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Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.

ABSTRACT: Monitoring neuronal electrical activity using fluorescent protein-based voltage sensors has been limited by small response magnitudes and slow kinetics of existing probes. Here we report the development of a fluorescent protein voltage sensor, named ArcLight, and derivative probes that exhibit large changes in fluorescence intensity in response to voltage changes. ArcLight consists of the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase and super ecliptic pHluorin that carries the point mutation A227D. The fluorescence intensity of ArcLight A242 decreases by 35% in response to a 100 mV depolarization when measured in HEK293 cells, which is more than five times larger than the signals from previously reported fluorescent protein voltage sensors. We show that the combination of signal size and response speed of these new probes allows the reliable detection of single action potentials and excitatory potentials in individual neurons and dendrites.

SUBMITTER: Jin L

PROVIDER: S-EPMC3439164 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.

Jin Lei L Han Zhou Z Platisa Jelena J Wooltorton Julian R A JR Cohen Lawrence B LB Pieribone Vincent A VA

Neuron 20120901 5

Monitoring neuronal electrical activity using fluorescent protein-based voltage sensors has been limited by small response magnitudes and slow kinetics of existing probes. Here we report the development of a fluorescent protein voltage sensor, named ArcLight, and derivative probes that exhibit large changes in fluorescence intensity in response to voltage changes. ArcLight consists of the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase and super ecliptic pHluorin that ...[more]

PMID: 22958819

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Project description:The spatial variation of the extracellular action potentials (EAP) of a single neuron contains information about the size and location of the dominant current source of its action potential generator, which is typically in the vicinity of the soma. Using this dependence in reverse in a three-component realistic probe + brain + source model, we solved the inverse problem of characterizing the equivalent current source of an isolated neuron from the EAP data sampled by an extracellular probe at multiple independent recording locations. We used a dipole for the model source because there is extensive evidence it accurately captures the spatial roll-off of the EAP amplitude, and because, as we show, dipole localization, beyond a minimum cell-probe distance, is a more accurate alternative to approaches based on monopole source models. Dipole characterization is separable into a linear dipole moment optimization where the dipole location is fixed, and a second, nonlinear, global optimization of the source location. We solved the linear optimization on a discrete grid via the lead fields of the probe, which can be calculated for any realistic probe + brain model by the finite element method. The global source location was optimized by means of Tikhonov regularization that jointly minimizes model error and dipole size. The particular strategy chosen reflects the fact that the dipole model is used in the near field, in contrast to the typical prior applications of dipole models to EKG and EEG source analysis. We applied dipole localization to data collected with stepped tetrodes whose detailed geometry was measured via scanning electron microscopy. The optimal dipole could account for 96% of the power in the spatial variation of the EAP amplitude. Among various model error contributions to the residual, we address especially the error in probe geometry, and the extent to which it biases estimates of dipole parameters. This dipole characterization method can be applied to any recording technique that has the capabilities of taking multiple independent measurements of the same single units.

Project description:Key pointsSome ion channels are known to behave as inductors and make up the parallel resonant circuit in the plasma membrane of neurons, which enables neurons to respond to current inputs with a specific frequency (so-called 'resonant properties'). Here, we report that heterologous expression of mouse Kv11 voltage-dependent K+ channels generate resonance and oscillation at depolarized membrane potentials in HEK293 cells; expressions of individual Kv11 subtypes generate resonance and oscillation with different frequency properties. Kv11.3-expressing HEK293 cells exhibited transient conductance changes that opposed the current changes induced by voltage steps; this probably enables Kv11 channels to behave like an inductor. The resonance and oscillation of inferior olivary neurons were impaired at the resting membrane potential in Kv11.3 knockout mice. This study helps to elucidate basic ion channel properties that are crucial for the frequency responses of neurons.AbstractThe plasma membranes of some neurons preferentially respond to current inputs with a specific frequency, and output as large voltage changes. This property is called resonance, and is thought to be mediated by ion channels that show inductor-like behaviour. However, details of the candidate ion channels remain unclear. In this study, we mainly focused on the functional roles of Kv11 potassium (K+ ) channels, encoded by ether-á-go-go-related genes, in resonance in mouse inferior olivary (IO) neurons. We transfected HEK293 cells with long or short splice variants of Kv11.1 (Merg1a and Merg1b) or Kv11.3, and examined membrane properties using whole-cell recording. Transfection with Kv11 channels reproduced resonance at membrane potentials depolarized from the resting state. Frequency ranges of Kv11.3-, Kv11.1(Merg1b)- and Kv11.1(Merg1a)-expressing cells were 2-6 Hz, 2-4 Hz, and 0.6-0.8 Hz, respectively. Responses of Kv11.3 currents to step voltage changes were essentially similar to those of inductor currents in the resistor-inductor-capacitor circuit. Furthermore, Kv11 transfections generated membrane potential oscillations. We also confirmed the contribution of HCN1 channels as a major mediator of resonance at more hyperpolarized potentials by transfection into HEK293 cells. The Kv11 current kinetics and properties of Kv11-dependent resonance suggested that Kv11.3 mediated resonance in IO neurons. This finding was confirmed by the impairment of resonance and oscillation at -30 to -60 mV in Kcnh7 (Kv11.3) knockout mice. These results suggest that Kv11 channels have important roles in inducing frequency-dependent responses in a subtype-dependent manner from resting to depolarized membrane potentials.

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Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.

Publications

Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.

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