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INNOVATIVE METHODOLOGY

Optical Recording of Fast Neuronal Membrane Potential Transients in Acute Mammalian Brain Slices by Second-Harmonic Generation Microscopy

¹School of Applied and Engineering Physics, Cornell University, Ithaca, New York; ²European Laboratory for Non-Linear Spectroscopy, University of Florence, Sesto Fiorentina, Florence, Italy; and ³Synthese et Electrosynthese Organiques, Centre National de la Recherche Scientifique, Institut de Chimie, Universite de Rennes 1, Rennes, France

Submitted 25 April 2005; accepted in final form 4 August 2005

Although nonlinear microscopy and fast (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 µm below the slice surface. The SHG signal provides a linear response to V_m of 7.5%/100 mV. By averaging repeated line scans (50), we show the ability to record action potentials (APs) optically with a signal-to-noise ratio (S/N) of 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 V_m provides the advantage that signal changes are not degraded by background and can be directly quantified in terms of 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.

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