Whether you are sauntering to the kitchen for a snack, or your stomach is churning to process your meal, all voluntary and involuntary movements need to be intricately regulated to keep you healthy and functional. Control over movements relies on cholinergic neurons in the spinal cord in all vertebrates. These neurons have been rigorously studied in the last 100 years. Yet, perhaps due to the lack of the right technical approach, the diversity of these neurons in has remained uninvestigated until now.

Researchers at the National Institutes of Health (NIH) in Bethesda, have developed a novel approach—targeted single nuclear RNA sequencing—and use it to identify an array of cholinergic interneurons that link the central nervous system to neurons that connect to internal organs (visceral motor neurons) and skeletal muscles (skeletal motor neurons). These findings are published in the article, “Single nucleus RNA-sequencing defines unexpected diversity of cholinergic neuron types in the adult mouse spinal cord” in the journal Nature Communications.

Skeletal cholinergic motor neurons include a subset that is susceptible to neurodegenerative diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis, or ALS. Research into these diseases and other studies have hinted at the presence of additional subtypes within the basic known categories of spinal neurons (motor neurons, visceral neurons and interneurons) and that some of these subtypes may be more vulnerable to neurodegenerative diseases than others. A granular systematic classification of cholinergic motor neuron subtypes will make it possible to pinpoint and treat specific affected neurons in these neurodegenerative diseases.

Claire Le Pichon, PhD, head of the Unit on the Development of Neurodegeneration at NIH’s Eunice Kennedy Shriver National Institute of Child Health and Human Development. [NICHD]

The team of researchers led by Claire Le Pichon, PhD, head of the Unit on the Development of Neurodegeneration at NIH’s Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) use the novel genetic sequencing technique to generate an adult mouse motor neuron atlas. This is openly available at spinalcordatlas.org. This transcriptomic analysis reveals 21 subtypes of neurons at discrete levels along the spinal cord and offers insight into how these neurons control movement, how they contribute to the functioning of organ systems and why some are disproportionately affected in neurodegenerative diseases.

“This study lays the groundwork for understanding these cell identities. By using the same approach to sequence motor neurons in disease states, we will now be able to identify which subtypes classified in this study are most susceptible, and hopefully identify the transcriptional drivers of this vulnerability,” says Le Pichon.

As part of the targeted single nuclear RNA sequencing approach adopted in the study, the authors use a Cre recombinase driven, nuclear envelope-tagged reporter to label and isolate cells using fluorescence activated cell sorting (FACs) prior to RNA sequencing. “We did this because it let us selectively enrich just the cholinergic neurons with a Chat-Cre mouse line,” says Le Pichon.

This enrichment step in the protocol prevents the loss of these cells that constitute only a small fraction of the total cells in the spinal cord. Moreover, these large cholinergic neurons are more difficult to capture using single-cell sequencing. “Using nuclei was key to capturing information from these large neurons,” says Le Pichon.

Spinal cord showing cholinergic neurons labelled using a green fluorescent protein [Claire Le Pichon/NICHD].

The study shows visceral motor neurons that provide autonomic control over glands and internal organs can be divided into more than a dozen classes based on the genes they express and that their location is anatomically specified along the spinal column. The team also shows that visceral motor neurons extend higher up along the spinal column than previously recognized. The authors believe these motor neurons may be newly discovered subtypes with unknown functions. In addition to visceral motor neurons, the researchers also classify subtypes of alpha, gamma and the elusive beta motor neurons that innervate skeletal motor neurons.

“It does seem that some of these subtypes may be more transcriptionally similar to each other than to others,” says Le Pichon. “We chose a UMAP (Uniform Manifold Approximation and Projection) representation to visualize it, in which transcriptional similarity determines how closely cells cluster relative to one other. We observed some subtypes clustering near each other, and others that were more distinct. Those that were distinct seemed to be located only in specific spinal cord levels, indicating that the distinct clusters correspond to innervation patterns.”

The relation between the different classes of cholinergic neurons that the study identifies, such as questions regarding developmental relatedness, will need a replication of the study design along the developmental trajectory.

Surprisingly, in these cholinergic motor neurons that control a variety of muscles and internal organs, the authors report no sex-specific differences. “The visceral motor neurons are unique in that they are preganglionic and so do not directly innervate organs and glands. They synapse onto ganglionic neurons that in turn directly innervate the glands and organs. Therefore, it is possible that this indirect motor control is conserved between sexes, and the differences may lay in the ganglionic neurons that directly innervate sexually dimorphic organs,” says Le Pichon.

This comprehensive transcriptomic description of cholinergic neurons that control physiology and movement and include neuronal types vulnerable to debilitating degenerative disorders presents a hitherto unknown level of complexity.

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