechanisms underlying complex forms of learning requiring the understanding of rules based on previous experience are not yet known. Previous studies have raised the intriguing possibility that improvement in complex learning tasks requires the long-term modulation of intrinsic neuronal excitability, induced by reducing the conductance of the slow calcium-dependent potassium current (sIAHP) simultaneously in most neurons in the relevant neuronal networks in several key brain areas. Such sIAHP reduction is expressed in attenuation of the postburst afterhyperpolarization (AHP) potential, and thus in enhanced repetitive action potential firing. Using complex olfactory discrimination (OD) learning as a model for complex learning, we show that brief activation of the GluK2 subtype glutamate receptor results in long-lasting enhancement of neuronal excitability in neurons from controls, but not from trained rats. Such an effect can be obtained by a brief tetanic synaptic stimulation or by direct application of kainate, both of which reduce the postburst AHP in pyramidal neurons. Induction of long-lasting enhancement of neuronal excitability is mediated via a metabotropic process that requires PKC and ERK activation. Intrinsic neuronal excitability cannot be modulated by synaptic activation in neurons from GluK2 knock-out mice. Accordingly, these mice are incapable of learning the complex OD task. Moreover, viral-induced overexpression of Gluk2 in piriform cortex pyramidal neurons results in remarkable enhancement of complex OD learning. Thus, signaling via kainate receptors has a central functional role in higher cognitive abilities.


Assuntos
Aprendizagem por Discriminação/fisiologia , Percepção Olfatória/fisiologia , Córtex Piriforme/fisiologia , Células Piramidais/fisiologia , Receptores de Ácido Caínico/metabolismo , Animais , Agonistas de Aminoácidos Excitatórios/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ácido Caínico/farmacologia , Masculino , Aprendizagem em Labirinto/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Córtex Piriforme/efeitos dos fármacos , Proteína Quinase C/metabolismo , Células Piramidais/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores de Ácido Caínico/genética , Técnicas de Cultura de Tecidos
11.
Glia ; 67(5): 791-801, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30715764

RESUMO

Peripheral sensory ganglia contain the somata of neurons mediating mechanical, thermal, and painful sensations from somatic, visceral, and oro-facial organs. Each neuronal cell body is closely surrounded by satellite glial cells (SGCs) that have properties and functions similar to those of central astrocytes, including expression of gap junction proteins and functional dye coupling. As shown in other pain models, after systemic pain induction by intra-peritoneal injection of lipopolysaccharide, dye coupling among SGCs in intact trigeminal ganglion was enhanced. Moreover, neuron-neuron and neuron-SGC coupling was also detected. To verify the presence of gap junction-mediated coupling between SGCs and sensory neurons, we performed dual whole cell patch clamp recordings from both freshly isolated and short term cultured cell pairs dissociated from mouse trigeminal ganglia. Bidirectional gap junction mediated electrical responses were frequently recorded between SGCs, between neurons and between neurons and SGCs. Polarization of SGC altered neuronal excitability, providing evidence that gap junction-mediated interactions between neurons and glia within sensory ganglia may contribute to integration of peripheral sensory responses, and to the modulation and coordinaton of neuronal activity.


Assuntos
Junções Comunicantes/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Transmissão Sináptica/fisiologia , Gânglio Trigeminal/citologia , Animais , Compostos de Boro/farmacologia , Carbenoxolona/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Ácido Flufenâmico/farmacologia , Junções Comunicantes/efeitos dos fármacos , Heptanol/farmacologia , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/patologia , Isoquinolinas/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Probenecid/farmacologia , Transmissão Sináptica/efeitos dos fármacos
12.
Artigo em Inglês | MEDLINE | ID: mdl-30723398

RESUMO

Our objective is to examine the layer and spectrotemporal architecture and laminar distribution of high-frequency oscillations (HFOs) in a neonatal freeze lesion model of focal cortical dysplasia (FCD) associated with a high prevalence of spontaneous spike-wave discharges (SWDs). Electrophysiological recording of local field potentials (LFPs) in control and freeze lesion animals were obtained with linear micro-electrode arrays to detect presence of HFOs as compared to changes in spectral power, signal coherence, and single-unit distributions during "hyper-excitable" epochs of anesthesia-induced burst-suppression (B-S). Result were compared to HFOs observed during spontaneous SWDs in animals during sleep. Micro-electrode array recordings from the malformed cortex indicated significant increases in the presence of HFOs above 100 Hz and associated increases in spectral power and altered LFP coherence of recorded signals across cortical lamina of freeze-lesioned animals with spontaneous bursts of high-frequency activity, confined predominately to granular and supragranular layers. Spike sorting of well-isolated single-units recorded from freeze-lesioned cortex indicated an increase in putative excitatory cell activity in the outer cortical layers that showed only a weak association with HFOs while deeper inhibitory units were strongly phase-locked to high-frequency ripple (HFR) oscillations (300-800 Hz). Both SWDs and B-S show increases in HFR activity that were phase-locked to the high-frequency spike pattern occurring at the trough of low frequency oscillations. The spontaneous cyclic spiking of cortical inhibitory cells appears to be the driving substrate behind the HFO patterns associated with SWDs and a hyperexcitable supragranular layer near the malformed cortex may play a key role in epileptogenesis in our model. These data, derived from a mouse model with a distinct focal cortical malformation, support recent clinical data that HFOs, particularly fast ripples, is a biomarker to help define the cortical seizure zone, and provide limited insights toward understanding cellular level changes underlying the HFOs.


Assuntos
Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Malformações do Desenvolvimento Cortical/patologia , Malformações do Desenvolvimento Cortical/fisiopatologia , Potenciais da Membrana/fisiologia , Animais , Animais Recém-Nascidos , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Modelos Animais de Doenças , Feminino , Congelamento/efeitos adversos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Malformações do Desenvolvimento Cortical/etiologia , Camundongos , Optogenética , Sono , Transdução Genética , Vigília
13.
Nitric Oxide ; 86: 54-62, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30797972

RESUMO

Under normal conditions, connexin (Cx) hemichannels have a low open probability, which can increase under pathological conditions. Since hemichannels are permeable to relatively large molecules, their exacerbated activity has been linked to cell damage. Cx46 is highly expressed in the lens and its mutations have been associated to cataract formation, but it is unknown whether Cx46 has a role in non-genetic cataract formation (i.e. aging and diabetes). Nitric oxide (NO) is a key element in non-genetic cataract formation and Cx46 hemichannels have been shown to be sensitive to NO. The molecular mechanisms of the effects of NO on Cx46 are unknown, but are likely to result from Cx46 S-nitrosation (also known as S-nitrosylation). In this work, we found that lens opacity was correlated with Cx46 S-nitrosation in an animal model of cataract. Consistent with this result, a NO donor increased Cx46 S-nitrosation and hemichannel opening in HLE-B3 cells (cell line derived from human lens epithelial cells). Mutagenesis studies point to the cysteine located in the fourth transmembrane helix (TM4; human C212, rat C218) as the NO sensor. Electrophysiological studies performed in Xenopus oocytes revealed that rat Cx46 hemichannels are sensitive to different NO donors, and that the presence of C218 is necessary to observe the NO donors' effects. Unexpectedly, gap junctions formed by Cx46 were insensitive to NO or the reducing agent dithiothreitol. We propose that increased hemichannel opening and/or changes in their electrophysiological properties of human Cx46 due to S-nitrosation of the cysteine in TM4 could be an important factor in cataract formation.


Assuntos
Catarata/etiologia , Conexinas/metabolismo , Cisteína/química , Óxido Nítrico/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Conexinas/química , Cricetulus , Junções Comunicantes/metabolismo , Humanos , Masculino , Potenciais da Membrana/fisiologia , Mesocricetus , Camundongos , Nitrosação , Conformação Proteica em alfa-Hélice , Processamento de Proteína Pós-Traducional , Ratos Sprague-Dawley , Alinhamento de Sequência , Xenopus laevis , Peixe-Zebra
14.
J Physiol Sci ; 69(3): 543-551, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30758781

RESUMO

We have developed a concave-shaped transparent electrode unit that enables the placement of several electrodes within the optical sampling area on the spherical surface of the rat brain. This concave-shaped transparent electrode unit consists of an insulator base (a plano-concave lens) and a gallium-doped zinc oxide film that is a transparent conductor coating the base. Most of the unit is wrapped in an insulator film made of silicon dioxide, and the few areas left unwrapped act as electrodes. In the study reported here this newly developed transparent electrode unit worked well within the optical detection area without affecting optical recording. We applied this unit to our multiple-site optical recording system for membrane potential in order to eliminate pulsation artifacts and succeeded in optically recording spontaneous neural activity, including small changes in membrane potential, in the cerebral cortex in a single-sweep recording.


Assuntos
Córtex Cerebral/fisiologia , Potenciais da Membrana/fisiologia , Animais , Estimulação Elétrica/métodos , Eletrodos , Feminino , Fenômenos Fisiológicos do Sistema Nervoso , Ratos , Ratos Sprague-Dawley
15.
Nanoscale ; 11(4): 1949-1958, 2019 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-30644958

RESUMO

Cell-penetrating peptides (CPPs) are frequently employed as drug delivery agents with rapid cellular uptake, however, the uptake mechanism and the detailed translocation pathway are at present not completely understood. Both endocytosis and direct translocation through membrane pores have been observed in experiments and simulations under different conditions. Here we report the molecular dynamics simulations providing evidence for the direct translocation of CPPs across the membrane driven by the membrane electrostatic potential. The local membrane potential can be produced by the ion concentration imbalance across the membrane, which is ubiquitous in biological environments. Moreover, if positively charged CPPs are adsorbed on the membrane, this further enhances the membrane potential, opening membrane pores through which CPPs can be instantly transported in a chain-like configuration. The classical nucleation theory is applied to estimate the translocation time by calculating the changes in the free energy upon transferring CPPs across the membrane at different potentials, showing good agreement with available experimental measurements. The revealed CPP translocation mechanism can be broadly relevant for cellular processes in biology.


Assuntos
Peptídeos Penetradores de Células/metabolismo , Sequência de Aminoácidos , Peptídeos Penetradores de Células/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Potenciais da Membrana/fisiologia , Simulação de Dinâmica Molecular , Eletricidade Estática , Termodinâmica
16.
Bull Exp Biol Med ; 166(3): 404-408, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30627894

RESUMO

Differential high-resolution ECG (V1-V2) and pelvic electric potential measured between the coccyx and perineum were recorded simultaneously in resting supine position in men with autonomic nervous system disorders (N=37). In healthy volunteers (N=23), the effective (rms) value of PEP presented by median and interdecile range was 30 (20-80) µV within the frequency band of 0.03-80 Hz. In patients, the corresponding value was significantly higher: 140 (80-280) µV. In both groups, the amplitude harmonic spectrum of pelvic electric potential decreased monotonically with frequency according to 1/f1.6 law. In some patients (N=16), rare single or grouped high-amplitude impulses (up to 1 mV) of pelvic electric potential with total duration of about 1 sec were observed; of them, some persons (N=7) demonstrated practically one-to-one synchronous relations between these impulses and arrhythmia episodes indicating abnormal activity of the autonomic nervous system as their most probable common cause. The high-amplitude pelvic electric potential impulses were also observed in ECG records as interference signals with an amplitude attaining 50 µV. Thus, high-resolution ECG and pelvic electric potential can reveal the risk of abnormal neurogenic influences on the heart. The data obtained are discussed in relation to diagnostics of the autonomic nervous system disorders, neurogenic arrhythmias, and risk of sudden cardiac death.


Assuntos
Arritmias Cardíacas/diagnóstico por imagem , Doenças do Sistema Nervoso Autônomo/diagnóstico por imagem , Eletrocardiografia/métodos , Coração/diagnóstico por imagem , Potenciais da Membrana/fisiologia , Pelve/diagnóstico por imagem , Adulto , Arritmias Cardíacas/fisiopatologia , Doenças do Sistema Nervoso Autônomo/fisiopatologia , Biomarcadores/análise , Estudos de Casos e Controles , Cóccix/diagnóstico por imagem , Cóccix/inervação , Cóccix/fisiopatologia , Morte Súbita Cardíaca/prevenção & controle , Coração/inervação , Coração/fisiopatologia , Frequência Cardíaca/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Pelve/inervação , Pelve/fisiopatologia , Períneo/diagnóstico por imagem , Períneo/inervação , Períneo/fisiopatologia , Risco
17.
Proc Natl Acad Sci U S A ; 116(3): 1053-1058, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30598447

RESUMO

Activation of most primary sensory neurons results in transduction currents that are carried by cations. One notable exception is the vertebrate olfactory receptor neuron (ORN), where the transduction current is carried largely by the anion [Formula: see text] However, it remains unclear why ORNs use an anionic current for signal amplification. We have sought to provide clarification on this topic by studying the so far neglected dynamics of [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text] in the small space of olfactory cilia during an odorant response. Using computational modeling and simulations we compared the outcomes of signal amplification based on either [Formula: see text] or [Formula: see text] currents. We found that amplification produced by [Formula: see text] influx instead of a [Formula: see text] efflux is problematic for several reasons: First, the [Formula: see text] current amplitude varies greatly, depending on mucosal ion concentration changes. Second, a [Formula: see text] current leads to a large increase in the ciliary [Formula: see text] concentration during an odorant response. This increase inhibits and even reverses [Formula: see text] clearance by [Formula: see text] exchange, which is essential for response termination. Finally, a [Formula: see text] current increases the ciliary osmotic pressure, which could cause swelling to damage the cilia. By contrast, a transduction pathway based on [Formula: see text] efflux circumvents these problems and renders the odorant response robust and reliable.


Assuntos
Sinalização do Cálcio/fisiologia , Canais de Cloreto/metabolismo , Potenciais da Membrana/fisiologia , Modelos Neurológicos , Neurônios/metabolismo , Receptores Odorantes/metabolismo , Animais , Cálcio/metabolismo , Camundongos , Neurônios/citologia , Potássio/metabolismo , Sódio/metabolismo
18.
Brain ; 142(2): 376-390, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30615093

RESUMO

Ion channel mutations can cause distinct neuropsychiatric diseases. We first studied the biophysical and neurophysiological consequences of four mutations in the human Na+ channel gene SCN8A causing either mild (E1483K) or severe epilepsy (R1872W), or intellectual disability and autism without epilepsy (R1620L, A1622D). Only combined electrophysiological recordings of transfected wild-type or mutant channels in both neuroblastoma cells and primary cultured neurons revealed clear genotype-phenotype correlations. The E1483K mutation causing mild epilepsy showed no significant biophysical changes, whereas the R1872W mutation causing severe epilepsy induced clear gain-of-function biophysical changes in neuroblastoma cells. However, both mutations increased neuronal firing in primary neuronal cultures. In contrast, the R1620L mutation associated with intellectual disability and autism-but not epilepsy-reduced Na+ current density in neuroblastoma cells and expectedly decreased neuronal firing. Interestingly, for the fourth mutation, A1622D, causing severe intellectual disability and autism without epilepsy, we observed a dramatic slowing of fast inactivation in neuroblastoma cells, which induced a depolarization block in neurons with a reduction of neuronal firing. This latter finding was corroborated by computational modelling. In a second series of experiments, we recorded three more mutations (G1475R, M1760I, G964R, causing intermediate or severe epilepsy, or intellectual disability without epilepsy, respectively) that revealed similar results confirming clear genotype-phenotype relationships. We found intermediate or severe gain-of-function biophysical changes and increases in neuronal firing for the two epilepsy-causing mutations and decreased firing for the loss-of-function mutation causing intellectual disability. We conclude that studies in neurons are crucial to understand disease mechanisms, which here indicate that increased or decreased neuronal firing is responsible for distinct clinical phenotypes.


Assuntos
Epilepsia/genética , Deficiência Intelectual/genética , Mutação de Sentido Incorreto/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Neurônios/fisiologia , Animais , Células Cultivadas , Humanos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Ratos
19.
Neuroscience ; 401: 73-83, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30654002

RESUMO

The amygdala is concerned with the emotional memory consolidation, and is known as a stress-vulnerable region of the brain. Slow network oscillation is considered to play roles in memory consolidation during sleep. We investigated the relationship between the sleep and oscillation in the basolateral nucleus (BL) of the amygdala, in which burst firing is preferentially observed during sleep and the slow inhibitory oscillation is recorded from projection neuron. We examined whether sleep deprivation (SD) alters the properties of the network inhibition by whole-cell recordings from BL projection neurons and interneurons of the slice preparation of the juvenile rats. The level of the oscillatory network inhibition, measured as summed power of the spectral density between 0.1 and 3 Hz of the synaptic currents in the projection neurons, was significantly attenuated by acute (3 h) SD in older (P20-24) but not in younger (P15-19) animals. This reduction was mainly derived from the reduced peak amplitude of periodic IPSC bursts. In inhibitory interneurons in BL, spontaneous firings were reduced in older SD rats. The spike threshold of interneurons was increased and the power of the periodic excitatory transmission was reduced in the SD rats. Moreover, a reduction in input resistance in projection neurons was observed in SD rats without significant difference in the excitability which was measured by the spike number induced by depolarizing currents. These results suggest that SD stress affects the network oscillatory property accompanied by changes of individual neuronal excitability and synaptic communications.


Assuntos
Complexo Nuclear Basolateral da Amígdala/fisiologia , Interneurônios/fisiologia , Privação do Sono/fisiopatologia , Potenciais de Ação/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Transmissão Sináptica/fisiologia
20.
BMC Neurosci ; 20(1): 3, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30611209

RESUMO

BACKGROUND: Transcranial ultrasonic stimulation is a novel noninvasive tool for neuromodulation, and has high spatial resolution and deep penetration. Although it can increase excitation of neurons, its effects on neuron are poorly understood. This study was to evaluate effect of ultrasonic stimulation (US) on neurons in vitro. In this paper, the effect of US on the excitability and voltage-dependent [Formula: see text] currents of CA1 pyramidal neurons in the rat hippocampus was studied using patch clamp. RESULTS: Our results suggest that US increased the spontaneous firing rate and inhibited transient outward potassium current ([Formula: see text]) and delayed rectifier potassium current ([Formula: see text]. Furthermore, US altered the activation of [Formula: see text] channels, inactivation and recovery properties of [Formula: see text] channels. After US, the [Formula: see text] activation curves significantly moved to the negative voltage direction and increased its slope factor. Moreover, the data showed that US moved the inactivation curve of [Formula: see text] to the negative voltage and increased the slope factor. Besides, US delayed the recovery of [Formula: see text] channel. CONCLUSIONS: Our data indicate that US can increase excitation of neurons by inhibiting potassium currents. Different US decreased the voltage sensitivity of [Formula: see text] activation differentially. Moreover, the more time is needed for US to make the [Formula: see text] channels open again after inactivating. US may play a physiological role by inhibiting voltage-dependent potassium currents in neuromodulation. Our research can provide a theoretical basis for the future clinical application of ultrasound in neuromodulation.


Assuntos
Região CA1 Hipocampal/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Células Piramidais/metabolismo , Terapia por Ultrassom , Animais , Desenho de Equipamento , Masculino , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Potássio/metabolismo , Ratos Sprague-Dawley , Técnicas de Cultura de Tecidos , Terapia por Ultrassom/instrumentação , Ondas Ultrassônicas
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA