Overall, the project involved the combination of multidisciplinary approaches to assess how multisensory courtship cues are represented in the brain and the extent to which the two modalities interact.
The project first focused on investigating the contribution to multimodal stimuli in driving female mice preference. Adult females underwent two-choice preference test in which the preference for unimodal versus multimodal stimuli was assessed. The test was successfully implemented and allowed to assess, for the first time, female mice preference for multimodal stimuli and set the basis for further investigation on how brain areas differentially represent olfactory and acoustic courtship stimuli in a unimodal or multimodal context.
To this aim, I focused on the analysis of specific genes, called Immediate Early Genes (IEGs), whose expression is timely linked to the neuronal electrical activation. In order to characterize IEGs levels across the entire brain, I coupled advanced anatomical approaches, such as whole-brain immunolabelling and tissue clearing, with light-sheet fluorescent microscopy, a state-of-the-art optical approach to acquire images of tissue section across the entire brain. This approach generated large amounts of data which were then analyzed by improving available open source software. I first implemented the described analytical pipeline to be suited to address my scientific questions and then applied it to assess how brain regions were differentially activated by unimodal or multimodal courtship stimuli. Overall, this approach allowed to demonstrate its suitability in investigating brain representation ethologically relevant sensory stimuli under different conditions and it helped identify key brain areas involved in the integration of multimodal courtship cues.
In order to complement behavioural and anatomical results with physiological characterization of neuronal responses to courtship cues, I have implemented in the hosting laboratory a system to collect neuronal activation in targeted brain regions while simultaneously presenting olfactory and acoustic courtship cues alone or combined. To this end, I set up the system to perform in vivo one-photon functional imaging of neurons expressing fluorescence calcium indicators and I coupled it with commercial tools to precisely deliver odors and sounds to head-restrained animals, while simultaneously monitoring physiological signals such as pupil dilation as a readout of animal arousal state. This will complement previous data to establish how neurons represent courtship cues depending on the context.
Finally, to understand how olfaction shapes the representation of acoustic courtship songs in primary sensory brain regions of adult female, I set up a system to perform in vivo single cell electrophysiology coupled with simultaneous presentation of odors and sounds in awake mice.
