Neurons from the invertebrate Aplysia are cultured, observed electrophysiologically, stained with the fluorescent membrane dye DiI and imaged in the confocal light and high-voltage electron microscopes. The cells were subjected to a variety of osmotic challenges and their response in terms of membrane physiology and three-dimensional morphology were correlated. The cells were observed to change shape and size in accordance with osmotic pressure of the external media. The neurons were observed to maintain their altered shape and size until the osmolarity of the media was changed again i.e. they did not autoregulate as most cells do. Furthermore, they shrank at a much slower rate than they swelled. The volume change was demonstrated to be nonuniform i.e. some portions of the cell surface showed marked change while other regions did not change. It was postulated that plasma membrane infoldings accounted for the volume changes. Large surface blebs were also observed. Some of these structures changed volume in response to osmolarity while others did not. Since the neurons measure 50 to 150 fm, the HVEM is the only practical approach to studying their ultrastructure. Numerous areas along the plasma membrane showed extensive infoldings while other regions were single membrane layers. Surface structures or blebs observed at the light microscope level were shown to be membrane specializations which formed complex multiple membrane layers that undoubtedly influence the electrical properties of the cells. Some blebs were shown to be open to the external media and therefore not osmotically responsive while others contained normal cytoplasmic matrix.
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