The swelling behavior of fully neutralized sodium polyacrylate gels was investigated in aqueous solutions of alkali metal (LiCl, NaCl, KCl, CsCl) and alkaline earth metal salts (CaCl2, SrCl2, BaCl2). The total salt concentration and the ratio of monovalent to divalent cations were varied in the biologically significant range. It is found that the concentrations of both monovalent and divalent cations vary continuously and smoothly in the gel despite the abrupt change in the gel volume. The individual elastic, mixing, and ionic contributions to the free energy of the gel were separately determined as a function of the degree of network swelling to elucidate the thermodynamics of swelling. Shear modulus measurements performed at different Ca2+ concentrations suggest that Ca2+ does not form stable cross-links between the polymer chains. At low and moderate swelling ratios the concentration dependence of the shear modulus follows a power law behavior, G variation of phi n, with n = 0.34 +/- 0.03. At high swelling degrees, however, the shear modulus increases with increasing swelling. The value of the Flory-Huggins interaction parameter, chi, determined from osmotic swelling pressure and shear modulus measurements, strongly depends on the ionic composition of the equilibrium solution and increases with increasing Ca2+ concentration.

Video-enhanced contrast-differential interference contrast microscopy has revealed new features of axonal transport in the giant axon of the squid, where no movement had been detected previously by conventional microscopy. The newly discovered dominant feature is vast numbers of "submicroscopic" particles, probably 30- to 50-nanometer vesicles and other tubulovesicular elements, moving parallel to linear elements, primarily in the orthograde direction but also in a retrograde direction, at a range of steady velocities up to +/- 5 micrometers per second. Medium (0.2 to 0.6 micrometer) and large (0.8 micrometer) particles move more slowly and more intermittently with a tendency at times to exhibit elastic recoil. The behavior of the smallest particles and the larger particles during actual translocation suggests that the fundamental processes in the mechanisms of organelle movement in axonal transport are not saltatory but continuous.

Swelling of nerve fibers during the action potential was demonstrated by three different methods. Generation of a propagated nerve impulse in a crab nerve produced an outward movement of 50 to 100 angstroms of the nerve surfce and a rise in swelling pressure on the order of 5 dynes per square centimeter. In squid giant axons, the amplitude of the observed outward movement of the surface was small.

The volume transition induced by monovalent-divalent cation exchange of fully neutralized polyacrylate hydrogels was investigated in aqueous NaCl solutions. The variation of the osmotic swelling pressure, shear modulus, and mixing pressure was measured when Na(+) ions were substituted by divalent or trivalent cations. Alkali metal salts move freely throughout the entirely network, and alkaline earth metal salts (CaCl(2), SrCl(2)) promote aggregation of polyacrylate chains, but these aggregates are relatively weak. Transition metal salts (CoCl(2), NiCl(2)) form stronger interchain associates. Rare earth cations (La(3+) and Ce(3+)) bind practically irreversibly to the polymer. Experimental data indicate that transition metal cations modify both the elastic and mixing components of the free energy, while alkaline earth metal cations affect primarily the mixing term. The behavior of freely swollen gels was compared with similar gels subjected to uniaxial compression. In uniaxially compressed gels, volume transition occurs at lower cation concentrations than in the corresponding undeformed gels. The shift of the transition point increases with the deformation ratio and is larger for Co(2+) than for Ca(2+).

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.

1. Intracellular injection of tetraethylammonium chloride (TEA) into a giant axon of the squid prolongs the duration of the action potential without changing the resting potential (Fig. 3). The prolongation is sometimes 100-fold or more. 2. The action potential of a giant axon treated with TEA has an initial peak followed by a plateau (Fig. 3). The membrane resistance during the plateau is practically normal (Fig. 4). Near the end of the action potential, there is an apparent increase in the membrane resistance (Fig. 5D and Fig. 6, right). 3. The phenomenon of abolition of action potentials was demonstrated in the squid giant axon treated with TEA (Fig. 7). Following an action potential abolished in its early phase, there is no refractoriness (Fig. 8). 4. By the method of voltage clamp, the voltage-current relation was investigated on normal squid axons as well as on axons treated with TEA (Figs. 9 and 10). 5. The presence of stable states of the membrane was demonstrated by clamping the membrane potential with two voltage steps (Fig. 11). Experimental evidence was presented showing that, in an "unstable" state, the membrane conductance is not uniquely determined by the membrane potential. 6. The effect of low sodium water was investigated in the axon treated with TEA (Fig. 12). 7. The similarity between the action potential of a squid axon under TEA and that of the vertebrate cardiac muscle was stressed. The experimental results were interpreted as supporting the view that there are two stable states in the membrane. Initiation and abolition of an action potential were explained as transitions between the two states.

Mechanical and thermal changes associated with a propagated nerve impulse were determined using the garfish olfactory nerve. Production of an action potential was found to be accompanied by swelling of the nerve fibers. The swelling starts nearly at the onset of the action potential and reaches its peak at the peak of the action potential. There is a decrease in the length of the fibers while an impulse travels along the fibers. The time-course of the initial heat was determined at room temperature using heat-sensors with a response-time of 2-3 ms. Positive heat production was found to start and reach its peak nearly simultaneously with the action potential. The rise in temperature of the nerve was shown to be 23 (+/- 4) mu degrees C. In the range between 10 degrees and 20 degrees C, the temperature coefficient of heat production is negative, primarily due to prolongation of the period of positive heat production at low temperatures. The amount of heat absorbed during the negative phase varies widely between 45 and 85% of the heat evolved during the positive phase. It is suggested that both mechanical and thermal changes in the nerve fibers are associated with the release and re-binding of Ca-ions in the nerve associated with action potential production.

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