In the first three reporting periods we have first focused on WPA&B; aspects of the project related to pathology (WPC) are starting now that enough progress has been made on the two first WPs. Methodological approaches for (i) long-term and (ii) large-scale imaging have been implemented and are now used to address the neurodevelopment-related goals of the project.
(i) Regarding long-term imaging, a dedicated in vivo 2P-microscope for large-scale in vivo imaging has been successfully installed in the Cossart lab and pilot 3P-calcium imaging experiments were performed. Imaging of hippocampal and neocortical assemblies in the adult mouse across different behavioral states has been performed. A complete in vivo calcium imaging dataset of the activity from clonally-resolved CA1 neurons in the adult mouse across 2 consecutive weeks has been obtained (n=13 mice imaged and analyzed), as well as longitudinal imaging during development from P15 into adulthood (Leprince et al. biorxiv 2025, revised in Current Biology). The role of local interneurons in shaping CA1 assemblies in the adult has been dissected using optogenetics (Bocchio, Vorobyev et al. PLOS Biology 2024) and the involvement of long-range extrahippocampal inputs during development (Leprince et al. Neuron 2023). In vivo assembly imaging during the early perinatal period (P1-14) has been successfully developed in the mouse neocortex (Majnik et al. 2025, Zangila et al. in preparation). In particular, longitudinal tracking of activity from the same cell assemblies during early postnatal development has been achieved (Majnik et al. eLife 2025). A two-color 3-photon laser system was developed and validated at the Beaurepaire lab. This process has been delayed by unexpected technical failures experienced during this technical development. The exploration of 3P color microscopy has led to the unanticipated discovery of a new imaging modality, TSFG microscopy, which is a label-free technique sensitive to pigments absorption (Ferrer Ortas et al. Light Sci Appl 2023; Dees et al. submitted).
(ii) Regarding large-scale imaging, the Beaurepaire and Livet groups implemented a new, accelerated generation of ChroMS microscopy (Abdeladim et al. 2019), a key technology for the project enabling brain-wide color imaging. Among others, an adaptative correction strategy was introduced for compensation of chromatic aberrations over the entire field of view (Blanc et al. ACS Photonics 2023). We also implemented a dedicated image reconstruction/stitching workflow and developed a machine learning-based workflow for automated cell detection and color analysis in these large datasets. With this approach, we were able to obtain coregistered live / structural images of Calcium activity and Brainbow labels in CA1, unlocking a key difficulty of the project. In parallel, new tools adapted to the project are being developed for neural cell labeling (Marcireau, Kaddour et al, in preparation), functional perturbation (Medvedeva et al, in preparation) and circuit analysis (Phan et al, PLoS Comput Biol. 2022; Matho, Phan et al, in preparation).
