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1.
Nat Commun ; 11(1): 4395, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879322

RESUMO

The formation and maintenance of spatial representations within hippocampal cell assemblies is strongly dictated by patterns of inhibition from diverse interneuron populations. Although it is known that inhibitory synaptic strength is malleable, induction of long-term plasticity at distinct inhibitory synapses and its regulation of hippocampal network activity is not well understood. Here, we show that inhibitory synapses from parvalbumin and somatostatin expressing interneurons undergo long-term depression and potentiation respectively (PV-iLTD and SST-iLTP) during physiological activity patterns. Both forms of plasticity rely on T-type calcium channel activation to confer synapse specificity but otherwise employ distinct mechanisms. Since parvalbumin and somatostatin interneurons preferentially target perisomatic and distal dendritic regions respectively of CA1 pyramidal cells, PV-iLTD and SST-iLTP coordinate a reprioritisation of excitatory inputs from entorhinal cortex and CA3. Furthermore, circuit-level modelling reveals that PV-iLTD and SST-iLTP cooperate to stabilise place cells while facilitating representation of multiple unique environments within the hippocampal network.


Assuntos
Hipocampo/fisiologia , Interneurônios/metabolismo , Células Piramidais/fisiologia , Potenciais de Ação , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/fisiologia , Canais de Cálcio Tipo T/metabolismo , Channelrhodopsins/metabolismo , Hipocampo/citologia , Camundongos , Optogenética/métodos , Parvalbuminas/metabolismo , Técnicas de Patch-Clamp , Transdução de Sinais , Somatostatina/metabolismo , Sinapses/metabolismo
2.
Nat Commun ; 11(1): 4388, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873805

RESUMO

Presynaptic spike timing-dependent long-term depression (t-LTD) at hippocampal CA3-CA1 synapses is evident until the 3rd postnatal week in mice, disappearing during the 4th week. At more mature stages, we found that the protocol that induced t-LTD induced t-LTP. We characterized this form of t-LTP and the mechanisms involved in its induction, as well as that driving this switch from t-LTD to t-LTP. We found that this t-LTP is expressed presynaptically at CA3-CA1 synapses, as witnessed by coefficient of variation, number of failures, paired-pulse ratio and miniature responses analysis. Additionally, this form of presynaptic t-LTP does not require NMDARs but the activation of mGluRs and the entry of Ca2+ into the postsynaptic neuron through L-type voltage-dependent Ca2+ channels and the release of Ca2+ from intracellular stores. Nitric oxide is also required as a messenger from the postsynaptic neuron. Crucially, the release of adenosine and glutamate by astrocytes is required for t-LTP induction and for the switch from t-LTD to t-LTP. Thus, we have discovered a developmental switch of synaptic transmission from t-LTD to t-LTP at hippocampal CA3-CA1 synapses in which astrocytes play a central role and revealed a form of presynaptic LTP and the rules for its induction.


Assuntos
Astrócitos/metabolismo , Hipocampo/crescimento & desenvolvimento , Potenciação de Longa Duração/fisiologia , Transmissão Sináptica/fisiologia , Adenosina/metabolismo , Animais , Feminino , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Masculino , Camundongos , Técnicas de Patch-Clamp , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo
3.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(5): 676-682, 2020 May 30.
Artigo em Chinês | MEDLINE | ID: mdl-32897202

RESUMO

OBJECTIVE: To investigate the effects of etomidate on electrophysiological properties and nicotinic acetylcholine receptors (nAChRs) of ventral horn neurons in the spinal cord. METHODS: The spinal cord containing lumbosacral enlargement was isolated from 19 neonatal SD rats aged 7-12 days. The spinal cord were sliced and digested with papain (0.18 g/30 mL artificial cerebrospinal fluid) and incubated for 40 min. At the ventral horn, acute mechanical separation of neurons was performed with fire-polished Pasteur pipettes, and perforated patch-clamp recordings combined with pharmacological methods were employed on the adherent healthy neurons. In current-clamp mode, the spontaneous action potential (AP) of the ventral horn neurons in the spinal cord was recorded. The effects of pretreatment with different concentrations of etomidate on AP recorded in the ventral horn neurons were examined. In the voltage-clamp mode, nicotine was applied to induce inward currents in the ventral horn neurons, and the effect of pretreatment with etomidate on the inward currents induced by nicotine were examined with different etomidate concentrations, different holding potentials and different use time. RESULTS: The isolated ventral horn neurons were in good condition with large diverse somata and intact processes. The isolated spinal ventral horn neurons (n=21) had spontaneous action potentials, and were continuously perfused for 2 min with 0.3, 3.0 and 30.0 µmol/L etomidate. Compared with those before administration, the AP amplitude, spike potential amplitude and overshoot were concentration-dependently suppressed (P < 0.01), and spontaneous discharge frequency was obviously reduced (P < 0.01, n=12). The APs of the other 9 neurons were completely abolished by etomidate at 3.0 or 30 µmol/L. At the same holding potential (VH=-70 mV), pretreatment with 0.3, 3.0 or 30.0 µmol/L etomidate for 2 min concentration-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine (P < 0.01, n=7). At the holding potentials of - 30, - 50, and - 70 mV, pretreatment with 30.0 µmol/L etomidate for 2 min voltage-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine (P < 0.01, n=6 for each holding potential). During the 6 min of 30.0 µmol/L etomidate pretreatment, the clamped cells were exposed to 0.4 mmol/L nicotine for 4 times at 0, 2, 4, and 6 min (each exposure time was 2 s), and the nicotinic current amplitude decreased gradually as the number of exposures increased. But at the same concentration, two nicotine exposures (one at the beginning and the other at the end of the 6 min pretreatment) resulted in a significantly lower inhibition rate compared with 4 nicotine exposures (P < 0.01, n=6). CONCLUSIONS: etomidate reduces the excitability of the spinal ventral neurons in a concentration-dependent manner and suppresses the function of nAChR in a concentration-, voltage-, and use-dependent manner.


Assuntos
Neurônios , Animais , Animais Recém-Nascidos , Etomidato , Técnicas de Patch-Clamp , Ratos , Medula Espinal
4.
Nat Commun ; 11(1): 4510, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908143

RESUMO

With human median lifespan extending into the 80s in many developed countries, the societal burden of age-related muscle loss (sarcopenia) is increasing. mTORC1 promotes skeletal muscle hypertrophy, but also drives organismal aging. Here, we address the question of whether mTORC1 activation or suppression is beneficial for skeletal muscle aging. We demonstrate that chronic mTORC1 inhibition with rapamycin is overwhelmingly, but not entirely, positive for aging mouse skeletal muscle, while genetic, muscle fiber-specific activation of mTORC1 is sufficient to induce molecular signatures of sarcopenia. Through integration of comprehensive physiological and extensive gene expression profiling in young and old mice, and following genetic activation or pharmacological inhibition of mTORC1, we establish the phenotypically-backed, mTORC1-focused, multi-muscle gene expression atlas, SarcoAtlas (https://sarcoatlas.scicore.unibas.ch/), as a user-friendly gene discovery tool. We uncover inter-muscle divergence in the primary drivers of sarcopenia and identify the neuromuscular junction as a focal point of mTORC1-driven muscle aging.


Assuntos
Envelhecimento/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fibras Musculares Esqueléticas/patologia , Junção Neuromuscular/patologia , Sarcopenia/patologia , Envelhecimento/efeitos dos fármacos , Animais , Linhagem Celular , Modelos Animais de Doenças , Eletromiografia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Microdissecção e Captura a Laser , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Mioblastos , Junção Neuromuscular/efeitos dos fármacos , Técnicas de Patch-Clamp , RNA-Seq , Sarcopenia/genética , Sarcopenia/fisiopatologia , Sarcopenia/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirolimo/administração & dosagem
5.
Nature ; 585(7823): 129-134, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848250

RESUMO

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.


Assuntos
Simulação por Computador , Genes Sintéticos/genética , Canais Iônicos/química , Canais Iônicos/genética , Modelos Moleculares , Biologia Sintética , Linhagem Celular , Microscopia Crioeletrônica , Cristalografia por Raios X , Condutividade Elétrica , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrazinas , Canais Iônicos/metabolismo , Transporte de Íons , Lipossomos/metabolismo , Técnicas de Patch-Clamp , Porinas/química , Porinas/genética , Porinas/metabolismo , Engenharia de Proteínas , Estrutura Secundária de Proteína , Solubilidade , Água/química
6.
Mol Pharmacol ; 98(2): 143-155, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32616523

RESUMO

The two-pore domain potassium channel (K2P-channel) THIK-1 has several predicted protein kinase A (PKA) phosphorylation sites. In trying to elucidate whether THIK-1 is regulated via PKA, we expressed THIK-1 channels in a mammalian cell line (CHO cells) and used the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) as a pharmacological tool to induce activation of PKA. Using the whole-cell patch-clamp recording, we found that THIK-1 currents were inhibited by application of IBMX with an IC50 of 120 µM. Surprisingly, intracellular application of IBMX or of the second messenger cAMP via the patch pipette had no effect on THIK-1 currents. In contrast, extracellular application of IBMX produced a rapid and reversible inhibition of THIK-1. In patch-clamp experiments with outside-out patches, THIK-1 currents were also inhibited by extracellular application of IBMX. Expression of THIK-1 channels in Xenopus oocytes was used to compare wild-type channels with mutated channels. Mutation of the putative PKA phosphorylation sites did not change the inhibitory effect of IBMX on THIK-1 currents. Mutational analysis of all residues of the (extracellular) helical cap of THIK-1 showed that mutation of the arginine residue at position 92, which is in the linker between cap helix 2 and pore helix 1, markedly reduced the inhibitory effect of IBMX. This flexible linker region, which is unique for each K2P-channel subtype, may be a possible target of channel-specific blockers. SIGNIFICANCE STATEMENT: The potassium channel THIK-1 is strongly expressed in the central nervous system. We studied the effect of 3-isobutyl-1-methyl-xanthine (IBMX) on THIK-1 currents. IBMX inhibits breakdown of cAMP and thus activates protein kinase A (PKA). Surprisingly, THIK-1 current was inhibited when IBMX was applied from the extracellular side of the membrane, but not from the intracellular side. Our results suggest that IBMX binds directly to the channel and that the inhibition of THIK-1 current was not related to activation of PKA.


Assuntos
1-Metil-3-Isobutilxantina/farmacologia , Canais de Potássio de Domínios Poros em Tandem/química , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Arginina/genética , Sítios de Ligação/efeitos dos fármacos , Células CHO , Cricetulus , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Mutação , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/genética , Ratos , Xenopus
7.
Nat Commun ; 11(1): 3790, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728032

RESUMO

Menthol in mints elicits coolness sensation by selectively activating TRPM8 channel. Although structures of TRPM8 were determined in the apo and liganded states, the menthol-bounded state is unresolved. To understand how menthol activates the channel, we docked menthol to the channel and systematically validated our menthol binding models with thermodynamic mutant cycle analysis. We observed that menthol uses its hydroxyl group as a hand to specifically grab with R842, and its isopropyl group as legs to stand on I846 and L843. By imaging with fluorescent unnatural amino acid, we found that menthol binding induces wide-spread conformational rearrangements within the transmembrane domains. By Φ analysis based on single-channel recordings, we observed a temporal sequence of conformational changes in the S6 bundle crossing and the selectivity filter leading to channel activation. Therefore, our study suggested a 'grab and stand' mechanism of menthol binding and how menthol activates TRPM8 at the atomic level.


Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Mentol/farmacologia , Canais de Cátion TRPM/agonistas , Sítios de Ligação/genética , Células HEK293 , Humanos , Mentol/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese , Técnicas de Patch-Clamp , Mutação Puntual , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Canais de Cátion TRPM/química , Canais de Cátion TRPM/genética
8.
Nat Commun ; 11(1): 3661, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694504

RESUMO

The relationship between orexin/hypocretin and rapid eye movement (REM) sleep remains elusive. Here, we find that a proportion of orexin neurons project to the sublaterodorsal tegmental nucleus (SLD) and exhibit REM sleep-related activation. In SLD, orexin directly excites orexin receptor-positive neurons (occupying ~3/4 of total-population) and increases gap junction conductance among neurons. Their interaction spreads the orexin-elicited partial-excitation to activate SLD network globally. Besides, the activated SLD network exhibits increased probability of synchronized firings. This synchronized excitation promotes the correspondence between SLD and its downstream target to enhance SLD output. Using optogenetics and fiber-photometry, we consequently find that orexin-enhanced SLD output prolongs REM sleep episodes through consolidating brain state activation/muscle tone inhibition. After chemogenetic silencing of SLD orexin signaling, a ~17% reduction of REM sleep amounts and disruptions of REM sleep muscle atonia are observed. These findings reveal a stabilization role of orexin in REM sleep.


Assuntos
Tronco Encefálico/fisiologia , Orexinas/metabolismo , Privação do Sono/fisiopatologia , Sono REM/fisiologia , Potenciais de Ação/fisiologia , Animais , Comportamento Animal , Tronco Encefálico/citologia , Modelos Animais de Doenças , Eletrodos Implantados , Eletroencefalografia , Eletromiografia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Tono Muscular/fisiologia , Neurônios/metabolismo , Optogenética , Receptores de Orexina/metabolismo , Orexinas/genética , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Vigília/fisiologia
9.
Nature ; 583(7818): 819-824, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32699411

RESUMO

The thalamic reticular nucleus (TRN), the major source of thalamic inhibition, regulates thalamocortical interactions that are critical for sensory processing, attention and cognition1-5. TRN dysfunction has been linked to sensory abnormality, attention deficit and sleep disturbance across multiple neurodevelopmental disorders6-9. However, little is known about the organizational principles that underlie its divergent functions. Here we performed an integrative study linking single-cell molecular and electrophysiological features of the mouse TRN to connectivity and systems-level function. We found that cellular heterogeneity in the TRN is characterized by a transcriptomic gradient of two negatively correlated gene-expression profiles, each containing hundreds of genes. Neurons in the extremes of this transcriptomic gradient express mutually exclusive markers, exhibit core or shell-like anatomical structure and have distinct electrophysiological properties. The two TRN subpopulations make differential connections with the functionally distinct first-order and higher-order thalamic nuclei to form molecularly defined TRN-thalamus subnetworks. Selective perturbation of the two subnetworks in vivo revealed their differential role in regulating sleep. In sum, our study provides a comprehensive atlas of TRN neurons at single-cell resolution and links molecularly defined subnetworks to the functional organization of thalamocortical circuits.


Assuntos
Redes Reguladoras de Genes , Núcleos Talâmicos/citologia , Núcleos Talâmicos/metabolismo , Animais , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Hibridização in Situ Fluorescente , Metaloendopeptidases/metabolismo , Camundongos , Vias Neurais , Neurônios/metabolismo , Osteopontina/metabolismo , Técnicas de Patch-Clamp , RNA-Seq , Análise de Célula Única , Sono/genética , Sono/fisiologia , Núcleos Talâmicos/fisiologia , Transcriptoma
10.
J Pharmacol Sci ; 143(4): 325-329, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32487451

RESUMO

The effects of class I antiarrhythmic drugs on the automaticity of isolated guinea pig pulmonary vein myocardia were investigated using microelectrode and voltage clamp methods. All of the drugs examined reduced the maximum rate of rise of automatic action potentials. The firing frequency and rate of diastolic depolarization were decreased by aprindine, flecainide and propafenone, but not by cibenzoline, disopyramide and pilsicainide, which correlated with blockade of the sodium current component induced by ramp depolarization mimicking the diastolic depolarization. In conclusion, class I antiarrhythmic drugs which block the diastolic sodium current component inhibit the automaticity of the pulmonary vein myocardium.


Assuntos
Antiarrítmicos/farmacologia , Veias Pulmonares/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Antiarrítmicos/classificação , Cobaias , Técnicas In Vitro , Microeletrodos , Técnicas de Patch-Clamp , Veias Pulmonares/metabolismo , Sódio/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(25): 14493-14502, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513685

RESUMO

A high rate of synaptic vesicle (SV) release is required at cerebellar mossy fiber terminals for rapid information processing. As the number of release sites is limited, fast SV reloading is necessary to achieve sustained release. However, rapid reloading has not been observed directly. Here, we visualize SV movements near presynaptic membrane using total internal reflection fluorescence (TIRF) microscopy. Upon stimulation, SVs appeared in the TIRF-field and became tethered to the presynaptic membrane with unexpectedly rapid time course, almost as fast as SVs disappeared due to release. However, such stimulus-induced tethering was abolished by inhibiting exocytosis, suggesting that the tethering is tightly coupled to preceding exocytosis. The newly tethered vesicles became fusion competent not immediately but only 300 ms to 400 ms after tethering. Together with model simulations, we propose that rapid tethering leads to an immediate filling of vacated spaces and release sites within <100 nm of the active zone by SVs, which serve as precursors of readily releasable vesicles, thereby shortening delays during sustained activity.


Assuntos
Cerebelo/fisiologia , Modelos Neurológicos , Fibras Nervosas/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Cerebelo/citologia , Exocitose/fisiologia , Feminino , Microscopia Intravital , Masculino , Microscopia de Fluorescência , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismo , Ratos , Wisteria
12.
Proc Natl Acad Sci U S A ; 117(25): 14503-14511, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513712

RESUMO

The nanoscale co-organization of neurotransmitter receptors facing presynaptic release sites is a fundamental determinant of their coactivation and of synaptic physiology. At excitatory synapses, how endogenous AMPARs, NMDARs, and mGluRs are co-organized inside the synapse and their respective activation during glutamate release are still unclear. Combining single-molecule superresolution microscopy, electrophysiology, and modeling, we determined the average quantity of each glutamate receptor type, their nanoscale organization, and their respective activation. We observed that NMDARs form a unique cluster mainly at the center of the PSD, while AMPARs segregate in clusters surrounding the NMDARs. mGluR5 presents a different organization and is homogenously dispersed at the synaptic surface. From these results, we build a model predicting the synaptic transmission properties of a unitary synapse, allowing better understanding of synaptic physiology.


Assuntos
Modelos Neurológicos , Neurônios/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/fisiologia , Animais , Células Cultivadas , Embrião de Mamíferos , Feminino , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Hipocampo/diagnóstico por imagem , Hipocampo/fisiologia , Microscopia Intravital , Neurônios/ultraestrutura , Técnicas de Patch-Clamp , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Imagem Individual de Molécula
13.
Proc Natl Acad Sci U S A ; 117(25): 14512-14521, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513714

RESUMO

Large-conductance Ca2+ and voltage-activated K+ (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is composed of a voltage sensor domain (VSD), a central pore-gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+ sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+ activations interact, less is known about the mechanisms involved. We explore here these mechanisms by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the Horrigan, Cui, and Aldrich model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+ activation for both Ca2+ bowl and RCK1 Ca2+ sites, suggesting that both high-affinity Ca2+ sites transduce their effect, at least in part, through the αB helix. Mg2+ activation also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to wild type, without other notable differences in the CTD, indicating that structural changes from the mutation were confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca2+ binding and voltage depolarization to pore opening and that shared Ca2+ and voltage transduction pathways involving the αB helix may be involved.


Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Domínios Proteicos/genética , Regulação Alostérica , Animais , Cátions Bivalentes/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/ultraestrutura , Potenciais da Membrana , Mutagênese Sítio-Dirigida , Oócitos , Técnicas de Patch-Clamp , Prolina/genética , Conformação Proteica em alfa-Hélice/genética , Relação Estrutura-Atividade , Xenopus laevis
14.
Nat Commun ; 11(1): 3161, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32572024

RESUMO

Evolutionary theory and behavioral biology suggest that kinship is an organizing principle of social behavior. The neural mechanisms that mediate kinship behavior are, however, not known. Experiments confirm a sibling-approach preference in young rat pups and a sibling-avoidance-preference in older pups. Lesions of the lateral septum eliminate such kin preferences. In vivo juxta-cellular and whole-cell patch-clamp recordings in the lateral septum show multisensory neuronal responses to kin and non-kin stimuli. Non-kin odor-responsive neurons are located dorsally and kin-odor responsive neurons are located ventrally in the lateral septum. With development, the fraction of kin-responsive lateral septal neurons decrease and ongoing firing rates increase. Lesion effects, developmental changes and the ordered representation of response preferences according to kinship-an organization we refer to as nepotopy-point to a key role of the lateral septum in organizing mammalian kinship behavior.


Assuntos
Septo do Cérebro/fisiologia , Comportamento Social , Animais , Comportamento Animal/fisiologia , Masculino , Neurônios , Técnicas de Patch-Clamp , Ratos
15.
Nat Commun ; 11(1): 3208, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32587250

RESUMO

Inner hair cells (IHCs) are the primary receptors for hearing. They are housed in the cochlea and convey sound information to the brain via synapses with the auditory nerve. IHCs have been thought to be electrically and metabolically independent from each other. We report that, upon developmental maturation, in mice 30% of the IHCs are electrochemically coupled in 'mini-syncytia'. This coupling permits transfer of fluorescently-labeled metabolites and macromolecular tracers. The membrane capacitance, Ca2+-current, and resting current increase with the number of dye-coupled IHCs. Dual voltage-clamp experiments substantiate low resistance electrical coupling. Pharmacology and tracer permeability rule out coupling by gap junctions and purinoceptors. 3D electron microscopy indicates instead that IHCs are coupled by membrane fusion sites. Consequently, depolarization of one IHC triggers presynaptic Ca2+-influx at active zones in the entire mini-syncytium. Based on our findings and modeling, we propose that IHC-mini-syncytia enhance sensitivity and reliability of cochlear sound encoding.


Assuntos
Cóclea , Células Ciliadas Auditivas Internas , Audição/fisiologia , Animais , Sinalização do Cálcio , Cóclea/citologia , Cóclea/inervação , Nervo Coclear/metabolismo , Tomografia com Microscopia Eletrônica , Células Gigantes , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/fisiologia , Camundongos , Técnicas de Patch-Clamp , Roedores/fisiologia , Sinapses/metabolismo
16.
PLoS One ; 15(6): e0234080, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479525

RESUMO

In the present study we have characterized the biophysical properties of wild-type (WT) α1ß2 and α3ß2 GABAA receptors and probed the molecular basis for the observed differences. The activation and desensitization behavior and the residual currents of the receptors expressed in HEK293 cells were determined in whole-cell patch clamp recordings. Kinetic parameters of α1ß2 and α3ß2 activation differed significantly, with α1ß2 and α3ß2 exhibiting rise times (10-90%) of 24 ± 2 ms and 51 ± 7 ms, respectively. In contrast, the two receptors exhibited largely comparable desensitization behavior with decay currents that could be fitted to exponential functions with two or three components. Most notably, the two receptor compositions displayed different degrees of desentization, with the residual currents of α1ß2 and α3ß2 constituting 34 ± 2% and 21 ± 2% of the peak current, respectively. The respective contributions of the extracellular domains and the transmembrane/intracellular domains of the α-subunit to these physiological profiles were next assessed in recordings from cells expressing αß2 receptors comprising chimeric α-subunits. The rise times displayed by α1ECD/α3TMDß2 and α3ECD/α1TMDß2 receptors were intermediate to those of WT α1ß2 and WT α3ß2, and the distribution of the different components of the current decays exhibited by the two chimeric receptors followed the same pattern as the two WT receptors. The residual current exhibited by α1ECD/α3TMDß2 (23 ± 3%) was similar to that of α3ß2 but significantly different from that of α1ß2, whereas the residual current displayed by α3ECD/α1TMDß2 (27 ± 2%) was intermediate to and did not differ significantly from either of the WT receptors. This points to molecular differences in the transmembrane/intracellular domains of the α-subunit as the main determinants of the observed differences in receptor physiology between α1ß2 and α3ß2 receptors.


Assuntos
Receptores de GABA-A/metabolismo , Potenciais de Ação/efeitos dos fármacos , Células HEK293 , Humanos , Rim/fisiologia , Cinética , Técnicas de Patch-Clamp , Domínios Proteicos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de GABA-A/genética , Ácido gama-Aminobutírico/farmacologia
17.
PLoS One ; 15(6): e0234114, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32516325

RESUMO

We previously reported that a single 5 ns high intensity electric pulse (NEP) caused an E-field-dependent decrease in peak inward voltage-gated Na+ current (INa) in isolated bovine adrenal chromaffin cells. This study explored the effects of a pair of 5 ns pulses on INa recorded in the same cell type, and how varying the E-field amplitude and interval between the pulses altered its response. Regardless of the E-field strength (5 to 10 MV/m), twin NEPs having interpulse intervals ≥ than 5 s caused the inhibition of TTX-sensitive INa to approximately double relative to that produced by a single pulse. However, reducing the interval from 1 s to 10 ms between twin NEPs at E-fields of 5 and 8 MV/m but not 10 MV/m decreased the magnitude of the additive inhibitory effect by the second pulse in a pair on INa. The enhanced inhibitory effects of twin vs single NEPs on INa were not due to a shift in the voltage-dependence of steady-state activation and inactivation but were associated with a reduction in maximal Na+ conductance. Paradoxically, reducing the interval between twin NEPs at 5 or 8 MV/m but not 10 MV/m led to a progressive interval-dependent recovery of INa, which after 9 min exceeded the level of INa reached following the application of a single NEP. Disrupting lipid rafts by depleting membrane cholesterol with methyl-ß-cyclodextrin enhanced the inhibitory effects of twin NEPs on INa and ablated the progressive recovery of this current at short twin pulse intervals, suggesting a complete dissociation of the inhibitory effects of twin NEPs on this current from their ability to stimulate its recovery. Our results suggest that in contrast to a single NEP, twin NEPs may influence membrane lipid rafts in a manner that enhances the trafficking of newly synthesized and/or recycling of endocytosed voltage-gated Na+ channels, thereby pointing to novel means to regulate ion channels in excitable cells.


Assuntos
Células Cromafins/fisiologia , Eletricidade , Glândulas Suprarrenais/citologia , Animais , Bovinos , Células Cultivadas , Células Cromafins/citologia , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Canais de Sódio Disparados por Voltagem/metabolismo , beta-Ciclodextrinas/farmacologia
18.
J Oral Sci ; 62(3): 265-270, 2020 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-32493863

RESUMO

Orexins (Oxs) are multifunctional neuropeptides, secreted from the lateral hypothalamus, that stimulate feeding behavior and energy expenditure. In this study, the direct effects of Oxs on the membrane properties of trigeminal motoneurons (TMNs) were examined, which critically participate in the genesis of rhythmical oral motor activities underlying suckling and mastication. Sprague-Dawley rats (3-6 day-old) were used to obtain whole-cell patch-clamp recordings from TMNs. Bath application of Ox-A depolarized the membrane potential and induced inward current, wherein Na+ and Ca2+ were charge carriers. Transient receptor potential channel activation potentially contributed to current and voltage responses by way of Ox-A. Ox-A increased the peak amplitude and duration at half-amplitude of the medium-duration after hyperpolarization following the action potential. The interspike frequency of steady-state firings during repetitive discharge was increased, along with a shift in the frequency-current relationship occurring toward the left. Extracellular and intracellular Ca2+ were involved in regulating modulatory effects, but a requisite level of intracellular Ca2+ was not essential for Ox-induced upregulation of the interspike frequency. Ox-A also enhanced conditional bursting induced by N-methyl-d-aspartate and 5-HT, suggesting it participates in modulating TMNs' discharge patterns during various oral motor activities.


Assuntos
Neurônios Motores , Potenciais de Ação , Animais , Potenciais da Membrana , Orexinas , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley
19.
Proc Natl Acad Sci U S A ; 117(25): 14473-14481, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513737

RESUMO

Hypothalamic tanycytes are chemosensitive glial cells that contact the cerebrospinal fluid in the third ventricle and send processes into the hypothalamic parenchyma. To test whether they can activate neurons of the arcuate nucleus, we targeted expression of a Ca2+-permeable channelrhodopsin (CatCh) specifically to tanycytes. Activation of tanycytes ex vivo depolarized orexigenic (neuropeptide Y/agouti-related protein; NPY/AgRP) and anorexigenic (proopiomelanocortin; POMC) neurons via an ATP-dependent mechanism. In vivo, activation of tanycytes triggered acute hyperphagia only in the fed state during the inactive phase of the light-dark cycle.


Assuntos
Núcleo Arqueado do Hipotálamo/fisiopatologia , Células Ependimogliais/fisiologia , Hiperfagia/fisiopatologia , Neurônios/fisiologia , Proteína Relacionada com Agouti/metabolismo , Animais , Apetite/fisiologia , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/diagnóstico por imagem , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Metabolismo Energético/fisiologia , Comportamento Alimentar/fisiologia , Feminino , Genes Reporter , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Rede Nervosa/fisiologia , Neuropeptídeo Y/metabolismo , Imagem Óptica , Optogenética , Técnicas de Patch-Clamp , Pró-Opiomelanocortina/metabolismo , Técnicas Estereotáxicas
20.
Nature ; 582(7812): 443-447, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499642

RESUMO

TWIK-related acid-sensitive potassium (TASK) channels-members of the two pore domain potassium (K2P) channel family-are found in neurons1, cardiomyocytes2-4 and vascular smooth muscle cells5, where they are involved in the regulation of heart rate6, pulmonary artery tone5,7, sleep/wake cycles8 and responses to volatile anaesthetics8-11. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli12-15. Unlike other K2P channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation16. In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K2P channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate-which we designate as an 'X-gate'-created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues (243VLRFMT248) that are essential for responses to volatile anaesthetics10, neurotransmitters13 and G-protein-coupled receptors13. Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders.


Assuntos
Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/química , Anestésicos/farmacologia , Animais , Cristalografia por Raios X , Condutividade Elétrica , Feminino , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Modelos Moleculares , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Xenopus laevis
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