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Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.

1. The degree to which mammalian brain cells swell in hypotonic environments has not previously been determined. We exposed hippocampal tissue slices prepared from anaesthetized rats to artificial cerebrospinal fluid from which varying amounts of NaCl had been deleted. Interstitial volume (ISV) change was determined from the volume of dilution of the marker ions tetramethylammonium (TMA+) or tetraethylammonium (TEA+). Tissue electrical resistance was measured as the voltage generated by constant current pulses. 2. ISV decreased as a function of lowered extracellular osmolality (osmotic pressure, pi o), indicating cell swelling. After reaching a minimum, ISV recovered partially, suggesting regulatory volume decrease of cells. After restoring normal pi o the ISV expanded, indicating post-hypotonic cell shrinkage. The electrical resistance of the tissue (Ro) increased when pi o was lowered, due to the reduced ionic strength, as well as restricted ISV. 3. To control for low NaCl concentration, reduced NaCl was replaced by mannitol or fructose. In isosmotic, NaCl-deficient solution, ISV showed inconsistent change, and Ro corrected for ionic strength tended to decrease. 4. Extracellular K+ concentration decreased slightly in low pi o except when spreading depression caused it to increase. Extracellular Ca2+ concentration decreased substantially, consistently and reversibly. Administration of isosmotic low-NaCl concentration solutions caused a similar decrease in extracellular Ca2+ concentrations. We propose that low Na+ concentration in extracellular fluid impaired the extrusion of Ca2+. 5. In severely hypotonic solution, ISV was reduced to 25% of its control volume, corresponding to a mean cell volume increase of at least 11%, probably more. From plotting relative changes in ISV against osmolarity we concluded that, within the range tested, hypotonic cell swelling was not opposed by the close approach of plasma membranes of neighbouring cells.

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