Calcium-dependent volume reduction in regenerating ganglion cell axons in vitro.

The effects of increasing [Ca2+]i on volume regulatory behavior was investigated by phase-contrast videomicroscopy in immature axons regenerating from goldfish retinal explants in vitro. Elevating [Ca2+]i by using EGTA-buffered, ionomycin-containing bathing media with either greater than or equal to 100 microM [Ca2+]o or 1 microM [Ca2+]o with N-methylglucamine substituted for Na+ caused axons to undergo a "syneresis." The syneresis was characterized by a marked loss in volume and condensation of axoplasm, accompanied by a proliferation of lateral processes, which resulted ultimately in an arrest of visible particle transport. The random appearance of dynamic phase-lucent axial protrusions in the distal axon, apparently caused by microtubules, was a frequent early manifestation. Syneresis was also produced by increasing the tonicity of the Cortland saline with sorbitol or treating axons with either valinomycin or with permeant cyclic AMP analogs in normal Cortland saline. In the latter case, extracellular Ca2+ was required. Preterminal axons showed an increase in phalloidin fluorescence after syneresis, suggesting polymerization and/or rearrangement of the actin cytoskeleton. Digitonin-permeabilized axonal field models, which maintained good morphology and particle transport, failed to develop a syneresis even when [Ca2+]o was increased to 250 microM. Cytochalasin D did not interfere with the development of a syneresis, but did suppress the proliferation of lateral processes. Syneresis could be blocked by high [K+]o, putative antagonists of Ca2(+)-activated K+ channels, or by calmidazolium, a calmodulin antagonist. The experimental findings suggest that cytoskeletal changes associated with volume reduction in growing retinal ganglion cell axons are secondary to a loss of cell water and that calcium/calmodulin-activated K+ channels very likely play a primary role in dehydration through the loss of K+ and osmotically obligated water.