Optical measurement of presynaptic calcium currents

Measurements of presynaptic calcium currents are vital to understanding the control of transmitter release. However, most presynaptic boutons in the vertebrate central nervous system are too small to allow electrical recordings of presynaptic calcium currents (I-Ca(pre)). We therefore tested the possibility of measuring I-Ca(pre) optically in boutons loaded with calcium-sensitive fluorophores. From a theoretical treatment of a system containing an endogenous buffer and an indicator, we determined the conditions necessary for the derivative of the stimulus-evoked change in indicator fluorescence to report I-Ca(pre) accurately. Matching the calcium dissociation rates of the endogenous buffer and indicator allows the most precise optical measurements of I-Ca(pre). We tested our ability to measure I-Ca(pre) in granule cells in rat cerebellar slices. The derivatives of stimulus-evoked fluorescence transients from slices loaded with the low-affinity calcium indicators magnesium green and mag-fura-5 had the same time courses and were unaffected by changes in calcium influx or indicator concentration. Thus both of these indicators were well suited to measuring I-Ca(pre). In contrast, the high-affinity indicator fura-2 distorted I-Ca(pre). The optically determined I-Ca(pre) was well approximated by a Gaussian with a half-width of 650 mu s at 24 degrees C and 340 mu s at 34 degrees C.