The effect of tonicity, d-mannitol, d-sorbitol, and choline chloride on the water content of mouse brain slices, and on the compartmentation of the space indicated by ‘extracellular space markers’

Abstract

(1) Mouse cerebrum slices were incubated with ‘extracellular space markers’ in either HEPES-( N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffered media with various concentrations of NaCl, or standard HEPES-buffered medium with 400 mM choline chloride, 800 mM d-mannitol, or 800 mM d-sorbitol added. Nominal tonicities of the media ranged from 170 to 1100 mosM. (2) Tissue swells extensively in media ranging from hypotonic to hypertonic containing various salt concentrations. Swelling is less in the choline chloride medium, and is slight in the d-mannitol or d-sorbitol medium. (3) In media with various salt concentrations, the penetration of inulin, sucrose, d-mannitol, d-sorbitol, and sulfate into tissue is greater at 37°C than at 0°C, showing the presence of a second marker (inulin) space for these substances; i.e., a tissue compartment which is accessible to the marker substance at 37°C but not at 0dgC (Cohen et al. 6). At both temperatures, the smaller the marker molecule, the larger the indicated ‘extracellular space’; and the greater the NaCl concentration, the greater the marker penetration. The size of the second space is only slightly dependent on the marker, or on the salt concentration. For some markers, the second space is reduced or absent in the sugar alcohol or choline chloride medium. (4) Several ‘extracellular space markers’ may enter some cellular compartment. The 800 mM sugar alcohols increase the permeability of cells to markers. Choline chloride, d-mannitol, and d-sorbitol, in high concentrations, enter most tissue water but are not actively concentrated. (5) The entry of thiocyanate is anomalous, being consistently greater at 0°C than 37°C. (6) In high NaCl medium, d-glutamate at 0°C penetrates tissue to the same extent as inulin at 37°C thus indicating the continuing existence at 0°C of a tissue compartment corresponding to the 37°C inulin space. Several other amino acids give larger tissue spaces at 0°C, with concentrative uptake of l-histidine and l-leucine occurring by 20 min incubation, and glycine by 60 min incubation. (7) It has been suggested that the second marker space may consist of a ramifying system of microtubules extending throughout a cell body and communicating with the extracellular space through a micropore. The effects of the incubation medium on the second space and on the total tissue water may be due in part to changes in the size of these tubules.