Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy

Year: 2005

Authors: Dombeck D.A., Sacconi L., Blanchard-Desce M., Webb W.W.

Autors Affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, New York;
European Laboratory for Non-Linear Spectroscopy, University of Florence, Sesto Fiorentino, Florence, Italy;
Synthese et Electrosynthese Organiques, Centre National de la Recherche Scientifique, Institut de Chimie, Universite de Rennes 1, Rennes, France.

Abstract: Although nonlinear microscopy and fast ( similar to 1 ms) membrane potential (V-m) recording have proven valuable for neuroscience applications, their potentially powerful combination has not yet been shown for studies of V-m activity deep in intact tissue. We show that laser illumination of neurons in acute rat brain slices intracellularly filled with FM4-64 dye generates an intense second-harmonic generation (SHG) signal from somatic and dendritic plasma membranes with high contrast > 125 mu m below the slice surface. The SHG signal provides a linear response to Delta V-m of similar to 7.5%/100 mV. By averaging repeated line scans ( similar to 50), we show the ability to record action potentials (APs) optically with a signal-to-noise ratio (S/N) of similar to 7 – 8. We also show recording of fast V-m steps from the dendritic arbor at depths inaccessible with previous methods. The high membrane contrast and linear response of SHG to Delta V-m provides the advantage that signal changes are not degraded by background and can be directly quantified in terms of Delta V-m. Experimental comparison of SHG and two-photon fluorescence V m recording with the best known probes for each showed that the SHG technique is superior for V-m recording in brain slice applications, with FM4-64 as the best tested SHG V-m probe.


Volume: 94 (5)      Pages from: 3628  to: 3636

KeyWords: SHG
DOI: 10.1152/jn.00416.2005

Citations: 110
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