Background: Fast Ca(2+) imaging using low-affinity fluorescent indicators allows tracking Ca(2+) neuronal influx at high temporal resolution. In some systems, where the Ca(2+)-bound indicator is linear with Ca(2+) entering the cell, the Ca(2+) current has same kinetics of the fluorescence time derivative. In other systems, like cerebellar Purkinje neuron dendrites, the time derivative strategy fails since fluorescence kinetics is affected by Ca(2+) binding proteins sequestering Ca(2+) from the indicator.
New method: Our novel method estimates the kinetics of the Ca(2+) current in cells where the time course of fluorescence is not linear with Ca(2+) influx. The method is based on a two-buffer and two-indicator model, with three free parameters, where Ca(2+) sequestration from the indicator is mimicked by Ca(2+)-binding to the slower buffer. We developed a semi-automatic protocol to optimise the free parameters and the kinetics of the input current to match the experimental fluorescence change with the simulated curve of the Ca(2+)-bound indicator.
Results: We show that the optimised input current is a good estimate of the real Ca(2+) current by validating the method both using computer simulations and data from real neurons. We report the first estimates of Ca(2+) currents associated with climbing fibre excitatory postsynaptic potentials in Purkinje neurons.
Comparison with existing methods: The present method extends the possibility of studying Ca(2+) currents in systems where the existing time derivative approach fails.
Conclusions: The information available from our technique allows investigating the physiological behaviour of Ca(2+) channels under all possible conditions.
Keywords: CA1 hippocampal pyramidal neuron; Calcium binding proteins; Calcium currents; Calcium imaging; Purkinje neuron.
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