Karima Ait Ouares1, Nadia Jaafari1, Marco Canepari2. 1. 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. 2. 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.
Abstract
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.
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.
Authors: Naomi Ak Hanemaaijer; Marko A Popovic; Xante Wilders; Sara Grasman; Oriol Pavón Arocas; Maarten Hp Kole Journal: Elife Date: 2020-06-17 Impact factor: 8.140