Optophysiological approach to resolve neuronal action potentials with high spatial and temporal resolution in cultured neurons

Stéphane Pagès

doi:10.3389/fncel.2011.00020

Abstract

Cell to cell communication in the central nervous system is encoded into transient and local membrane potential changes (ΔVm). Deciphering the rules that govern synaptic transmission and plasticity entails to be able to perform Vm recordings throughout the entire neuronal arborization. Classical electrophysiology is, in most cases, not able to do so within small and fragile neuronal subcompartments. Thus, optical techniques based on the use of fluorescent voltage-sensitive dyes (VSDs) have been developed. However, reporting spontaneous or small ΔVm from neuronal ramifications has been challenging, in part due to the limited sensitivity and phototoxicity of VSD-based optical measurements. Here we demonstrate the use of water soluble VSD, ANNINE-6plus, with laser-scanning microscopy to optically record ΔVm in cultured neurons. We show that the sensitivity (>10% of fluorescence change for 100 mV depolarization) and time response (sub millisecond) of the dye allows the robust detection of action potentials (APs) even without averaging, allowing the measurement of spontaneous neuronal firing patterns. In addition, we show that back-propagating APs can be recorded, along distinct dendritic sites and within dendritic spines. Importantly, our approach does not induce any detectable phototoxic effect on cultured neurons. This optophysiological approach provides a simple, minimally invasive, and versatile optical method to measure electrical activity in cultured neurons with high temporal (ms) resolution and high spatial (μm) resolution.

Highlights

The capacity to make local ΔV m measurements should ideally be on time scales expanding over three orders of magnitude: millisecond time scale to resolve a single action potentials (APs) and second to minute time scale to record spontaneous activity
Concerning the ability to record membrane potentials in small compartments, there are, to our knowledge, only three studies reporting optical detection of ΔV m in dendritic spines
This approach is based on two-photon voltage-dependent light scattering properties of an second harmonic generation (SHG) reporter (FM4-64) bound to plasma membrane

Summary

METHODS

Reviewed by: Knut Holthoff, Universitätsklinikum Jena, Germany Srdjan D. Laser-scanning systems are limited in speed for generating a full neuron image, but are ideal, with the line-scanning mode, for sampling small compartments at high speed and minimizing photodamage by illuminating only a tiny fraction of the neuron under investigation This results in a point-recording technique for which, in contrast to classical electrophysiology, the point can be moved all across the dendritic or axonal arborization of neurons. We show that line-scanning measurements of ANNINE-6plus, passively introduced into neuronal membranes via brief incubation, can readily resolve individual APs in the soma, dendrites, and even spines, without photodamage or any impact on the electrophysiological properties of the investigated neuron It can resolve low amplitude depolarization (down to 4 mV) with averaging. Our study provides a simple approach to non-invasively study spontaneous ΔV m with high spatial and temporal resolutions in cultured neurons

RESULTS

DISCUSSION

MATERIALS AND METHODS