The “dorsal pons”, or “dorsal pontine tegmentum” (dPnTg), is part of the brainstem. It is a complex, densely packed region whose nuclei are involved in regulating many vital functions. Notable among them are the parabrachial nucleus, the Kölliker Fuse, the Barrington nucleus, the locus coeruleus, and the laterodorsal, ventral, and dorsal tegmental nuclei. In this study, we applied single-nucleus RNA-seq (snRNA-seq) to resolve neuronal subtypes based on their unique transcriptional profiles and then used multiplexed error robust fluorescence in situ hybridization (MERFISH) to map them spatially. We sampled ~1 million cells across the dPnTg and defined the spatial distribution of over 120 neuronal subtypes. Our analysis identified an unpredicted high transcriptional diversity in this region and pinpointed many neuronal subtypes' unique marker genes. We also demonstrated that many neuronal subtypes were transcriptionally similar between humans and mice, enhancing this study's translational value. Finally, we developed a freely accessible, GPU and CPU-powered dashboard (http://harvard.heavy.ai:6273/) that combines interactive visual analytics and hardware-accelerated SQL into a data science framework to allow the scientific community to query and gain insights into the data.

The study includes data from two single-cell transcriptomics technologies: snRNAseq,10X + DroNc-seq, which both are droplet-based single-nucleus RNA-seq techniques, and MERFISH, which is an image-based spatial transcriptomics technique at subcellular resolution. The dashboard contains the following datasets that have been clustered using a bioinformatic pipeline that integrates Seurat v3.2.3 and Harmony v1.1:

1.     “pons_all_cells_snrnaseq" includes all nuclei from neuronal/non-neuronal/glial cell types from our ROI, the dPnTg, after QC.  “pons_exc_neurons_snrnaseq” includes glutamatergic (Slc17a6, Slc17a7, and Slc17a8), noradrenergic (Th/slc18a2), cholinergic (Chat/Slc5a7), serotoninergic (Tph2/Slc6a4) and “hybrid” (Slc32a1/Slc17a6).neuron types.   “pons_inh_neurons_snrnaseq” includes GABAergic (Slc32a1) and  Glycinergic (Slc32a1/Slc6a5) neuron types.  

2.   "pons_all_cells_merfish" includes all nuclei from neuronal/non-neuronal/glial cell types from our ROI, the dPnTg.  "pons_exc_neurons_merfish" includes glutamatergic (Slc17a6, Slc17a7, and Slc17a8), noradrenergic (Th/slc18a2), cholinergic (Chat/Slc5a7), serotoninergic (Tph2/Slc6a4)  and “hybrid” (Slc32a1/Slc17a6) neuron types.   pons_inh_neurons_merfish" includes GABAergic (Slc32a1) and  Glycinergic (Slc32a1/Slc6a5) neuron types.

3.     Finally, we divided the dPnTg (our initial ROI) into 4 anatomical subregions to provide transcriptional resolution on a spatial scale that is of specific interest to investigators.    “atlas_1” includes the KF; “atlas_2” includes the PB; “atlas_3” includes the pre-LC, LC, Bar, and MTN; and “atlas_4” includes LDTgV, LDTg, VTg, DTgC, DTgP, PDTg, CGA, CGB, Sph, O, and CGPn.