Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 37.297
Filtrar
1.
J Enzyme Inhib Med Chem ; 34(1): 1465-1473, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31411081

RESUMO

In this investigation, we studied a family of compounds with an oxathiazolidine-4-one-2,2-dioxide skeleton and their amide synthetic precursors as new anticonvulsant drugs. The cyclic structures were synthesized using a three-step protocol that include solvent-free reactions and microwave-assisted heating. The compounds were tested in vivo through maximal electroshock seizure test in mice. All the structures showed activity at the lower doses tested (30 mg/Kg) and no signs of neurotoxicity were detected. Compound encoded as 1g displayed strong anticonvulsant effects in comparison with known anticonvulsants (ED50 = 29 mg/Kg). First approximations about the mechanisms of action of the cyclic structures were proposed by docking simulations and in vitro assays against sodium channels (patch clamp methods).


Assuntos
Anticonvulsivantes/química , Anticonvulsivantes/farmacologia , Desenho de Drogas , Imidas/química , Imidas/farmacologia , Tiazóis/química , Animais , Anticonvulsivantes/administração & dosagem , Anticonvulsivantes/síntese química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Imidas/síntese química , Masculino , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.1/efeitos dos fármacos , Óxidos/química , Técnicas de Patch-Clamp , Espectroscopia de Prótons por Ressonância Magnética
2.
Zhonghua Xin Xue Guan Bing Za Zhi ; 47(8): 608-613, 2019 Aug 24.
Artigo em Chinês | MEDLINE | ID: mdl-31434431

RESUMO

Objective: To investigate the effects and mechanism of digoxin on atrium electrical remodeling and susceptibility of atrial fibrillation (AF) in aged rabbits. Methods: Twenty aged male New Zealand rabbits were divided into aged group and aged plus digoxin group (n=10 each). Electrical parameters including heart rate (HR), RR and QT interval, ST segment and P wave dispersion from normal Ⅱ electrocardiogram, and the maximum upstroke velocity (Max(dv/dt)), plateau potential (plateau P), action potential duration of 10%, 20% and 90% (APD(10), APD(20), APD(90)) from recording of monophasic action potential (MAP), as well as atrial effective refractory period (AERP(200)) and dispersion (dERP(200)) with 200 ms of basic cycle length (BCL), and frequency self adaptation of AERP with 300 ms and 150 ms of BCLs (fERP) were recorded and compared between the 2 groups. BCLs and inducibility of AF post programmed electrical stimulation and Burst-pacing in left atrium tissue of rabbits in vivo were also analyzed. The L-type calcium current (I(Ca-L)) in 2 groups were recorded via whole-cell patch clamp technique, and the fluorescence intensity of intracellular free Ca(2+) was detected with Flup-3/AM loading by the laser scanning confocal microscope in enzymatically dissociated single rabbit atrial myocytes. Results: Compared with aged group, the heart rate was faster, RR and QT interval were obvious shorter, ST segment was raised and P wave dispersion was significantly increased in aged plus digoxin group (all P<0.05). Moreover, compared with aged group, the Max(dv/dt) and plateau P were obviously increased, APD(10) and APD(20) were significantly prolongated, and APD(9)0 was significantly shorter in aged plus digoxin group (all P<0.01). Otherwise, the fERP was markedly increased (0.81±0.15 vs. 0.67±0.05), and the induced rate of AF was obviously higher in aged plus digoxin group than in aged group (6/8 vs. 4/9) (all P<0.01). With voltage clamp model, digoxin significantly increased I(Ca-L) of atrial myocytes of aged rabbits, When command potential was 10 mV, the current densities of I(Ca-L) were significantly higher in digoxin group than that in aged group ((15.45±2.38) pA/pF vs. (7.03±1.69) pA/pF, P<0.01). Otherwise, the I-V curve of I(Ca-L) was downward shifted of all I-V curves in digoxin perfused aged atrial cells of rabbits. Moreover, the fluorescence intensities of intracellular free Ca(2+) was significantly higher in aged plus digoxin group than in aged group ((1 748±173) µmol/L vs. (478.13±87.63) µmol/L, P<0.01). Conclusion: Digoxin could aggravate the atrial electrical remodeling in atrium of aged rabbits, facilitate susceptibility of atrial fibrillation in aged rabbit, increased current density of I(Ca-L) and concentration of intracellular free Ca(2+), followed Ca(2+) overload and oscillations might be part of the underlying mechanisms.


Assuntos
Fibrilação Atrial , Remodelamento Atrial , Potenciais de Ação , Animais , Digoxina , Átrios do Coração , Masculino , Técnicas de Patch-Clamp , Coelhos
3.
Br J Anaesth ; 123(4): 439-449, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31383364

RESUMO

BACKGROUND: Nerve growth factor (NGF) has been implicated in hyperalgesia by sensitising nociceptors. A role for NGF in modulating myocardial injury through ischaemic nociceptive signalling is plausible. We examined whether inhibition of spinal NGF attenuates myocardial ischaemia-reperfusion injury and explored the underlying mechanisms. METHODS: In adult rats, lentivirus-mediated short-hairpin RNA targeted at reducing NGF gene expression (NGF-shRNA) or a transient receptor potential vanilloid 1 (TRPV1) antagonist (capsazepine) was injected intrathecally before myocardial ischaemia-reperfusion. Infarct size (expressed as the ratio of area at risk) and risk of arrhythmias were quantified. Whole-cell clamp patch electrophysiology was used to record capsaicin currents in primary dorsal root ganglion neurones. The co-expression of substance P (SP) and calcitonin gene-related peptide (CGRP), plus activation of TRPV1, protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) were also quantified. RESULTS: NGF levels increased by 2.95 (0.34)-fold in dorsal root ganglion and 2.12 (0.27)-fold in spinal cord after myocardial ischaemia-reperfusion injury. Intrathecal injection of NGF-shRNA reduced infarct area at risk from 0.58 (0.02) to 0.37 (0.02) (P<0.01) and reduced arrhythmia score from 3.67 (0.33) to 1.67 (0.33) (P<0.01). Intrathecal capsazepine was similarly cardioprotective. NGF-shRNA suppressed expression of SP/CGRP and activation of Akt/ERK and TRPV1 in spinal cord. NGF increased capsaicin current amplitude from 144 (42) to 840 (132) pA (P<0.05), which was blocked by the TRPV1 antagonist 5'-iodoresiniferatoxin. Exogenous NGF enhanced capsaicin-induced Akt/ERK and TRPV1 activation in PC12 neuroendocrine tumour cells in culture. CONCLUSIONS: Spinal NGF contributes to myocardial ischaemia-reperfusion injury by mediating nociceptive signal transmission.


Assuntos
Terapia Genética/métodos , Lentivirus/genética , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Fator de Crescimento Neural/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/uso terapêutico , Animais , Arritmias Cardíacas/prevenção & controle , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Cardiotônicos/administração & dosagem , Cardiotônicos/uso terapêutico , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Injeções Espinhais , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/prevenção & controle , Fator de Crescimento Neural/biossíntese , Células PC12 , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo
4.
Fitoterapia ; 137: 104272, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31326417

RESUMO

In the current study effects of fungal extracts on the G-protein-activated inwardly rectifying potassium channel (GIRK1/4) were screened using the automated patch-clamp method. 40 organic (n-hexane, chloroform, and 50% methanol) and aqueous extracts were prepared from 10 mushroom species native to Hungary. Among the examined fungal fractions of different polarities some n-hexane and chloroform extracts exerted considerable ion channel activity. One of the most active fungal species, Hypholoma lateritium was selected for further detailed examination to determine the compounds responsible for the observed pharmacological property. Evaluation of the ion channel activity of mushroom metabolites 1-10 revealed that lanosta-7,9(11)-diene-12ß,21α-epoxy-2α,3ß,24ß,25-tetraol (5) demonstrates remarkable blocking activity on GIRK current (IC50 395.1 ±â€¯31.8 nM). Investigation of the selectivity of the GIRK inhibitory effect proved that lanosta-7,9(11)-diene-12ß,21α-epoxy-2α,3ß,24ß,25-tetraol (5) has only weak inhibitory activity on hERG channel (7.9 ±â€¯2.8% at 100 µM), exerting more than three orders of magnitude lower blocking activity on hERG channel than on GIRK channel.


Assuntos
Agaricales/química , Canal de Potássio ERG1/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/antagonistas & inibidores , Células HEK293 , Humanos , Hungria , Estrutura Molecular , Técnicas de Patch-Clamp
5.
Life Sci ; 232: 116620, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31291594

RESUMO

AIMS: Cell-based biological pacemakers aim to overcome limitations and side effects of electronic pacemaker devices. We here developed and tested different approaches to achieve nodal-type differentiation using human adipose- and bone marrow-derived mesenchymal stem cells (haMSC, hbMSC). MAIN METHODS: haMSC and hbMSC were differentiated using customized protocols. Quantitative RT-PCR was applied for transcriptional pacemaker-gene profiling. Protein membrane expression was analyzed by immunocytochemistry. Pacemaker current (If) was studied in haMSC with and without lentiviral HCN4-transduction using patch clamp recordings. Functional characteristics were evaluated by co-culturing with neonatal rat ventricular myocytes (NRVM). KEY FINDINGS: Culture media-based differentiation for two weeks generated cells with abundant transcription of ion channel genes (Cav1.2, NCX1), transcription factors (TBX3, TBX18, SHOX2) and connexins (Cx31.9 and Cx45) characteristic for cardiac pacemaker tissue, but lack adequate HCN transcription. haMSC-derived cells revealed transcript levels, which were closer related to sinoatrial nodal cells than hbMSC-derived cells. To substitute for the lack of If, we performed lentiviral HCN4-transduction of haMSC resulting in stable If. Co-culturing with NRVM demonstrated that differentiated haMSC expressing HCN4 showed earlier onset of spontaneous contractions and higher beating regularity, synchrony and rate compared to co-cultures with non-HCN4-transduced haMSC or HCN4-transduced, non-differentiated haMSC. Confocal imaging indicated increased membrane expression of cardiac gap junctional proteins in differentiated haMSC. SIGNIFICANCE: By differentiation haMSC, rather than hbMSC attain properties favorable for cardiac pacemaking. In combination with lentiviral HCN4-transduction, a cellular phenotype was generated that sustainably controls and stabilizes rate in co-culture with NRVM.


Assuntos
Relógios Biológicos/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas Musculares/metabolismo , Canais de Potássio/metabolismo , Tecido Adiposo/fisiologia , Animais , Células da Medula Óssea/fisiologia , Diferenciação Celular/fisiologia , Técnicas de Cocultura , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Células-Tronco Mesenquimais/metabolismo , Células Musculares/metabolismo , Proteínas Musculares/fisiologia , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/fisiologia , Ratos , Nó Sinoatrial
6.
Forensic Sci Int ; 301: 289-298, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31195250

RESUMO

BACKGROUND: Multiple genome-wide association studies (GWAS) and targeted gene sequencing have identified common variants in SCN10A in cases of PR and QRS duration abnormalities, atrial fibrillation and Brugada syndrome. The New York City Office of Chief Medical Examiner has now also identified five SCN10A variants of uncertain significance in six separate cases within a cohort of 330 sudden unexplained death events. The gene product of SCN10A is the Nav1.8 sodium channel. The purpose of this study was to characterize effects of these variants on Nav1.8 channel function to provide better information for the reclassification of these variants. METHODS AND RESULTS: Patch clamp studies were performed to assess effects of the variants on whole-cell Nav1.8 currents. We also performed RNA-seq analysis and immunofluorescence confocal microcopy to determine Nav1.8 expression in heart. We show that four of the five rare 'variants of unknown significance' (L388M, L867F, P1102S and V1518I) are associated with altered functional phenotypes. The R756W variant behaved similar to wild-type under our experimental conditions. We failed to detect Nav1.8 protein expression in immunofluorescence microscopy in rat heart. Furthermore, RNA-seq analysis failed to detect full-length SCN10A mRNA transcripts in human ventricle or mouse specialized cardiac conduction system, suggesting that the effect of Nav1.8 on cardiac function is likely to be extra-cardiac in origin. CONCLUSIONS: We have demonstrated that four of five SCN10A variants of uncertain significance, identified in unexplained death, have deleterious effects on channel function. These data extend the genetic testing of SUD cases, but significantly more clinical evidence is needed to satisfy the criteria needed to associate these variants with the onset of SUD.


Assuntos
Morte Súbita/etiologia , Variação Genética , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Adulto , Animais , Western Blotting , Pré-Escolar , Feminino , Genética Forense , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Masculino , Microscopia de Fluorescência , Miocárdio/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Ratos , Adulto Jovem
7.
Nat Commun ; 10(1): 2431, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160566

RESUMO

Contextual modulation of neuronal responses by surrounding environments is a fundamental attribute of sensory processing. In the mammalian retina, responses of On-Off direction selective ganglion cells (DSGCs) are modulated by motion contexts. However, the underlying mechanisms are unknown. Here, we show that posterior-preferring DSGCs (pDSGCs) are sensitive to discontinuities of moving contours owing to contextually modulated cholinergic excitation from starburst amacrine cells (SACs). Using a combination of synapse-specific genetic manipulations, patch clamp electrophysiology and connectomic analysis, we identified distinct circuit motifs upstream of On and Off SACs that are required for the contextual modulation of pDSGC activity for bright and dark contrasts. Furthermore, our results reveal a class of wide-field amacrine cells (WACs) with straight, unbranching dendrites that function as "continuity detectors" of moving contours. Therefore, divergent circuit motifs in the On and Off pathways extend the information encoding of On-Off DSGCs beyond their direction selectivity during complex stimuli.


Assuntos
Acetilcolina/metabolismo , Células Amácrinas/metabolismo , Percepção de Movimento/fisiologia , Células Ganglionares da Retina/metabolismo , Sinapses/metabolismo , Visão Ocular/fisiologia , Ácido gama-Aminobutírico/metabolismo , Células Amácrinas/fisiologia , Animais , Conectoma , Dendritos/metabolismo , Ácido Glutâmico/metabolismo , Camundongos , Técnicas de Patch-Clamp , Receptores de GABA-A/genética , Células Ganglionares da Retina/fisiologia , Percepção Visual/fisiologia
8.
Nat Neurosci ; 22(7): 1110-1121, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31160741

RESUMO

Learning to predict rewards based on environmental cues is essential for survival. The orbitofrontal cortex (OFC) contributes to such learning by conveying reward-related information to brain areas such as the ventral tegmental area (VTA). Despite this, how cue-reward memory representations form in individual OFC neurons and are modified based on new information is unknown. To address this, using in vivo two-photon calcium imaging in mice, we tracked the response evolution of thousands of OFC output neurons, including those projecting to VTA, through multiple days and stages of cue-reward learning. Collectively, we show that OFC contains several functional clusters of neurons distinctly encoding cue-reward memory representations, with only select responses routed downstream to VTA. Unexpectedly, these representations were stably maintained by the same neurons even after extinction of the cue-reward pairing, and supported behavioral learning and memory. Thus, OFC neuronal activity represents a long-term cue-reward associative memory to support behavioral adaptation.


Assuntos
Adaptação Psicológica/fisiologia , Aprendizagem por Associação/fisiologia , Sinalização do Cálcio , Condicionamento Clássico/fisiologia , Memória de Longo Prazo/fisiologia , Neurônios/fisiologia , Córtex Pré-Frontal/fisiologia , Recompensa , Estimulação Acústica , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Sinais (Psicologia) , Comportamento de Ingestão de Líquido/fisiologia , Extinção Psicológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/fisiologia , Neurônios/enzimologia , Optogenética , Técnicas de Patch-Clamp , Córtex Pré-Frontal/citologia , Análise de Célula Única , Área Tegmentar Ventral/fisiologia
9.
Nat Neurosci ; 22(7): 1132-1139, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31182867

RESUMO

An approaching predator and self-motion toward an object can generate similar looming patterns on the retina, but these situations demand different rapid responses. How central circuits flexibly process visual cues to activate appropriate, fast motor pathways remains unclear. Here we identify two descending neuron (DN) types that control landing and contribute to visuomotor flexibility in Drosophila. For each, silencing impairs visually evoked landing, activation drives landing, and spike rate determines leg extension amplitude. Critically, visual responses of both DNs are severely attenuated during non-flight periods, effectively decoupling visual stimuli from the landing motor pathway when landing is inappropriate. The flight-dependence mechanism differs between DN types. Octopamine exposure mimics flight effects in one, whereas the other probably receives neuronal feedback from flight motor circuits. Thus, this sensorimotor flexibility arises from distinct mechanisms for gating action-specific descending pathways, such that sensory and motor networks are coupled or decoupled according to the behavioral state.


Assuntos
Drosophila melanogaster/fisiologia , Reação de Fuga/fisiologia , Voo Animal/fisiologia , Atividade Motora/fisiologia , Vias Neurais/fisiologia , Neurônios/fisiologia , Desempenho Psicomotor/fisiologia , Percepção Visual/fisiologia , Potenciais de Ação , Animais , Vias Eferentes/fisiologia , Octopamina/farmacologia , Técnicas de Patch-Clamp , Estimulação Luminosa
10.
Nat Neurosci ; 22(7): 1182-1195, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209381

RESUMO

Understanding the diversity of cell types in the brain has been an enduring challenge and requires detailed characterization of individual neurons in multiple dimensions. To systematically profile morpho-electric properties of mammalian neurons, we established a single-cell characterization pipeline using standardized patch-clamp recordings in brain slices and biocytin-based neuronal reconstructions. We built a publicly accessible online database, the Allen Cell Types Database, to display these datasets. Intrinsic physiological properties were measured from 1,938 neurons from the adult laboratory mouse visual cortex, morphological properties were measured from 461 reconstructed neurons, and 452 neurons had both measurements available. Quantitative features were used to classify neurons into distinct types using unsupervised methods. We established a taxonomy of morphologically and electrophysiologically defined cell types for this region of the cortex, with 17 electrophysiological types, 38 morphological types and 46 morpho-electric types. There was good correspondence with previously defined transcriptomic cell types and subclasses using the same transgenic mouse lines.


Assuntos
Conjuntos de Dados como Assunto , Neurônios/classificação , Córtex Visual/citologia , Potenciais de Ação , Animais , Forma Celular , Bases de Dados Factuais , Genes Reporter , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Transcriptoma , Córtex Visual/fisiologia
11.
Cell Physiol Biochem ; 53(1): 36-48, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31169990

RESUMO

BACKGROUND/AIMS: Ivabradine lowers the heart rate by inhibition of hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels mediating the 'funny' pacemaker current If in the sinoatrial node. It is clinically approved for the treatment of heart failure and angina pectoris. Due to its proposed high selectivity for If administration of ivabradine is not associated with the side effects commonly observed following the application of other heart rate lowering agents. Recent evidence, however, has shown significant affinity of ivabradine towards Kv11.1 (ether-a-go-go related gene, ERG) potassium channels. Despite the inhibition of Kv11.1 channels by ivabradine, cardiac action potential (AP) duration and heart rate corrected QT interval (QTc) of the human electrocardiogram (ECG) were not prolonged. We thus surmised that compensatory mechanisms might counteract the drug's inhibitory action on Kv11.1. METHODS: The effects of ivabradine on human Kv11.1 and Kv7.1 potassium, Cav1.2 calcium, and Nav1.5 sodium channels, heterologously expressed in tsA-201 cells, were studied in the voltage-clamp mode of the whole cell patch clamp technique. In addition, changes in action potential parameters of human induced pluripotent stem cell (iPSC) derived cardiomyocytes upon application of ivabradine were studied with current-clamp experiments. RESULTS: Here we show that ivabradine exhibits significant affinity towards cardiac ion channels other than HCN. We demonstrate for the first time inhibition of human voltage-gated Nav1.5 sodium channels at therapeutically relevant concentrations. Within this study we also confirm recent findings of human Kv11.1 inhibition by low µM concentrations of ivabradine and observed no prolongation of ventricular-like APs in cardiomyocytes derived from iPSCs. CONCLUSION: Our results provide an explanation why ivabradine, despite its affinity for Kv11.1 channels, does not prolong the cardiac AP and QTc interval. Furthermore, our results suggest the inhibition of voltage-gated Nav1.5 sodium channels to underlie the recent observations of slowed atrioventricular conduction by increased atrial-His bundle intervals upon administration of ivabradine.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Fármacos Cardiovasculares/farmacologia , Canais Iônicos/metabolismo , Ivabradina/farmacologia , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Canais Iônicos/antagonistas & inibidores , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp
12.
Life Sci ; 231: 116567, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31202839

RESUMO

AIMS: Metabotropic glutamate receptor 5 (mGluR5), a member of group I mGluR, exerts its effect via elevation of intracellular Ca2+ level. We here characterized Ca2+ signals in the tsA201 cells transfected with mGluR5 and investigated the role of passages for mGluR5-induced Ca2+ signals in synaptic plasticity. MAIN METHODS: Using a genetically encoded Ca2+ indicator, GCamp2, Ca2+ signals were reliably induced by bath application of (S)-3,5-dihydroxyphenylglycine, the group I mGluR agonist, in the tsA201 cells transfected with mGluR5. Using whole-cell recordings in the substantia gelatinosa (SG) neurons of the spinal trigeminal subnucleus caudalis (Vc), excitatory postsynaptic currents were recorded by stimulating the trigeminal tract. KEY FINDINGS: Ca2+ signals were mediated by "classical" or "canonical" transient receptor potential (TRPC) channels, particularly TRPC1/3/4/6, but not TRPC5, naturally existing in the tsA201 cells. Interestingly, the induction of Ca2+ signals was independent of the phospholipase C signaling pathway; instead, it critically involves the cyclic adenosine diphosphate ribose/ryanodine receptor-dependent signaling pathway and only partially protein kinase C. On the other hand, both TRPC3 and TRPC4 mediated mGluR1/5-induced long-lasting potentiation of excitatory synaptic transmission from the trigeminal primary afferents to the SG neurons of the Vc. SIGNIFICANCE: This study demonstrates that endogenous TRPC channels contribute to mGluR5-induced Ca2+ signals in tsA201 cells and synaptic plasticity at excitatory synapses.


Assuntos
Sinalização do Cálcio/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Receptor de Glutamato Metabotrópico 5/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores , Feminino , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/metabolismo , Transmissão Sináptica , Nervo Trigêmeo/metabolismo , Núcleo Espinal do Trigêmeo/metabolismo
13.
Nat Neurosci ; 22(7): 1061-1065, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209378

RESUMO

A key assumption of optogenetics is that light only affects opsin-expressing neurons. However, illumination invariably heats tissue, and many physiological processes are temperature-sensitive. Commonly used illumination protocols increased the temperature by 0.2-2 °C and suppressed spiking in multiple brain regions. In the striatum, light delivery activated an inwardly rectifying potassium conductance and biased rotational behavior. Thus, careful consideration of light-delivery parameters is required, as even modest intracranial heating can confound interpretation of optogenetic experiments.


Assuntos
Córtex Cerebral/fisiologia , Corpo Estriado/fisiologia , Hipocampo/fisiologia , Neurônios/fisiologia , Temperatura Ambiente , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/efeitos da radiação , Animais , Compostos de Bário/farmacologia , Córtex Cerebral/citologia , Cloretos/farmacologia , Corpo Estriado/citologia , Hipocampo/citologia , Temperatura Alta , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/efeitos da radiação , Luz , Camundongos , Atividade Motora/efeitos da radiação , Neurônios/efeitos dos fármacos , Neurônios/efeitos da radiação , Optogenética/métodos , Técnicas de Patch-Clamp , Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/efeitos da radiação , Projetos de Pesquisa
14.
Nat Commun ; 10(1): 2315, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127098

RESUMO

Encoding and retrieval of contextual memories is initially mediated by sparsely activated neurons, so-called engram cells, in the hippocampus. Subsequent memory persistence is thought to depend on network-wide changes involving progressive contribution of cortical regions, a process referred to as systems consolidation. Using a viral-based TRAP (targeted recombination in activated populations) approach, we studied whether consolidation of contextual fear memory by neurons in the medial prefrontal cortex (mPFC) is modulated by memory strength and CREB function. We demonstrate that activity of a small subset of mPFC neurons is sufficient and necessary for remote memory expression, but their involvement depends on the strength of conditioning. Furthermore, selective disruption of CREB function in mPFC engram cells after mild conditioning impairs remote memory expression. Together, our data demonstrate that memory consolidation by mPFC engram cells requires CREB-mediated transcription, with the functionality of this network hub being gated by memory strength.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Medo/fisiologia , Consolidação da Memória/fisiologia , Memória de Longo Prazo/fisiologia , Córtex Pré-Frontal/fisiologia , Animais , Comportamento Animal/fisiologia , Condicionamento (Psicologia)/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Modelos Animais , Neurônios/metabolismo , Técnicas de Patch-Clamp , Córtex Pré-Frontal/citologia , Técnicas Estereotáxicas
15.
Mol Med Rep ; 19(6): 5185-5194, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059080

RESUMO

Sphincter of Oddi dysfunction (SOD) is a benign obstructive disorder predominantly resulting from spasms of the SO. Pharmacological therapies aim to induce SO relaxation; the hypercholesterolemic (HC) rabbit is the only SOD model available for study. In the present study, SO muscle strips, intracellular calcium ion concentrations and the mRNA expression levels of the α1C subunit of the L­type calcium channel in the SO muscle cells of HC rabbits were employed to investigate the effects of paeoniflorin (PF). Alterations in L­type calcium channel α subunit 1C mRNA and protein expression in SO cells with HC following the application of different concentrations of PF were determined by reverse transcription­quantitative polymerase chain reaction and western blotting. The whole cell patch clamp technique was used to observe the effects of different concentrations of paeoniflorin on L­type calcium channel current. The results of the present study demonstrated that PF induced the relaxation of SO muscle strips and reduced the intracellular calcium concentration in the SO muscle cells of HC rabbits. In addition, PF decreased the mRNA expression levels of the α1C subunit of the L­type calcium channel and reduced the L­type calcium channel current in SO cells. These results suggested that the mechanism underlying the relaxation of the SO muscle by PF may be associated with the reduction of calcium ion influx via L­type calcium channels.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Canais de Cálcio Tipo L/metabolismo , Glucosídeos/farmacologia , Hipercolesterolemia/patologia , Monoterpenos/farmacologia , Músculos/efeitos dos fármacos , Esfíncter da Ampola Hepatopancreática/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Colesterol/sangue , Modelos Animais de Doenças , Feminino , Glucosídeos/uso terapêutico , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/metabolismo , Masculino , Monoterpenos/uso terapêutico , Tono Muscular/efeitos dos fármacos , Músculos/fisiologia , Técnicas de Patch-Clamp , Coelhos
16.
Nat Neurosci ; 22(7): 1140-1147, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31110322

RESUMO

Most neurons transmit information digitally using spikes that trigger the release of synaptic vesicles with low probability. The first stages of vision and hearing are distinct in that they operate with analog signals, but it is unclear how these are recoded for synaptic transmission. By imaging the release of glutamate in live zebrafish, we demonstrate that ribbon synapses of retinal bipolar cells encode contrast through changes in both the frequency and amplitude of release events. Higher contrasts caused multiple vesicles to be released within an event, and such coding by amplitude often continued after the rate code had reached a maximum frequency. Glutamate packets equivalent to five vesicles transmitted four times as many bits of information per vesicle compared with those released individually. By discretizing analog signals into sequences of numbers up to about 11, ribbon synapses can increase the dynamic range, temporal precision and efficiency with which visual information is transmitted.


Assuntos
Células Bipolares da Retina/fisiologia , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/fisiologia , Vias Visuais/fisiologia , Potenciais de Ação , Animais , Genes Reporter , Ácido Glutâmico/fisiologia , Fusão de Membrana , Técnicas de Patch-Clamp , Detecção de Sinal Psicológico , Peixe-Zebra/fisiologia
17.
Nat Neurosci ; 22(7): 1122-1131, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31133689

RESUMO

A remarkable feature of motor control is the ability to coordinate movements across distinct body parts into a consistent, skilled action. To reach and grasp an object, 'gross' arm and 'fine' dexterous movements must be coordinated as a single action. How the nervous system achieves this coordination is currently unknown. One possibility is that, with training, gross and fine movements are co-optimized to produce a coordinated action; alternatively, gross and fine movements may be modularly refined to function together. To address this question, we recorded neural activity in the primary motor cortex and dorsolateral striatum during reach-to-grasp skill learning in rats. During learning, the refinement of fine and gross movements was behaviorally and neurally dissociable. Furthermore, inactivation of the primary motor cortex and dorsolateral striatum had distinct effects on skilled fine and gross movements. Our results indicate that skilled movement coordination is achieved through emergent modular neural control.


Assuntos
Corpo Estriado/fisiologia , Modelos Neurológicos , Córtex Motor/fisiologia , Destreza Motora/fisiologia , Desempenho Psicomotor/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Condicionamento Operante , Corpo Estriado/efeitos dos fármacos , Membro Anterior/fisiologia , Trombose Intracraniana/fisiopatologia , Aprendizagem/fisiologia , Muscimol/farmacologia , Técnicas de Patch-Clamp , Ratos , Reforço (Psicologia)
18.
Life Sci ; 227: 74-81, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31002920

RESUMO

AIMS: Benidipine is a dihydropyridine (DHP) derived Ca2+ antagonist, can block triple Ca2+ channels (L, N, and T). It has been used as a safety anti-hypertensive drug because of its long-acting relaxant effect on vascular smooth muscle (VSM). However, whether benidipine has similar pharmacological actions in airway smooth muscle (ASM) is unknown. This research aims to reveal the relaxant property and Ca2+ antagonistic effect of benidipine on ASM. MAIN METHODS: The relaxant property of mouse ASM was investigated by tissue tension tests, and Ca2+ antagonistic effect was evaluated through patch-clamp techniques. KEY FINDINGS: Benidipine caused dose-dependent relaxations on high K+ (80 mM) induced precontraction in mouse ASM, which relied on inhibition of extracellular Ca2+ influx, and 1 µM benidipine totally blocked L-type voltage-dependent Ca2+ channels (LVDCCs) currents in airway smooth muscle cells (ASMCs). Benidipine also showed dose-dependent inhibition of ACh-induced precontraction with or without the LVDCCs blocker nifedipine, and 100 µM benidipine blocked ACh-stimulated Ca2+ influx through not only LVDCCs but also non-selective cation channels (NSCCs). SIGNIFICANCE: Benidipine blocked LVDCCs and NSCCs to abolish these channels-mediated Ca2+ influx, which relaxed precontracted ASM. This study represented benidipine with a new potential medicinal value for ASM hypercontractility.


Assuntos
Di-Hidropiridinas/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Animais , Anti-Hipertensivos/farmacologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Di-Hidropiridinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Técnicas de Patch-Clamp , Sistema Respiratório/efeitos dos fármacos
19.
Biol Bull ; 236(2): 108-114, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30933638

RESUMO

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.


Assuntos
Células Fotorreceptoras/fisiologia , Strongylocentrotus purpuratus/fisiologia , Animais , Escuridão , Luz , Técnicas de Patch-Clamp/métodos , Strongylocentrotus purpuratus/citologia
20.
Int J Mol Sci ; 20(8)2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022885

RESUMO

Transient receptor potential melastatin member 4 (TRPM4) and 5 (TRPM5) channels are Ca2+-activated nonselective cation channels. Intracellular Ca2+ is the most important regulator for them to open, though PI(4,5)P2, a membrane phosphoinositide, has been reported to regulate their Ca2+-sensitivities. We previously reported that negatively-charged amino acid residues near and in the TRP domain are necessary for the normal Ca2+ sensitivity of TRPM4. More recently, a cryo-electron microscopy structure of Ca2+-bound (but closed) TRPM4 was reported, proposing a Ca2+-binding site within an intracellular cavity formed by S2 and S3. Here, we examined the functional effects of mutations of the amino acid residues related to the proposed Ca2+-binding site on TRPM4 and also TRPM5 using mutagenesis and patch clamp techniques. The mutations of the amino acid residues of TRPM4 and TRPM5 reduced their Ca2+-sensitivities in a similar way. On the other hand, intracellular applications of PI(4,5)P2 recovered Ca2+-sensitivity of desensitized TRPM4, but its effect on TRPM5 was negligible. From these results, the Ca2+-binding sites of TRPM4 and TRPM5 were shown to be formed by the same amino acid residues by functional analyses, but the impact of PI(4,5)P2 on the regulation of TRPM5 seemed to be smaller than that on the regulation of TRPM4.


Assuntos
Cálcio/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Cátion TRPM/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Ratos , Canais de Cátion TRPM/química , Canais de Cátion TRPM/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA