Detecting brain areas recruited by multimodal courtship cues using ABBA toolbox

Advanced optical and computational approaches, combined with immediate early gene expression analysis, enable to analyse whole-brain activity with single-neuron resolution. These techniques facilitate the extraction of key insights from large datasets, linking functional networks to their anatomical circuits. Specifically, using a whole-brain approach, the laboratory where I performed my thesis, is interested in identifying the brain regions involved in the integration of olfactory and auditory cues during courtship behavior. Indeed, despite extensive research on how olfactory and acoustic signals drive mate preference, the neural circuits responsible for integrating these sensory stimuli remain poorly understood. To this aim, the laboratory previously collected brains from mice exposed to combined olfactory and auditory stimuli, and performed whole-brain c-Fos immunolabeling, iDISCO tissue clearing, and light-sheet imaging. Whole-brain analysis revealed the recruitment of several brain regions in different conditions, with the infralimbic area being strongly activated by multisensory stimuli. The infralimbic cortex is known for its role in social novelty and decision-making, suggesting its involvement in processing and integrating multimodal sexual cues during courtship, I thus focused my work on this specific brain area. Specifically, the scope of my work was to develop a comprehensive pipeline for analysing light-sheet microscopy images using an alternative pipeline that includes the use of two toolbox named ABBA and QuPath, in order to assess the consistency of whole brain data and complement the analysis with detailed description of c-Fos expression in subregions. By taking advantage of whole brain images acquired using light-sheet microscopy I implemented this analytical pipeline, and identified an increase in c-Fos expression within the infralimbic area, which is part of the medial prefrontal cortex, in response to multimodal sensory stimulation. Different parameters, such as the choice of different detection thresholds have been evaluated and results have been assessed using multiple scale, from macro the region to single layers. Moreover, I used the same pipeline to perform detailed analysis of additional brain regions, which are known to be recruited by single sensory modalities in order to confirm the robustness of the analysis. These findings provide new insights into the neural mechanisms underlying mate preference and highlight key brain areas that process complex social stimuli.