title: A generalised method to estimate the kinetics of fast Ca(2+) currents from Ca(2+) imaging experiments.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  The present method extends the possibility of studying Ca(2+) currents in systems where the existing time derivative approach fails.
  
  
  
  The information available from our technique allows investigating the physiological behaviour of Ca(2+) channels under all possible conditions.
authors.affiliation: Laboratory for Interdisciplinary Physics, UMR 5588, Université Grenoble Alpes and CNRS, 38402 Saint Martin d'Hères, France; Laboratories of Excellence, Ion Channel Science and Therapeutics, France.
authors.forename: Karima
authors.initials: K
authors.lastname: Ait Ouares
authors.affiliation: Laboratory for Interdisciplinary Physics, UMR 5588, Université Grenoble Alpes and CNRS, 38402 Saint Martin d'Hères, France; Laboratories of Excellence, Ion Channel Science and Therapeutics, France.
authors.forename: Nadia
authors.initials: N
authors.lastname: Jaafari
authors.affiliation: Laboratory for Interdisciplinary Physics, UMR 5588, Université Grenoble Alpes and CNRS, 38402 Saint Martin d'Hères, France; Laboratories of Excellence, Ion Channel Science and Therapeutics, France; Institut National de la Santé et Recherche Médicale (INSERM), France. Electronic address: marco.canepari@univ-grenoble-alpes.fr.
authors.forename: Marco
authors.initials: M
authors.lastname: Canepari
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism; D003198:Computer Simulation; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology*; D008810:Mice, Inbred C57BL; D008959:Models, Neurological; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D017711:Nonlinear Dynamics; D010363:Pattern Recognition, Automated / Q000379:methods; D012815:Signal Processing, Computer-Assisted; D046509:Tissue Culture Techniques; D056969:Voltage-Sensitive Dye Imaging / Q000379:methods*
chemical_list: D015220:Calcium Channels; D002118:Calcium
keywords: CA1 hippocampal pyramidal neuron; Calcium binding proteins; Calcium currents; Calcium imaging; Purkinje neuron