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1.
Nat Commun ; 11(1): 4361, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32868773

RESUMO

The sensory responses of cortical neuronal populations following training have been extensively studied. However, the spike firing properties of individual cortical neurons following training remain unknown. Here, we have combined two-photon Ca2+ imaging and single-cell electrophysiology in awake behaving mice following auditory associative training. We find a sparse set (~5%) of layer 2/3 neurons in the primary auditory cortex, each of which reliably exhibits high-rate prolonged burst firing responses to the trained sound. Such bursts are largely absent in the auditory cortex of untrained mice. Strikingly, in mice trained with different multitone chords, we discover distinct subsets of neurons that exhibit bursting responses specifically to a chord but neither to any constituent tone nor to the other chord. Thus, our results demonstrate an integrated representation of learned complex sounds in a small subset of cortical neurons.


Assuntos
Córtex Auditivo/fisiologia , Percepção Auditiva/fisiologia , Neurônios/fisiologia , Estimulação Acústica/métodos , Córtex Auditivo/citologia , Sinalização do Cálcio , Eletrofisiologia/métodos , Aprendizagem/fisiologia , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neurônios/metabolismo , Análise de Célula Única/métodos
2.
Nature ; 585(7824): 245-250, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32884146

RESUMO

Adaptive behaviour crucially depends on flexible decision-making, which in mammals relies on the frontal cortex, specifically the orbitofrontal cortex (OFC)1-9. How OFC encodes decision variables and instructs sensory areas to guide adaptive behaviour are key open questions. Here we developed a reversal learning task for head-fixed mice, monitored the activity of neurons of the lateral OFC using two-photon calcium imaging and investigated how OFC dynamically interacts with primary somatosensory cortex (S1). Mice learned to discriminate 'go' from 'no-go' tactile stimuli10,11 and adapt their behaviour upon reversal of stimulus-reward contingency ('rule switch'). Imaging individual neurons longitudinally across all behavioural phases revealed a distinct engagement of S1 and lateral OFC, with S1 neural activity reflecting initial task learning, whereas lateral OFC neurons responded saliently and transiently to the rule switch. We identified direct long-range projections from lateral OFC to S1 that can feed this activity back to S1 as value prediction error. This top-down signal updated sensory representations in S1 by functionally remapping responses in a subpopulation of neurons that was sensitive to reward history. Functional remapping crucially depended on top-down feedback as chemogenetic silencing of lateral OFC neurons disrupted reversal learning, as well as plasticity in S1. The dynamic interaction of lateral OFC with sensory cortex thus implements computations critical for value prediction that are history dependent and error based, providing plasticity essential for flexible decision-making.


Assuntos
Plasticidade Neuronal/fisiologia , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/fisiologia , Reversão de Aprendizagem/fisiologia , Córtex Somatossensorial/citologia , Córtex Somatossensorial/fisiologia , Percepção do Tato/fisiologia , Adaptação Psicológica , Animais , Mapeamento Encefálico , Sinalização do Cálcio , Tomada de Decisões/fisiologia , Discriminação Psicológica/fisiologia , Masculino , Camundongos , Estimulação Física , Células Receptoras Sensoriais/metabolismo
3.
Curr Protoc Plant Biol ; 5(3): e20116, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32813335

RESUMO

Elevation of the cytosolic free calcium ion (Ca2+ ) concentration ([Ca2+ ]cyt ) is one of the earliest responses to biotic and abiotic stress in plant cells. Among the various Ca2+ detection systems available, aequorin-based luminescence Ca2+ imaging systems provide a relatively amenable and robust method that facilitates large-scale genetic-mutant screening based on [Ca2+ ]cyt responses. Compared to that mediated by chemical elicitors, mechanical stimulation-induced elevation of [Ca2+ ]cyt is considerably more rapid, occurring within 10 s following stimulation. Therefore, its assessment using aequorin-based Ca2+ imaging systems represents a notable challenge, given that a time interval of ≥1 min is required to reduce the background light before operating the photon imaging detector. In this context, we designed a device that can rotate automatically within the confines of an enclosed dark box, and using this, we can record [Ca2+ ]cyt dynamics immediately after plants had been rotated to induce mechanical stimulation. This tool can facilitate the study of perception and early signal transduction in response to mechanical stimulation on a large scale based on [Ca2+ ]cyt responses. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Detection of background luminance signals in aequorin-transformed Arabidopsis seedlings using a photon imaging detector Basic Protocol 2: Construction of the rotatory device Basic Protocol 3: Calcium measurement in Arabidopsis seedlings after rotatory stimulation Basic Protocol 4: Data analysis and processing.


Assuntos
Equorina , Arabidopsis , Cálcio , Sinalização do Cálcio , Citosol
4.
Nat Commun ; 11(1): 4276, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848151

RESUMO

The structural organization of excitatory inputs supporting spike-timing-dependent plasticity (STDP) remains unknown. We performed a spine STDP protocol using two-photon (2P) glutamate uncaging (pre) paired with postsynaptic spikes (post) in layer 5 pyramidal neurons from juvenile mice. Here we report that pre-post pairings that trigger timing-dependent LTP (t-LTP) produce shrinkage of the activated spine neck and increase in synaptic strength; and post-pre pairings that trigger timing-dependent LTD (t-LTD) decrease synaptic strength without affecting spine shape. Furthermore, the induction of t-LTP with 2P glutamate uncaging in clustered spines (<5 µm apart) enhances LTP through a NMDA receptor-mediated spine calcium accumulation and actin polymerization-dependent neck shrinkage, whereas t-LTD was dependent on NMDA receptors and disrupted by the activation of clustered spines but recovered when separated by >40 µm. These results indicate that synaptic cooperativity disrupts t-LTD and extends the temporal window for the induction of t-LTP, leading to STDP only encompassing LTP.


Assuntos
Espinhas Dendríticas/fisiologia , Plasticidade Neuronal/fisiologia , Potenciais de Ação/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Técnicas In Vitro , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica , Modelos Neurológicos , Células Piramidais/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia
5.
Proc Natl Acad Sci U S A ; 117(33): 20088-20099, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32732436

RESUMO

T lymphocyte motility and interaction dynamics with other immune cells are vital determinants of immune responses. Regulatory T (Treg) cells prevent autoimmune disorders by suppressing excessive lymphocyte activity, but how interstitial motility patterns of Treg cells limit neuroinflammation is not well understood. We used two-photon microscopy to elucidate the spatial organization, motility characteristics, and interactions of endogenous Treg and Th17 cells together with antigen-presenting cells (APCs) within the spinal cord leptomeninges in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Th17 cells arrive before the onset of clinical symptoms, distribute uniformly during the peak, and decline in numbers during later stages of EAE. In contrast, Treg cells arrive after Th17 cells and persist during the chronic phase. Th17 cells meander widely, interact with APCs, and exhibit cytosolic Ca2+ transients and elevated basal Ca2+ levels before the arrival of Treg cells. In contrast, Treg cells adopt a confined, repetitive-scanning motility while contacting APCs. These locally confined but highly motile Treg cells limit Th17 cells from accessing APCs and suppress Th17 cell Ca2+ signaling by a mechanism that is upstream of store-operated Ca2+ entry. Finally, Treg cell depletion increases APC numbers in the spinal cord and exaggerates ongoing neuroinflammation. Our results point to fundamental differences in motility characteristics between Th17 and Treg cells in the inflamed spinal cord and reveal three potential cellular mechanisms by which Treg cells regulate Th17 cell effector functions: reduction of APC density, limiting access of Th17 cells to APCs, and suppression of Th17 Ca2+ signaling.


Assuntos
Sinalização do Cálcio/fisiologia , Medula Espinal/metabolismo , Células Th17/metabolismo , Animais , Autoantígenos , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Fluorescência Verde , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Bainha de Mielina , Linfócitos T Reguladores
7.
Chin J Physiol ; 63(4): 187-194, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32859886

RESUMO

Thioridazine, belonging to first-generation antipsychotic drugs, is a prescription used to treat schizophrenia. However, the effect of thioridazine on intracellular Ca2+ concentration ([Ca2+]i) and viability in human liver cancer cells is unclear. This study examined whether thioridazine altered Ca2+ signaling and viability in HepG2 human hepatocellular carcinoma cells. Ca2+ concentrations in suspended cells were measured using the fluorescent Ca2+-sensitive dye fura-2. Cell viability was examined by WST-1 assay. Thioridazine at concentrations of 25-100 µM induced [Ca2+]i rises. Ca2+ removal reduced the signal by 20%. Thioridazine (100 µM) induced Mn2+ influx suggesting of Ca2+ entry. Thioridazine-induced Ca2+ entry was inhibited by 20% by protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate) and inhibitor (GF109203X) and by three inhibitors of store-operated Ca2+ channels: nifedipine, econazole, and SKF96365. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) abolished thioridazine-evoked [Ca2+]i rises. On the other hand, thioridazine preincubation completely inhibited the [Ca2+]i rises induced by TG. Furthermore, U73122 totally suppressed the [Ca2+]i rises induced by thioridazine via inhibition of phospholipase C (PLC). Regarding cytotoxicity, at 30-80 µM, thioridazine reduced cell viability in a concentration-dependent fashion. This cytotoxicity was not prevented by preincubation with 1,2-bis (2-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM) (a Ca2+ chelator). To conclude, thioridazine caused concentration-dependent [Ca2+]i rises in HepG2 human hepatoma cells by inducing Ca2+ release from the endoplasmic reticulum via PLC-associated pathways and Ca2+ influx from extracellular medium through PKC-sensitive store-operated Ca2+ entry. In addition, thioridazine induced cytotoxicity in a Ca2+-independent manner.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Apoptose , Cálcio , Sinalização do Cálcio , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Tioridazina , Fosfolipases Tipo C
8.
Nature ; 585(7823): 91-95, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32788726

RESUMO

Signalling between cells of the neurovascular unit, or neurovascular coupling, is essential to match local blood flow with neuronal activity. Pericytes interact with endothelial cells and extend processes that wrap capillaries, covering up to 90% of their surface area1,2. Pericytes are candidates to regulate microcirculatory blood flow because they are strategically positioned along capillaries, contain contractile proteins and respond rapidly to neuronal stimulation3,4, but whether they synchronize microvascular dynamics and neurovascular coupling within a capillary network was unknown. Here we identify nanotube-like processes that connect two bona fide pericytes on separate capillary systems, forming a functional network in the mouse retina, which we named interpericyte tunnelling nanotubes (IP-TNTs). We provide evidence that these (i) have an open-ended proximal side and a closed-ended terminal (end-foot) that connects with distal pericyte processes via gap junctions, (ii) carry organelles including mitochondria, which can travel along these processes, and (iii) serve as a conduit for intercellular Ca2+ waves, thus mediating communication between pericytes. Using two-photon microscope live imaging, we demonstrate that retinal pericytes rely on IP-TNTs to control local neurovascular coupling and coordinate light-evoked responses between adjacent capillaries. IP-TNT damage following ablation or ischaemia disrupts intercellular Ca2+ waves, impairing blood flow regulation and neurovascular coupling. Notably, pharmacological blockade of Ca2+ influx preserves IP-TNTs, rescues light-evoked capillary responses and restores blood flow after reperfusion. Our study thus defines IP-TNTs and characterizes their critical role in regulating neurovascular coupling in the living retina under both physiological and pathological conditions.


Assuntos
Nanotubos , Acoplamento Neurovascular , Pericitos/metabolismo , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Cálcio/metabolismo , Sinalização do Cálcio , Capilares/fisiopatologia , Capilares/efeitos da radiação , Comunicação Celular , Feminino , Junções Comunicantes/metabolismo , Hemodinâmica , Masculino , Camundongos , Mitocôndrias/metabolismo , Acoplamento Neurovascular/fisiologia , Pericitos/citologia , Pericitos/patologia , Retina/citologia , Retina/patologia
9.
PLoS Genet ; 16(8): e1008505, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776934

RESUMO

Dynamic gene expression in neurons shapes fundamental processes in the nervous systems of animals. However, how neuronal activation by different stimuli can lead to distinct transcriptional responses is not well understood. We have been studying how microbial metabolites modulate gene expression in chemosensory neurons of Caenorhabditis elegans. Considering the diverse environmental stimuli that can activate chemosensory neurons of C. elegans, we sought to understand how specific transcriptional responses can be generated in these neurons in response to distinct cues. We have focused on the mechanism of rapid (<6 min) and selective transcriptional induction of daf-7, a gene encoding a TGF-ß ligand, in the ASJ chemosensory neurons in response to the pathogenic bacterium Pseudomonas aeruginosa. DAF-7 is required for the protective behavioral avoidance of P. aeruginosa by C. elegans. Here, we define the involvement of two distinct cyclic GMP (cGMP)-dependent pathways that are required for daf-7 expression in the ASJ neuron pair in response to P. aeruginosa. We show that a calcium-independent pathway dependent on the cGMP-dependent protein kinase G (PKG) EGL-4, and a canonical calcium-dependent signaling pathway dependent on the activity of a cyclic nucleotide-gated channel subunit CNG-2, function in parallel to activate rapid, selective transcription of daf-7 in response to P. aeruginosa metabolites. Our data suggest that fast, selective early transcription of neuronal genes require PKG in shaping responses to distinct microbial stimuli in a pair of C. elegans chemosensory neurons.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Células Quimiorreceptoras/metabolismo , GMP Cíclico/metabolismo , Pseudomonas aeruginosa/metabolismo , Fator de Crescimento Transformador beta/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Sinalização do Cálcio , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Ativação Transcricional , Fator de Crescimento Transformador beta/metabolismo
10.
PLoS Genet ; 16(8): e1008644, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776941

RESUMO

Correct regulation of cell contractility is critical for the function of many biological systems. The reproductive system of the hermaphroditic nematode C. elegans contains a contractile tube of myoepithelial cells known as the spermatheca, which stores sperm and is the site of oocyte fertilization. Regulated contraction of the spermatheca pushes the embryo into the uterus. Cell contractility in the spermatheca is dependent on actin and myosin and is regulated, in part, by Ca2+ signaling through the phospholipase PLC-1, which mediates Ca2+ release from the endoplasmic reticulum. Here, we describe a novel role for GSA-1/Gαs, and protein kinase A, composed of the catalytic subunit KIN-1/PKA-C and the regulatory subunit KIN-2/PKA-R, in the regulation of Ca2+ release and contractility in the C. elegans spermatheca. Without GSA-1/Gαs or KIN-1/PKA-C, Ca2+ is not released, and oocytes become trapped in the spermatheca. Conversely, when PKA is activated through either a gain of function allele in GSA-1 (GSA-1(GF)) or by depletion of KIN-2/PKA-R, the transit times and total numbers, although not frequencies, of Ca2+ pulses are increased, and Ca2+ propagates across the spermatheca even in the absence of oocyte entry. In the spermathecal-uterine valve, loss of GSA-1/Gαs or KIN-1/PKA-C results in sustained, high levels of Ca2+ and a loss of coordination between the spermathecal bag and sp-ut valve. Additionally, we show that depleting phosphodiesterase PDE-6 levels alters contractility and Ca2+ dynamics in the spermatheca, and that the GPB-1 and GPB-2 Gß subunits play a central role in regulating spermathecal contractility and Ca2+ signaling. This work identifies a signaling network in which Ca2+ and cAMP pathways work together to coordinate spermathecal contractions for successful ovulations.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Sinalização do Cálcio , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Contração Muscular , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Mutação com Ganho de Função , Células Musculares/metabolismo , Células Musculares/fisiologia , Oócitos/fisiologia
11.
PLoS Genet ; 16(8): e1008925, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32790785

RESUMO

Taste receptor cells use multiple signaling pathways to detect chemicals in potential food items. These cells are functionally grouped into different types: Type I cells act as support cells and have glial-like properties; Type II cells detect bitter, sweet, and umami taste stimuli; and Type III cells detect sour and salty stimuli. We have identified a new population of taste cells that are broadly tuned to multiple taste stimuli including bitter, sweet, sour, and umami. The goal of this study was to characterize these broadly responsive (BR) taste cells. We used an IP3R3-KO mouse (does not release calcium (Ca2+) from internal stores in Type II cells when stimulated with bitter, sweet, or umami stimuli) to characterize the BR cells without any potentially confounding input from Type II cells. Using live cell Ca2+ imaging in isolated taste cells from the IP3R3-KO mouse, we found that BR cells are a subset of Type III cells that respond to sour stimuli but also use a PLCß signaling pathway to respond to bitter, sweet, and umami stimuli. Unlike Type II cells, individual BR cells are broadly tuned and respond to multiple stimuli across different taste modalities. Live cell imaging in a PLCß3-KO mouse confirmed that BR cells use this signaling pathway to respond to bitter, sweet, and umami stimuli. Short term behavioral assays revealed that BR cells make significant contributions to taste driven behaviors and found that loss of either PLCß3 in BR cells or IP3R3 in Type II cells caused similar behavioral deficits to bitter, sweet, and umami stimuli. Analysis of c-Fos activity in the nucleus of the solitary tract (NTS) also demonstrated that functional Type II and BR cells are required for normal stimulus induced expression.


Assuntos
Papilas Gustativas/citologia , Paladar , Vias Aferentes/citologia , Animais , Sinalização do Cálcio , Células Cultivadas , Feminino , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipase C beta/metabolismo , Núcleo Solitário/citologia , Núcleo Solitário/metabolismo , Núcleo Solitário/fisiologia , Papilas Gustativas/metabolismo , Papilas Gustativas/fisiologia , Percepção Gustatória
12.
Nat Commun ; 11(1): 3240, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632168

RESUMO

Astrocytic Ca2+ signaling has been intensively studied in health and disease but has not been quantified during natural sleep. Here, we employ an activity-based algorithm to assess astrocytic Ca2+ signals in the neocortex of awake and naturally sleeping mice while monitoring neuronal Ca2+ activity, brain rhythms and behavior. We show that astrocytic Ca2+ signals exhibit distinct features across the sleep-wake cycle and are reduced during sleep compared to wakefulness. Moreover, an increase in astrocytic Ca2+ signaling precedes transitions from slow wave sleep to wakefulness, with a peak upon awakening exceeding the levels during whisking and locomotion. Finally, genetic ablation of an important astrocytic Ca2+ signaling pathway impairs slow wave sleep and results in an increased number of microarousals, abnormal brain rhythms, and an increased frequency of slow wave sleep state transitions and sleep spindles. Our findings demonstrate an essential role for astrocytic Ca2+ signaling in regulating slow wave sleep.


Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio , Sono de Ondas Lentas/fisiologia , Animais , Camundongos , Vigília/fisiologia
13.
PLoS Comput Biol ; 16(7): e1008048, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658888

RESUMO

Heart failure (HF) is associated with an increased propensity for atrial fibrillation (AF), causing higher mortality than AF or HF alone. It is hypothesized that HF-induced remodelling of atrial cellular and tissue properties promotes the genesis of atrial action potential (AP) alternans and conduction alternans that perpetuate AF. However, the mechanism underlying the increased susceptibility to atrial alternans in HF remains incompletely elucidated. In this study, we investigated the effects of how HF-induced atrial cellular electrophysiological (with prolonged AP duration) and tissue structural (reduced cell-to-cell coupling caused by atrial fibrosis) remodelling can have an effect on the generation of atrial AP alternans and their conduction at the cellular and one-dimensional (1D) tissue levels. Simulation results showed that HF-induced atrial electrical remodelling prolonged AP duration, which was accompanied by an increased sarcoplasmic reticulum (SR) Ca2+ content and Ca2+ transient amplitude. Further analysis demonstrated that HF-induced atrial electrical remodelling increased susceptibility to atrial alternans mainly due to the increased sarcoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ reuptake, modulated by increased phospholamban (PLB) phosphorylation, and the decreased transient outward K+ current (Ito). The underlying mechanism has been suggested that the increased SR Ca2+ content and prolonged AP did not fully recover to their previous levels at the end of diastole, resulting in a smaller SR Ca2+ release and AP in the next beat. These produced Ca2+ transient alternans and AP alternans, and further caused AP alternans and Ca2+ transient alternans through Ca2+→AP coupling and AP→Ca2+ coupling, respectively. Simulation of a 1D tissue model showed that the combined action of HF-induced ion channel remodelling and a decrease in cell-to-cell coupling due to fibrosis increased the heart tissue's susceptibility to the formation of spatially discordant alternans, resulting in an increased functional AP propagation dispersion, which is pro-arrhythmic. These findings provide insights into how HF promotes atrial arrhythmia in association with atrial alternans.


Assuntos
Remodelamento Atrial , Insuficiência Cardíaca/fisiopatologia , Potenciais de Ação , Algoritmos , Animais , Fibrilação Atrial/fisiopatologia , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Simulação por Computador , Cães , Condutividade Elétrica , Átrios do Coração/fisiopatologia , Ventrículos do Coração/fisiopatologia , Humanos , Camundongos , Modelos Cardiovasculares , Contração Miocárdica , Miócitos Cardíacos/patologia , Fosforilação , Retículo Sarcoplasmático/metabolismo
14.
PLoS Comput Biol ; 16(7): e1008015, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32678848

RESUMO

Calmodulin-dependent kinase II (CaMKII) has long been known to play an important role in learning and memory as well as long term potentiation (LTP). More recently it has been suggested that it might be involved in the time averaging of synaptic signals, which can then lead to the high precision of information stored at a single synapse. However, the role of the scaffolding molecule, neurogranin (Ng), in governing the dynamics of CaMKII is not yet fully understood. In this work, we adopt a rule-based modeling approach through the Monte Carlo method to study the effect of Ca2+ signals on the dynamics of CaMKII phosphorylation in the postsynaptic density (PSD). Calcium surges are observed in synaptic spines during an EPSP and back-propagating action potential due to the opening of NMDA receptors and voltage dependent calcium channels. Using agent-based models, we computationally investigate the dynamics of phosphorylation of CaMKII monomers and dodecameric holoenzymes. The scaffolding molecule, Ng, when present in significant concentration, limits the availability of free calmodulin (CaM), the protein which activates CaMKII in the presence of calcium. We show that Ng plays an important modulatory role in CaMKII phosphorylation following a surge of high calcium concentration. We find a non-intuitive dependence of this effect on CaM concentration that results from the different affinities of CaM for CaMKII depending on the number of calcium ions bound to the former. It has been shown previously that in the absence of phosphatase, CaMKII monomers integrate over Ca2+ signals of certain frequencies through autophosphorylation (Pepke et al, Plos Comp. Bio., 2010). We also study the effect of multiple calcium spikes on CaMKII holoenzyme autophosphorylation, and show that in the presence of phosphatase, CaMKII behaves as a leaky integrator of calcium signals, a result that has been recently observed in vivo. Our models predict that the parameters of this leaky integrator are finely tuned through the interactions of Ng, CaM, CaMKII, and PP1, providing a mechanism to precisely control the sensitivity of synapses to calcium signals. Author Summary not valid for PLOS ONE submissions.


Assuntos
Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Neurogranina/metabolismo , Potenciais de Ação , Animais , Área Sob a Curva , Biologia Computacional , Simulação por Computador , Potenciação de Longa Duração , Camundongos , Método de Monte Carlo , Plasticidade Neuronal , Fosforilação , Densidade Pós-Sináptica/metabolismo , Ligação Proteica , Receptores de N-Metil-D-Aspartato/metabolismo , Software , Sinapses/fisiologia , Fatores de Tempo
15.
PLoS Comput Biol ; 16(7): e1008078, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32701987

RESUMO

Animals remember temporal links between their actions and subsequent rewards. We previously discovered a synaptic mechanism underlying such reward learning in D1 receptor (D1R)-expressing spiny projection neurons (D1 SPN) of the striatum. Dopamine (DA) bursts promote dendritic spine enlargement in a time window of only a few seconds after paired pre- and post-synaptic spiking (pre-post pairing), which is termed as reinforcement plasticity (RP). The previous study has also identified underlying signaling pathways; however, it still remains unclear how the signaling dynamics results in RP. In the present study, we first developed a computational model of signaling dynamics of D1 SPNs. The D1 RP model successfully reproduced experimentally observed protein kinase A (PKA) activity, including its critical time window. In this model, adenylate cyclase type 1 (AC1) in the spines/thin dendrites played a pivotal role as a coincidence detector against pre-post pairing and DA burst. In particular, pre-post pairing (Ca2+ signal) stimulated AC1 with a delay, and the Ca2+-stimulated AC1 was activated by the DA burst for the asymmetric time window. Moreover, the smallness of the spines/thin dendrites is crucial to the short time window for the PKA activity. We then developed a RP model for D2 SPNs, which also predicted the critical time window for RP that depended on the timing of pre-post pairing and phasic DA dip. AC1 worked for the coincidence detector in the D2 RP model as well. We further simulated the signaling pathway leading to Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation and clarified the role of the downstream molecules of AC1 as the integrators that turn transient input signals into persistent spine enlargement. Finally, we discuss how such timing windows guide animals' reward learning.


Assuntos
Sinalização do Cálcio , Corpo Estriado/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Dopamina/fisiologia , Aprendizagem , Plasticidade Neuronal , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Simulação por Computador , Dendritos/fisiologia , Espinhas Dendríticas/fisiologia , Cinética , Camundongos , Neurônios/fisiologia , Receptores de Dopamina D2 , Recompensa
16.
Proc Natl Acad Sci U S A ; 117(30): 18068-18078, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661165

RESUMO

Mast cells and basophils are main drivers of allergic reactions and anaphylaxis, for which prevalence is rapidly increasing. Activation of these cells leads to a tightly controlled release of inflammatory mediators stored in secretory granules. The release of these granules is dependent on intracellular calcium (Ca2+) signals. Ca2+ release from endolysosomal compartments is mediated via intracellular cation channels, such as two-pore channel (TPC) proteins. Here, we uncover a mechanism for how TPC1 regulates Ca2+ homeostasis and exocytosis in mast cells in vivo and ex vivo. Notably, in vivo TPC1 deficiency in mice leads to enhanced passive systemic anaphylaxis, reflected by increased drop in body temperature, most likely due to accelerated histamine-induced vasodilation. Ex vivo, mast cell-mediated histamine release and degranulation was augmented upon TPC1 inhibition, although mast cell numbers and size were diminished. Our results indicate an essential role of TPC1 in endolysosomal Ca2+ uptake and filling of endoplasmic reticulum Ca2+ stores, thereby regulating exocytosis in mast cells. Thus, pharmacological modulation of TPC1 might blaze a trail to develop new drugs against mast cell-related diseases, including allergic hypersensitivity.


Assuntos
Anafilaxia/etiologia , Anafilaxia/metabolismo , Canais de Cálcio/deficiência , Suscetibilidade a Doenças , Mastócitos/imunologia , Mastócitos/metabolismo , Biomarcadores , Sinalização do Cálcio , Degranulação Celular , Citocinas/metabolismo , Predisposição Genética para Doença , Histamina/metabolismo , Imunoglobulina E/imunologia , Mediadores da Inflamação/metabolismo
17.
Nat Commun ; 11(1): 3527, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669538

RESUMO

Ca2+ signaling in pulmonary arterial smooth muscle cells (PASMCs) plays an important role in pulmonary hypertension (PH). However, the underlying specific ion channel mechanisms remain largely unknown. Here, we report ryanodine receptor (RyR) channel activity and Ca2+ release both are increased, and association of RyR2 by FK506 binding protein 12.6 (FKBP12.6) is decreased in PASMCs from mice with chronic hypoxia (CH)-induced PH. Smooth muscle cell (SMC)-specific RyR2 knockout (KO) or Rieske iron-sulfur protein (RISP) knockdown inhibits the altered Ca2+ signaling, increased nuclear factor (NF)-κB/cyclin D1 activation and cell proliferation, and CH-induced PH in mice. FKBP12.6 KO or FK506 treatment enhances CH-induced PH, while S107 (a specific stabilizer of RyR2/FKBP12.6 complex) produces an opposite effect. In conclusion, CH causes RISP-dependent ROS generation and FKBP12.6/RyR2 dissociation, leading to PH. RISP inhibition, RyR2/FKBP12.6 complex stabilization and Ca2+ release blockade may be potentially beneficial for the treatment of PH.


Assuntos
Ciclina D1/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Hipertensão Pulmonar/metabolismo , NF-kappa B/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Sinalização do Cálcio , Proliferação de Células , Citosol/metabolismo , Humanos , Hipóxia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Miócitos de Músculo Liso/metabolismo , Oxigênio/metabolismo , Artéria Pulmonar/patologia , Espécies Reativas de Oxigênio/metabolismo , Transtornos Respiratórios/metabolismo , Transdução de Sinais
18.
Nat Commun ; 11(1): 3624, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681036

RESUMO

TRIM family proteins play integral roles in the innate immune response to virus infection. MG53 (TRIM72) is essential for cell membrane repair and is believed to be a muscle-specific TRIM protein. Here we show human macrophages express MG53, and MG53 protein expression is reduced following virus infection. Knockdown of MG53 in macrophages leads to increases in type I interferon (IFN) upon infection. MG53 knockout mice infected with influenza virus show comparable influenza virus titres to wild type mice, but display increased morbidity accompanied by more accumulation of CD45+ cells and elevation of IFNß in the lung. We find that MG53 knockdown results in activation of NFκB signalling, which is linked to an increase in intracellular calcium oscillation mediated by ryanodine receptor (RyR). MG53 inhibits IFNß induction in an RyR-dependent manner. This study establishes MG53 as a new target for control of virus-induced morbidity and tissue injury.


Assuntos
Influenza Humana/imunologia , Interferon beta/metabolismo , Proteínas de Membrana/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Animais , Sinalização do Cálcio/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/virologia , Interferon beta/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , RNA Interferente Pequeno , Transdução de Sinais/imunologia , Proteínas com Motivo Tripartido/genética
19.
J Pharmacol Sci ; 144(2): 83-88, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32709559

RESUMO

Astrocytes generate robust intracellular Ca2+ signals that are assumed to be key regulators of astrocytic function. Among various Ca2+ mobilization mechanisms, Ca2+ release from the endoplasmic reticulum (ER) via the inositol 1,4,5-trisphosphate receptor (IP3R) has attracted attention as a major component of astrocytic Ca2+ signaling. Manipulation of astrocytic IP3-Ca2+ signaling, such as genetic deletion of the type 2 IP3R, has revealed multifaceted roles of astrocytic ER Ca2+ release in health and disease. Recent developments in Ca2+ imaging techniques including ER intraluminal Ca2+ imaging have been indispensable in determining the physiological and pathophysiological significance of astrocytic ER Ca2+ release via IP3Rs. Beneficial and detrimental roles of IP3R-dependent Ca2+ release in astrocytes have been revealed in wide variety of disorders in the brain, strongly suggesting astrocytic IP3-Ca2+ signaling as a novel and promising therapeutic target.


Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Animais , Encefalopatias/etiologia , Encefalopatias/genética , Encefalopatias/terapia , Deleção de Genes , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Imagem Molecular , Terapia de Alvo Molecular
20.
Proc Natl Acad Sci U S A ; 117(31): 18849-18857, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690691

RESUMO

One of the major events of early plant immune responses is a rapid influx of Ca2+ into the cytosol following pathogen recognition. Indeed, changes in cytosolic Ca2+ are recognized as ubiquitous elements of cellular signaling networks and are thought to encode stimulus-specific information in their duration, amplitude, and frequency. Despite the wealth of observations showing that the bacterial elicitor peptide flg22 triggers Ca2+ transients, there remain limited data defining the molecular identities of Ca2+ transporters involved in shaping the cellular Ca2+ dynamics during the triggering of the defense response network. However, the autoinhibited Ca2+-ATPase (ACA) pumps that act to expel Ca2+ from the cytosol have been linked to these events, with knockouts in the vacuolar members of this family showing hypersensitive lesion-mimic phenotypes. We have therefore explored how the two tonoplast-localized pumps, ACA4 and ACA11, impact flg22-dependent Ca2+ signaling and related defense responses. The double-knockout aca4/11 exhibited increased basal Ca2+ levels and Ca2+ signals of higher amplitude than wild-type plants. Both the aberrant Ca2+ dynamics and associated defense-related phenotypes could be suppressed by growing the aca4/11 seedlings at elevated temperatures. Relocalization of ACA8 from its normal cellular locale of the plasma membrane to the tonoplast also suppressed the aca4/11 phenotypes but not when a catalytically inactive mutant was used. These observations indicate that regulation of vacuolar Ca2+ sequestration is an integral component of plant immune signaling, but also that the action of tonoplast-localized Ca2+ pumps does not require specific regulatory elements not found in plasma membrane-localized pumps.


Assuntos
Proteínas de Arabidopsis , Sinalização do Cálcio/fisiologia , ATPases Transportadoras de Cálcio , Cálcio/metabolismo , Imunidade Vegetal/fisiologia , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/metabolismo , Vacúolos/metabolismo
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