Optical measurement of presynaptic calcium currents

ABSTRACT 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 (ICa pre). We therefore tested the possibility of measuring ICa 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 ICa pre accurately. Matching the calcium dissociation rates of the endogenous buffer and indicator allows the most precise optical measurements of ICa pre. We tested our ability to measure ICa 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 ICa pre. In contrast, the high-affinity indicator fura-2 distorted ICa pre. The optically determined ICa pre was well approximated by a Gaussian with a half-width of 650 ms at 24°C and 340 ms at 34°C