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1.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi ; 38(4): 695-702, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34459169

RESUMO

Patch clamp is a technique that can measure weak current in the level of picoampere (pA). It has been widely used for cellular electrophysiological recording in fundamental medical researches, such as membrane potential and ion channel currents recording, etc. In order to obtain accurate measurement results, both the resistance and capacitance of the pipette are required to be compensated. Capacitance compensations are composed of slow and fast capacitance compensation. The slow compensation is determined by the lipid bilayer of cell membrane, and its magnitude usually ranges from a few picofarads (pF) to a few microfarads (µF), depending on the cell size. The fast capacitance is formed by the distributed capacitance of the glass pipette, wires and solution, mostly ranging in a few picofarads. After the pipette sucks the cells in the solution, the positions of the glass pipette and wire have been determined, and only taking once compensation for slow and fast capacitance will meet the recording requirements. However, when the study needs to deal with the temperature characteristics, it is still necessary to make a recognition on the temperature characteristic of the capacitance. We found that the time constant of fast capacitance discharge changed with increasing temperature of bath solution when we studied the photothermal effect on cell membrane by patch clamp. Based on this phenomenon, we proposed an equivalent circuit to calculate the temperature-dependent parameters. Experimental results showed that the fast capacitance increased in a positive rate of 0.04 pF/℃, while the pipette resistance decreased. The fine data analysis demonstrated that the temperature rises of bath solution determined the kinetics of the fast capacitance mainly by changing the inner solution resistance of the glass pipette. This result will provide a good reference for the fine temperature characteristic study related to cellular electrophysiology based on patch clamp technique.


Assuntos
Temperatura , Membrana Celular , Capacitância Elétrica , Potenciais da Membrana , Técnicas de Patch-Clamp
2.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 37(4): 445-448, 2021 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-34374268

RESUMO

Objective: To introduce a method of marking neurons using patch clamp technique. Methods: The brain slices of the target area was cut with a vibrating microtome. The glass microelectrode was perfused with the electrode liquid containing NeurobiotinTM Tracer, and the whole-cell patch-clamp recording was performed. After recording, the brain slices were fixed and rinsed with 4% paraformaldehyde. After stained in phosphate buffer with Streptavidin-Texas Red and Triton X-100 for at least 2 hours, the neurons can be observed under a fluorescence microscope. Results: The cell membrane voltage was clamped at -70 mV, and the neuron showed a gradually increasing membrane current after step stimulation. When recording in the current clamp mode, the step stimulus caused the neuron to depolarize to the threshold potential and then burst into action potentials. The morphology of intact neurons with clear cell body and protrusions of a neuron could be observed under a fluorescence microscope. Conclusion: This method is suitable for observing the morphological features of the recorded neuron after patch clamp experiments, which is easy to operate, and the image is intuitive and clear.


Assuntos
Encéfalo , Neurônios , Potenciais de Ação , Técnicas de Patch-Clamp
3.
Nat Commun ; 12(1): 4171, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234116

RESUMO

Here we report the pharmacologic blockade of voltage-gated sodium ion channels (NaVs) by a synthetic saxitoxin derivative affixed to a photocleavable protecting group. We demonstrate that a functionalized saxitoxin (STX-eac) enables exquisite spatiotemporal control of NaVs to interrupt action potentials in dissociated neurons and nerve fiber bundles. The photo-uncaged inhibitor (STX-ea) is a nanomolar potent, reversible binder of NaVs. We use STX-eac to reveal differential susceptibility of myelinated and unmyelinated axons in the corpus callosum to NaV-dependent alterations in action potential propagation, with unmyelinated axons preferentially showing reduced action potential fidelity under conditions of partial NaV block. These results validate STX-eac as a high precision tool for robust photocontrol of neuronal excitability and action potential generation.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Saxitoxina/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Células CHO , Células Cultivadas , Corpo Caloso/citologia , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/metabolismo , Cricetulus , Embrião de Mamíferos , Feminino , Hipocampo/citologia , Masculino , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Técnicas de Patch-Clamp , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saxitoxina/análogos & derivados , Saxitoxina/efeitos da radiação , Análise de Célula Única , Análise Espaço-Temporal , Raios Ultravioleta , Bloqueadores do Canal de Sódio Disparado por Voltagem/efeitos da radiação
4.
Biomed Pharmacother ; 139: 111615, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243598

RESUMO

BACKGROUND: Severe acidosis deteriorates cardiac injury. Rat coronary arteries (RCAs) are unusually hypercontractive to extracellular (o) acidosis (EA). TMEM16A-encoded anoctamin 1 (ANO1), a Ca2+-activated chloride channel (CaCC), plays an important role in regulating coronary arterial tension. PURPOSE: We tested the possibility that the activation of CaCCs in the arterial smooth muscle cell (ASMC) contributes to EA-induced RCA constriction. METHODS: ANO1 expression was detected with immunofluorescence staining and Western blot. TMEM16A mRNA was assessed with quantitative Real-Time PCR. Cl- currents and membrane potentials were quantified with a patch clamp. The vascular tension was recorded with a myograph. Intracellular (i) level of Cl- and Ca2+ was measured with fluorescent molecular probes. RESULTS: ANO1 was expressed in all tested arterial myocytes, but was much more abundant in RCA ASMCs as compared with ASMCs isolated from rat cerebral basilar, intrarenal and mesenteric arteries. EA reduced [Cl-]i levels, augmented CaCC currents exclusively in RCA ASMCs and depolarized RCA ASMCs to a greater extent. Cl- deprivation, which depleted [Cl-]i by incubating the arteries or their ASMCs in Cl--free bath solution, decreased EA-induced [Cl-]i reduction, diminished EA-induced CaCC augmentation and time-dependently depressed EA-induced RCA constriction. Inhibitor studies showed that these EA-induced effects including RCA constriction, CaCC current augmentation, [Cl-]i reduction and/or [Ca2+]i elevation were depressed by various Cl- channel blockers, [Ca2+]i release inhibitors and L-type voltage-gated Ca2+ channel inhibitor nifedipine. ANO1 antibody attenuated all observed changes induced by EA in RCA ASMCs. CONCLUSION: The greater activity of RCA ASMC CaCCs complicated with an enhanced Ca2+ mobilization from both [Ca2+]i release and [Ca2+]o influx plays a pivotal role in the distinctive hypercontractility of RCAs to acidosis. Translation of these findings to human beings may lead to a new conception in our understanding and treating cardiac complications in severe acidosis.


Assuntos
Acidose/metabolismo , Anoctamina-1/metabolismo , Vasos Coronários/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Vasoconstrição/fisiologia , Acidose/tratamento farmacológico , Animais , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Vasos Coronários/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Nifedipino/farmacologia , Técnicas de Patch-Clamp/métodos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacos
5.
Cell Physiol Biochem ; 55(S3): 157-170, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34318654

RESUMO

BACKGROUND/AIMS: The Amyloid Precursor Protein (APP) is involved in the regulation of multiple cellular functions via protein-protein interactions and has been most studied with respect to Alzheimer's disease (AD). Abnormal processing of the single transmembrane-spanning C99 fragment of APP contributes to the formation of amyloid plaques, which are causally related to AD. Pathological C99 accumulation is thought to associate with early cognitive defects in AD. Here, unexpectedly, sequence analysis revealed that C99 exhibits 24% sequence identity with the KCNE1 voltage-gated potassium (Kv) channel ß subunit, comparable to the identity between KCNE1 and KCNE2-5 (21-30%). This suggested the possibility of C99 regulating Kv channels. METHODS: We quantified the effects of C99 on Kv channel function, using electrophysiological analysis of subunits expressed in Xenopus laevis oocytes, biochemical and immunofluorescence techniques. RESULTS: C99 isoform-selectively inhibited (by 30-80%) activity of a range of Kv channels. Among the KCNQ (Kv7) family, C99 isoform-selectively inhibited, shifted the voltage dependence and/or slowed activation of KCNQ2, KCNQ3, KCNQ2/3 and KCNQ5, with no effects on KCNQ1, KCNQ1-KCNE1 or KCNQ4. C99/APP co-localized with KCNQ2 and KCNQ3 in adult rat sciatic nerve nodes of Ranvier. Both C99 and full-length APP co-immunoprecipitated with KCNQ2 in vitro, yet unlike C99, APP only weakly affected KCNQ2/3 activity. Finally, C99 altered the effects on KCNQ2/3 function of inhibitors tetraethylammounium and XE991, but not openers retigabine and ICA27243. CONCLUSION: Our findings raise the possibility of C99 accumulation early in AD altering cellular excitability by modulating Kv channel activity.


Assuntos
Precursor de Proteína beta-Amiloide/farmacologia , Canais de Potássio KCNQ/genética , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ3/genética , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Antracenos/farmacologia , Expressão Gênica , Humanos , Canais de Potássio KCNQ/metabolismo , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Nós Neurofibrosos/efeitos dos fármacos , Nós Neurofibrosos/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tetraetilamônio/farmacologia , Xenopus laevis
6.
Methods Mol Biol ; 2352: 183-199, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324188

RESUMO

Direct reprogramming is an emerging research field where you can generate neurons from a somatic cell, such as a skin or glial cell by overexpressing neurogenic transcription factors. This technique allows fast generation of subtype-specific and functional neurons from both human and mouse cells. Despite the fact that neurons have been successfully generated both in vitro and in vivo, a more extensive analysis of the induced neurons including phenotypic functional identity or gradual maturity is still lacking. This is an important step for a further development of induced neurons towards cell therapy or disease modeling of neurological diseases. In this protocol, we describe a method for functional assessment of direct reprogrammed neuronal cells both in vitro and in vivo. Using a synapsin-driven reporter, our protocol allows for a direct identification of the reprogrammed neurons that permits functional assessment using patch-clamp electrophysiology. For in vitro reprogramming we further provide an optimized coating condition that allows a long-term maturation of human induced neurons in vitro.


Assuntos
Diferenciação Celular/genética , Reprogramação Celular , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , Reprogramação Celular/genética , Técnicas de Reprogramação Celular , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Vetores Genéticos/isolamento & purificação , Humanos , Lentivirus/genética , Camundongos , Técnicas de Patch-Clamp , Fatores de Transcrição/genética , Transdução Genética
7.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201658

RESUMO

The death of photoreceptor cells is induced by continuous light exposure. However, it is unclear whether light damage was induced in retinal ganglion cells with photosensitivity by transduction of optogenetic genes. In this study, we evaluated the phototoxicities of continuous light exposure on retinal ganglion cells after transduction of the optogenetic gene mVChR1 using an adeno-associated virus vector. Rats were exposed to continuous light for a week, and visually evoked potentials (VEPs) were recorded. The intensities of continuous light (500, 1000, 3000, and 5000 lx) increased substantially after VEP recordings. After the final recording of VEPs, retinal ganglion cells (RGCs) were retrogradely labeled with a fluorescein tracer, FluoroGold, and the number of retinal ganglion cells was counted under a fluorescent microscope. There was no significant reduction in the amplitudes of VEPs and the number of RGCs after exposure to any light intensity. These results indicated that RGCs were photosensitive after the transduction of optogenetic genes and did not induce any phototoxicity by continuous light exposure.


Assuntos
Optogenética/métodos , Células Ganglionares da Retina/fisiologia , Rodopsina/genética , Animais , Dependovirus/genética , Potenciais Evocados Visuais , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Luz/efeitos adversos , Técnicas de Patch-Clamp , Estimulação Luminosa , Ratos , Células Ganglionares da Retina/patologia , Rodopsina/metabolismo , Estilbamidinas/química , Estilbamidinas/metabolismo , Transdução Genética , Volvox/genética
8.
Methods Mol Biol ; 2320: 111-119, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302653

RESUMO

Induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) have been shown to have great potential to play a key role in investigating cardiac diseases in vitro. Multielectrode array (MEA) system is sometimes preferable to patch-clamp in electrophysiological experiments in terms of several advantages. Here we show our protocol of electrophysiological examinations using MEA.


Assuntos
Bioensaio/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Análise Serial de Tecidos/métodos , Células Cultivadas , Fenômenos Eletrofisiológicos/fisiologia , Humanos , Técnicas de Patch-Clamp/métodos
9.
Methods Mol Biol ; 2320: 121-133, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302654

RESUMO

Electrophysiological analysis of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using a patch-clamp technique enables the most precise evaluation of electrophysiological properties in single cells. Compared to multielectrode array (MEA) and membrane voltage imaging, patch-clamp recordings offer quantitative measurements of action potentials, and the relevant ionic currents which are essential for the research of disease modeling of inherited arrhythmias, safety pharmacology, and drug discovery using hiPSC-CMs. In this chapter, we describe the detail flow of patch-clamp recordings in hiPSC-CMs.


Assuntos
Fenômenos Eletrofisiológicos/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Técnicas de Patch-Clamp/métodos , Análise Serial de Tecidos/métodos , Potenciais de Ação/fisiologia , Arritmias Cardíacas/terapia , Células Cultivadas , Humanos
10.
Methods Mol Biol ; 2320: 135-149, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302655

RESUMO

Human iPSC-derived cardiomyocytes (hiPSC-CMs) are expected to be used in regenerative therapies and drug discovery for heart failure. hiPSC-CMs are a mixture of mainly ventricular CMs (VCMs) and also of atrial CMs (ACMs) and pacemaker cells. Here we describe a method to enrich VCM and ACM differentiation and to characterize these subtypes by gene expression analysis using qRT-PCR and by electrophysiological properties using the patch-clamp method. The differentiated VCMs and ACMs highly express VCM and ACM marker genes, respectively. Furthermore, both subtypes show specific properties of action potentials.


Assuntos
Ventrículos do Coração/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Potenciais de Ação/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Fenômenos Eletrofisiológicos/fisiologia , Átrios do Coração/citologia , Humanos , Técnicas de Patch-Clamp/métodos
11.
Nat Commun ; 12(1): 4482, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301959

RESUMO

Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia - histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism.


Assuntos
Conexinas/metabolismo , Desacetilase 6 de Histona/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Acetilação , Animais , Células Cultivadas , Conexinas/genética , Conexinas/fisiologia , Células HEK293 , Desacetilase 6 de Histona/genética , Humanos , Células Jurkat , Lisina/genética , Lisina/metabolismo , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Técnicas de Patch-Clamp , Receptores Adrenérgicos alfa 1/genética , Transdução de Sinais/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
12.
Zhonghua Yi Xue Za Zhi ; 101(26): 2055-2059, 2021 Jul 13.
Artigo em Chinês | MEDLINE | ID: mdl-34275238

RESUMO

Objective: To confirm the direct projection pathway between the medial vestibular nucleus (MVN) and vestibular efferent (VE) neurons and explore its electrophysiological characteristics. Methods: Newborn [(9±1) day-old] male and female Wistar rats were used in the study. The postsynaptic currents of VE were recorded after stimulating neurons in MVN by the whole-cell patch clamp recording technique. The action potentials (APs) of the afferent neurons in MVN were recorded retrogradely after stimulating the area of VE neurons distribution medial to genu of facial nerve (g7), and the position and shape of the recorded neurons were determined by biocytin staining. Results: The resting membrane potentials of VE neurons located medial to g7 ranged between -70 mV and -55 mV in current clamp recordings. Excitatory postsynaptic currents (EPSCs) were recorded in the VE neurons medial to the g7 evoked by single-pulse (0.08 mA, 0.1 Hz, 100 µs) electrical stimulation of MVN. The mean values of amplitude and duration were (195.6±23.7) pA and (23.9±5.9) ms, respectively. APs were recorded in MVN after stimulating the distribution area of VE neurons. The mean amplitude of the action potentials was (62.0±4.3) mV, and the mean duration was (94.9±4.7) ms. Biocytin staining indicated that the recorded neurons located in MVN and the axons' terminals went into the area medial to g7 in which VE neurons located. Conclusions: There is a direct excitatory pathway projecting from MVN to VE neurons medial to g7. Its physiological function may be related to the feedback regulation of vestibular center to peripheral vestibular afferent signals.


Assuntos
Neurônios Eferentes , Núcleos Vestibulares , Animais , Feminino , Masculino , Neurônios , Técnicas de Patch-Clamp , Ratos , Ratos Wistar
13.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205953

RESUMO

The ionotropic P2X receptor, P2X7, is believed to regulate and/or generate nociceptive pain, and pain in several neuropathological diseases. Although there is a known relationship between P2X7 receptor activity and pain sensing, its detailed functional properties in trigeminal ganglion (TG) neurons remains unclear. We examined the electrophysiological and pharmacological characteristics of the P2X7 receptor and its functional coupling with other P2X receptors and pannexin-1 (PANX1) channels in primary cultured rat TG neurons, using whole-cell patch-clamp recordings. Application of ATP and Bz-ATP induced long-lasting biphasic inward currents that were more sensitive to extracellular Bz-ATP than ATP, indicating that the current was carried by P2X7 receptors. While the biphasic current densities of the first and second components were increased by Bz-ATP in a concentration dependent manner; current duration was only affected in the second component. These currents were significantly inhibited by P2X7 receptor antagonists, while only the second component was inhibited by P2X1, 3, and 4 receptor antagonists, PANX1 channel inhibitors, and extracellular ATPase. Taken together, our data suggests that autocrine or paracrine signaling via the P2X7-PANX1-P2X receptor/channel complex may play important roles in several pain sensing pathways via long-lasting neuronal activity driven by extracellular high-concentration ATP following tissue damage in the orofacial area.


Assuntos
Conexinas/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Receptores Purinérgicos P2X7/genética , Gânglio Trigeminal/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Cultura Primária de Células , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ratos , Gânglio Trigeminal/efeitos dos fármacos
14.
Life Sci ; 282: 119761, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34217764

RESUMO

AIMS: Eugenol is a natural compound found in the essential oils of many aromatic plants. The compound is used as a local anesthetic because of its inhibitory effect on the voltage-gated Na+ channels (Nav), which are expressed in the nociceptive neurons. Eugenol has shown wide range of activities in the cardiovascular system; most of these activities are attributed to the modulation of voltage-sensitive Ca2+ channels. However, its action on Nav1.5, the main subtype of Nav expressed in the mammalian myocardium, is unknown. The interaction of eugenol with Nav1.5 could also contribute to its antiarrhythmic properties in vitro and ex vivo. We investigated the compound's effect on sodium current (INa) and its possible cardiac antiarrhythmic activity. METHODS: The effect of eugenol on cardiac contractility was investigated using isolated atrium from guinea pig (for isometric force measurements). The compound's effect on INa was evaluated using human embryonic cell transiently expressing human Nav1.5 and patch-clamp technique. KEY FINDINGS: Eugenol caused negative inotropic and chronotropic effects in the atria. In the ex vivo arrhythmia model, eugenol decreased atrial pacing disturbance induced by ouabain. Eugenol reduced the INa in a concentration-dependent manner. Furthermore, the compound left-shifted the stationary inactivation curve, delayed recovery from inactivation of the INa, and preferentially blocked the channel in the inactivated state. Importantly, eugenol was able to attenuate the late sodium current. All these aspects are considered to be antiarrhythmic. SIGNIFICANCE: Overall, our findings demonstrate that eugenol has antiarrhythmic activity due, at least in part, to its interaction with Nav1.5.


Assuntos
Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Eugenol/uso terapêutico , Coração/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Feminino , Cobaias , Células HEK293 , Coração/fisiopatologia , Humanos , Masculino , Técnicas de Patch-Clamp
15.
Int J Mol Sci ; 22(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205237

RESUMO

A substantial proportion of neurons undergoes programmed cell death (apoptosis) during early development. This process is attenuated by increased levels of neuronal activity and enhanced by suppression of activity. To uncover whether the mere level of activity or also the temporal structure of electrical activity affects neuronal death rates, we optogenetically controlled spontaneous activity of synaptically-isolated neurons in developing cortical cultures. Our results demonstrate that action potential firing of primary cortical neurons promotes neuronal survival throughout development. Chronic patterned optogenetic stimulation allowed to effectively modulate the firing pattern of single neurons in the absence of synaptic inputs while maintaining stable overall activity levels. Replacing the burst firing pattern with a non-physiological, single pulse pattern significantly increased cell death rates as compared to physiological burst stimulation. Furthermore, physiological burst stimulation led to an elevated peak in intracellular calcium and an increase in the expression level of classical activity-dependent targets but also decreased Bax/BCL-2 expression ratio and reduced caspase 3/7 activity. In summary, these results demonstrate at the single-cell level that the temporal pattern of action potentials is critical for neuronal survival versus cell death fate during cortical development, besides the pro-survival effect of action potential firing per se.


Assuntos
Neocórtex/citologia , Neurônios/fisiologia , Optogenética , Potenciais de Ação , Animais , Células Cultivadas , Proteínas Luminescentes , Camundongos , Técnicas de Patch-Clamp
16.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281161

RESUMO

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for genetic models of cardiac diseases. We report an arrhythmia syndrome consisting of Early Repolarization Syndrome (ERS) and Short QT Syndrome (SQTS). The index patient (MMRL1215) developed arrhythmia-mediated syncope after electrocution and was found to carry six mutations. Functional alterations resulting from these mutations were examined in patient-derived hiPSC-CMs. Electrophysiological recordings were made in hiPSC-CMs from MMRL1215 and healthy controls. ECG analysis of the index patient showed slurring of the QRS complex and QTc = 326 ms. Action potential (AP) recordings from MMRL1215 myocytes showed slower spontaneous activity and AP duration was shorter. Field potential recordings from MMRL1215 hiPSC-CMs lack a "pseudo" QRS complex suggesting reduced inward current(s). Voltage clamp analysis of ICa showed no difference in the magnitude of current. Measurements of INa reveal a 60% reduction in INa density in MMRL1215 hiPSC-CMs. Steady inactivation and recovery of INa was unaffected. mRNA analysis revealed ANK2 and SCN5A are significantly reduced in hiPSC-CM derived from MMRL1215, consistent with electrophysiological recordings. The polygenic cause of ERS/SQTS phenotype is likely due to a loss of INa due to a mutation in PKP2 coupled with and a gain of function in IK,ATP due to a mutation in ABCC9.


Assuntos
Arritmias Cardíacas/genética , Miócitos Cardíacos/metabolismo , Potenciais de Ação/genética , Trifosfato de Adenosina/metabolismo , Anquirinas/genética , Anquirinas/metabolismo , Arritmias Cardíacas/fisiopatologia , Fenômenos Eletrofisiológicos , Variação Genética/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp/métodos , Placofilinas/genética , Potássio/metabolismo , Sódio/metabolismo , Receptores de Sulfonilureias/genética
17.
Int J Mol Sci ; 22(13)2021 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-34281255

RESUMO

Midazolam (MDZ) could affect lymphocyte immune functions. However, the influence of MDZ on cell's K+ currents has never been investigated. Thus, in the present study, the effects of MDZ on Jurkat T lymphocytes were studied using the patch-clamp technique. Results showed that MDZ suppressed the amplitude of delayed-rectifier K+ current (IK(DR)) in concentration-, time-, and state-dependent manners. The IC50 for MDZ-mediated reduction of IK(DR) density was 5.87 µM. Increasing MDZ concentration raised the rate of current-density inactivation and its inhibitory action on IK(DR) density was estimated with a dissociation constant of 5.14 µM. In addition, the inactivation curve of IK(DR) associated with MDZ was shifted to a hyperpolarized potential with no change on the slope factor. MDZ-induced inhibition of IK(DR) was not reversed by flumazenil. In addition, the activity of intermediate-conductance Ca2+-activated K+ (IKCa) channels was suppressed by MDZ. Furthermore, inhibition by MDZ on both IK(DR) and IKCa-channel activity appeared to be independent from GABAA receptors and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes. In conclusion, MDZ suppressed current density of IK(DR) in concentration-, time-, and state-dependent manners in Jurkat T-lymphocytes and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes.


Assuntos
Canais de Potássio de Retificação Tardia/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Midazolam/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Citocinas/metabolismo , Canais de Potássio de Retificação Tardia/metabolismo , Relação Dose-Resposta a Droga , Flumazenil/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Humanos , Hipnóticos e Sedativos/administração & dosagem , Hipnóticos e Sedativos/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Células Jurkat , Cinética , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Microscopia Confocal , Midazolam/administração & dosagem , Técnicas de Patch-Clamp , Fito-Hemaglutininas/farmacologia , Linfócitos T/imunologia
18.
Nat Commun ; 12(1): 4509, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301949

RESUMO

The capacity of the brain to encode multiple types of sensory input is key to survival. Yet, how neurons integrate information from multiple sensory pathways and to what extent this influences behavior is largely unknown. Using two-photon Ca2+ imaging, optogenetics and electrophysiology in vivo and in vitro, we report the influence of auditory input on sensory encoding in the somatosensory cortex and show its impact on goal-directed behavior. Monosynaptic input from the auditory cortex enhanced dendritic and somatic encoding of tactile stimulation in layer 2/3 (L2/3), but not layer 5 (L5), pyramidal neurons in forepaw somatosensory cortex (S1). During a tactile-based goal-directed task, auditory input increased dendritic activity and reduced reaction time, which was abolished by photoinhibition of auditory cortex projections to forepaw S1. Taken together, these results indicate that dendrites of L2/3 pyramidal neurons encode multisensory information, leading to enhanced neuronal output and reduced response latency during goal-directed behavior.


Assuntos
Potenciais de Ação/fisiologia , Córtex Auditivo/fisiologia , Dendritos/fisiologia , Células Piramidais/fisiologia , Córtex Somatossensorial/fisiologia , Animais , Córtex Auditivo/citologia , Estimulação Elétrica , Eletromiografia/métodos , Objetivos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Optogenética/métodos , Técnicas de Patch-Clamp , Células Piramidais/citologia , Córtex Somatossensorial/citologia , Tato/fisiologia
19.
eNeuro ; 8(4)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34257077

RESUMO

Patch-clamp instruments including amplifier circuits and pipettes affect the recorded voltage signals. We hypothesized that realistic and complete in silico representation of recording instruments together with detailed morphology and biophysics of small recorded structures will reveal signal distortions and provide a tool that predicts native, instrument-free electrical signals from distorted voltage recordings. Therefore, we built a model that was verified by small axonal recordings. The model accurately recreated actual action potential (AP) measurements with typical recording artefacts and predicted the native electrical behavior. The simulations verified that recording instruments substantially filter voltage recordings. Moreover, we revealed that instrumentation directly interferes with local signal generation depending on the size of the recorded structures, which complicates the interpretation of recordings from smaller structures, such as axons. However, our model offers a straightforward approach that predicts the native waveforms of fast voltage signals and the underlying conductances even from the smallest neuronal structures.


Assuntos
Axônios , Neurônios , Potenciais de Ação , Simulação por Computador , Condução Nervosa , Técnicas de Patch-Clamp
20.
eNeuro ; 8(4)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34312222

RESUMO

Patch clamp electrophysiology is a common technique used in neuroscience to understand individual neuron behavior, allowing one to record current and voltage changes with superior spatiotemporal resolution compared with most electrophysiology methods. While patch clamp experiments produce high fidelity electrophysiology data, the technique is onerous and labor intensive. Despite the emergence of patch clamp systems that automate key stages in the typical patch clamp procedure, full automation remains elusive. Patch clamp pipettes can miss the target cell during automated experiments because of positioning errors in the robotic manipulators, which can easily exceed the diameter of a neuron. Further, when patching in acute brain slices, the inherent light scattering from non-uniform brain tissue can complicate pipette tip identification. We present a convolutional neural network (CNN), based on ResNet101, to identify and correct pipette positioning errors before each patch clamp attempt, thereby preventing the deleterious effects of and accumulation of positioning errors. This deep-learning-based pipette detection method enabled superior localization of the pipette within 0.62 ± 0.58 µm, resulting in improved cell detection success rate and whole-cell patch clamp success rates by 71% and 59%, respectively, compared with the state-of-the-art cross-correlation method. Furthermore, this technique reduced the average time for pipette correction by 81%. This technique enables real-time correction of pipette position during patch clamp experiments with similar accuracy and quality of recording to manual patch clamp, making notable progress toward full human-out-of-the-loop automation for patch clamp electrophysiology.


Assuntos
Fenômenos Eletrofisiológicos , Neurônios , Automação , Humanos , Aprendizado de Máquina , Técnicas de Patch-Clamp
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