Calcium transients in identified leech glial cells in situ evoked by high potassium concentrations and...

We have recorded the fluorescence of Fura-2-loaded, identified glial cells in the neuropile of the central nervous system of the leech Hirudo medicinalis using the ratio of emission at 350 nm excitation to that at 380 nm excitation as an indicator of intracellular Ca2+ concentration ([Ca2+]i). The neuropile glial cells were exposed by mechanically removing the overlying ganglionic capsule and the neuronal cell bodies and were then impaled using a microelectrode under visual control to inject Fura-2 ionophoretically. The resting Ca2+ level was measured using digitonin or Triton to permeabilize the cell membrane at different external concentrations of Ca2+; it was found to vary between 5 and 79 nmol l-1 and averaged 32 +/- 23 nmol l-1 (+/- S.D., N = 7). Raising the external K+ concentration from 4 to 20 mmol l-1 or adding 50 mumol l-1 5-hydroxytryptamine (5-HT) produced a rapid, reversible rise in [Ca2+]i. During prolonged exposure to high [K+] or 5-HT, [Ca2+]i remained high. Upon restoring normal external [K+] or removing 5-HT, [Ca2+]i returned to its initial resting value within 1-2 min. The responses of [Ca2+]i to high [K+] and 5-HT were greatly reduced in nominally Ca(2+)-free saline, suggesting that the [Ca2+]i transients required an influx of Ca2+ into the cells. In the presence of 5-HT, the rise in [Ca2+]i was accompanied by a decrease in the resistance and an increase in the responsiveness to K+ of the glial cell membrane, indicating the existence of a Ca(2+)-dependent K+ conductance elicited by 5-HT.