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1.
Life Sci ; 244: 117333, 2020 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-31962132

RESUMEN

AIMS: Detect the antiarrhythmic effect of crotonoside (Cro). MAIN METHODS: We used whole-cell patch-clamp techniques to detect the effects of Cro on action potentials (APs) and transmembrane ion currents in isolated rabbit left ventricular myocytes. We also verified the effect of Cro on ventricular arrhythmias caused by aconitine in vivo. KEY FINDINGS: Cro reduced the maximum depolarization velocity (Vmax) of APs and shortened the action potential duration (APD) in a concentration-dependent manner, but it had no significant effect on the resting membrane potential (RMP) or action potential amplitude (APA). It also inhibited the peak sodium current (INa) and L-type calcium current (ICaL) in a concentration-dependent manner with half-maximal inhibitory concentrations (IC50) of 192 µmol/L and 159 µmol/L, respectively. However, Cro had no significant effects on the inward rectifier potassium current (IK1) or rapidly activating delayed rectifier potassium current (IKr). Sea anemone toxin II (ATX II) increased the late sodium current (INaL), but Cro abolished this effect. Moreover, Cro significantly abolished ATX II-induced early afterdepolarizations (EADs) and high extracellular Ca2+ concentration (3.6 mmol/L)-induced delayed afterdepolarizations (DADs). We also verified that Cro effectively delayed the onset time and reduced the incidence of ventricular arrhythmias caused by aconitine in vivo. SIGNIFICANCE: These results revealed that Cro effectively inhibits INa, INaL, and ICaL in ventricular myocytes. Cro has antiarrhythmic potential and thus deserves further study.


Asunto(s)
Guanina/farmacología , Miocitos Cardíacos/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Antiarrítmicos/metabolismo , Antiarrítmicos/farmacología , Arritmias Cardíacas/fisiopatología , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , China , Femenino , Guanina/metabolismo , Ventrículos Cardíacos/metabolismo , Técnicas de Placa-Clamp/métodos , Conejos , Sodio/metabolismo , Canales de Sodio/efectos de los fármacos
2.
Nat Protoc ; 14(7): 2015-2035, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31168087

RESUMEN

Measurements of a single entity underpin knowledge of the heterogeneity and stochastics in the behavior of molecules, nanoparticles, and cells. Electrochemistry provides a direct and fast method to analyze single entities as it probes electron/charge-transfer processes. However, a highly reproducible electrochemical-sensing nanointerface is often hard to fabricate because of a lack of control of the fabrication processes at the nanoscale. In comparison with conventional micro/nanoelectrodes with a metal wire inside, we present a general and easily implemented protocol that describes how to fabricate and use a wireless nanopore electrode (WNE). Nanoscale metal deposition occurs at the tip of the nanopipette, providing an electroactive sensing interface. The WNEs utilize a dynamic ionic flow instead of a metal wire to sense the interfacial redox process. WNEs provide a highly controllable interface with a 30- to 200-nm diameter. This protocol presents the construction and characterization of two types of WNEs-the open-type WNE and closed-type WNE-which can be used to achieve reproducible electrochemical measurements of single entities. Combined with the related signal amplification mechanisms, we also describe how WNEs can be used to detect single redox molecules/ions, analyze the metabolism of single cells, and discriminate single nanoparticles in a mixture. This protocol is broadly applicable to studies of living cells, nanomaterials, and sensors at the single-entity level. The total time required to complete the protocol is ~10-18 h. Each WNE costs ~$1-$3.


Asunto(s)
Técnicas Electroquímicas/instrumentación , Electrodos , Nanoporos , Nanotecnología/métodos , Técnicas Electroquímicas/métodos , Diseño de Equipo , Humanos , Células MCF-7 , Nanopartículas/análisis , Oxidación-Reducción , Técnicas de Placa-Clamp/instrumentación , Técnicas de Placa-Clamp/métodos , Análisis de la Célula Individual/instrumentación , Análisis de la Célula Individual/métodos , Tecnología Inalámbrica
3.
Psychopharmacology (Berl) ; 236(11): 3291-3300, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31201475

RESUMEN

It has been shown that dystrobrevin-binding protein 1 gene that encodes the protein dysbindin-1 is associated with risk for cognitive deficits, and studies have shown decreases in glutamate and correlated decreases in dysbindin-1 protein in the prefrontal cortex (PFC) and hippocampus of post-mortem tissue from schizophrenia patients. The PFC and the hippocampus have been shown to play a fundamental role in cognition, and studies in dysbindin-1 null mice have shown alterations in NMDAR located in pyramidal neurons as well as perturbation in LTP and cognitive deficits. The balance between excitatory and inhibitory transmission is crucial for normal cognitive functions; however, there is a dearth of information regarding the effects of loss of dysbindin-1 in GABAergic transmission. Using in vitro whole-cell clamp recordings, Western blots, and immunohistochemistry, we report here that dysbindin-1-deficient mice exhibit a significant decrease in the frequency of sIPSCs and in the amplitude of mIPSCs and significant decreases in PV staining and protein level. These results suggest that loss of dysbindin-1 affects GABAergic transmission at pre- and postsynaptic level and decreases parvalbumin markers.


Asunto(s)
Disbindina/deficiencia , Neuronas GABAérgicas/metabolismo , Corteza Prefrontal/metabolismo , Transmisión Sináptica/fisiología , Animales , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Placa-Clamp/métodos , Células Piramidales/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo
4.
Cell Biol Int ; 43(8): 965-975, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31141273

RESUMEN

Transient receptor potential vanilloid 6 (TRPV6) channels are key players in calcium metabolism of healthy and cancerous cells. Nevertheless, the mechanisms controlling abundance of these channels in plasma membrane of the cells to regulate Ca2+ transport is still poorly understood. In this study, we provide the first evidence that TRPV6 calcium channels and Ca 2+ influx in Jurkat T cell line are modulated by cholesterol, a main lipid component of the plasma membrane. Using patch-clamp technique, we found that activity of TRPV6 channels decreased by cholesterol sequestration with methyl-ß-cyclodextrin (MßCD). Continuous measurement of intracellular Ca2+ revealed a reduction of Ca2+ influx into Jurkat cells following cholesterol depletion. Immunofluorescence and immunoelectron microscopy analyses of MßCD-treated cells detected the lower surface expression of the TRPV6 proteins in comparison with control cells. In general, our data showed that cholesterol regulates TRPV6 channel activity and TRPV6-mediated Ca2+ influx in cells, apparently affecting the localization and density of the calcium channels in the plasma membrane of Jurkat T cells.


Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Colesterol/deficiencia , Canales Catiónicos TRPV/metabolismo , Transporte Biológico , Humanos , Células Jurkat , Técnicas de Placa-Clamp/métodos , beta-Ciclodextrinas/química
5.
Int J Mol Sci ; 20(10)2019 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-31137894

RESUMEN

In contrast to the long-standing notion that the role of individual neurons in population activity is vanishingly small, recent studies have shown that electrical activation of only a single cortical neuron can have measurable effects on global brain state, movement, and perception. Although highly important for understanding how neuronal activity in cortex is orchestrated, the cellular and network mechanisms underlying this phenomenon are unresolved. Here, we first briefly review the current state of knowledge regarding the phenomenon of single-cell induced network modulation and discuss possible underpinnings. Secondly, we show proof of principle for an experimental approach to elucidate the mechanisms of single-cell induced changes in cortical activity. The setup allows simultaneous recordings of the spiking activity of multiple neurons across all layers of the cortex using a multi-electrode array, while manipulating the activity of one individual neuron in close proximity to the array. We demonstrate that single cells can be recorded and stimulated reliably for hundreds of trials, conferring high statistical power even for expectedly small effects of single-neuron spiking on network activity. Preliminary results suggest that single-cell stimulation on average decreases the firing rate of local network units. We expect that characterization of the spatiotemporal spread of single-cell evoked activity across layers and columns will yield novel insights into intracortical processing.


Asunto(s)
Electrofisiología/métodos , Neuronas/fisiología , Corteza Somatosensorial/fisiología , Potenciales de Acción , Animales , Estudios de Factibilidad , Técnicas de Placa-Clamp/métodos , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Ratas Wistar , Análisis de la Célula Individual/métodos , Corteza Somatosensorial/citología
6.
Biol Bull ; 236(2): 108-114, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30933638

RESUMEN

Sea urchins can detect and respond to light, and many species of sea urchins are negatively phototaxic. Light detection is hypothesized to occur via photoreceptors located on sea urchin tube feet, and opsins have been detected in tube feet, spines, and the test. However, the molecular mechanisms underlying light detection are, for the most part, unknown. Individual tube feet disc cells were isolated from purple sea urchins (Strongylocentrotus purpuratus), and the electrical responses of these cells to varying levels of illumination were quantified using the patch clamp technique. No currents were observed under bright illumination, whereas under dark conditions, large, slowly activating currents were consistently observed. Two types of cells were functionally identified based on their responses to darkness. Type I cells sustained currents indefinitely in the dark, whereas Type II cell currents spontaneously decayed after several seconds. The large currents observed were composed of the summation of many smaller events that were characterized by a rapid onset and an exponentially decaying component, which may be indicative of direct vesicular release from the tube feet disc cells in response to the dark conditions.


Asunto(s)
Células Fotorreceptoras/fisiología , Strongylocentrotus purpuratus/fisiología , Animales , Oscuridad , Luz , Técnicas de Placa-Clamp/métodos , Strongylocentrotus purpuratus/citología
7.
Methods Mol Biol ; 1987: 83-97, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31028675

RESUMEN

The family of transient receptor potential (TRPs) channels contains 28 mammalian members, each a unique cellular sensor that responds to a wide variety of external and internal signals. TRP channels are expressed by most mammalian cells, where they are involved in many different physiological functions. Canonical TRP channels (TRPCs) form a family of nonselective cationic channels, although with greater selectivity for Ca2+. This family is made up of seven members (TRPC1-7), all of which contain a TRP box in the carboxyl terminal and 3-4 ankyrin repeats in the amino terminal. While these channels share some similar properties, they display diverse gating mechanisms and are involved in different signaling pathways (Gees M et al., Compr Physiol, 2012). The activation or inhibition of these channels has been studied using different approaches and techniques. Here, we characterize the activation of the TRPC5 channel expressed in a heterologous system, using calcium imaging and the patch-clamp technique in whole-cell configuration.


Asunto(s)
Calcio/análisis , Técnicas de Placa-Clamp/métodos , Canales Catiónicos TRPC/metabolismo , Fura-2/química , Células HEK293 , Humanos , Microscopía Fluorescente , Concentración Osmolar
8.
Methods Mol Biol ; 1987: 125-141, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31028678

RESUMEN

Patch-clamp recording combined with biophysical modeling and mutagenic perturbations provides an effective means to study structural functions of ion channels. The methodology has been successful for studying ligand- or voltage-gated channels and brought about much of the knowledge we know today on how ligand or voltage gates an ion channel. The approach, when applied to thermal channels, however, has faced unique challenges. For one problem, thermal channels can operate at high temperatures, and for these channels, prolonged temperature stimulation incurs excessive thermal stress to destabilize patches. For another problem, conventional temperature controls are slow and limit the attainment of high resolution data such as time-resolved activations of thermal channels. Due to these issues, thermal channels have been less accessible to biophysical studies at mechanistic levels. In this chapter we address the problems and demonstrate fast temperature controls enabling recording of time-resolved responses of thermal channels at high temperatures.


Asunto(s)
Técnicas de Placa-Clamp/métodos , Canales Receptores Transitorios de Potencial/metabolismo , Animales , Células HEK293 , Calor , Humanos , Cinética , Ratones , Nociceptores/fisiología , Ratas , Termorreceptores/fisiología
9.
Methods Mol Biol ; 1942: 131-139, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30900181

RESUMEN

Performing electrophysiological recordings from human neurons that have been differentiated in vitro, as compared to primary cultures, raises many challenges. However, patch-clamp recording from neurons derived from stem cells provides an abundance of valuable information, reliably and fast. Here, we describe a protocol that is used successfully in our lab for recording from both control and Fragile X neurons, derived in vitro from human embryonic stem cells.


Asunto(s)
Diferenciación Celular , Fenómenos Electrofisiológicos , Síndrome del Cromosoma X Frágil/fisiopatología , Células Madre Embrionarias Humanas/fisiología , Neuronas/fisiología , Técnicas de Placa-Clamp/métodos , Células Cultivadas , Células Madre Embrionarias Humanas/citología , Humanos , Neuronas/citología
10.
Methods Mol Biol ; 1952: 143-156, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30825173

RESUMEN

Divalent cations can change the actual electrical potential at the outer surface of the plasma membrane. They do so by the so-called Gouy-Chapman-Stern effect which is due to the electrical "masking" that certain ions, especially divalents, can exert onto the electrically negative charged polar heads of the membrane phospholipids.Chondroitin sulfates can chelate free calcium ions to a different extent based on the spatial arrangement of their sulfate groups and can thus alter the actual availability of screening divalent ions at the outer membrane surface.Voltage-dependent ion channels sense the actual potential difference between the two sides of the plasma membrane and are thus exquisite and extremely sensitive "devices" able to react to changes in the electrical potential across the membrane.Hence, by recording the shift in the activation curve of well-known voltage-dependent ionic channels it will be possible to study the physical effect of ECM chondroitin sulfates on membrane conductances.


Asunto(s)
Sulfatos de Condroitina/metabolismo , Matriz Extracelular/metabolismo , Activación del Canal Iónico , Canales Iónicos/metabolismo , Potenciales de la Membrana , Animales , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Técnicas de Placa-Clamp/métodos , Xenopus laevis
11.
Methods Mol Biol ; 1941: 107-135, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30707431

RESUMEN

Development of the whole-cell patch-clamp electrophysiology technique has allowed for enhanced visualization and experimentation of ionic currents in neurons of mammalian tissue with high spatial and temporal resolution. Electrophysiology has become an exceptional tool for identifying single cellular mechanisms underlying behavior. Specifically, the role of glutamatergic signaling through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors underlying behavior has been extensively studied. Here we will discuss commonly used protocols and techniques for performing whole-cell patch-clamp recordings and exploring AMPA and NMDA receptor-mediated glutamatergic responses and alterations in the context of substance abuse.


Asunto(s)
Encéfalo/metabolismo , Electrofisiología/métodos , Ácido Glutámico/metabolismo , Técnicas de Placa-Clamp/métodos , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Trastornos Relacionados con Sustancias/metabolismo , Animales , Encéfalo/citología , Trastornos Relacionados con Sustancias/patología
12.
Methods Mol Biol ; 1941: 139-154, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30707432

RESUMEN

Brain circuit assemblies comprise different cellular subpopulations that exhibit morphological, electrophysiological, and molecular diversity. Here we describe a protocol which, combined with whole-cell patch-clamp recording and morphological reconstruction, allows the transcriptomic analysis of the recorded cell. This protocol provides recipes on how to detect simultaneously the expression of 24 genes/markers at the single-cell level using polymerase chain reaction (PCR), how to design gene-specific probes, and how to validate them. This technique provides multiplexed expression data that cannot be easily obtained by other approaches such as immunological co-labeling.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Neuronas/metabolismo , Técnicas de Placa-Clamp/métodos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Análisis de la Célula Individual/métodos , Células Cultivadas , Humanos , Neuronas/citología
13.
J Vis Exp ; (143)2019 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-30735182

RESUMEN

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions under various membrane lipid compositions. Among them, the droplet interface bilayer has gained popularity; however, the large membrane size hinders the recording of low electrical background noise. We have established a contact bubble bilayer (CBB) method that combines the benefits of planar lipid bilayer and patch-clamp methods, such as the ability to vary the lipid composition and to manipulate the bilayer mechanics, respectively. Using the setup for conventional patch-clamp experiments, CBB-based experiments can be readily performed. In brief, an electrolyte solution in a glass pipette is blown into an organic solvent phase (hexadecane), and the pipette pressure is maintained to obtain a stable bubble size. The bubble is spontaneously lined with a lipid monolayer (pure lipids or mixed lipids), which is provided from liposomes in the bubbles. Next, the two monolayer-lined bubbles (~50 µm in diameter) at the tip of the glass pipettes are docked for bilayer formation. Introduction of channel-reconstituted liposomes into the bubble leads to the incorporation of channels in the bilayer, allowing for single-channel current recording with a signal-to-noise ratio comparable to that of patch-clamp recordings. CBBs with an asymmetric lipid composition are readily formed. The CBB is renewed repeatedly by blowing out the previous bubbles and forming new ones. Various chemical and physical perturbations (e.g., membrane perfusion and bilayer tension) can be imposed on the CBBs. Herein, we present the basic procedure for CBB formation.


Asunto(s)
Membrana Dobles de Lípidos/química , Técnicas de Placa-Clamp/métodos , Vidrio , Lípidos/química , Liposomas , Membranas , Canales de Potasio/metabolismo
14.
J Vis Exp ; (143)2019 01 18.
Artículo en Inglés | MEDLINE | ID: mdl-30735185

RESUMEN

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.


Asunto(s)
Neuronas/fisiología , Técnicas de Placa-Clamp/métodos , Sustancia Gelatinosa/citología , Potenciales de Acción/fisiología , Animales , Masculino , Ratas Sprague-Dawley , Transmisión Sináptica/fisiología
15.
Methods Mol Biol ; 1925: 75-86, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30674018

RESUMEN

Mitochondria accumulate significant amounts of calcium when cytosolic calcium is elevated above the resting levels of 50-100 nM during signaling events. This calcium uptake is primarily mediated by a macromolecular protein assembly called mitochondrial calcium uniporter (MCU) that resides in the mitochondrial inner membrane. In 2004, we applied patch-clamp technique for the first time to record calcium currents from the mitochondrial inner membrane and proved unequivocally that MCU is a highly selective calcium channel. This chapter describes how patch-clamp technique can be applied to record the Ca2+ uniporter currents from the mitochondrial inner membrane, isolation of mitochondria from the heart tissue, and preparation of mitoplast using French Press. We also discuss advantages of patch-clamp technique as compared to other methods of determining mitochondrial uniporter activity and important considerations in applying patch-clamp technique to such a small subcellular organelle. With small variations in the bath and pipette solution composition, the same methodology can be applied to study any other currents (e.g., H+ or Cl-) from the mitochondrial inner membrane.


Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio , Calcio/metabolismo , Mitocondrias Cardíacas/metabolismo , Membranas Mitocondriales/metabolismo , Técnicas de Placa-Clamp/métodos , Animales , Cationes Bivalentes/metabolismo , Potencial de la Membrana Mitocondrial , Ratones Endogámicos C57BL
16.
ChemMedChem ; 14(5): 570-582, 2019 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-30676691

RESUMEN

We previously reported that a lipophilic N-(4'-hydroxy-3',5'-di-tert-butylbenzyl) derivative (1) of the voltage-gated sodium channel blocker mexiletine, was a more potent sodium channel blocker in vitro and in vivo. We demonstrate that replacing the chiral methylethylene linker between the amine and di-tert-butylphenol with an achiral 1,3-propylene linker (to give (2)) maintains potency in vitro. We synthesized 25 analogues bearing the 1,3-propylene linker and found that minor structural changes resulted in pronounced changes in state dependence of blocking human NaV 1.2 and 1.6 channels by high-throughput patch-clamp analysis. Compared to mexiletine, compounds 1 and 2 are highly selective NaV 1.2 inhibitors and >500 times less potent in inhibiting NaV 1.6 channels. On the other hand, a derivative (compound 4) bearing 2,6-dimethoxy groups in place of the 2,6-dimethyl groups found in mexiletine was found to be the most potent inhibitor, but is nonselective against both channels in the tonic, frequency-dependent and inactivated states. In a kindled mouse model of refractory epilepsy, compound 2 inhibited seizures induced by 6 Hz 44 mA electrical stimulation with an IC50 value of 49.9±1.6 mg kg-1 . As established sodium channel blockers do not suppress seizures in this mouse model, this indicates that 2 could be a promising candidate for treating pharmaco-resistant epilepsy.


Asunto(s)
Bencilaminas/síntesis química , Convulsiones/tratamiento farmacológico , Bloqueadores del Canal de Sodio Activado por Voltaje/síntesis química , Canales de Sodio Activados por Voltaje/metabolismo , Animales , Bencilaminas/metabolismo , Estabilidad de Medicamentos , Estimulación Eléctrica , Humanos , Mexiletine/metabolismo , Ratones , Estructura Molecular , Técnicas de Placa-Clamp/métodos , Relación Estructura-Actividad , Bloqueadores del Canal de Sodio Activado por Voltaje/metabolismo
17.
J Vis Exp ; (143)2019 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-30663667

RESUMEN

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. Although the molecular mechanisms for the biological responses to fluid flow/shear force are not fully understood, fluid flow-mediated regulation of ion channel gating may contribute critically. Therefore, fluid flow/shear force sensitivity of ion channels has been studied using the patch-clamp technique. However, depending on the experimental protocol, the outcomes and interpretation of data can be erroneous. Here, we present experimental and theoretical evidence for fluid flow-related errors and provide methods for estimating, preventing, and correcting these errors. Changes in junction potential between the Ag/AgCl reference electrode and bathing fluid were measured with an open pipette filled with 3 M KCl. Fluid flow could then shift the liquid/metal junction potential to approximately 7 mV. Conversely, by measuring the voltage shift induced by fluid flow, we estimated the ion concentration in the unstirred boundary layer. In the static condition, the real ion concentrations adjacent to the Ag/AgCl reference electrode or ion channel inlet at the cell-membrane surface can reach as low as approximately 30% of that in the flow condition. Placing an agarose 3 M KCl bridge between the bathing fluid and reference electrode may have prevented this problem of junction potential shifting. However, the unstirred layer effect adjacent to the cell membrane surface could not be fixed in this way. Here, we provide a method for measuring real ion concentrations in the unstirred boundary layer with an open patch-clamp pipette, emphasizing the importance of using an agarose salt-bridge while studying fluid flow-induced regulation of ion currents. Therefore, this novel approach, which takes into consideration the real concentrations of ions in the unstirred boundary layer, may provide useful insight on the experimental design and data interpretation related to fluid shear stress regulation of ion channels.


Asunto(s)
Canales Iónicos/metabolismo , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp/métodos , Animales
18.
PLoS Pathog ; 15(1): e1007570, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30695069

RESUMEN

Glutamate-gated chloride channel receptors (GluClRs) mediate inhibitory neurotransmission at invertebrate synapses and are primary targets of parasites that impact drastically on agriculture and human health. Ivermectin (IVM) is a broad-spectrum pesticide that binds and potentiates GluClR activity. Resistance to IVM is a major economic and health concern, but the molecular and synaptic mechanisms of resistance are ill-defined. Here we focus on GluClRs of the agricultural endoparasite, Haemonchus contortus. We demonstrate that IVM potentiates inhibitory input by inducing a tonic current that plateaus over 15 minutes and by enhancing post-synaptic current peak amplitude and decay times. We further demonstrate that IVM greatly enhances the active durations of single receptors. These effects are greatly attenuated when endogenous IVM-insensitive subunits are incorporated into GluClRs, suggesting a mechanism of IVM resistance that does not affect glutamate sensitivity. We discovered functional groups of IVM that contribute to tuning its potency at different isoforms and show that the dominant mode of access of IVM is via the cell membrane to the receptor.


Asunto(s)
Canales de Cloruro/metabolismo , Haemonchus/efectos de los fármacos , Ivermectina/farmacología , Animales , Canales de Cloruro/antagonistas & inhibidores , Antagonistas de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/farmacología , Células HEK293 , Haemonchus/metabolismo , Humanos , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Receptores de Glutamato/metabolismo , Xenopus laevis/embriología
19.
Anat Sci Int ; 94(2): 199-208, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30600446

RESUMEN

Neurons are classified into several morphological types according to the locations of their somata and the branching patterns of their axons and dendrites. Recent studies suggest that these morphological features are related to their physiological properties, including firing characteristics, responses to neuromodulators, and wiring patterns. Therefore, rapid morphological identification of electrophysiologically recorded neurons promises to advance our understanding of neuronal circuits. One of the most common anatomical cell identification methods is neuronal reconstruction with biocytin delivered through whole-cell patch-clamp pipettes. However, conventional reconstruction methods usually take longer than 24 h and limit the throughput of electrophysiological experiments. Here, we developed a quick, simple cell reconstruction method by optimizing the tissue clearing protocol ScaleSQ. We found that adding 200 mM NaCl almost entirely prevented tissue swelling without compromising optical clearing ability. This solution, termed IsoScaleSQ, allowed us to increase the transparency of the gray matter of 500-µm-thick slices within 30 min, meaning that the total time required to reconstruct whole-cell recorded neurons was reduced to 1 h. This novel method will improve the efficacy and effectiveness of electrophysiological experiments linked to cell morphology.


Asunto(s)
Encéfalo/citología , Electrofisiología/métodos , Neuronas/citología , Neuronas/fisiología , Técnicas de Placa-Clamp/métodos , Animales , Lisina/análogos & derivados , Potenciales de la Membrana , Ratones Endogámicos ICR , Ratones Transgénicos
20.
Neuron ; 101(1): 76-90.e4, 2019 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-30472076

RESUMEN

The structure of the neocortex varies across the neocortical mantle to govern the physical size of principal neurons. What impact such anatomical variation has on the computational operations of principal neurons remains unknown. Here, we demonstrate within a functionally defined area that neocortical thickness governs the anatomical, electrophysiological, and computational properties of the principal output neurons of the neocortex. We find that neocortical thickness and the size of layer 5B pyramidal neurons changes as a gradient across the rostro-caudal axis of the rat primary visual cortex. Simultaneous somato-dendritic whole-cell recordings and compartmental modeling revealed that the electrical architecture of principal neurons was not preserved; rather, primary visual cortex site-dependent differences in intracellular resistivity accentuated a gradient of the electrical behavior of layer 5B pyramidal neurons to influence the emergence of active dendritic computations. Our findings therefore reveal an exquisite relationship between neocortical structure and neuronal computation.


Asunto(s)
Potenciales de Acción/fisiología , Dendritas/fisiología , Neocórtex/citología , Neocórtex/fisiología , Técnicas de Placa-Clamp/métodos , Células Piramidales/fisiología , Animales , Masculino , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar
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