A characterization of muscarinic receptor‐mediated intracellular Ca2+ mobilization in cultured rat hippocampal neurones

Abstract

1. The properties of muscarinic receptor-mediated Ca2+ mobilization were investigated in hippocampal cultures using fluorescent imaging techniques. 2. Somatic responses to carbachol (1-10 microM) were observed in 21 % of neurones under control conditions (5.4 mM K+, 1. 8 mM Ca2+, 0.5-1 microM tetrodotoxin). Smaller responses were observed in Ca2+-free medium. 3. In cells where responses to carbachol were absent under control conditions, responses were often observed following depolarization with high extracellular K+ (16. 2-25 mM). These responses decreased in magnitude with time after the depolarizing episode. Mobilization of Ca2+ from stores using caffeine (50 mM) exhibited similar properties. 4. Carbachol responses were greatly facilitated in the presence of moderate elevations in extracellular K+ or Ca2+ levels (2- or 3-fold, respectively). These conditions were usually, but not always, associated with a small increase in cytosolic Ca2+ levels (< 50 nM). 5. Muscarinic responses in 10.8 mM K+ were inhibited by 80-95 % in the presence of the L-type voltage-gated Ca2+ channel antagonists nitrendipine (2-5 microM) or nifedipine (10 microM). Depletion of intracellular Ca2+ stores with thapsigargin (2-10 microM) blocked responses. 6. Oscillatory Ca2+ mobilizing responses were observed in some cells. Their expression was facilitated by moderate cytosolic Ca2+ elevations and by increasing the duration of carbachol exposure. 7. Ca2+ mobilizing responses were also observed in dendritic regions. These were smaller than somatic responses, but had faster decay kinetics. 8. In conclusion, muscarinic receptor-mediated Ca2+ mobilization in cultured hippocampal neurones shows a strong Ca2+ dependence. Moderate intracellular Ca2+ rises greatly facilitate muscarinic responses and uncover, in some cells, oscillatory Ca2+ mobilization. These effects appear to reflect the loading state of intracellular Ca2+ stores.