Silvestro Micera, Maria Pasquini, Francesco Saverio Pavone
Genetically encoded calcium indicators (GECIs) can be used to monitor the intracellular concentration of free calcium in populations of neurons. 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 such as channelrhodopsin. The aim of this work is to evaluate the performances of four red-shifted GECIs (jRCaMP1a, jRCaMP1b, jRGECO1a, jRGECO1b) using both ex vivo and in vivo experimental techniques. Cortical neurons were infected with adeno-associated virus (AAV) expressing one of the red GECI variants. First we characterized the transfection ex vivo in terms of extension and intensity of the indicator. Next, we monitored the neuronal activity over the right cortical hemisphere of a jRCaMP1a-transfected mouse during a goal-directed motor task. To this aim, we combined wide-field fluorescence microscopy with a robotic device 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 simultaneously over multiple functional areas, and can be successfully used to perform longitudinal imaging sessions in awake mice.
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