Ca2+ Uncaging in Nerve Terminals: A Three-Point Calibration Procedure.

ABSTRACT

Ca(2+) uncaging can be used to create a spatially homogenous elevation of the intracellular free Ca(2+) concentration, [Ca(2+)]i, in cells. When applied to nerve terminals or secretory cells, this technique allows one to elicit transmitter release with a [Ca(2+)]i signal of measurable amplitude, and therefore to directly relate the rate of transmitter release to the measured [Ca(2+)]i. When combined with patch-clamp measurements, Ca(2+) uncaging is done by introducing a Ca(2+)-loaded photolyzable Ca(2+) chelator (like DM-nitrophen) into the cell via the whole-cell patch-pipette. A brief light pulse from a flash lamp or a pulsed laser is used to photolyze the DM-nitrophen. The resulting increase in [Ca(2+)]i is measured with ratiometric fluorescent indicators of suitable Ca(2+) affinity, such as Fura-2, Fura-4F, Fura-2FF, or Fura-6F, depending on the postflash [Ca(2+)]i values. To quantitatively measure [Ca(2+)]i, an accurate calibration of the fluorescent indicator in the presence of the photolyzable Ca(2+) chelator is necessary, which will be described here. Ca(2+) uncaging in nerve terminals has proven useful for investigating Ca(2+)-dependent functions like transmitter release, short-term plasticity, and exocytosis-endocytosis coupling in the presynaptic compartment of neurons.