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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.
J Physiol ; 597(13): 3251-3252, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31111490
3.
J Neurosci ; 39(11): 1969-1981, 2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30630881

RESUMO

In cerebellar Purkinje neuron dendrites, the transient depolarization associated with a climbing fiber (CF) EPSP activates voltage-gated Ca2+ channels (VGCCs), voltage-gated K+ channels (VGKCs), and Ca2+-activated SK and BK K+ channels. The resulting membrane potential (V m) and Ca2+ transients play a fundamental role in dendritic integration and synaptic plasticity of parallel fiber inputs. Here we report a detailed investigation of the kinetics of dendritic Ca2+ and K+ channels activated by CF-EPSPs, based on optical measurements of V m and Ca2+ transients and on a single-compartment NEURON model reproducing experimental data. We first measured V m and Ca2+ transients associated with CF-EPSPs at different initial V m, and we analyzed the changes in the Ca2+ transients produced by the block of each individual VGCCs, of A-type VGKCs and of SK and BK channels. Then, we constructed a model that includes six active ion channels to accurately match experimental signals and extract the physiological kinetics of each channel. We found that two different sets of channels are selectively activated. When the dendrite is hyperpolarized, CF-EPSPs mainly activate T-type VGCCs, SK channels, and A-type VGKCs that limit the transient V m ∼ <0 mV. In contrast, when the dendrite is depolarized, T-type VGCCs and A-type VGKCs are inactivated and CF-EPSPs activate P/Q-type VGCCs, high-voltage activated VGKCs, and BK channels, leading to Ca2+ spikes. Thus, the potentially activity-dependent regulation of A-type VGKCs, controlling the activation of this second set of channels, is likely to play a crucial role in signal integration and plasticity in Purkinje neuron dendrites.SIGNIFICANCE STATEMENT The climbing fiber synaptic input transiently depolarizes the dendrite of cerebellar Purkinje neurons generating a signal that plays a fundamental role in dendritic integration. This signal is mediated by two types of Ca2+ channels and four types of K+ channels. Thus, understanding the kinetics of all of these channels is crucial for understanding PN function. To obtain this information, we used an innovative strategy that merges ultrafast optical membrane potential and Ca2+ measurements, pharmacological analysis, and computational modeling. We found that, according to the initial membrane potential, the climbing fiber depolarizing transient activates two distinct sets of channels. Moreover, A-type K+ channels limit the activation of P/Q-type Ca2+ channels and associated K+ channels, thus preventing the generation of Ca2+ spikes.

4.
Bioorg Med Chem ; 27(1): 247-253, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30529150

RESUMO

The scorpion toxin AmmTx3 is a specific blocker of Kv4 channels. It was shown to have interesting potential for neurological disorders. In this study, we report the first chemical synthesis of AmmTx3 by using the native chemical ligation strategy and validate its biological activity. We determined its 3D structure by nuclear magnetic resonance spectroscopy, and pointed out that AmmTx3 possesses the well-known CSαß structural motif, which is found in a large number of scorpion toxins. Overall, this study establishes an easy synthetic access to biologically active AmmTx3 toxin.


Assuntos
Bloqueadores dos Canais de Potássio/química , Venenos de Escorpião/química , Sequência de Aminoácidos , Animais , Cerebelo/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/farmacologia , Conformação Proteica em alfa-Hélice , Venenos de Escorpião/síntese química , Venenos de Escorpião/farmacologia , Escorpiões/química
5.
Eur J Neurosci ; 2018 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-30387216

RESUMO

Optogenetics is based on the selective expression of exogenous opsins by neurons allowing experimental control of their electrical activity using visible light. The interpretation of the results of optogenetic experiments is based on the assumption that light stimulation selectively acts on those neurons expressing the exogenous opsins without perturbing the activity of naive ones. Here, we report that light stimulation, of wavelengths and power in the range of those normally used in optogenetic experiments, consistently reduces the firing activity of naive Mitral Cells (MCs) and Tufted Neurons in the olfactory bulb as well as in Medium Spiny Neurons (MSNs) in the striatum. No such effect was observed for cerebellar Purkinje and hippocampal CA1 neurons. The effects on MC firing appear to be mainly due to a light-induced increase in tissue temperature, between 0.1 and 0.4°C, associated with the generation of a hyperpolarizing current and a modification of action potential (AP) shape. Therefore, light in the visible range can affect neuronal physiology in a cell-specific manner. Beside the implications for optogenetic studies, our results pave the way to investigating the use of visible light for therapeutic purposes in pathologies associated with neuronal hyperexcitability.

6.
Brain Struct Funct ; 223(7): 3011-3043, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29748872

RESUMO

Imaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-ß plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals.

7.
J Biophotonics ; 11(3)2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29165917

RESUMO

In brain slices, resolving fast Ca2+ fluorescence signals from submicron structures is typically achieved using 2-photon or confocal scanning microscopy, an approach that limits the number of scanned points. The novel multiplexing confocal system presented here overcomes this limitation. This system is based on a fast spinning disk, a multimode diode laser and a novel high-resolution CMOS camera. The spinning disk, running at 20 000 rpm, has custom-designed spiral pattern that maximises light collection, while rejecting out-of-focus fluorescence to resolve signals from small neuronal compartments. Using a 60× objective, the camera permits acquisitions of tens of thousands of pixels at resolutions of ~250 nm per pixel in the kHz range with 14 bits of digital depth. The system can resolve physiological Ca2+ transients from submicron structures at 20 to 40 µm below the slice surface, using the low-affinity Ca2+ indicator Oregon Green BAPTA-5N. In particular, signals at 0.25 to 1.25 kHz were resolved in single trials, or through averages of a few recordings, from dendritic spines and small parent dendrites in cerebellar Purkinje neurons. Thanks to an unprecedented combination of temporal and spatial resolution with relatively simple implementation, it is expected that this system will be widely adopted for multisite monitoring of Ca2+ signals.

8.
Neurophotonics ; 4(3): 031211, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28523281

RESUMO

Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor electrical subthreshold events as they travel from synapses on distal dendrites and summate at particular locations to initiate action potentials. It is now possible to carry out these measurements at the scale of individual dendritic spines using voltage imaging. In these measurements, the voltage-sensitive probes can be thought of as transmembrane voltmeters with a linear scale, which directly monitor electrical signals. Grinvald et al. were important early contributors to the methodology of voltage imaging, and they pioneered some of its significant results. We combined voltage imaging and glutamate uncaging using computer-generated holography. The results demonstrated that patterned illumination, by reducing the surface area of illuminated membrane, reduces photodynamic damage. Additionally, region-specific illumination practically eliminated the contamination of optical signals from individual spines by the scattered light from the parent dendrite. Finally, patterned illumination allowed one-photon uncaging of glutamate on multiple spines to be carried out in parallel with voltage imaging from the parent dendrite and neighboring spines.

9.
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
10.
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
12.
Adv Exp Med Biol ; 859: 57-101, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26238049

RESUMO

A central question in neuronal network analysis is how the interaction between individual neurons produces behavior and behavioral modifications. This task depends critically on how exactly signals are integrated by individual nerve cells functioning as complex operational units. Regional electrical properties of branching neuronal processes which determine the input-output function of any neuron are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin (synaptic contacts on distal dendrites) and summate at particular locations to influence action potential initiation. It became possible recently to carry out this type of measurement using high-resolution multisite recording of membrane potential changes with intracellular voltage-sensitive dyes. This chapter reviews the development and foundation of the method of voltage-sensitive dye recording from individual neurons. Presently, this approach allows monitoring membrane potential transients from all parts of the dendritic tree as well as from axon collaterals and individual dendritic spines.


Assuntos
Axônios/fisiologia , Espinhas Dendríticas/fisiologia , Corantes Fluorescentes/química , Potenciais da Membrana/fisiologia , Imagens com Corantes Sensíveis à Voltagem/métodos , Animais , Axônios/ultraestrutura , Bivalves , Espinhas Dendríticas/ultraestrutura , Lasers , Luz , Camundongos , Rede Nervosa/fisiologia , Rede Nervosa/ultraestrutura , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Sinapses/fisiologia , Sinapses/ultraestrutura , Fatores de Tempo , Imagens com Corantes Sensíveis à Voltagem/instrumentação
13.
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
14.
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.

15.
Front Cell Neurosci ; 8: 311, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25339864

RESUMO

Information processing in the central nervous system makes use of densely woven networks of neurons with complex dendritic and axonal arborizations. Studying signaling in such a network requires precise control over the activity of specific neurons and an understanding how the synaptic signals are integrated. We established a system using a recently published red-shifted voltage sensitive dye in slices from mice expressing channelrhodopsin (Ch) in GABAergic neurons. Using a focused 473 nm laser for Ch activation and 635 nm laser wide field illumination for voltage sensitive dye excitation we were able to simultaneously measure dendritic voltage transients and stimulate inhibitory synaptic connections. The combination of these techniques provides excellent spatiotemporal control over neuron activation and high resolution information on dendritic signal processing.

16.
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
17.
Front Cell Neurosci ; 8: 181, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25018699
18.
J Neurosci ; 34(4): 1280-92, 2014 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-24453319

RESUMO

Little is known about how neuron firing recorded in vivo retrogradely influences synaptic strength. We injected the firing of a rat hippocampal neurogliaform cell (NGFC), a widely expressed GABAergic neuron type, detected in vivo during theta rhythm, into NGFCs of rat or neuronal nitric oxide synthase (nNOS)-Cre-tdTomato mouse recorded in vitro. We found that the "in vivo firing pattern" produced a transient firing-induced suppression of synaptic inhibition (FSI) evoked by a presynaptic NGFC. Imaging experiments demonstrate that FSI was associated with action potential backpropagation (bAP) and a supralinear increase in dendritic Ca(2+). The application of the L-type Ca(2+) channel antagonist nimodipine blocked FSI. Further pharmacological experiments, such as the application of a nitric oxide-sensitive guanylyl cyclase (NO-sGC) receptor antagonist, a NOS inhibitor, and NO donors, suggested that NO released from postsynaptic cells mediated FSI and likely activated presynaptic receptors to inhibit GABA release. The in vivo firing pattern modulated the size of unitary EPSPs impinging on NGFCs through FSI and not via a direct effect on excitatory synaptic transmission. Our data demonstrate: (1) retrograde signaling initiated by in vivo firing pattern, (2) interneuron bAPs detected with fast temporal resolution, and (3) a novel role for NO expressed by specific interneuron types.


Assuntos
Hipocampo/fisiologia , Interneurônios/fisiologia , Inibição Neural/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Masculino , Camundongos Transgênicos , Neurotransmissores/metabolismo , Óxido Nítrico/metabolismo , Técnicas de Patch-Clamp , Ratos
19.
Cold Spring Harb Protoc ; 2013(12): 1125-31, 2013 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24298025

RESUMO

Ca(2+) imaging is a commonly used approach for measuring Ca(2+) signals at high spatial resolution. The method is often combined with electrode recordings to correlate electrical and chemical signals or to investigate Ca(2+) signals following an electrical stimulation. To obtain information on electrical activity at the same spatial resolution, Ca(2+) imaging must be combined with membrane potential imaging. Similarly, stimulation of subcellular compartments requires photostimulation. Thus, combining Ca(2+) imaging with an additional optical technique facilitates the study of a number of physiological questions. The aim of this article is to introduce some basic principles regarding the combination of Ca(2+) imaging with other optical techniques. We discuss the design of the optics, the design of experimental protocols, the optical characteristics of Ca(2+) indicators used in combination with an optical probe, and the affinity of the Ca(2+) indicator in relation to the type of measurement. This information will enable the reader to devise an optimal strategy for combined optical experiments.


Assuntos
Cálcio/análise , Fenômenos Eletrofisiológicos , Imagem Óptica/métodos , Coloração e Rotulagem/métodos , Sinalização do Cálcio , Potenciais da Membrana
20.
Cold Spring Harb Protoc ; 2013(12): 1161-4, 2013 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24298027

RESUMO

The ability to monitor Ca(2+) signals and membrane potential simultaneously at multiple locations on the same neuron facilitates further progress in our understanding of neuronal function. In particular, this method allows correlation of electrical and chemical signals from multiple sites, including those inaccessible to microelectrodes. This protocol describes a procedure for loading cells with two indicators, a Ca(2+)-sensitive Fura dye and voltage-sensitive JPW1114, together with the equipment required for detecting and imaging the two signals. Potential problems are discussed as well as the capabilities and limitations of the technique.


Assuntos
Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Potenciais da Membrana , Neurônios/fisiologia , Imagem Óptica/métodos , Animais , Fenômenos Eletrofisiológicos , Corantes Fluorescentes/metabolismo , Neurônios/metabolismo , Coloração e Rotulagem/métodos
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