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1.
Adv Exp Med Biol ; 1131: 73-91, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646507

RESUMO

Imaging techniques may overcome the limitations of electrode techniques to measure locally not only membrane potential changes, but also ionic currents. Here, we review a recently developed approach to image native neuronal Ca2+ currents from brain slices. The technique is based on combined fluorescence recordings using low-affinity Ca2+ indicators possibly in combination with voltage sensitive dyes. We illustrate how the kinetics of a Ca2+ current can be estimated from the Ca2+ fluorescence change and locally correlated with the change of membrane potential, calibrated on an absolute scale, from the voltage fluorescence change. We show some representative measurements from the dendrites of CA1 hippocampal pyramidal neurons, from olfactory bulb mitral cells and from cerebellar Purkinje neurons. We discuss the striking difference in data analysis and interpretation between Ca2+ current measurements obtained using classical electrode techniques and the physiological currents obtained using this novel approach. Finally, we show how important is the kinetic information on the native Ca2+ current to explore the potential molecular targets of the Ca2+ flux from each individual Ca2+ channel.


Assuntos
Canais de Cálcio , Neuroimagem , Animais , Cálcio/metabolismo , Canais de Cálcio/fisiologia , Dendritos/fisiologia , Humanos , Potenciais da Membrana/fisiologia , Imagem Óptica , Células Piramidais/fisiologia
2.
Sci Rep ; 8(1): 4155, 2018 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-29515177

RESUMO

AMPA receptor (AMPAR) trafficking is a key determinant of synaptic strength and synaptic plasticity. Under basal conditions, constitutive trafficking maintains surface AMPARs by internalization into the endosomal system, where the majority are sorted and targeted for recycling back to the plasma membrane. NMDA receptor (NMDAR)-dependent Long-Term Depression (LTD) is characterised by a reduction in synaptic strength, and involves endosomal sorting of AMPARs away from recycling pathways to lysosomes. The mechanisms that determine whether AMPARs are trafficked to lysosomes or to recycling endosomes, especially in response to NMDAR stimulation, are unclear. Here, we define a role for the actin-regulatory protein cortactin as a mediator of AMPAR endosomal sorting by direct interaction with the GluA2 subunit. Disrupting GluA2-cortactin binding in neurons causes the targeting of GluA2/A3-containing receptors to lysosomes and their consequent degradation, resulting in a loss of surface and synaptic GluA2 under basal conditions and an occlusion of subsequent LTD expression. Furthermore, we show that NMDAR stimulation causes a dissociation of endogenous cortactin from GluA2 via tyrosine phosphorylation of cortactin. These results demonstrate that cortactin maintains GluA2/A3 levels by directing receptors away from lysosomes, and that disrupting GluA2-cortactin interactions to target GluA2/A3 to lysosomes is an essential component of LTD expression.

3.
J Neurosci Methods ; 268: 66-77, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27163479

RESUMO

BACKGROUND: Fast Ca(2+) imaging using low-affinity fluorescent indicators allows tracking Ca(2+) neuronal influx at high temporal resolution. In some systems, where the Ca(2+)-bound indicator is linear with Ca(2+) entering the cell, the Ca(2+) current has same kinetics of the fluorescence time derivative. In other systems, like cerebellar Purkinje neuron dendrites, the time derivative strategy fails since fluorescence kinetics is affected by Ca(2+) binding proteins sequestering Ca(2+) from the indicator. NEW METHOD: Our novel method estimates the kinetics of the Ca(2+) current in cells where the time course of fluorescence is not linear with Ca(2+) influx. The method is based on a two-buffer and two-indicator model, with three free parameters, where Ca(2+) sequestration from the indicator is mimicked by Ca(2+)-binding to the slower buffer. We developed a semi-automatic protocol to optimise the free parameters and the kinetics of the input current to match the experimental fluorescence change with the simulated curve of the Ca(2+)-bound indicator. RESULTS: We show that the optimised input current is a good estimate of the real Ca(2+) current by validating the method both using computer simulations and data from real neurons. We report the first estimates of Ca(2+) currents associated with climbing fibre excitatory postsynaptic potentials in Purkinje neurons. COMPARISON WITH EXISTING METHODS: The present method extends the possibility of studying Ca(2+) currents in systems where the existing time derivative approach fails. CONCLUSIONS: The information available from our technique allows investigating the physiological behaviour of Ca(2+) channels under all possible conditions.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Potenciais da Membrana/fisiologia , Neurônios/metabolismo , Processamento de Sinais Assistido por Computador , Imagens com Corantes Sensíveis à Voltagem/métodos , Animais , Cerebelo/citologia , Cerebelo/metabolismo , Simulação por Computador , Hipocampo/citologia , Hipocampo/metabolismo , Cinética , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Neurônios/citologia , Dinâmica não Linear , Reconhecimento Automatizado de Padrão/métodos , Técnicas de Cultura de Tecidos
4.
J Physiol ; 594(4): 967-83, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26634988

RESUMO

KEY POINTS: In neurons, the Ca(2+) signal associated with the dendritic back-propagating action potential codes a chemical message to the different dendritic sites, playing a crucial role in electrical signalling, synaptic transmission and synaptic plasticity. The study of the underlying Ca(2+) current, mediated by different types of voltage-gated Ca(2+) channels, cannot be achieved by using the patch clamp technique. In this article, we used a recently developed cutting-edge optical technique to investigate the physiological behaviour of local Ca(2+) currents along the apical dendrite of CA1 hippocampal pyramidal neurons. We directly measure, for the first time, the synergistic activation and deactivation of the diverse dendritic voltage-gated Ca(2+) channels operating during bursts of back-propagating action potentials to precisely control the Ca(2+) signal. We demonstrate that the Ca(2+) loss via high-voltage-activated channels is compensated by the Ca(2+) entry via the other channels translating in high fidelity of Ca(2+) signalling. ABSTRACT: In CA1 hippocampal pyramidal neurons, the dendritic Ca(2+) signal associated with somatic firing represents a fundamental activation code for several proteins. This signal, mediated by voltage-gated Ca(2+) channels (VGCCs), varies along the dendrites. In this study, using a recent optical technique based on the low-affinity indicator Oregon Green 488 BAPTA-5N, we analysed how activation and deactivation of VGCCs produced by back-propagating action potentials (bAPs) along the apical dendrite shape the Ca(2+) signal at different locations in CA1 hippocampal pyramidal neurons of the mouse. We measured, at multiple dendritic sites, the Ca(2+) transients and the changes in membrane potential associated with bAPs at 50 µs temporal resolution and we estimated the kinetics of the Ca(2+) current. We found that during somatic bursts, the bAPs decrease in amplitude along the apical dendrite but the amplitude of the associated Ca(2+) signal in the initial 200 µm dendritic segment does not change. Using a detailed pharmacological analysis, we demonstrate that this effect is due to the perfect compensation of the loss of Ca(2+) via high-voltage-activated (HVA) VGCCs by a larger Ca(2+) component via low-voltage-activated (LVA) VGCCs, revealing a mechanism coupling the two VGCC families of K(+) channels. More distally, where the bAP does not activate HVA-VGCCs, the Ca(2+) signal is variable during the burst. Thus, we demonstrate that HVA- and LVA-VGCCs operate synergistically to stabilise Ca(2+) signals associated with bAPs in the most proximal 200 µm dendritic segment.


Assuntos
Potenciais de Ação , Região CA1 Hipocampal/metabolismo , Canais de Cálcio/metabolismo , Dendritos/metabolismo , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/fisiologia , Dendritos/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Canais de Potássio , Células Piramidais/metabolismo , Células Piramidais/fisiologia
5.
Adv Exp Med Biol ; 859: 103-25, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26238050

RESUMO

Membrane potential imaging using voltage-sensitive dyes can be combined with other optical techniques for a variety of applications. Combining voltage imaging with Ca2+ imaging allows correlating membrane potential changes with intracellular Ca2+ signals or with Ca2+ currents. Combining voltage imaging with uncaging techniques allows analyzing electrical signals elicited by photorelease of a particular molecule. This approach is also a useful tool to calibrate the change in fluorescence intensity in terms of membrane potential changes from different sites permitting spatial mapping of electrical activity. Finally, combining voltage imaging with optogenetics, in particular with channelrhodopsin stimulation, opens the gate to novel investigations of brain circuitries by allowing measurements of synaptic signals mediated by specific sets of neurons. Here we describe in detail the methods of membrane potential imaging in combination with other optical techniques and discus some important applications.


Assuntos
Sinalização do Cálcio/fisiologia , Corantes Fluorescentes/química , Potenciais da Membrana/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Cálcio/metabolismo , Channelrhodopsins , Ácido Glutâmico/metabolismo , Camundongos , Rede Nervosa/fisiologia , Rede Nervosa/ultraestrutura , Neurônios/ultraestrutura , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Optogenética/instrumentação , Optogenética/métodos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Sinapses/ultraestrutura , Imagens com Corantes Sensíveis à Voltagem/instrumentação , Imagens com Corantes Sensíveis à Voltagem/métodos
6.
Neurophotonics ; 2(2): 021010, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26158000

RESUMO

The combination of fluorescence measurements of membrane potential and intracellular [Formula: see text] concentration allows correlating the electrical and calcium activity of a cell with spatial precision. The technical advances allowing this type of measurement were achieved only recently and represent an important step in the progress of the voltage imaging approach pioneered over 40 years ago by Lawrence B. Cohen. Here, we show how this approach can be used to investigate the function of [Formula: see text] channels using the foreseen possibility to extract [Formula: see text] currents from imaging experiments. The kinetics of the [Formula: see text] current, mediated by voltage-gated [Formula: see text] channels, can be accurately derived from the [Formula: see text] fluorescence measurement using [Formula: see text] indicators with [Formula: see text] that equilibrate in [Formula: see text]. In this respect, the imaging apparatus dedicated to this application is described in detail. Next, we illustrate the mathematical procedure to extract the current from the [Formula: see text] fluorescence change, including a method to calibrate the signal to charge flux density. Finally, we show an example of simultaneous membrane potential and [Formula: see text] optical measurement associated with an action potential at a CA1 hippocampal pyramidal neuron from a mouse brain slice. The advantages and limitations of this approach are discussed.

7.
Biophys J ; 107(6): 1280-8, 2014 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-25229136

RESUMO

The current understanding of Ca(2+) channel function is derived from the use of the patch-clamp technique. In particular, the measurement of fast cellular Ca(2+) currents is routinely achieved using whole-cell voltage-clamp recordings. However, this experimental approach is not applicable to the study of local native Ca(2+) channels during physiological changes of membrane potential in complex cells, since the voltage-clamp configuration constrains the membrane potential to a given value. Here, we report for the first time to our knowledge that Ca(2+) currents from individual cells can be quantitatively measured beyond the limitations of the voltage-clamp approach using fast Ca(2+) imaging with low-affinity indicators. The optical measurement of the Ca(2+) current was correlated with the membrane potential, simultaneously measured with a voltage-sensitive dye to investigate the activation of Ca(2+) channels along the apical dendrite of the CA1 hippocampal pyramidal neuron during the back-propagation of an action potential. To validate the method, we analyzed the voltage dependence of high- and low-voltage-gated Ca(2+) channels. In particular, we measured the Ca(2+) current component mediated by T-type channels, and we investigated the mechanisms of recovery from inactivation of these channels. This method is expected to become a reference approach to investigate Ca(2+) channels in their native physiological environment.


Assuntos
Cálcio/metabolismo , Fenômenos Eletrofisiológicos , Imagem Óptica/métodos , Animais , Canais de Cálcio Tipo T/metabolismo , Eletrodos , Hipocampo/citologia , Hipocampo/fisiologia , Espaço Intracelular/metabolismo , Camundongos , Imagem Óptica/instrumentação
8.
J Biol Chem ; 289(10): 6681-94, 2014 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-24425870

RESUMO

Inhibitory GABAB receptors (GABABRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABABRs are heterodimers of GABAB1 and GABAB2 subunits and here we show that the trafficking and surface expression of GABABRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABABR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABABRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABAB1 but decreases GABAB2 surface expression. The increase in surface GABAB1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABAB2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABABR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival.


Assuntos
Neurônios/metabolismo , Receptores de GABA-B/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Transporte Proteico , Ratos , Transdução de Sinais , Estresse Fisiológico
9.
Neuron ; 79(2): 293-307, 2013 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-23889934

RESUMO

Inhibition of Arp2/3-mediated actin polymerization by PICK1 is a central mechanism to AMPA receptor (AMPAR) internalization and long-term depression (LTD), although the signaling pathways that modulate this process in response to NMDA receptor (NMDAR) activation are unknown. Here, we define a function for the GTPase Arf1 in this process. We show that Arf1-GTP binds PICK1 to limit PICK1-mediated inhibition of Arp2/3 activity. Expression of mutant Arf1 that does not bind PICK1 leads to reduced surface levels of GluA2-containing AMPARs and smaller spines in hippocampal neurons, which occludes subsequent NMDA-induced AMPAR internalization and spine shrinkage. In organotypic slices, NMDAR-dependent LTD of AMPAR excitatory postsynaptic currents is abolished in neurons expressing mutant Arf1. Furthermore, NMDAR stimulation downregulates Arf1 activation and binding to PICK1 via the Arf-GAP GIT1. This study defines Arf1 as a critical regulator of actin dynamics and synaptic function via modulation of PICK1.


Assuntos
Fator 1 de Ribosilação do ADP/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/fisiologia , Actinas/metabolismo , Proteínas de Transporte/fisiologia , Plasticidade Neuronal/fisiologia , Proteínas Nucleares/fisiologia , Sinapses/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Actinas/fisiologia , Animais , Células COS , Células Cultivadas , Cercopithecus aethiops , Células HEK293 , Humanos , Técnicas de Cultura de Órgãos , Polimerização , Ratos , Ratos Wistar
10.
J Biomed Opt ; 18(6): 60505, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23764747

RESUMO

ABSTRACT. In recent years, the use of light emitting diodes (LEDs) has become commonplace in fluorescence microscopy. LEDs are economical and easy to couple to commercial microscopes, and they provide powerful and stable light that can be triggered by transistor-transistor logic pulses in the range of tens of microseconds or shorter. LEDs are usually installed on the epifluorescence port of the microscope to obtain whole-field illumination, which is ideal for fluorescence imaging. In contrast, photolysis or channelrhodopsin stimulation often requires localized illumination, typically achieved using lasers. Here we show that insertion of a long-pass (>411 nm) filter with an appropriately sized pinhole in the epifluorescence pathway, combined with dual UV/visible illumination, can produce efficient whole-field visible illumination and spot UV illumination of 15 to 20 µm. We tested our system by performing calcium imaging experiments combined with L-glutamate or N-methyl-D-aspartic acid (NMDA) photorelease in hippocampal neurons from brain slices or dissociated cultures, demonstrating the ability to obtain local activation of NMDA receptors exclusively in the illuminated spot. The very inexpensive and simple system that we report here will allow many laboratories with limited budgets to run similar experiments in a variety of physiological applications.


Assuntos
Microscopia de Fluorescência/métodos , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cálcio/metabolismo , Fluorescência , Hipocampo/metabolismo , Hipocampo/patologia , Luz , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/economia , N-Metilaspartato/química , Neurônios/metabolismo , Óptica e Fotônica/economia , Óptica e Fotônica/instrumentação , Óptica e Fotônica/métodos , Fotólise , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Rodopsina/química
11.
PLoS One ; 8(1): e52345, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23326329

RESUMO

Multiple pathways participate in the AMPA receptor trafficking that underlies long-term potentiation (LTP) of synaptic transmission. Here we demonstrate that protein SUMOylation is required for insertion of the GluA1 AMPAR subunit following transient glycine-evoked increase in AMPA receptor surface expression (ChemLTP) in dispersed neuronal cultures. ChemLTP increases co-localisation of SUMO-1 and the SUMO conjugating enzyme Ubc9 and with PSD95 consistent with the recruitment of SUMOylated proteins to dendritic spines. In addition, we show that ChemLTP increases dendritic levels of SUMO-1 and Ubc9 mRNA. Consistent with activity dependent translocation of these mRNAs to sites near synapses, levels of the mRNA binding and dendritic transport protein CPEB are also increased by ChemLTP. Importantly, reducing the extent of substrate protein SUMOylation by overexpressing the deSUMOylating enzyme SENP-1 or inhibiting SUMOylation by expressing dominant negative Ubc9 prevent the ChemLTP-induced increase in both AMPAR surface expression and dendritic SUMO-1 mRNA. Taken together these data demonstrate that SUMOylation of synaptic protein(s) involved in AMPA receptor trafficking is necessary for activity-dependent increases in AMPAR surface expression.


Assuntos
Espinhas Dendríticas/efeitos dos fármacos , Glicina/farmacologia , Neurônios/efeitos dos fármacos , Receptores de AMPA/fisiologia , Animais , Células Cultivadas , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/fisiologia , Proteína 4 Homóloga a Disks-Large , Endopeptidases/genética , Endopeptidases/metabolismo , Hipocampo/citologia , Hipocampo/fisiologia , Immunoblotting , Hibridização in Situ Fluorescente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Neurônios/metabolismo , Neurônios/fisiologia , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Wistar , Receptores de AMPA/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Sumoilação , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
12.
J Neurosci ; 32(34): 11618-30, 2012 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-22915106

RESUMO

The number and subunit composition of postsynaptic AMPA receptors (AMPARs) is a key determinant of synaptic transmission. The vast majority of AMPARs contain GluA2 subunit, which renders the channel impermeable to calcium. However, a small proportion are GluA2 lacking and therefore calcium permeable (CP-AMPARs). It has been proposed recently that long-term potentiation (LTP) involves not only an increase in the total number of AMPARs at the synapse but also a transient switch to CP-AMPARs in the first few minutes after LTP induction. The molecular mechanisms that underlie this switch to CP-AMPARs and the subsequent switch back to calcium-impermeable AMPARs are unknown. Here, we show that endogenous GluA1 is rapidly inserted at the synaptic plasma membrane of rat hippocampal neurons immediately after stimulation with elevated glycine, a treatment known to induce LTP. In contrast, GluA2 is restricted from trafficking to the cell surface by a glycine-induced increase in PICK1-GluA2 binding on endosomal compartments. Between 5 and 20 min after stimulus, activation of CP-AMPARs triggers a release of GluA2 from PICK1, allowing GluA2-containing AMPARs to traffic to the synaptic plasma membrane. These results define a PICK1-dependent mechanism that underlies transient alterations in the subunit composition and calcium permeability of synaptic AMPARs that is important during the early phase after stimulation with glycine and therefore is likely to be important during the expression of LTP.


Assuntos
Proteínas de Transporte/metabolismo , Glicina/farmacologia , Neurônios/fisiologia , Proteínas Nucleares/metabolismo , Receptores de AMPA/metabolismo , Animais , Biotinilação , Cálcio/metabolismo , Proteínas de Transporte/genética , Células Cultivadas , Proteína 4 Homóloga a Disks-Large , Embrião de Mamíferos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipocampo/citologia , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Proteínas Nucleares/genética , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Receptores de AMPA/deficiência , Receptores de AMPA/genética , Espermina/análogos & derivados , Espermina/farmacologia , Sinapses/metabolismo , Fatores de Tempo , Transfecção , Transferrina/metabolismo , Proteínas de Transporte Vesicular/metabolismo
13.
J Biol Chem ; 287(27): 22781-8, 2012 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-22582390

RESUMO

Homeostatic scaling allows neurons to alter synaptic transmission to compensate for changes in network activity. Here, we show that suppression of network activity with tetrodotoxin, which increases surface expression of AMPA receptors (AMPARs), dramatically reduces levels of the deSUMOylating (where SUMO is small ubiquitin-like modifier) enzyme SENP1, leading to a consequent increase in protein SUMOylation. Overexpression of the catalytic domain of SENP1 prevents this scaling effect, and we identify Arc as a SUMO substrate involved in the tetrodotoxin-induced increase in AMPAR surface expression. Thus, protein SUMOylation plays an important and previously unsuspected role in synaptic trafficking of AMPARs that underlies homeostatic scaling.


Assuntos
Endopeptidases/metabolismo , Hipocampo/fisiologia , Homeostase/fisiologia , Neurônios/fisiologia , Sumoilação/fisiologia , Sinapses/metabolismo , Animais , Proteínas do Citoesqueleto/metabolismo , Endopeptidases/genética , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Células HEK293 , Hipocampo/citologia , Humanos , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/fisiologia , Técnicas de Cultura de Órgãos , Transporte Proteico/fisiologia , Ratos , Receptores de AMPA/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Sumoilação/efeitos dos fármacos , Tetrodotoxina/farmacologia
14.
J Cereb Blood Flow Metab ; 32(1): 17-22, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21989481

RESUMO

Here, we show that oxygen and glucose deprivation (OGD) causes increased small ubiquitin-like modifier (SUMO)-1 and SUMO-2/3 conjugation to substrate proteins in cultured hippocampal neurones. Surprisingly, the SUMO protease SENP-1, which removes SUMO from conjugated proteins, was also increased by OGD, suggesting that the neuronal response to OGD involves a complex interplay between SUMOylation and deSUMOylation. Importantly, decreasing global SUMOylation in cultured hippocampal neurones by overexpression of the catalytic domain of SENP-1 increased neuronal vulnerability to OGD-induced cell death. Taken together, these results suggest a neuroprotective role for neuronal SUMOylation after OGD.


Assuntos
Endopeptidases/metabolismo , Glucose/metabolismo , Neurônios/metabolismo , Oxigênio/metabolismo , Proteína SUMO-1/metabolismo , Sumoilação , Animais , Domínio Catalítico , Morte Celular , Células Cultivadas , Clonagem Molecular , Endopeptidases/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Mutação , Neurônios/patologia , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína SUMO-1/genética , Vírus Sindbis/genética
15.
Proc Natl Acad Sci U S A ; 108(49): 19772-7, 2011 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-22089239

RESUMO

The surface expression and regulated endocytosis of kainate (KA) receptors (KARs) plays a critical role in neuronal function. PKC can modulate KAR trafficking, but the sites of action and molecular consequences have not been fully characterized. Small ubiquitin-like modifier (SUMO) modification of the KAR subunit GluK2 mediates agonist-evoked internalization, but how KAR activation leads to GluK2 SUMOylation is unclear. Here we show that KA stimulation causes rapid phosphorylation of GluK2 by PKC, and that PKC activation increases GluK2 SUMOylation both in vitro and in neurons. The intracellular C-terminal domain of GluK2 contains two predicted PKC phosphorylation sites, S846 and S868, both of which are phosphorylated in response to KA. Phosphomimetic mutagenesis of S868 increased GluK2 SUMOylation, and mutation of S868 to a nonphosphorylatable alanine prevented KA-induced SUMOylation and endocytosis in neurons. Infusion of SUMO-1 dramatically reduced KAR-mediated currents in HEK293 cells expressing WT GluK2 or nonphosphorylatable S846A mutant, but had no effect on currents mediated by the S868A mutant. These data demonstrate that agonist activation of GluK2 promotes PKC-dependent phosphorylation of S846 and S868, but that only S868 phosphorylation is required to enhance GluK2 SUMOylation and promote endocytosis. Thus, direct phosphorylation by PKC and GluK2 SUMOylation are intimately linked in regulating the surface expression and function of GluK2-containing KARs.


Assuntos
Endocitose , Neurônios/metabolismo , Proteína Quinase C/metabolismo , Receptores de Ácido Caínico/metabolismo , Alanina/genética , Alanina/metabolismo , Substituição de Aminoácidos , Animais , Western Blotting , Células COS , Células Cultivadas , Cercopithecus aethiops , Células HEK293 , Humanos , Ácido Caínico/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Mutação , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de Ácido Caínico/agonistas , Receptores de Ácido Caínico/genética , Proteína SUMO-1/metabolismo , Serina/genética , Serina/metabolismo , Sumoilação/efeitos dos fármacos
16.
J Neurosci ; 31(33): 11941-52, 2011 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-21849555

RESUMO

Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca(2+), resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 min OGD protocol, a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by metabotropic glutamate receptor 1 (mGluR1) or A(3) receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC, or chelation of intracellular Ca(2+) also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalization of AMPARs after OGD. We also show that a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A(3) receptor antagonists, indicating that AMPARs are degraded following internalization. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotection.


Assuntos
Região CA3 Hipocampal/metabolismo , Glucose/deficiência , Inibição Neural/fisiologia , Oxigênio/metabolismo , Receptor A3 de Adenosina/fisiologia , Receptores de AMPA/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/fisiologia , Sinapses/metabolismo , Animais , Animais Recém-Nascidos , Região CA3 Hipocampal/citologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Masculino , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Receptor A3 de Adenosina/metabolismo , Receptores de AMPA/metabolismo
17.
Biochem Biophys Res Commun ; 409(4): 657-62, 2011 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-21616059

RESUMO

G-protein coupled receptor interacting scaffold protein (GISP) is a multi-domain, brain-specific protein derived from the A-kinase anchoring protein (AKAP)-9 gene. Using yeast two-hybrid screens to identify GISP interacting proteins we isolated the SUMO conjugating enzyme Ubc9. GISP interacts with Ubc9 in vitro, in heterologous cells and in neurons. SUMOylation is a post-translational modification in which the small protein SUMO is covalently conjugated to target proteins, modulating their function. Consistent with its interaction with Ubc9, we show that GISP is SUMOylated by both SUMO-1 and SUMO-2 in both in vitro SUMOylation assays and in mammalian cells. Intriguingly, SUMOylation of GISP in neurons occurs in an activity-dependent manner in response to chemical LTP. These data suggest that GISP is a novel neuronal SUMO substrate whose SUMOylation status is modulated by neuronal activity.


Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sumoilação , Animais , Encéfalo/citologia , Células COS , Cercopithecus aethiops , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Ratos , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Enzimas de Conjugação de Ubiquitina/metabolismo
18.
EMBO J ; 30(4): 719-30, 2011 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-21252856

RESUMO

Activity-dependent remodelling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the precise molecular mechanisms that regulate this process are unclear. Activators of Arp2/3-mediated actin polymerisation are required for spine enlargement; however, during long-term depression (LTD), spines shrink via actin depolymerisation and Arp2/3 inhibitors in this process have not yet been identified. Here, we show that PICK1 regulates spine size in hippocampal neurons via inhibition of the Arp2/3 complex. PICK1 knockdown increases spine size, whereas PICK1 overexpression reduces spine size. NMDA receptor activation results in spine shrinkage, which is blocked by PICK1 knockdown or overexpression of a PICK1 mutant that cannot bind Arp2/3. Furthermore, we show that PICK1-Arp2/3 interactions are required for functional hippocampal LTD. This work demonstrates that PICK1 is a novel regulator of spine dynamics. Via Arp2/3 inhibition, PICK1 has complementary yet distinct roles during LTD to regulate AMPA receptor trafficking and spine size, and therefore functions as a crucial factor in both structural and functional plasticity.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Proteínas de Transporte/fisiologia , Espinhas Dendríticas/fisiologia , Plasticidade Neuronal , Proteínas Nucleares/fisiologia , Sinapses/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Embrião de Mamíferos , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Tamanho das Organelas/efeitos dos fármacos , Tamanho das Organelas/fisiologia , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo
19.
Eur J Pharmacol ; 599(1-3): 121-5, 2008 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-18835556

RESUMO

Tachykinins are a family of neuropeptides, involved in a variety of physiological and pathological processes occurring in the gastrointestinal tract. They act via three distinct types of receptors, tachykinin NK(1), NK(2), and NK(3) receptors, which belong to the family of G protein-coupled receptors. The aim of the present study was to characterize, for the first time in the healthy human colon, the TACR(1), TACR(2) and TACR(3) mRNAs encoding the three different tachykinin receptors and to measure their relative expression by quantitative reverse transcription-PCR assay. Our results confirm the broad distribution of the tachykinin receptors but evidenced significant differences in the expression level of their respective mRNAs. A higher expression level of the TACR2 mRNA alpha isoform, the gene encoding the functional tachykinin NK(2) receptor, was observed in comparison to TACR1 and TACR3 mRNAs genes encoding for NK(1) and NK(3) receptors respectively. The prevalence of the TACR2 mRNA alpha isoform strongly suggests a major involvement of tachykinin NK(2) receptor in the regulation of human colonic functions.


Assuntos
Colo/fisiologia , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-2/genética , Receptores da Neurocinina-3/genética , Adulto , Idoso , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
J Comp Neurol ; 507(4): 1542-58, 2008 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-18219665

RESUMO

The involvement of NK2 receptors (NK2r) in the neuroregulation of human colonic motility has been mainly assessed by using pharmacological approaches. The aim of this study was to characterize the intramural neurons and nerve varicosities expressing NK2r in human colonic neuronal pathways. Neuronal coding in the myenteric plexus and external muscle layers was identified on the basis of the patterns of colocalization of tachykinins (TK), vesicular acetylcholine transporter (VAChT), nitric oxide synthase (NOS), glutamate decarboxylase (GAD), and vasoactive intestinal peptide (VIP) with NK2r immunoreactivity. The proportions of chemically defined synaptophysin-immunoreactive nerve varicosities were accurately determined by using specific software. NK2r immunoreactivity was detected in the soma of many myenteric neurons (71.8%). A large proportion of these neurons was immunoreactive to VAChT (39.3%), TK (30%), and GAD (23.5%) and, to a lesser extent, to NOS and VIP. The proportions of nerve varicosities expressing NK2r showed significant regional differences: the highest proportion (59.8%) was located in the myenteric plexus. High proportions of the myenteric nerve varicosities expressing NK2r were immunoreactive to VIP (80.9%) and NOS (77.9%), and lower proportions were recorded with VAChT, TK, and GAD. In the circular and longitudinal muscle layers, the proportions of nerve varicosities expressing NK2r were 49.6% and 45.3%, respectively. The chemically defined intramuscular varicosities were closely apposed to smooth muscle cells expressing NK2r. In conclusion, the data obtained in this study, in which the expression of NK2r was mapped in the human colonic neuronal pathways, confirm that these receptors are involved in the neuroneuronal and neuromuscular processes regulating human colonic motility.


Assuntos
Colo/metabolismo , Mucosa Intestinal/metabolismo , Vias Neurais/metabolismo , Neurônios/metabolismo , Receptores de Taquicininas/biossíntese , Idoso , Western Blotting , Feminino , Imunofluorescência , Motilidade Gastrointestinal/fisiologia , Humanos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Plexo Mientérico/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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