Periodic Reporting for period 1 - OLF-ACtion (Multimodal integration of olfactory and acoustic cues in mouse courtship communication)
Reporting period: 2023-01-01 to 2024-12-31
The ability to find, attract, and select a suitable mating partner is one of the fundamental processes driving evolution. Animals have evolved species-specific stereotyped strategies, collectively referred to as courtship behaviors, to attract individuals of the opposite sex. Despite the vast variability in courtship displays across species, a common feature is their multimodal nature, where courtship behaviors involve at least two sensory modalities. This multisensory aspect has long intrigued scientists across disciplines, including ethology and neuroscience. However, most studies have investigated the role of individual sensory modalities in isolation, without exploring their combined information content in a multimodal context. Moreover, how these multisensory stimuli are integrated and represented in brain neural circuits remains largely unexplored.
OLF-ACtion addresses these gaps through interdisciplinary approaches combining neuroanatomy and brain physiology. The overall aim of the action is to understand the neuronal basis of multimodal communication in naturalistic contexts. In particular, the project has two main objectives: 1) identify key brain areas involved in the integration of multimodal courtship cues in adult female mice; 2) investigate the interaction between olfactory and acoustic sensory modalities in primary sensory areas.
Project’s results aim to shine light on the role of multimodal communication during courtship behaviour and the underlying neuronal mechanisms involved in the selection of potential mating partners. Understanding the circuits involved in the integration of multimodal information is among the main challenges in the neuroscience field. Past studies have been focusing on brain mechanisms merging information from different sensory modalities by using artificial and simplified paradigm of stimuli presentation. This project goes beyond the state-of-the-art and investigates these mechanisms under naturalistic conditions involving complex and ethologically relevant sensory stimuli. Moreover, by assessing which brain areas play a role in the representation of multisensory courtship cues, the project will help move forward our knowledge on the role of multimodal communication during courtship.
OLF-ACtion addresses these gaps through interdisciplinary approaches combining neuroanatomy and brain physiology. The overall aim of the action is to understand the neuronal basis of multimodal communication in naturalistic contexts. In particular, the project has two main objectives: 1) identify key brain areas involved in the integration of multimodal courtship cues in adult female mice; 2) investigate the interaction between olfactory and acoustic sensory modalities in primary sensory areas.
Project’s results aim to shine light on the role of multimodal communication during courtship behaviour and the underlying neuronal mechanisms involved in the selection of potential mating partners. Understanding the circuits involved in the integration of multimodal information is among the main challenges in the neuroscience field. Past studies have been focusing on brain mechanisms merging information from different sensory modalities by using artificial and simplified paradigm of stimuli presentation. This project goes beyond the state-of-the-art and investigates these mechanisms under naturalistic conditions involving complex and ethologically relevant sensory stimuli. Moreover, by assessing which brain areas play a role in the representation of multisensory courtship cues, the project will help move forward our knowledge on the role of multimodal communication during courtship.
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.
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.
Overall, the project has highlighted important results which go beyond the state of the art.
By comparing animal behavioural preferences for unimodal or multimodal stimuli, I identified a clear preference for multimodal cues, suggesting a synergistic effect of multisensory communication during courtship. These results pushed the project forward in assessing how olfactory and acoustic courtship cues were represented in the brain either in unimodal or multimodal contexts.
Whole-brain anatomical analysis of IEGs indicate that, as expected, olfaction plays a major role in brain activation in both contexts, while acoustic cues were only able to trigger local brain activation in primary sensory regions. However, we found that only the combination of acoustic and olfactory stimuli were able to activate a specific region of the mouse medial prefrontal cortex, the Infralimbic Area, known to be involved in social behavior and kin recognition. In addition, we reliably identified the activation of a deep hypothalamic nucleus, called the Supramammillary Nucleus, only in the multimodal context. By performing anatomical tracing analysis we demonstrated a direct link between the Infralimbic area and the Supramammillary, opening up new directions for future experiments aimed in assessing the role of this Cortical-Hypothalamic circuits in the multimodal communication of female and male mice during courtship.
Finally, to better investigate how acoustic courtship cues are represented, I performed in vivo two-photon recordings in young mice during the presentation of male ultrasonic vocalizations. This led to a publication where I demonstrated that ultrasound vocalization are represented in the primary auditory areas as soon as the mice start hearing.
By comparing animal behavioural preferences for unimodal or multimodal stimuli, I identified a clear preference for multimodal cues, suggesting a synergistic effect of multisensory communication during courtship. These results pushed the project forward in assessing how olfactory and acoustic courtship cues were represented in the brain either in unimodal or multimodal contexts.
Whole-brain anatomical analysis of IEGs indicate that, as expected, olfaction plays a major role in brain activation in both contexts, while acoustic cues were only able to trigger local brain activation in primary sensory regions. However, we found that only the combination of acoustic and olfactory stimuli were able to activate a specific region of the mouse medial prefrontal cortex, the Infralimbic Area, known to be involved in social behavior and kin recognition. In addition, we reliably identified the activation of a deep hypothalamic nucleus, called the Supramammillary Nucleus, only in the multimodal context. By performing anatomical tracing analysis we demonstrated a direct link between the Infralimbic area and the Supramammillary, opening up new directions for future experiments aimed in assessing the role of this Cortical-Hypothalamic circuits in the multimodal communication of female and male mice during courtship.
Finally, to better investigate how acoustic courtship cues are represented, I performed in vivo two-photon recordings in young mice during the presentation of male ultrasonic vocalizations. This led to a publication where I demonstrated that ultrasound vocalization are represented in the primary auditory areas as soon as the mice start hearing.