DISCRETE AND REVERSIBLE VACUOLE-LIKE DILATIONS INDUCED BY OSMOMECHANICAL PERTURBATION OF NEURONS

In cultured Lymnaea stagnalis neurons, osmolarity increases (upshocks) rapidly elicited large membranous dilations that could be dislodged a nd pushed around inside the cell with a microprobe. Subsequent osmolar ity decreases (downshocks) caused these vacuole-like dilations (VLDs) to disappear. Additional upshock/downshock perturbations resulted in r epeated appearance/disappearance (formation/reversal) of VLDs at discr ete sites. Confocal microscopy indicated that VLDs formed as invaginat ions of the substrate-adherent surface of the neuron: extracellular rh odamine-dextran entered VLDs as they formed and was expelled during re versal. Our standard VLD-inducing perturbation was: 2-4 min downshock to distilled water, upshock to normal saline, However, a wide range of other osmotic perturbations (involving osmolarities up to 3.5x normal , perturbations with or without Ca2+, replacement of ions by sucrose) were also used. We concluded that mechanical, not chemical, aspects of the osmo-mechanical shocks drove the VLD formation and reversal dynam ics and that extracellular Ca2+ was not required. Following a standard perturbation, VLDs grew from invisible to their full diameter (>10 mu m) in just over a minute. Over the next 0.5-3 hr in normal saline, ne urons recovered. Recovery eliminated any visible VLDs and was accompan ied by cytoplasmic turmoil around the VLDs. Recovery was prevented by cytochalasin B, brefeldin A and N-ethylmaleimide but not by nocodazole . In striking contrast, these drugs did not prevent repeated VLD forma tion and reversal in response to standard osmo-mechanical perturbation s; VLD disappearance during reversal and during recovery are different . The osmo-mechanical changes that elicited VLDs may, in an exaggerate d fashion, mimic tension changes in extending and retracting neurites. In this context we postulate: (a) the trafficking or disposition of m embrane between internal stores and plasma membrane is mechanosensitiv e, (b) normally, this mechanosensitivity provides an ''on demand'' sys tem by which neurons can accommodate stretch/release perturbations and control cell shape but, (c) given sudden extreme mechanical stimuli, it yields VLDs.