Subaxolemmal filamentous network in the giant nerve fiber of the squid (Loligo pealei L.) and its possible role in excitability.

Abstract

A new technique utilizing the squid [L. peali] giant nerve fiber was developed which permitted direct examination of the inner face of the axolemma by scanning electron microscopy [SEM]. The axoplasm was removed sequentially in a 15-mm long segment of the fiber by intracellular perfusion with a solution of KF, KCl, Ca2+-containing seawater, or with pronase. The action potential of the fibers was monitored during these treatments. After brief prefixation in 1% paraformaldehyde and 1% glutaraldehyde, the perfused segment was opened by a longitudinal cut with a razor bade. The overall view of the opened perfusion zone could be related to information on the detailed morphology of the cytoplasmic face of the axolemma and the ectoplasm. The results obtained by SEM were further substantiated by transmission electron microscopy of thin sections. Living axons were studied with polarized light during the axoplasm removal, and the identification of actin by heavy meromyosin labeling and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was accomplished. A three-dimensional network of interwoven filaments, consisting partaly of an actinlike protein, was firmly attached to the axolemma. The axoplasmic face of fibers in which the filaments were removed partially after perfusion with pronase displayed smooth membranous blebs and large profiles which apposed the axolemma. In fibers where the excitability was suppressed by pronase perfusion, about 1/3 of the inner face of the axolemma in the perfusion zone was free of filaments. Attachment of axoplasma filaments to the axolemma may have a role in the maintenance of the normal morphology of the axolemma and thus, in some aspect of excitability.