Protocol for using the MCA toolbox in ImageJ for single cell calcium imaging analysis

Here, we provide a description of the behavioral response and dataset used for this protocol pipeline. Following the loss of environmental illumination, zebrafish will engage in a circling behavior in an attempt to locate a source of light. On repeated observations of this behavior, zebrafish and other fish species will preferentially circle in a consistent direction, exhibiting a persistent motor bias for either leftward or rightward circling. This behavior is controlled by a subset of approximately 60 neurons in the lateral thalamus referred to as asymmetry maintaining neurons (AMNs). The AMNs are made up of two bilateral clusters that maintain an individual’s motor bias direction. Following the loss of illumination, AMNs will exhibit larger calcium flux in the AMN cluster from the hemisphere contralateral to the individual’s bias. For example, a rightward circling zebrafish will exhibit stronger responses to the loss of illumination from the AMN cluster in the left hemisphere of the brain. This correlation between behavior, neurophysiology, and neuron type provides a powerful system to resolve and evaluate functional imaging data. Moreover, this dataset allows for the application of motion correction, cell segmentation using nuclear localized GCaMP, and ROI segmentation. For the purpose of this protocol, the calcium imaging dataset used to determine this functional thalamic asymmetry will be used as a sample dataset. However, MCA is compatible with a variety of functional imaging techniques and is viable across multiple GEIs, model organisms, and imaging methods such as confocal and multiphoton based functional imaging. This protocol is targeted towards computers and laptops running the Microsoft Windows 11 operating system; however, it is also applicable to Linux and Mac operating systems.