Search

Fluorescence calcium imaging using a range of microscopy approaches, such as two-photon excitation or head-mounted "miniscopes," is one of the preferred methods to record neuronal activity and glial signals in various experimental settings, including acute brain slices, brain organoids, and behaving animals. Because changes in the fluorescence intensity of genetically encoded or chemical calcium indicators correlate with action potential firing in neurons, data analysis is based on inferring such spiking from changes in pixel intensity values across time within different regions of interest. However, the algorithms necessary to extract biologically relevant information from these fluorescent signals are complex and require significant expertise in programming to develop robust analysis pipelines. For decades, the only way to perform these analyses was for individual laboratories to write their custom code. These routines were typically not well annotated and lacked intuitive graphical user interfaces (GUIs), which made it difficult for scientists in other laboratories to adopt them. Although the panorama is changing with recent tools like CaImAn , Suite2P , and others, there is still a barrier for many laboratories to adopt these packages, especially for potential users without sophisticated programming skills. As two-photon microscopes are becoming increasingly affordable, the bottleneck is no longer the hardware, but the software used to analyze the calcium data optimally and consistently across different groups. We addressed this unmet need by incorporating recent software solutions, namely NoRMCorre and CaImAn, for motion correction, segmentation, signal extraction, and deconvolution of calcium imaging data into an open-source, easy to use, GUI-based, intuitive and automated data analysis software package, which we named EZcalcium ., (Copyright © 2020 Cantu, Wang, Gongwer, He, Goel, Suresh, Kourdougli, Arroyo, Zeiger and Portera-Cailliau.)

The first three postnatal weeks in rodents are a time when sensory experience drives the maturation of brain circuits, an important process that is not yet well understood. Alterations in this critical period of experience-dependent circuit assembly and plasticity contribute to several neurodevelopmental disorders, such as autism, epilepsy, and schizophrenia. Therefore, techniques for recording network activity and tracing neuronal connectivity over this time period are necessary for delineating circuit refinement in typical development and how it deviates in disease. Calcium imaging with GCaMP6 and other genetically encoded indicators is rapidly becoming the preferred method for recording network activity at the single-synapse and single-cell level in vivo , especially in genetically identified neuronal populations. We describe a protocol for intracortical injection of recombinant adeno-associated viruses in P1 neonatal mice and demonstrate its use for longitudinal imaging of GCaMP6s in the same neurons over several weeks to characterize the developmental desynchronization of cortical network activity. Our approach is ideally suited for chronic in vivo two-photon calcium imaging of neuronal activity from synapses to entire networks during the early postnatal period.