Summary. The fluorescence of dyes added to squid giant axons was studied during action potentials and voltage-clamp steps. One goal was to find fluorescence changes related to the increases in membrane conductance that underlie propagation. A second goal was to find large changes in fluorescence that would allow optical monitoring of membrane potential in neurons and other cells. Attempts were made to measure fluorescence changes using over 300 different fluorescent molecules and positive results were obtained with more than half of these. No evidence was found that would relate any of the fluorescence changes to the increases in membrane conductance that accompany depolarization; most, instead, were correlated with the changes in membrane potential. The fluorescence changes of several dyes were relatively large; the largest changes during an action potential were 10 -3 of the resting intensity. They could be measured with a signal-to-noise ratio of better than 10:1 in a single sweep.Changes in the fluorescence of molecules added to axons have been studied in an effort to learn about alterations in membrane structure that occur during excitation (Tasaki, Carnay &Watanabe, 1969;Cohen, Landowne, Shrivastav &Ritchie, 1970; Conti, Tasaki &Wanke, 1971). Although it was suggested that ANS and TNS fluorescence changes were related to increases in membrane conductance, our experiments (Davila, Cohen, Salzberg &Shrivastav, 1974) indicated that these fluorescence changes were, in fact, potential dependent. With the hope that other dyes would provide information about the structural basis of the conductance

SUMMARY1. To obtain information about structural events that occur in axons, changes in light scattering from squid giant axons were measured during action potentials and voltage-clamp steps.2. The scattering changes were measured at several scattering angles. Because the changes in scattering divided by the resting scattering were between 106 and 10-5, signal-averaging techniques were used to increase the signal-to-noise ratio.3. The scattering changes during the action potential were different at different angles. Two types were found, one at 10-30°(forward angles) and the other at 60-120°(right angles).4. At forward angles, there was a transient scattering decrease during the action potential. The time course of the change was similar to that of the action potential; this change was thought to be potential-dependent.5. At right angles, there was a transient scattering increase during the action potential followed later by a second, longer-lasting increase. Indirect evidence indicated that neither component could be totally potentialdependent.6. To further analyse these effects, scattering was measured during voltage-clamp steps. The changes seen during hyperpolarizing steps were presumed to be potential-dependent; again two different changes were found, one at forward angles and one at right angles.7. The potential-dependent change at right angles occurred with a time course that could be approximated by a single exponential with a time constant r = 24 /ssec. The change at forward angles required two exponentials, r1 = 23 usec, r2 = 900 Utsec, to represent its time course. 702 L. B. COHEN, R. D. KEYNES AND D. LANDOWNE 8. The size of both potential-dependent changes was proportional to the square of potential. The change at right angles, but not that at forward angles, was increased in size by the addition of butanol or octanol to the bathing solution.

SUMMARY1. An analysis has been made of the change in optical retardation of the membrane elicited by the application of voltage-clamp pulses in squid giant axons.2. The retardation response consists of three separate voltage-dependent components. For freshly mounted axons, defined as being in state 1, hyperpolarizing pulses give a rapid increase in the light intensity measured with crossed polarizers which has been termed the fast phase. This is followed by a rather slow return towards the base line termed the rebound. On treatment of the axon with certain agents that include tetrodotoxin, high calcium and terbium, the rebound disappears and the fast phase slows down, increases in size, and has a new slow component added to it. This transition from state 1 to a second state, 2, appears to be irreversible.3. In state 1, the time constant of the fast phase is 20-40 tsec at 13°C; it has a very large negative temperature coefficient (Q10 = Ca.j).The size of the retardation change is independent of temperature and * National Science Foundation post-doctoral fellow, 1966-68. 6. A tenfold increase in external calcium concentration had no discernible effect on the fast and slow phases, but reversibly reduced the amplitude of the rebound nearly to half.7. In experiments on perfused axons, the retardation response was not measurably altered by any of the modifications made to the composition of the perfusing fluid.8. There was some indication of the possible existence of a small current-or conductance-dependent component of the retardation response. 9. These phenomena seem likely to originate either from molecular relaxation processes analogous with the Kerr effect, or from changes in membrane thickness under the influence of the pressure exerted by the electric field. However, the specific molecules involved in the retardation response cannot yet be identified.