Multimodal mapping of cell types and projections in the Substantia Nigra
1 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany
2 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany and Institute of Reconstructive
3 Neurobiology, Medical Faculty, University of Bonn, Bonn, Germany
Midbrain dopaminergic (mDA) neurons are known to have an essential role in motor activity, reward and aversion processing, motivated behavior and learning. To this date, it remains unclear how one neurotransmitter can encode so many different functions. The largest populations of mDA neurons are localized in the ventral tegmental area (VTA), substantia nigra (SN) and the retrorubral field (RRF). Regardless of their different axonal projections, mDA neurons have traditionally been regarded as a relatively homogenous group of neurons. However, recent studies have shown that these neurons are quite heterogenous, as they do not only have different anatomical locations and projection targets but also diverse developmental origins, gene expression profiles and electrophysiological properties, which could explain their wide variety of functions. For example, it is well established that mDA neurons in the SN respond to reward, aversion, and locomotion, but it is not known whether all of these behavioral patterns are encoded by the same neurons or whether they are encoded separately by different groups of SN mDA neurons. Although molecularly distinct subpopulations of mDA neurons have been identified by single-cell gene expression profiling, fundamental features of these molecular subtypes, such as their projection patterns or their specific functions in encoding behavior have not been systematically elucidated. The present study aims to bridge this gap by determining the molecular identity and anatomical distribution of SN-mDA neurons projecting to distinct output targets in the striatum. To investigate this, we implemented a multimodal mapping approach by combining 5-plex fluorescent retrograde labeling with a spatial transcriptomic technique called “expansion-assisted iterative fluorescence in situ hybridization“ (EASI-FISH) in 100 µm-thick tissue sections. Our results provide an increased understanding of the organization of mDA neurons across the SN including their molecular, spatial, morphological and connectional diversity. Overall, we show a novel approach to systematically map gene expression onto distinct projection pathways of the mDA system in thick tissue volumes, allowing us to identify whether individual neurons from a genetically-defined population project to a specific or multiple brain regions. In future experiments, we plan to combine this approach with deep-brain calcium imaging during appetitive and aversive learning to determine the relationship of molecular marker expression with functional profiles of individual mDA neurons.