Imaging of cortical neuronal-activity with red-shifted functional indicators during motor task execution

Intracellular concentration of free calcium ions in neuronal populations can be longitudinally evaluated by using fluorescent protein indicators, called genetically encoded calcium indicators (GECIs). GECIs with long emission wavelengths are particularly attractive for deep tissue microscopy in vivo, and have the additional advantage of avoiding spectral overlap with commonly used neuronal actuators like Channelrhodopsin. Here we investigated the performances of selected red-shifted GECIs through an ex vivo characterization and in vivo imaging of cortical mouse activity during motor task execution. Cortical neurons were infected with adeno-associated virus (AAV) expressing one of the red GECI variants (jRCaMP1a, jRCaMP1b, jRGECO1a, jRGECO1b). First we characterized the transfection in terms of extension and intensity using wide-field fluorescence microscopy. Next, we used RCaMP1a to analyse the cortical neuronal activity during motor behaviour. To that end, wide-field fluorescent microscopy and a robotic device for motor control were combined for simultaneous recording of cortical neuronal-activity, force applied and forelimb position during task execution. Our results show that jRCaMP1a has sufficient sensitivity to monitor in vivo neuronal activity over multiple functional areas, and can be successfully used to perform longitudinal imaging in awake mice.