Changes in intrinsic optical signals could be related to cell swelling; however, the evidence is not compelling. We measured light transmittance, ECS volume fraction (alpha), and extracellular K+ in rat spinal cord slices during electrical stimulation and the application of elevated potassium, NMDA, or anisoosmotic solutions. Dorsal root stimulation (10 Hz/1 min) induced an elevation in extracellular K+ to 6-8 mM, a light transmittance increase of 6-8%, and a relative ECS volume decrease of less than 5%; all of these changes had different time courses. The application of 6 or 10 mM K+ or NMDA (10(-5) M) had no measurable effect on alpha, but light transmittance increased by 20-25%. The application of 50 or 80 mM K+ evoked a 72% decrease in alpha while the light transmittance increase remained as large as that in 6 or 10 mM K+. While the change in alpha persisted throughout the 45-min application, light transmittance, after peaking in 6-8 min, quickly returned to control levels and decreased below them. Astrocytic hypertrophy was observed in 6, 10, and 50 mM K+. The same results followed the application of 10(-4) M NMDA or hypotonic solution (160 mmol/kg). The elevation of extracellular K+ after NMDA application, corresponding to increased neuronal activity, had a similar time course as the light transmittance changes. Furosemide, Cl(-)-free, or Ca(2+)-free solution blocked or slowed down the decreases in alpha, while the light transmittance increases were unaffected. In hypertonic solution (400 mmol/kg), alpha increased by 30-40%, while light transmittance decreased by 15-20%. Thus, light transmittance changes do not correlate with changes in ECS volume but are associated with neuronal activity and morphological changes in astrocytes.