Activity associated ultrastructural changes in peripheral nodes of Ranvier are independent of fixation

We recently reported structural changes in nodes of Ranvier of frog dorsal roots that are associated with activation at 20 or 50 Hz for 15 min. The structural effects co-occur with alterations in the latency, amplitude, and waveform of the compound action potential. The alterations in nodal morphology include formation of large extracellular vacuoles separating myelin terminal lammelae of the paranodal apparatus. Vacuolization was not apparent when the nerve was allowed to recover for 30 min prior to fixation and processing for transmission electron microscopy, indicating that it is a transient phenomenon. We first observed this effect in axons from nerve roots fixed with aldehydes and processed conventionally for TEM. The possibility that the changes noted actually occur later during tissue processing, reflecting differences in sensitivity to fixation or dehydration, remained. To address this concern we examined activated frog nodes processed by three different methods: (i) rapid freezing/freeze-substitution, (ii) direct osmication, and (iii) primary fixation in a mixture of aldehydes and osmium. Under these conditions paranodal vacuolization is still quite evident, but there are subtle differences, primarily in the appearance of the vacuoles. Also the nonstimulated control nodes fixed by direct osmication and by mixed aldehyde-osmium tended to have more vacuolized paranodes than did either the conventionally fixed or freeze-substituted preparations. Regardless, the changes are similar to those seen previously and very likely reflect changes occurring in situ. We interpret these results in the context of a functional model involving accumulation of potassium in the interstices of the compact myelin during impulse propagation. During periods of extended activity this system may be overloaded promoting focal edema and mechanical disruption of some paranodal structures. These changes could then bias subsequent nodal functions such as activation threshold, firing patterns, and conduction velocity.