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.

Summary Sensory perturbations in visual, auditory and tactile perception are core problems in Fragile X Syndrome (FXS). In the Fmr1 knockout mouse model of FXS, the maturation of synapses and circuits during critical period (CP) development in the somatosensory cortex is delayed, but it is unclear how this contributes to altered tactile sensory processing in the mature CNS. Here we demonstrate that inhibiting the juvenile chloride co-transporter NKCC1, which contributes to altered chloride homeostasis in developing cortical neurons of FXS mice, rectifies the chloride imbalance in layer IV somatosensory cortex neurons and corrects the development of thalamocortical excitatory synapses during the CP. Comparison of protein abundances demonstrated that NKCC1 inhibition during early development caused a broad remodeling of the proteome in the barrel cortex. In addition, the abnormally large size of whisker-evoked cortical maps in adult Fmr1 knockout mice was corrected by rectifying the chloride imbalance during the early CP. These data demonstrate that correcting the disrupted driving force through GABAA receptors during the CP in cortical neurons restores their synaptic development, has an unexpectedly large effect on differentially expressed proteins, and produces a long-lasting correction of somatosensory circuit function in FXS mice.

Imaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-β plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals.

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.

Fluorescence calcium imaging using a range of microscopy approaches, such as 2-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 own 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 2-photon microscopes are becoming increasingly affordable, 2 the bottleneck is no longer the hardware, but the software used to analyze the calcium data in an optimal manner and consistently across different groups. We addressed this unmet need by incorporating recent software solutions 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, which we named EZcalcium.