| Co-Investigator(Kenkyū-buntansha) |
SALZBERG Brian m. University of Pennsylvania School of Medicine, Professor., 医学部, 教授
COHEN Lawrence b. Yale University School of Medicine, Professor, 医学部, 教授
HIROTA Akihiko Tokyo Medical and Dental University, School of Medicine, Assistant Professor., 医学部, 助教授 (50156717)
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| Research Abstract |
Extrinsic (requiring voltage-sensitive dyes) optical signals have provided a powerful means for monitoring electrical activity of living cells. Indeed, using optical techniques and voltage-sensitive dyes, we have been able to overcome several obstacles to the electrophysiological study of nervous and cardiac systems. Therefore, much progress has been achieved in the development of voltage-sensitive dyes for measurements of rapid changes in membrane potential. On the other hand, in using of conventional voltage-sensitive dyes, there are several limitations and difficulties in their voltage-sensitivity and physicochemical properties. Thus, we have tried to search for new voltage-sensitive dyes, and we have obtained the following results.
(1) Extensive dye screening led to the choice of di-8-ANEPPS as the most suitable voltage-sensitive dye for monitoring cardiac action potentials : it is internalized slowly and permits optical recording with signal-to-noise ratio as high as 40 : 1 (measur
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ed in peak-to-peak) and average fractional fluorescence change of 15 % per 100 mV of the membrane potential. [with B.M.Salzberg in the MBL and University of Pennsylvania].
(2) We tested seven styryl dyes di4- and di8-ANEPPS (negative fixed charge), di2-, di8-, di12-, and di16-ANEPPQ (positive fixed charge), in the embryonic spinal cord. The dyes were pressure-injected into ventral roots using a microelectrode filled with a concentrated dye solution. All the more hydrophobic dyes (<greater than or equal> di8) appeared to specifically label motor neurons and their process. The obtained results suggest that relatively large optical signals may be obtained from specially labeled populations of central neurons. [with L.B.Cohen in the MBL and Yale Un
(3) To evaluate the suitability of a variety of fast voltage-sensitive dyes for optical monitoring of rapid transmembrane potential activity in the embryonic nervous system, we screened over twenty dyes, including several newly synthesized probes. And then, several promising new merocyanine-rhodanine dyes for embryonic nervous systems were found. [in our laboratory] Less
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