Assessment of membrane permeability in primary cultures of neurons and glia in response to osmotic perturbation

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Abstract
In primary cultures of neurons and astrocytes from rat brain, the cellular contents of K+ and Na+ were used to assess plasma membrane permeability in response to osmotic shock. In contrast to trypan blue exclusion, the measure of ionic content reflected both transient and permanent membrane damage, and was also applicable to aggregates of cells. In steady state, the neurons and glia exhibited a K+ to Na+ ratio of 3–5, and the mean cellular contents (μEq/mg protein) of K+ and Na+ were 0.72 and 0.17 for astrocytes and 0.78 and 0.23 for neurons. Both hypo‐ and hypertonicity resulted in marked efflux of cell K+, but elevation of cellular Na+ occurred only under severe hypertonic conditions. Relative to neurons, astrocytes displayed considerably higher resistence to osmotic shock. During subsequent isotonic incubation, these cells were able to completely recover from transient membrane damage caused by a 10‐min exposure to fourfold hypertonicity. Permanent changes in glial permeability were obtained only after a 20‐min hypertonic shock. In contrast, 5 min hypertonic treatment of neurons decreased the ratio of cellular K+ to Na+ from 4.5 to 1. This ratio was restored twofold by isotonic incubation, but decreased permanently to below 1 after 10 min of hypertonic shock. The results describe marked differences in the osmotic fragility of neurons and glia and demonstrate that the determination of cellular K+ and Na+ provides a sensitive and accurate indicator of membrane permeability in neural cells propagated as surface‐growing cultures. The approach has wide‐ranging applicability.