Riassunto analitico
The understanding of the cytoarchitectural and functional organization of the hippocampus crucial to unveil its crucial role in storing long-term memories and in spatiotemporal learning and to investigate its involvement in the emergence of severe neurological diseases. In this context, the knowledge about neuronal morphology, localization, connectivity in the hippocampus, is not yet fully available. These data are particularly important to generate computational models of hippocampal circuits. To create in fact full-scale models of extended brain regions, precise information about neuronal distribution and connectivity is required. The final aim of this experimental work was to obtain morpho-anatomical data to generate high-resolution mathematical models of hippocampal regions.
In this thesis work, I provide an experimental workflow to map the distribution of neuronal cells in mouse hippocampus. The mouse brain underwent to surgical procedures to allow viral expression of fluorescent neuronal markers, such as GCaMP6f and TdTomato, only in hippocampal region. The mouse brain was also cleared according to the latest version of the iDISCO+ method (Jin et al., 2022). During this procedure, we were able to mark the neuronal hippocampal population identifying inhibitory neurons using GAD61 a selective antibody. The cleared procedure allowed to distribute within the brain a medium with a uniform refractive index and therefore unable to absorb light. The cleared brains were then optically reconstructed by using custom-made Light-Sheet Microscope Fluorescence (LSFM). A specific algorithm to count neurons in the brain was developed and applied to LSFM images obtained from hippocampus and an automatic 3D image analysis to highlight the neuronal population distribution was performed. The procedure allowed to identify a total neuronal population of 28.535 neurons in the CA1 and CA3 hippocampal areas with an average neuronal distribution of 57 neurons/mm³. These data, which fit into the range reported in the literature will allow to reconstruct 3D hippocampi and could be used as a benchmark for predicting neuronal distribution and connections.
In sum, this experimental work carried out during the training period allowed to establish biological and optical procedures for mapping neuronal population in the mouse hippocampus. Future steps will be to replicate this modus operandi in human samples.
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