Cytoplasmic gel and water relations of axon.

A previous method of measuring the swelling pressure (delta IIg) of the cytoplasmic gel of the giant axon of Loligo vulgaris was refined. The estimates of delta IIg made with the improved method were consistent with those made with the earlier method. In these methods the activity of the solvent in the gel is measured by increasing the activity of the solvent in the internal phase of the gel by application of hydrostatic pressure to the gel directly. Comparable values for the activity of the solvent in the gel were obtained also by an alternate method, in which the deswelling of the gel is measured upon decreasing the activity of the solvent in the external phase by addition of a nonpenetrating high mol wt polymer (i.e., Ficoll). Additional support was obtained for the earlier suggestion that delta IIg contributes to the swelling and shrinkage pattern of the whole axon. In part, the new evidence involved two consecutive direct measurements of intraxonal pressure. The first measurement was that of a mixed pressure composed of delta IIg and delta IIm (delta IIm being the effective osmotic pressure due to the intra-extraxonal gradient in the activity of mobile solutes). The subsequent measurement was that of delta IIg alone. The latter measurement was made feasible by destroying the axolemma, thereby eliminating the contribution of delta IIm. An estimate of delta IIm was obtained by subtracting delta IIg from the total pressure measured initially. The delta IIm determined by the above method was two orders of magnitude smaller than the theoretical osmotic pressure. This is consistent with the delta IIm determined previously, where osmotic intra-extraxonal filtration coefficients were compared to the hydrostatic. The mixed pressure experiments lend credence to the idea that the substantial contribution of delta IIg to the water relations of the whole axon is due to delta IIg being of the same order of magnitude as delta IIm. The degree of free swelling of axoplasmic gels was studied as a function of pH, salt concentration, and hydration radius of the anion of the salt used. The swelling increased with an increase in the reciprocal of the hydration radius, a decrease in salt concentration, and at pH below or above similar to 4.5. The nature of the constraints to the free swelling of axoplasm in axons immersed in seawater was studied. With the seawater employed, these constraints appeared to be due more to the retractive forces of the sheath than to delta IIm.

Water fluxes in nerve fiber.

The hydrostatic (Lp) and osmotic (LPD) filtration coefficients and the efflux rates of tritiated water were measured in the giant axon of Loligo vulgaris. The Lp was 8 to 14 X 10(-8) cm/sec/cm H2O and the LPD was two orders of magnitude smaller (3 to 6 X 10(-10) cm/sec/cm H2O). In axons whose diameter was approximately 500 micron, the time (t1/2) required for a reduction in the axonal labeled water activity to one half its initial value was 38 to 48 sec. The rate limiting structure for solute flux was made ineffective by (1) storing the axon in isosmotoc KF at 0-2 degrees C for one month to one year or by (2) fixing the axon in 2-4% glutaraldehyde for 3 to 7 hr. The criteria of ineffectiveness of the rate limiting structure for solute flux were (1) a reduction of LPD to immeasurably low values, (2) the absence of electrical properties characteristic of plasmalemma, and (3) a marked increase in the rate of efflux of Na22. In such impaired axons the Lp and the t 1/2 of tritiated water efflux were unaffected. This independence of solute and solvent flux in conjunction with the finding that the hydraulic conductivity determined by bulk osmotic and hydrostatic pressure gradients is not equivalent (i.e., LPD/LP less than 1) indicate that the rate limiting structures for solute and solvent flux are in series. Solvent fluxes appear to be surface-limited, not bulk-limited. We have been unable to resolve whether the surface structure involved in limiting solvent flux is the sheath (Schwann layer and adhering connective tissue) and/or the cortical layer of the axoplasmic gel.

Osmotic relations of nerve fiber.

The volumetric elastic modulus of the sheath and the osmotic swelling pressure of the axoplasmic polymer network of the giant axon of Loligo vulgaris were measured. Evidence was obtained that (1) the elastic modulus of the sheath, (2) the swelling pressure of axoplasm, and (3) the effective osmotic pressure difference due to mobile solutes determine axonal volume. The contributions of the sheath and the axoplasm were significant because the effective osmotic pressure due to mobile solutes was a small fraction of the theoretical bulk osmotic pressure due to these solutes. The giant axon was converted from an imperfect to a near perfect osmometer by minimizing the contribution of the sheath and the axoplasmic gel.