Single nucleus RNA-sequencing defines unexpected diversity of cholinergic neuron types in the adult mouse spinal cord

Abstract

In vertebrates, motor control relies on cholinergic neurons in the spinal cord that have been extensively studied over the past hundred years, yet the full heterogeneity of these neurons and their different functional roles in the adult remain to be defined. Here, we developed a targeted single nuclear RNA sequencing approach and used it to identify an array of cholinergic interneurons, visceral and skeletal motor neurons. Our data expose markers for distinguishing these classes of cholinergic neurons and their extremely rich diversity. Specifically, visceral motor neurons, which provide autonomic control, could be divided into more than a dozen transcriptomic classes with anatomically restricted localization along the spinal cord. The complexity of the skeletal motor neurons was also reflected in our analysis with alpha, beta, and gamma subtypes clearly distinguished. In combination, our data provide a comprehensive transcriptomic description of this important population of neurons that control many aspects of physiology and movement and encompass the cellular substrates for debilitating degenerative disorders.

A harmonized atlas of mouse spinal cord cell types and their spatial organization

“…Sixteen major types were identified that represent all known classes of spinal cord cell types. These cell types are: (1) oligodendrocyte precursor cells; (2-3) two maturational stages of oligodendrocyte progenitors; (4-5) two types of oligodendrocytes that likely correspond to myelinating and mature cell types and that blend into each other; (6) Schwann cells; (7) peripheral glia; (8)(9) two types of meninges that likely correspond to vascular leptomeningeal cells and arachnoid barrier cells; (10) ependymal cells that surround the central canal; (11)(12) two types of astrocytes that likely correspond to a major population of regular astrocytes and a minor population of Gfap-expressing proliferating/activated/white matter astrocytes; (13)(14) two types of vascular cells that likely correspond to endothelial cells and pericytes; (15) microglia; and (16) neurons, which are discussed in detail below.…”

“…In the postnatal mouse spinal cord, there have been several papers profiling single-cell RNA expression that, combined, cover a range of biological parameters, including age, tissue region, developmental lineage, and circuit features [1][2][3][4][5][6][7][8][9][10][11] . These studies provide a powerful and multi-faceted perspective on spinal cord cell types, yet despite this significant effort and a rich literature of spinal cord cell type characterization (see reviews [12][13][14][15][16][17][18][19] ), there is still no consensus cell type atlas of the spinal cord 20 .…”

A Single Cell Atlas of Spared Tissue Below a Spinal Cord Injury Reveals Cellular Mechanisms of Repair

“…There are several limitations to consider in this work. In this study, nuclei were analyzed instead of whole cells because it has been shown that profiling nuclei avoids experimentally-induced gene expression and selective cell death common in single cell profiling [60][61][62] , though the sequencing depth is lower than for cells 63 . More broadly, while single nucleus sequencing is increasingly transforming our understanding on the biology of disease and trauma, it is important to note that it provides a particular perspective on these complex processes.…”

A Harmonized Atlas of Spinal Cord Cell Types and Their Computational Classification

“…As examples, future work could incorporate embryonic single cell data 7 and lineage tracing to link together developmental origin with postnatal cell types or could focus deeply on specific spinal cord regions and cell types. Indeed, forthcoming work has revealed an impressive diversity of PGC visceral motoneurons that are enriched in either the thoracic or sacral spinal segments 21,22 . Relatedly, the in situ hybridization experiments here are also limited in scope, being specific to the adult lumbar spinal cord.…”