Single Nuclei Analyses Reveal Transcriptional Profiles and Marker Genes for Diverse Supraspinal Populations

Abstract

The mammalian brain contains numerous neurons distributed across forebrain, midbrain, and hindbrain that project axons to the lower spinal cord and work in concert to control movement and achieve homeostasis. Extensive work has mapped the anatomic location of supraspinal cell types and continues to establish specific physiological functions. The patterns of gene expression that typify and distinguish these disparate populations, however, are mostly unknown. Here, using adult mice of mixed sex, we combined retrograde labeling of supraspinal cell nuclei with fluorescence-activated nuclei sorting and single-nuclei RNA sequencing analyses to transcriptionally profile neurons that project axons from the brain to lumbar spinal cord. We identified 14 transcriptionally distinct cell types and used a combination of established and newly identified marker genes to assign an anatomic location to each. To validate the putative marker genes, we visualized selected transcripts and confirmed selective expression within lumbar-projecting neurons in discrete supraspinal regions. Finally, we illustrate the potential utility of these data by examining the expression of transcription factors that distinguish different supraspinal cell types and by surveying the expression of receptors for growth and guidance cues that may be present in the spinal cord. Collectively, these data establish transcriptional differences between anatomically defined supraspinal populations, identify a new set of marker genes of use in future experiments, and provide insight into potential differences in cellular and physiological activity across the supraspinal connectome.SIGNIFICANCE STATEMENTThe brain communicates with the body through a wide variety of neuronal populations with distinct functions and differential sensitivity to damage and disease. We have used single-nuclei RNA sequencing technology to distinguish patterns of gene expression within a diverse set of neurons that project axons from the mouse brain to the lumbar spinal cord. The results reveal transcriptional differences between populations previously defined on the basis of anatomy, provide new marker genes to facilitate rapid identification of cell type in future work, and suggest distinct responsiveness of different supraspinal populations to external growth and guidance cues.

Descending GABAergic Neurons of the RVM That Mediate Widespread Bilateral Antinociception

“…In general, most of the neurons in the RVM that we could retrogradely label from the spinal cord with AAV2retro contained either vGAT or vGluT2 mRNA. However, we found that inhibitory neurons were a small proportion of the total population of descending projection neurons in the ventral hindbrain, in agreement with recent transcriptional profiling studies 31 .…”

An integrated single‐cell RNA‐seq atlas of the mouse hypothalamic paraventricular nucleus links transcriptomic and functional types

“…Preautonomic neurons cocluster with the Adarb2 + neurons. (A) Violin plots showing expression of Adarb2, Reln, Snca, and Ntng1 in the paraventricular nucleus (PVN) atlas and retrogradely traced PVN neuron from Beine et al 26 (B) The minimally distorting embedding (MDE) representation, with cells from Beine et al 26 highlighted in red. (C) Immunofluorescent staining of RELN in the rat PVN, showing colocalization with retrograde tracing from the intermediolateral nucleus.…”

“…(E) Zoomed‐in MDE representation, showing the Adarb2 + clusters. Rows show cells from Beine et al 26 and the rest of the dataset, and columns are split by expression of Avp, Crh, Oxt, and Penk.…”

“…To identify preautonomic neurons in our data, we integrated a recent single-cell dataset that utilized retrograde tracing from the IML. 26 From this dataset, all neurons identified by the authors to be from the PVN were integrated into our PVN atlas. After integration, the traced neurons were embedded within the populations using Adarb2 as secondary marker, here referred to as the Adarb2 + populations (Figure 3A).…”

Recent advances in understanding neuronal diversity and neural circuit complexity across different brain regions using single-cell sequencing

“…Neurons, the most critical structural as well as functional components of the nervous system, are divided into sensory neurons, motor neurons, and interneurons in the functional sense ( Peng et al, 2021 ; Zeng, 2022 ). Due to their genetic diversity and wide distribution across brain regions, specific types of neurons generally have distinct functions ( Beine et al, 2022 ; Breton-Provencher et al, 2022 ). Interestingly, these neurons do not work individually.…”