Fig. 7. Low Na⁺ buffer, ethacrynic acid and protease inhibitors prevent NMDA-induced dendritic varicosity formation. (A) Representative confocal images of dendrites of CA1 pyramidal cells in untreated slices (a) or 30 µM NMDA-treated slices in normal (b) or low Na⁺ solution (c). Culture medium was changed to low Na⁺ buffer 30 minutes before 10 minutes exposure to NMDA. No varicosity was generated under low Na⁺ conditions. (B) Effects of various manipulations and drugs on NMDA-induced varicosity formation. The culture medium was changed to lowered Na⁺ solution or 200 mM sucrose-containing (hyperosmotic) medium 30 minutes before NMDA exposure. Tetrodotoxin (1 µM), HgCl₂ (100 µM), amiloride (100 µM), furosemide (100 µM) and ethacrynic acid (400 µM) were added 30 minutes before NMDA exposure. A mixture of protease inhibitors (1:1000) was applied 12 hours prior to NMDA exposure. Slices received 10 minutes treatment with 0 µM (open columns) or 30 µM NMDA (filled columns), immediately followed by fixation with 4% paraformaldehyde. Low Na⁺ buffer, ethacrynic acid and protease inhibitors significantly blocked the varicosity formation, whereas tetrodotoxin, amiloride and furosemide induced varicosity formation. Data are expressed as the means±s.e.m. of 9-12 slices. **P<0.01 versus intact slices (None and Control), ^##P<0.01 versus NMDA alone (None and NMDA).