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J Neurophysiol 99: 1545-1553, 2008. First published January 2, 2008; doi:10.1152/jn.00929.2007
0022-3077/08 $8.00
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INNOVATIVE METHODOLOGY

Two-Photon Excitation of Potentiometric Probes Enables Optical Recording of Action Potentials From Mammalian Nerve Terminals In Situ

Jonathan A. N. Fisher1, Jonathan R. Barchi2, Cristin G. Welle3, Gi-Ho Kim3, Paul Kosterin3, Ana Lía Obaid3, Arjun G. Yodh1, Diego Contreras3 and Brian M. Salzberg3,4

1Department of Physics and Astronomy, University of Pennsylvania School of Arts and Sciences, Philadelphia; 2Department of Bioengineering, University of Pennsylvania School of Engineering and Applied Science, Philadelphia; and 3Departments of Neuroscience and 4Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Submitted 17 August 2007; accepted in final form 2 January 2008

We report the first optical recordings of action potentials, in single trials, from one or a few (~1–2 µm) mammalian nerve terminals in an intact in vitro preparation, the mouse neurohypophysis. The measurements used two-photon excitation along the "blue" edge of the two-photon absorption spectrum of di-3-ANEPPDHQ (a fluorescent voltage-sensitive naphthyl styryl-pyridinium dye), and epifluorescence detection, a configuration that is critical for noninvasive recording of electrical activity from intact brains. Single-trial recordings of action potentials exhibited signal-to-noise ratios of ~5:1 and fractional fluorescence changes of up to ~10%. This method, by virtue of its optical sectioning capability, deep tissue penetration, and efficient epifluorescence detection, offers clear advantages over linear, as well as other nonlinear optical techniques used to monitor voltage changes in localized neuronal regions, and provides an alternative to invasive electrode arrays for studying neuronal systems in vivo.

Address for reprint requests and other correspondence: B. M. Salzberg, Dept. of Neuroscience, Univ. of Pennsylvania School of Medicine, 234 Stemmler Hall, Philadelphia, PA 19104-6074 (E-mail: bmsalzbe{at}mail.med.upenn.edu)




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