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1.
BMC Biol ; 17(1): 63, 2019 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-31412898

RESUMEN

BACKGROUND: Voltage-gated sodium (Nav) channels have traditionally been considered a trademark of excitable cells. However, recent studies have shown the presence of Nav channels in several non-excitable cells, such as astrocytes and macrophages, demonstrating that the roles of these channels are more diverse than was previously thought. Despite the earlier discoveries, the presence of Nav channel-mediated currents in the cells of retinal pigment epithelium (RPE) has been dismissed as a cell culture artifact. We challenge this notion by investigating the presence and possible role of Nav channels in RPE both ex vivo and in vitro. RESULTS: Our work demonstrates that several subtypes of Nav channels are found in human embryonic stem cell (hESC)-derived and mouse RPE, most prominently subtypes Nav1.4, Nav1.6, and Nav1.8. Whole cell patch clamp recordings from the hESC-derived RPE monolayers showed that the current was inhibited by TTX and QX-314 and was sensitive to the selective blockers of the main Nav subtypes. Importantly, we show that the Nav channels are involved in photoreceptor outer segment phagocytosis since blocking their activity significantly reduces the efficiency of particle internalization. Consistent with this role, our electron microscopy results and immunocytochemical analysis show that Nav1.4 and Nav1.8 accumulate on phagosomes and that pharmacological inhibition of Nav channels as well as silencing the expression of Nav1.4 with shRNA impairs the phagocytosis process. CONCLUSIONS: Taken together, our study shows that Nav channels are present in RPE, giving this tissue the capacity of fast electrical signaling. The channels are critical for the physiology of RPE with an important role in photoreceptor outer segment phagocytosis.


Asunto(s)
Fagocitosis/genética , Epitelio Pigmentado de la Retina/fisiología , Transducción de Señal/genética , Canales de Sodio/fisiología , Animales , Células Madre Embrionarias Humanas , Humanos , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp
2.
PLoS Comput Biol ; 15(5): e1007042, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-31150383

RESUMEN

The conduction of electrical signals through cardiac tissue is essential for maintaining the function of the heart, and conduction abnormalities are known to potentially lead to life-threatening arrhythmias. The properties of cardiac conduction have therefore been the topic of intense study for decades, but a number of questions related to the mechanisms of conduction still remain unresolved. In this paper, we demonstrate how the so-called EMI model may be used to study some of these open questions. In the EMI model, the extracellular space, the cell membrane, the intracellular space and the cell connections are all represented as separate parts of the computational domain, and the model therefore allows for study of local properties that are hard to represent in the classical homogenized bidomain or monodomain models commonly used to study cardiac conduction. We conclude that a non-uniform sodium channel distribution increases the conduction velocity and decreases the time delays over gap junctions of reduced coupling in the EMI model simulations. We also present a theoretical optimal cell length with respect to conduction velocity and consider the possibility of ephaptic coupling (i.e. cell-to-cell coupling through the extracellular potential) acting as an alternative or supporting mechanism to gap junction coupling. We conclude that for a non-uniform distribution of sodium channels and a sufficiently small intercellular distance, ephaptic coupling can influence the dynamics of the sodium channels and potentially provide cell-to-cell coupling when the gap junction connection is absent.


Asunto(s)
Sistema de Conducción Cardíaco/fisiología , Modelos Cardiovasculares , Animales , Arritmias Cardíacas/fisiopatología , Membrana Celular/fisiología , Biología Computacional , Simulación por Computador , Fenómenos Electrofisiológicos , Espacio Extracelular/fisiología , Uniones Comunicantes/fisiología , Humanos , Espacio Intracelular/fisiología , Contracción Miocárdica/fisiología , Miocitos Cardíacos/fisiología , Canales de Sodio/fisiología
3.
BMC Dev Biol ; 19(1): 12, 2019 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-31226923

RESUMEN

BACKGROUND: Alterations of bioelectrical properties of cells and tissues are known to function as wide-ranging signals during development, regeneration and wound-healing in several species. The Drosophila follicle-cell epithelium provides an appropriate model system for studying the potential role of electrochemical signals, like intracellular pH (pHi) and membrane potential (Vmem), during development. Therefore, we analysed stage-specific gradients of pHi and Vmem as well as their dependence on specific ion-transport mechanisms. RESULTS: Using fluorescent indicators, we found distinct alterations of pHi- and Vmem-patterns during stages 8 to 12 of oogenesis. To determine the roles of relevant ion-transport mechanisms in regulating pHi and Vmem and in establishing stage-specific antero-posterior and dorso-ventral gradients, we used inhibitors of Na+/H+-exchangers and Na+-channels (amiloride), V-ATPases (bafilomycin), ATP-sensitive K+-channels (glibenclamide), voltage-dependent L-type Ca2+-channels (verapamil), Cl--channels (9-anthroic acid) and Na+/K+/2Cl--cotransporters (furosemide). Either pHi or Vmem or both parameters were affected by each tested inhibitor. While the inhibition of Na+/H+-exchangers (NHE) and amiloride-sensitive Na+-channels or of V-ATPases resulted in relative acidification, inhibiting the other ion-transport mechanisms led to relative alkalisation. The most prominent effects on pHi were obtained by inhibiting Na+/K+/2Cl--cotransporters or ATP-sensitive K+-channels. Vmem was most efficiently hyperpolarised by inhibiting voltage-dependent L-type Ca2+-channels or ATP-sensitive K+-channels, whereas the impact of the other ion-transport mechanisms was smaller. In case of very prominent effects of inhibitors on pHi and/or Vmem, we also found strong influences on the antero-posterior and dorso-ventral pHi- and/or Vmem-gradients. For example, inhibiting ATP-sensitive K+-channels strongly enhanced both pHi-gradients (increasing alkalisation) and reduced both Vmem-gradients (increasing hyperpolarisation). Similarly, inhibiting Na+/K+/2Cl--cotransporters strongly enhanced both pHi-gradients and reduced the antero-posterior Vmem-gradient. To minor extents, both pHi-gradients were enhanced and both Vmem-gradients were reduced by inhibiting voltage-dependent L-type Ca2+-channels, whereas only both pHi-gradients were reduced (increasing acidification) by inhibiting V-ATPases or NHE and Na+-channels. CONCLUSIONS: Our data show that in the Drosophila follicle-cell epithelium stage-specific pHi- and Vmem-gradients develop which result from the activity of several ion-transport mechanisms. These gradients are supposed to represent important bioelectrical cues during oogenesis, e.g., by serving as electrochemical prepatterns in modifying cell polarity and cytoskeletal organisation.


Asunto(s)
Epitelio/fisiología , Transporte Iónico/fisiología , Folículo Ovárico/citología , Simportadores de Cloruro de Sodio-Potasio/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Membrana Celular/metabolismo , Drosophila melanogaster , Electroquímica , Femenino , Concentración de Iones de Hidrógeno , Potenciales de la Membrana/fisiología , Oogénesis , Canales de Potasio/fisiología , Canales de Sodio/fisiología
4.
PLoS Comput Biol ; 15(6): e1007154, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31226124

RESUMEN

Neurons utilize bursts of action potentials as an efficient and reliable way to encode information. It is likely that the intrinsic membrane properties of neurons involved in burst generation may also participate in preserving its temporal features. Here we examined the contribution of the persistent and resurgent components of voltage-gated Na+ currents in modulating the burst discharge in sensory neurons. Using mathematical modeling, theory and dynamic-clamp electrophysiology, we show that, distinct from the persistent Na+ component which is important for membrane resonance and burst generation, the resurgent Na+ can help stabilize burst timing features including the duration and intervals. Moreover, such a physiological role for the resurgent Na+ offered noise tolerance and preserved the regularity of burst patterns. Model analysis further predicted a negative feedback loop between the persistent and resurgent gating variables which mediate such gain in burst stability. These results highlight a novel role for the voltage-gated resurgent Na+ component in moderating the entropy of burst-encoded neural information.


Asunto(s)
Modelos Neurológicos , Neuronas/fisiología , Canales de Sodio/fisiología , Potenciales de Acción/fisiología , Animales , Biología Computacional , Retroalimentación Fisiológica , Ratones
5.
PLoS Comput Biol ; 15(3): e1006476, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30830905

RESUMEN

Coincidence detector neurons transmit timing information by responding preferentially to concurrent synaptic inputs. Principal cells of the medial superior olive (MSO) in the mammalian auditory brainstem are superb coincidence detectors. They encode sound source location with high temporal precision, distinguishing submillisecond timing differences among inputs. We investigate computationally how dynamic coupling between the input region (soma and dendrite) and the spike-generating output region (axon and axon initial segment) can enhance coincidence detection in MSO neurons. To do this, we formulate a two-compartment neuron model and characterize extensively coincidence detection sensitivity throughout a parameter space of coupling configurations. We focus on the interaction between coupling configuration and two currents that provide dynamic, voltage-gated, negative feedback in subthreshold voltage range: sodium current with rapid inactivation and low-threshold potassium current, IKLT. These currents reduce synaptic summation and can prevent spike generation unless inputs arrive with near simultaneity. We show that strong soma-to-axon coupling promotes the negative feedback effects of sodium inactivation and is, therefore, advantageous for coincidence detection. Furthermore, the feedforward combination of strong soma-to-axon coupling and weak axon-to-soma coupling enables spikes to be generated efficiently (few sodium channels needed) and with rapid recovery that enhances high-frequency coincidence detection. These observations detail the functional benefit of the strongly feedforward configuration that has been observed in physiological studies of MSO neurons. We find that IKLT further enhances coincidence detection sensitivity, but with effects that depend on coupling configuration. For instance, in models with weak soma-to-axon and weak axon-to-soma coupling, IKLT in the axon enhances coincidence detection more effectively than IKLT in the soma. By using a minimal model of soma-to-axon coupling, we connect structure, dynamics, and computation. Although we consider the particular case of MSO coincidence detectors, our method for creating and exploring a parameter space of two-compartment models can be applied to other neurons.


Asunto(s)
Axones , Neuronas/citología , Potenciales de Acción , Animales , Activación del Canal Iónico , Neuronas/fisiología , Canales de Sodio/fisiología , Complejo Olivar Superior/fisiología , Sinapsis/fisiología
6.
Neuroscience ; 404: 371-386, 2019 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-30703508

RESUMEN

Transcranial random noise electrical stimulation (tRNS) of the human brain is a non-invasive technique that can be employed to increase the excitability of the cerebral cortex; however, the physiological mechanisms remain unclear. Here we report for the first time the effects of short-term (250 ms) random noise electrical stimulation (RNS) on in-vitro acutely-isolated brain pyramidal neurons from the somatosensory and auditory cerebral cortex. We analyzed the correlation between the peak amplitude of the Na+ current and its latency for different levels of RNS. We found three groups of neurons. The first group exhibited a positive correlation, the second, a negative correlation, and the third group of neurons did not exhibit correlation. In the first group, both the peak amplitude of a TTX-sensitive Na+ current and its inverse of latency followed similar inverted U-like functions relative to the electrical RNS level. In this group, the RNS levels in which the maximal values of the inverted U-like functions occurred were the same. In the second group, the maximal values of the inverted U-like functions occurred at different levels. In the third group, only the peak amplitude of the Na+ current exhibited a clear inverted U-like function, but the inverse of the latency versus the electrical RNS, did not exhibit a clear inverted U-like function. A Hodgkin-Huxley neuron model reproduces our experimental results and shows that the observed behavior in the Na+ current could be due to the impact of RNS on the kinetics of activation and inactivation of the Na+ channels.


Asunto(s)
Corteza Cerebral/citología , Corteza Cerebral/fisiología , Ruido , Células Piramidales/fisiología , Animales , Estimulación Eléctrica/métodos , Distribución Aleatoria , Ratas , Ratas Wistar , Canales de Sodio/fisiología , Factores de Tiempo
7.
J Neurosci ; 39(4): 584-595, 2019 01 23.
Artículo en Inglés | MEDLINE | ID: mdl-30674614

RESUMEN

In the mammalian olfactory bulb, the inhibitory axonless granule cells (GCs) feature reciprocal synapses that interconnect them with the principal neurons of the bulb, mitral, and tufted cells. These synapses are located within large excitable spines that can generate local action potentials (APs) upon synaptic input ("spine spike"). Moreover, GCs can fire global APs that propagate throughout the dendrite. Strikingly, local postsynaptic Ca2+ entry summates mostly linearly with Ca2+ entry due to coincident global APs generated by glomerular stimulation, although some underlying conductances should be inactivated. We investigated this phenomenon by constructing a compartmental GC model to simulate the pairing of local and global signals as a function of their temporal separation Δt. These simulations yield strongly sublinear summation of spine Ca2+ entry for the case of perfect coincidence Δt = 0 ms. Summation efficiency (SE) sharply rises for both positive and negative Δt. The SE reduction for coincident signals depends on the presence of voltage-gated Na+ channels in the spine head, while NMDARs are not essential. We experimentally validated the simulated SE in slices of juvenile rat brain (both sexes) by pairing two-photon uncaging of glutamate at spines and APs evoked by somatic current injection at various intervals Δt while imaging spine Ca2+ signals. Finally, the latencies of synaptically evoked global APs and EPSPs were found to correspond to Δt ≈ 10 ms, explaining the observed approximately linear summation of synaptic local and global signals. Our results provide additional evidence for the existence of the GC spine spike.SIGNIFICANCE STATEMENT Here we investigate the interaction of local synaptic inputs and global activation of a neuron by a backpropagating action potential within a dendritic spine with respect to local Ca2+ signaling. Our system of interest, the reciprocal spine of the olfactory bulb granule cell, is known to feature a special processing mode, namely, a synaptically triggered action potential that is restricted to the spine head. Therefore, coincidence detection of local and global signals follows different rules than in more conventional synapses. We unravel these rules using both simulations and experiments and find that signals coincident within ≈±7 ms around 0 ms result in sublinear summation of Ca2+ entry because of synaptic activation of voltage-gated Na+ channels within the spine.


Asunto(s)
Neuronas/fisiología , Bulbo Olfatorio/citología , Potenciales de Acción/fisiología , Algoritmos , Animales , Señalización del Calcio/fisiología , Simulación por Computador , Dendritas/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Masculino , Modelos Neurológicos , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Canales de Sodio/fisiología
8.
J Ethnopharmacol ; 233: 56-63, 2019 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-30599222

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Strychnos nux-vomica L. (Loganiaceae) is grown extensively in South Asian. The dried seed of this plant, nux vomica, has been clinically used in Chinese medicine for relieving rheumatic pain, reducing swelling and treating cancer. Brucine, the second abundant alkaloid constituent of nux vomica, shows excellent clinical therapeutic effect, especially in relieving pain, but mechanism of brucine in relieving pain is still unclear. AIM OF THE STUDY: Explore the analgesic effect of brucine, reveal the molecular mechanism of brucine analgesia. MATERIALS AND METHODS: Antinociceptive effects of brucine were assessed in acute and chronic pain mice model. Electrophysiological experiments were used to evaluate the effects of brucine on neuronal activity and sodium channel function. RESULTS: In acute pain models, brucine significantly inhibits response induced by nociceptive heat and mechanical stimulation. Furthermore, thermal hypersensitivity and mechanical allodynia were also alleviated by brucine treatment in a chronic constriction injury (CCI) mouse model. Sodium channel plays a crucial role in neuropathic pain. Electrophysiological results show that brucine inhibits the excitability of DRG neurons directly, the number of action potential (AP) was significantly reduced after brucine treatment, and this kind of inhibition is due to brucine inhibits both tetrodotoxin-sensitive (TTXs) and tetrodotoxin-resistant (TTXr) sodium channel. CONCLUSIONS: Taken together, brucine is a novel drug candidate in treating acute and chronic pain diseases, which might be attributed to inhibition the excitability of sodium channel directly.


Asunto(s)
Analgésicos/farmacología , Analgésicos/uso terapéutico , Neuralgia/tratamiento farmacológico , Canales de Sodio/fisiología , Estricnina/análogos & derivados , Potenciales de Acción/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Células Cultivadas , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiología , Masculino , Ratones Endogámicos C57BL , Neuralgia/fisiopatología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Estricnina/farmacología , Estricnina/uso terapéutico
9.
Biochim Biophys Acta Biomembr ; 1861(1): 100-109, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30463693

RESUMEN

A kinetic model accounting for all salient features of the Na+ channel of the squid giant axon is provided. The model furnishes explanations for the Cole-Moore-like effect, the rising phase of the ON gating current and the slow 'intermediate component' of its decaying phase, as well as the gating charge immobilization. Experimental ON ionic currents are semi-quantitatively simulated by the use of only three free parameters, upon assuming that the Na+ channel opening proceeds along with the stepwise aggregation of its four domains, while they are moving their gating charge outward under depolarizing conditions. The inactivation phase of the ON ionic current is interpreted by a progressive electrostatic attraction between the positively charged 'hinged lid' containing the hydrophobic IFM triad and its receptor inside the channel pore, as the stepwise outward movement of the S4 segments of the Na+ channel progressively increases the negative charge attracting the triad to its receptor. The Na+ channel closing is assumed to proceed by repolarization-induced disaggregation of its domains, accompanied by inward movement of their gating charge. The phenomenon of 'gating charge immobilization' can be explained by assuming that gradual structural changes of the receptor over the time course of depolarization strengthen the interaction between the IFM triad and its receptor, causing a slow release of the gating charge during the subsequent repolarization.


Asunto(s)
Axones/fisiología , Canales de Sodio/fisiología , Animales , Cristalografía por Rayos X , Decapodiformes , Activación del Canal Iónico/fisiología , Iones , Cinética , Potenciales de la Membrana/fisiología , Modelos Biológicos , Sodio , Electricidad Estática , Relación Estructura-Actividad
10.
Eur J Pharmacol ; 844: 241-252, 2019 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-30571955

RESUMEN

A series of amino-2-cyclohexyl ester derivatives were studied for their ion channel blocking and antiarrhythmic actions in the rat and a structure-activity analysis was conducted. The compounds are similar in chemical structure except for ionizable amine groups (pK values 6.1-8.9) and the positional arrangements of aromatic naphthyl moieties. Ventricular arrhythmias were produced in rats by coronary-artery occlusion or electrical stimulation. The electrophysiological effects of these compounds on rat heart sodium channels (Nav1.5) expressed in Xenopus laevis oocytes and transient outward potassium currents (Kv4.3) from isolated rat ventricular myocytes were examined. The compounds reduced the incidence of ischemia-related arrhythmias and increased current threshold for induction of ventricular fibrillo-flutter (VFt) dose-dependently. As pK increased compounds showed a diminished effectiveness against ischemia-induced arrhythmias, and were less selective for ischemia- versus electrically-induced arrhythmias. Where tested, compounds produced a concentration-dependent tonic block of Nav1.5 channels. An increased potency for inhibition of Nav1.5 occurred when the external pH (pHo) was reduced to 6.5. Some compounds inhibited Kv4.3 in a pH-independent manner. Overall, the differences in antiarrhythmic and ion channel blocking properties in this series of compounds can be explained by differences in chemical structure. Antiarrhythmic activity for the amino-2-cyclohexyl ester derivatives is likely a function of mixed ion channel blockade in ischemic myocardium. These studies show that drug inhibition of Nav1.5 occurred at lower concentrations than Kv4.3 and was more sensitive to changes in the ionizable amine groups rather than on positional arrangements of the naphthyl constituents. These results offer insight into antiarrhythmic mechanisms of drug-ion channel interactions.


Asunto(s)
Antiarrítmicos/uso terapéutico , Arritmias Cardíacas/tratamiento farmacológico , Bloqueadores de los Canales de Potasio/uso terapéutico , Bloqueadores de los Canales de Sodio/uso terapéutico , Animales , Antiarrítmicos/química , Antiarrítmicos/farmacología , Ésteres/química , Ésteres/farmacología , Ésteres/uso terapéutico , Corazón/efectos de los fármacos , Corazón/fisiología , Masculino , Isquemia Miocárdica/complicaciones , Oocitos/fisiología , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Ratas Sprague-Dawley , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/fisiología , Relación Estructura-Actividad , Xenopus laevis
11.
Biomed Pharmacother ; 111: 427-435, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30594781

RESUMEN

Decades of focus on selective ion channel blockade has been dismissed as an effective approach to antiarrhythmic drug development. In that context many older antiarrhythmic drugs lacking ion channel selectivity may serve as tools to explore mixed ion channel blockade producing antiarrhythmic activity. This study investigated the non-clinical electrophysiological and antiarrhythmic actions of bisaramil and penticainide using in vitro and in vivo methods. In isolated cardiac myocytes both drugs directly block sodium currents with IC50 values of 13µM (bisaramil) and 60µM (penticainide). Both drugs reduced heart rate but prolonged the P-R, QRS and Q-T intervals of the ECG (due to sodium and potassium channel blockade) in intact rats. They reduced cardiac conduction velocity in isolated rat hearts, increased the threshold currents for capture and fibrillation (indices of sodium channel blockade) and reduced the maximum following frequency as well as prolonged the effective refractory period (indices of potassium channel blockade) of electrically stimulated rat hearts. Both drugs reduced ventricular arrhythmias and eliminated mortality due to VF in ischemic rat hearts. The index of cardiac electrophysiological balance (iCEB) did not change significantly over the dose range evaluated; however, different drug effects resulted when changes in BP and HR were considered. While bisaramil is a more potent sodium channel blocker compared to penticainide, both produce a spectrum of activity against ventricular arrhythmias due to mixed cardiac ion channel blockade. Antiarrhythmic drugs exhibiting mixed ion channel blockade may serve as tools for development of safer mixed ion channel blocking antiarrhythmic drugs.


Asunto(s)
Antiarrítmicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Clorobencenos/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Propilaminas/farmacología , Piridinas/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Animales , Antiarrítmicos/química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Células Cultivadas , Clorobencenos/química , Relación Dosis-Respuesta a Droga , Frecuencia Cardíaca/fisiología , Masculino , Técnicas de Cultivo de Órganos , Bloqueadores de los Canales de Potasio/química , Canales de Potasio/fisiología , Propilaminas/química , Piridinas/química , Ratas , Ratas Sprague-Dawley , Bloqueadores de los Canales de Sodio/química , Canales de Sodio/fisiología
12.
J Neurosci Methods ; 309: 1-5, 2018 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-30107209

RESUMEN

BACKGROUND: Peripheral nerves carry afferent and efferent signals between the central nervous system and the periphery of the body. When nerves are strained above physiological levels, conduction blocks occur, resulting in debilitating loss of motor and sensory function. Understanding the effects of strain on nerve function requires knowledge of the multi-scale mechanical behaviour of the tissue, and how this is transferred to the cellular environment. NEW METHOD: The aim of this work was to establish a technique to measure the partitioning of strain between tissue and axons in axially loaded peripheral nerves. This was achieved by staining extracellular domains of sodium channels clustered at nodes of Ranvier, without altering tissue mechanical properties by fixation or permeabilisation. RESULTS: Stained nerves were imaged by multi-photon microscopy during in situ tensile straining, and digital image correlation was used to measure axonal strain with increasing tissue strain. Strain was partitioned between tissue and axon scales by an average factor of 0.55. COMPARISONS WITH EXISTING METHODS: This technique allows non-invasive probing of cell-level strain within the physiological tissue environment. CONCLUSIONS: This technique can help understand the mechanisms behind the onset of conduction blocks in injured peripheral nerves, as well as to evaluate changes in multi-scale mechanical properties in diseased nerves.


Asunto(s)
Axones/fisiología , Nódulos de Ranvier/fisiología , Canales de Sodio/fisiología , Animales , Masculino , Imagen Óptica/métodos , Estimulación Física , Ratas Sprague-Dawley , Nervio Ciático/citología , Nervio Ciático/metabolismo
13.
Toxins (Basel) ; 10(9)2018 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-30134593

RESUMEN

Spider venoms are complex mixtures of biologically active components with potentially interesting applications for drug discovery or for agricultural purposes. The spider Phoneutria nigriventer is responsible for a number of envenomations with sometimes severe clinical manifestations in humans. A more efficient treatment requires a comprehensive knowledge of the venom composition and of the action mechanism of the constituting components. PnTx2-1 (also called δ-ctenitoxin-Pn1a) is a 53-amino-acid-residue peptide isolated from the venom fraction PhTx2. Although PnTx2-1 is classified as a neurotoxin, its molecular target has remained unknown. This study describes the electrophysiological characterization of PnTx2-1 as a modulator of voltage-gated sodium channels. PnTx2-1 is investigated for its activity on seven mammalian NaV-channel isoforms, one insect NaV channel and one arachnid NaV channel. Furthermore, comparison of the activity of both PnTx2-1 and PnTx2-6 on NaV1.5 channels reveals that this family of Phoneutria toxins modulates the cardiac NaV channel in a bifunctional manner, resulting in an alteration of the inactivation process and a reduction of the sodium peak current.


Asunto(s)
Activación del Canal Iónico/efectos de los fármacos , Neuropéptidos/toxicidad , Neurotoxinas/toxicidad , Canales de Sodio/fisiología , Venenos de Araña/toxicidad , Animales , Femenino , Insectos , Masculino , Oocitos , Isoformas de Proteínas/fisiología , Arañas , Xenopus laevis
14.
PLoS Comput Biol ; 14(7): e1006293, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-30020934

RESUMEN

Bladder small DRG neurons, which are putative nociceptors pivotal to urinary bladder function, express more than a dozen different ionic membrane mechanisms: ion channels, pumps and exchangers. Small-conductance Ca2+-activated K+ (SKCa) channels which were earlier thought to be gated solely by intracellular Ca2+ concentration ([Ca]i) have recently been shown to exhibit inward rectification with respect to membrane potential. The effect of SKCa inward rectification on the excitability of these neurons is unknown. Furthermore, studies on the role of KCa channels in repetitive firing and their contributions to different types of afterhyperpolarization (AHP) in these neurons are lacking. In order to study these phenomena, we first constructed and validated a biophysically detailed single compartment model of bladder small DRG neuron soma constrained by physiological data. The model includes twenty-two major known membrane mechanisms along with intracellular Ca2+ dynamics comprising Ca2+ diffusion, cytoplasmic buffering, and endoplasmic reticulum (ER) and mitochondrial mechanisms. Using modelling studies, we show that inward rectification of SKCa is an important parameter regulating neuronal repetitive firing and that its absence reduces action potential (AP) firing frequency. We also show that SKCa is more potent in reducing AP spiking than the large-conductance KCa channel (BKCa) in these neurons. Moreover, BKCa was found to contribute to the fast AHP (fAHP) and SKCa to the medium-duration (mAHP) and slow AHP (sAHP). We also report that the slow inactivating A-type K+ channel (slow KA) current in these neurons is composed of 2 components: an initial fast inactivating (time constant ∼ 25-100 ms) and a slow inactivating (time constant ∼ 200-800 ms) current. We discuss the implications of our findings, and how our detailed model can help further our understanding of the role of C-fibre afferents in the physiology of urinary bladder as well as in certain disorders.


Asunto(s)
Fenómenos Biofísicos , Simulación por Computador , Ganglios Espinales/citología , Neuronas/fisiología , Vejiga Urinaria/inervación , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Colorantes/metabolismo , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Potenciales de la Membrana/fisiología , Mitocondrias/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Canales de Potasio Calcio-Activados/fisiología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Canales de Sodio/fisiología
15.
J Neurosci ; 38(35): 7667-7682, 2018 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-30012693

RESUMEN

Spontaneous network activity (SNA) emerges in the spinal cord (SC) before the formation of peripheral sensory inputs and central descending inputs. SNA is characterized by recurrent giant depolarizing potentials (GDPs). Because GDPs in motoneurons (MNs) are mainly evoked by prolonged release of GABA, they likely necessitate sustained firing of interneurons. To address this issue we analyzed, as a model, embryonic Renshaw cell (V1R) activity at the onset of SNA (E12.5) in the embryonic mouse SC (both sexes). V1R are one of the interneurons known to contact MNs, which are generated early in the embryonic SC. Here, we show that V1R already produce GABA in E12.5 embryo, and that V1R make synaptic-like contacts with MNs and have putative extrasynaptic release sites, while paracrine release of GABA occurs at this developmental stage. In addition, we discovered that V1R are spontaneously active during SNA and can already generate several intrinsic activity patterns including repetitive-spiking and sodium-dependent plateau potential that rely on the presence of persistent sodium currents (INap). This is the first demonstration that INap is present in the embryonic SC and that this current can control intrinsic activation properties of newborn interneurons in the SC of mammalian embryos. Finally, we found that 5 µm riluzole, which is known to block INaP, altered SNA by reducing episode duration and increasing inter-episode interval. Because SNA is essential for neuronal maturation, axon pathfinding, and synaptogenesis, the presence of INaP in embryonic SC neurons may play a role in the early development of mammalian locomotor networks.SIGNIFICANCE STATEMENT The developing spinal cord (SC) exhibits spontaneous network activity (SNA) involved in the building of nascent locomotor circuits in the embryo. Many studies suggest that SNA depends on the rhythmic release of GABA, yet intracellular recordings of GABAergic neurons have never been performed at the onset of SNA in the SC. We first discovered that embryonic Renshaw cells (V1R) are GABAergic at E12.5 and spontaneously active during SNA. We uncover a new role for persistent sodium currents (INaP) in driving plateau potential in V1R and in SNA patterning in the embryonic SC. Our study thus sheds light on a role for INaP in the excitability of V1R and the developing SC.


Asunto(s)
Neuronas GABAérgicas/fisiología , Red Nerviosa/fisiología , Células de Renshaw/fisiología , Canales de Sodio/fisiología , Sodio/fisiología , Médula Espinal/embriología , Potenciales de Acción , Animales , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Técnicas de Sustitución del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/citología , Comunicación Paracrina , Técnicas de Placa-Clamp , Riluzol/farmacología , Médula Espinal/citología , Sinapsis/fisiología
16.
Biosens Bioelectron ; 117: 354-365, 2018 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-29940523

RESUMEN

Cardiac issues are always one of major health problems that attract wide attention by the public. It is urgent to explore a preclinical strategy to efficiently prevent the life-threatening arrhythmias by precisely assessing the cardiac excitation-contraction behavior. Conventional label-free asynchronous strategies are difficult to synchronously record and precisely match the excitation and contraction signals in vitro, while label-based strategies generally present pharmacological adverse effects and phototoxicity that significantly interfere the natural excitation and contraction signals. Both types of strategies preclude to exactly understand how cardiac excitation-contraction coupling changes in quantitative and coherent detail when dysfunctions occur. Here, we show a label-free synchronized electromechanical integration detection strategy that can synchronously monitor electrical and mechanical signals of cardiomyocytes over a long period of time by an integrated microelectrode-interdigitated electrode (ME-IDE). ME-IDE can detect subtle changes in electromechanical integration signals induced by drugs that target excitation-contraction coupling. Moreover, electromechanical integration delay is explored to specifically recognize the sodium channel inhibition. Furthermore, biomimetic electronic pacemaker function provides an alternative way to efficiently assess the drug-induced arrhythmia using refractory period of cardiomyocytes.


Asunto(s)
Arritmias Cardíacas/prevención & control , Técnicas Biosensibles/instrumentación , Microelectrodos , Miocitos Cardíacos/fisiología , Arritmias Cardíacas/diagnóstico , Humanos , Contracción Miocárdica , Canales de Sodio/fisiología
17.
Epilepsia ; 59(8): 1492-1506, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29953587

RESUMEN

OBJECTIVE: Pharmacoresistance is a problem affecting ∼30% of chronic epilepsy patients. An understanding of the mechanisms of pharmacoresistance requires a precise understanding of how antiepileptic drugs interact with their targets in control and epileptic tissue. Although the effects of (S)-licarbazepine (S-Lic) on sodium channel fast inactivation are well understood and have revealed maintained activity in epileptic tissue, it is not known how slow inactivation processes are affected by S-Lic in epilepsy. METHODS: We have used voltage clamp recordings in isolated dentate granule cells (DGCs) and cortical pyramidal neurons of control versus chronically epileptic rats (pilocarpine model of epilepsy) and in DGCs isolated from hippocampal specimens from temporal lobe epilepsy patients to examine S-Lic effects on sodium channel slow inactivation. RESULTS: S-Lic effects on entry into and recovery from slow inactivation were negligible, even at high concentrations of S-Lic (300 µmol/L). Much more pronounced S-Lic effects were observed on the voltage dependence of slow inactivation, with significant effects at 100 µmol/L S-Lic in DGCs from control and epileptic rats or temporal lobe epilepsy patients. For none of these effects of S-Lic could we observe significant differences either between sham-control and epileptic rats, or between human DGCs and the two animal groups. S-Lic was similarly effective in cortical pyramidal neurons from sham-control and epileptic rats. Finally, we show in expression systems that S-Lic effects on slow inactivation voltage dependence are only observed in Nav 1.2 and Nav 1.6 subunits, but not in Nav 1.1 and Nav 1.3 subunits. SIGNIFICANCE: From these data, we conclude that a major mechanism of action of S-Lic is an effect on slow inactivation, primarily through effects on slow inactivation voltage dependence of Nav 1.2 and Nav 1.6 channels. Second, we demonstrate that this main effect of S-Lic is maintained in both experimental and human epilepsy and applies to principal neurons of different brain areas.


Asunto(s)
Anticonvulsivantes/farmacología , Giro Dentado/patología , Dibenzazepinas/farmacología , Epilepsia/patología , Neuronas/efectos de los fármacos , Canales de Sodio/fisiología , Adulto , Análisis de Varianza , Animales , Anticonvulsivantes/uso terapéutico , Biofisica , Células Cultivadas , Dibenzazepinas/uso terapéutico , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Epilepsia/inducido químicamente , Femenino , Humanos , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Persona de Mediana Edad , Técnicas de Placa-Clamp , Pilocarpina/toxicidad , Ratas , Ratas Wistar
18.
Eur J Clin Invest ; 48 Suppl 2: e12964, 2018 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-29873837

RESUMEN

The recruitment of neutrophils to sites of inflammation, their battle against invading microorganisms through phagocytosis and the release of antimicrobial agents is a highly coordinated and tightly regulated process that involves the interplay of many different receptors, ion channels and signalling pathways. Changes in intracellular calcium levels, caused by cytosolic Ca2+ store depletion and the influx of extracellular Ca2+ via ion channels, play a critical role in synchronizing neutrophil activation and function. In this review, we provide an overview of how Ca2+ signalling is initiated in neutrophils and how changes in intracellular Ca2+ levels modulate neutrophil function.


Asunto(s)
Señalización del Calcio/fisiología , Infiltración Neutrófila/fisiología , Animales , Canales de Calcio/fisiología , Humanos , Canales Iónicos/fisiología , Ratones , Neutrófilos/fisiología , Canales de Potasio/fisiología , Receptores Purinérgicos P2X/fisiología , Canales de Sodio/fisiología , Canales Receptores Transitorios de Potencial/fisiología
19.
eNeuro ; 5(3)2018.
Artículo en Inglés | MEDLINE | ID: mdl-29876522

RESUMEN

Action potentials (APs) are electric phenomena that are recorded both intracellularly and extracellularly. APs are usually initiated in the short segment of the axon called the axon initial segment (AIS). It was recently proposed that at the onset of an AP the soma and the AIS form a dipole. We study the extracellular signature [the extracellular AP (EAP)] generated by such a dipole. First, we demonstrate the formation of the dipole and its extracellular signature in detailed morphological models of a reconstructed pyramidal neuron. Then, we study the EAP waveform and its spatial dependence in models with axonal AP initiation and contrast it with the EAP obtained in models with somatic AP initiation. We show that in the models with axonal AP initiation the dipole forms between somatodendritic compartments and the AIS, and not between soma and dendrites as in the classical models. The soma-dendrites dipole is present only in models with somatic AP initiation. Our study has consequences for interpreting extracellular recordings of single-neuron activity and determining electrophysiological neuron types, but also for better understanding the origins of the high-frequency macroscopic extracellular potentials recorded in the brain.


Asunto(s)
Potenciales de Acción , Segmento Inicial del Axón/fisiología , Modelos Neurológicos , Células Piramidales/fisiología , Animales , Fenómenos Electrofisiológicos , Células Piramidales/citología , Ratas , Canales de Sodio/fisiología
20.
Neuron ; 98(1): 156-165.e6, 2018 04 04.
Artículo en Inglés | MEDLINE | ID: mdl-29621485

RESUMEN

Fast-spiking, parvalbumin-expressing GABAergic interneurons (PV+-BCs) express a complex machinery of rapid signaling mechanisms, including specialized voltage-gated ion channels to generate brief action potentials (APs). However, short APs are associated with overlapping Na+ and K+ fluxes and are therefore energetically expensive. How the potentially vicious combination of high AP frequency and inefficient spike generation can be reconciled with limited energy supply is presently unclear. To address this question, we performed direct recordings from the PV+-BC axon, the subcellular structure where active conductances for AP initiation and propagation are located. Surprisingly, the energy required for the AP was, on average, only ∼1.6 times the theoretical minimum. High energy efficiency emerged from the combination of fast inactivation of Na+ channels and delayed activation of Kv3-type K+ channels, which minimized ion flux overlap during APs. Thus, the complementary tuning of axonal Na+ and K+ channel gating optimizes both fast signaling properties and metabolic efficiency.


Asunto(s)
Potenciales de Acción/fisiología , Axones/fisiología , Neuronas GABAérgicas/fisiología , Interneuronas/fisiología , Canales de Potasio Shaw/fisiología , Canales de Sodio/fisiología , Animales , Metabolismo Energético/fisiología , Femenino , Hipocampo/fisiología , Activación del Canal Iónico/fisiología , Masculino , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar
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