Anatomical and functional characterization of serotonergic innervation in the mouse brain

Serotonin, one of the major neuromodulators of the mammalian brain, can also exert its action as a neurotransmitter thus playing an important role in neuronal plasticity and development. The two main brain areas where serotonin is synthesized are the dorsal and median Raphe nuclei located deep in the brainstem. From these deep brain structures, serotonergic axons emerge and send axonal projections virtually throughout the whole brain, thus creating an evenly distributed serotonergic innervation of the central nervous system. This anatomical specificity may provide one explanation for the widespread effects of 5-HT and as to why they can influence simultaneously a broad array of brain areas and functions. Since the discovery of these neuronal clusters, a lot of extensive work has been focused on the innervation pattern of these cells, however, due to the complexity of their network and unreliable tracing methods, a satisfying map of the serotonergic innervation is still needed. Even though recent studies have been able to look at the Raphe architecture in more detail, they still fall short of giving us the full extent of the serotonergic innervation in the mammalian brain. The main issues arise at first from the heterogeneity of molecular markers serotonergic neurons have in common with other neuronal cell types which made them susceptible to false positives but also from the fact that serotonergic fiber bundles can appear to randomly innervate brain regions on their way to their target areas. This can also impede the development of a highly precise anatomical distribution map of the serotonergic system. To overcome these caveats, I drew a spatial map detailing the extent of serotonergic projections throughout the forebrain using a genetically modified mouse strain to highlight serotonergic fibers. Our results show that most of the highly labelled nuclei and cortical areas have pronounced anatomical connections to limbic structures with an abundance of serotonergic positive fibers in the mid-thalamic nuclei, in most hypothalamic nuclei with a few exceptions, three distinct streams of serotonergic fibers reaching the hippocampus bifurcating at the level of the septal nuclei before reaching their target. To dissociate between en passant fibers and the fibers innervating a specific brain area I performed retrograde tracing studies to quantify the number of serotonergic neurons that innervate these areas by taking advantage of the retrograde characteristics of the rabies virus aiming to identify a correlation between specific subpopulations of serotonergic neurons with their targets in the rostral brain. My results showed that the serotonergic innervation of distinct brain regions is highly heterogeneous, with cell bodies arising from distinct Raphe sub-nuclei that innervate one or more targets at once. To further investigate the type and function of these projections have on the activity of cellular ensembles in distinct brain areas I used two-photon (2ph) imaging in vivo, with which I can track the structural and functional modifications in distinct projection brain areas by imaging the activity of cellular ensembles. Using a combination of retrograde adenoassociated viral vectors, I was able to label serotonergic neurons that send projections to the distinct layers of the olfactory bulb while simultaneously imaging calcium transients and structural dendritic modifications at the cellular and subcellular levels using 2ph imaging from distinct regions of the bulbar network thus establishing a framework for exploring the relationship between serotonin, neural circuitry and behavior.