Changes in fluorescence, turbidity, and birefringence associated with nerve excitation.

The objective of this paper is to offer experimental evidence which shows that the process of excitation in the nerve is accompanied by a transient change in optical properties of the nervous tissue. The optical properties examined include fluorescence, turbidity, and birefringence. Changes in fluorescence were examined after nervous tissues were stained with the dye 8-anilinonaphthalene-1-sulfonic acid (ANS).1 Our search for fluorescence under these conditions was prompted by the work of Aronson, Detert, and Morales,2 who demonstrated that the fluorescence of ANS is extremely sensitive to conformational changes of various macromolecules. Our attempt to measure turbidity changes of the nerve during excitation was made with a view to extending the work reported by Cohen and Keynes3 by the use of monochromatic light. The observation by Inoue and Sato4 of rapid changes in the mitotic figure of the Pectinaria obcyte under a polarizing microscope aroused our interest in the birefringent properties of the nerve during excitation. Our experiments along this line were greatly accelerated when we encountered a very significant paper by Cohen et al.5 on this subject in the early stages of our investigation. Changes in the optical properties of the nerve during excitation are very small. In fluorescence and turbidity studies, optical signs of nerve excitation could not be measured without the use of a computer to average multiple responses. However, in birefringence studies, it was possible to record optical signs of nerve excitation directly on an oscillograph screen. Method.-Most of the present fluorescence studies were carried out with nerve trunks from the legs of lobsters (Homarus americanus) and spider crabs (Libinia emarginata) and with the fin nerves of squid (Loligo pealii). Nerves were immersed in sea water containing 0.05 mg/ml ANS (Baker Chemical Co. or Eastman Organic Chemicals) for a period of 15-20 min. The nerves were then transferred to a chamber made of black acrylic plastic filled with sea water without dye as shown diagrammatically at the top of Figure 1. The nerve chamber was provided with two pairs of platinum electrodes (not shown iuL the figure): one pair for delivering electric stimuli from a Grass stimulator to the overlying nerve, and the other pair for recording action potentials near the end of the nerve. Various types of light sources were used during the course of the present experiments; an Osram quartz-iodine lamp operated at d-c 15 volts and approximately 150 watts was used in the later stage of the present study as a source of near-visible ultraviolet light. Quartz lenses Li and L2 (Fig. 1) were used to condense the light on a 1-3-mm portion of the nerve. Optical filter F1, used to absorb visible light, was either a Corning glass filter (CS 7-83) or a Bausch and Lomb interference filter for 365 mg. Visible light emitted by the nerve was detected with a photomultiplier tube (RCA 4463) at a right angle to the direction of the incident light. Filter F2, used to absorb the incident light scattered by the nerve, was either a Corning filter CS 3-72 (transmitting visible light longer than 430 my in wavelength) or a Corning filter CS 5-75 (transmitting a narrow spectrum around 460 m).