title: Discrete and reversible vacuole-like dilations induced by osmomechanical perturbation of neurons.
abstract: In cultured Lymnaea stagnalis neurons, osmolarity increases (upshocks) rapidly elicited large membranous dilations that could be dislodged and pushed around inside the cell with a microprobe. Subsequent osmolarity decreases (downshocks) caused these vacuole-like dilations (VLDs) to disappear. Additional upshock/downshock perturbations resulted in repeated appearance/disappearance (formation/reversal) of VLDs at discrete sites. Confocal microscopy indicated that VLDs formed as invaginations of the substrate-adherent surface of the neuron: extracellular rhodamine-dextran entered VLDs as they formed and was expelled during reversal. 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 3.5 x 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 dynamics and that extracellular Ca2+ was not required. Following a standard perturbation, VLDs grew from invisible to their full diameter (> 10 microns) in just over a minute. Over the next 0.5-3 hr in normal saline, neurons recovered. Recovery eliminated any visible VLDs and was accompanied 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 formation and reversal in response to standard osmo-mechanical perturbations; VLD disappearance during reversal and during recovery are different. The osmo-mechanical changes that elicited VLDs may, in an exaggerated fashion, mimic tension changes in extending and retracting neurites. In this context we postulate: (a) the trafficking or disposition of membrane between internal stores and plasma membrane is mechanosensitive, (b) normally, this mechanosensitivity provides an "on demand" system by which neurons can accommodate stretch/release perturbations and control cell shape but, (c) given sudden extreme mechanical stimuli, it yields VLDs.
authors.affiliation: Loeb Institute, Ottawa Civic Hospital, Ontario, Canada.
authors.forename: C
authors.initials: C
authors.lastname: Reuzeau
authors.forename: L R
authors.initials: LR
authors.lastname: Mills
authors.forename: J A
authors.initials: JA
authors.lastname: Harris
authors.forename: C E
authors.initials: CE
authors.lastname: Morris
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002448:Cell Adhesion; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000502:physiology*; D008195:Lymnaea / Q000166:cytology / Q000502:physiology*; D018613:Microscopy, Confocal; D009474:Neurons / Q000502:physiology* / Q000648:ultrastructure; D009994:Osmolar Concentration; D013314:Stress, Mechanical
chemical_list: D002118:Calcium