Periodic Reporting for period 2 - HOPE (Reverse engineering the assembly of the hippocampal scaffold with novel optical and transgenic strategies)
Reporting period: 2023-01-01 to 2024-06-30
We aim at shedding a unique light on hippocampal function at circuit level through the design of a novel method to trace the construction of brain circuits in health and disease based on hybrid multiphoton in vivo longitudinal colour imaging of the dynamics and structure of genetically-tagged neuronal clones from birth into adulthood. This is made possible by the exceptional interdisciplinary collaboration of three labs with expertise in advanced optical microscopy, genetic engineering and systems developmental neuroscience. Our novel methodology, applied here to the hippocampus, will pioneer a new way to track the emergence and plasticity of heterogeneous neuronal circuits as these progressively give rise to function in physiological and pathological contexts, thus bridging the gap between early development and adult circuit physio-pathology. This will enable us to revisit adult hippocampal function from a developmental perspective. The hippocampus is classically described as providing a cognitive map of space, involved in navigation, learning, and episodic memory. However, a more computational and less representational vision of its role presently emerges in which its circuits are best described as producing sequences of neuronal activation arising from the interaction between external contextual inputs and internally-generated preconfigured dynamics. Converging work including ours leads us to hypothesize that internally preconfigured assemblies, shaped by their developmental journey, are the basic modules of hippocampal function. In this context, we will use our new approach to address three interrelated questions: 1) Are assemblies shaped by specific circuits in the adult hippocampus (objective A)? 2) Are they prewired or shaped by experience (objective B)? 3) What is their patho-physiological significance (objective C)? Addressing these major issues raises a timely challenge in both optics and genetic engineering at the core of this synergistic proposal.gineering at the core of this synergistic proposal.
In the first two reporting periods we have principally focused on WPA&B as planned (in agreement with our workplan, the aspects of the project related to pathology (WPC) are scheduled to start after month 42). Methodological approaches for (i) long-term and (ii) large-scale imaging are being implemented and validated.
(i) Regarding long-term imaging, a new in vivo 2P-microscope allowing 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 (including different stages of sleep, WPA1.3). 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 being analyzed, WPB2.2) as well as longitudinal imaging during development from P15 into adulthood (WPB1.2 LePrince et al. in progress). The role of local interneurons in shaping CA1 assemblies in the adult has been dissected using optogenetics (WPA2.4 Bocchio, Vorobyev et al. submitted) and the involvement of long-range extrahippocampal inputs during development (WP2.6 Leprince et al. Neuron 2023). On the methodological side, first tests using Vivid-Cre have been initiated (WPA1.2). In vivo assembly imaging during the early perinatal period (P1-4) has been successfully developed in the mouse neocortex (Zangila, Platel et al. in progress). Additionally, a robust version of a two-color 3-photon laser system was validated at the Beaurepaire lab using laser prototypes loaned by the Amplitude laser company. The development 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 hemoglobin absorption (Light Sci App 2023).
(ii) Regarding large-scale imaging, The Beaurepaire and Livet groups successfully developed Fast-ChroMS, a key enabling technology for the project. ChroMS (Abdeladim 2019) enables brain-wide color imaging based on serial two-photon acquisition and wavelength mixing. In the new fast-ChroMS version, the acquisition throughput has been accelerated more than 100 times. We also implemented and validated a dedicated image reconstruction/stitching workflow. Several datasets have been successfully recorded. In particular, brains imaged in vivo in the Cossart lab were reimaged post-mortem at large scale using ChroMS. Finally, we have implemented machine a learning-based workflow for automated cell detection and color analysis in these large datasets. The benchmarking and optimization are underway. We are also working on registration strategies to register in-vivo data, ex-vivo data and reference brain atlases.
(i) Regarding long-term imaging, a new in vivo 2P-microscope allowing 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 (including different stages of sleep, WPA1.3). 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 being analyzed, WPB2.2) as well as longitudinal imaging during development from P15 into adulthood (WPB1.2 LePrince et al. in progress). The role of local interneurons in shaping CA1 assemblies in the adult has been dissected using optogenetics (WPA2.4 Bocchio, Vorobyev et al. submitted) and the involvement of long-range extrahippocampal inputs during development (WP2.6 Leprince et al. Neuron 2023). On the methodological side, first tests using Vivid-Cre have been initiated (WPA1.2). In vivo assembly imaging during the early perinatal period (P1-4) has been successfully developed in the mouse neocortex (Zangila, Platel et al. in progress). Additionally, a robust version of a two-color 3-photon laser system was validated at the Beaurepaire lab using laser prototypes loaned by the Amplitude laser company. The development 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 hemoglobin absorption (Light Sci App 2023).
(ii) Regarding large-scale imaging, The Beaurepaire and Livet groups successfully developed Fast-ChroMS, a key enabling technology for the project. ChroMS (Abdeladim 2019) enables brain-wide color imaging based on serial two-photon acquisition and wavelength mixing. In the new fast-ChroMS version, the acquisition throughput has been accelerated more than 100 times. We also implemented and validated a dedicated image reconstruction/stitching workflow. Several datasets have been successfully recorded. In particular, brains imaged in vivo in the Cossart lab were reimaged post-mortem at large scale using ChroMS. Finally, we have implemented machine a learning-based workflow for automated cell detection and color analysis in these large datasets. The benchmarking and optimization are underway. We are also working on registration strategies to register in-vivo data, ex-vivo data and reference brain atlases.
HOPE aims to shed new light on the function of the hippocampus and the role of its neuronal circuits through the design of a new, non-invasive and universal method to monitor the growth and construction of brain circuits located deep in the brain, from their neurogenesis to adulthood, under normal and pathological conditions. We have already made key progress in this direction.