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Single-Cell Transcriptomes Reveal Neuronal Cell Types in Mouse Brain Region

NEW YORK (GenomeWeb) – Using single-cell transcriptomes, a team from the University of Connecticut, the Jackson Laboratory for Genomic Medicine, and elsewhere has identified clusters representing dozens of inhibitory and excitatory neuron cell types in a mouse brain region called the lateral hypothalamic area (LHA) that has been implicated in everything from feeding and stress response to sleep-wake cycles and other behaviors.

"This study lays the groundwork for better understanding the circuit-level underpinnings of LHA function," co-corresponding authors Alexander Jackson and Paul Robson, at the University of Connecticut and the Jackson Laboratory for Genomic Medicine, respectively, and their colleagues wrote, noting that the "census provides a detailed resource for precisely deconstructing the biology of genetically defined LHA circuits and their orchestration of innate behavior."

As they reported online in Nature Neuroscience this week, Jackson, Robson, and their colleagues used droplet-based single-cell RNA sequencing to assess thousands of individual cells in micro-dissected LHA samples from male and female mice, uncovering 15 populations apiece of new and known glutamatergic and GABAergic neurons. For their follow-up analyses, they dug into populations of neurons expressing the hormone somatostatin, documenting roles for these neurons in a specific type of repetitive mouse locomotion.

Although past studies have spelled out the nature of certain glutamatergic and GABAergic neurons in the LHA, the team explained, more work is needed to find and characterize the full suite of neurons at work in this brain region.

"It is likely that within these broad classes of neurons, there are molecularly distinct populations that differ in functional connectivity and behavioral state-dependent engagement," the authors explained. "Yet, cellular heterogeneity among these populations remains poorly defined."

For their single-cell RNA-seq experiments, the researchers generated and amplified complementary DNA from 3,784 individual cells from male mouse LHA brain regions and another 3,434 cells from the LHA of female mice, isolated from micro-dissected brain slices and single-cell 10x Genomics Chromium Controller systems.

They went on to verify their findings with approaches such as fluorescence in situ hybridization, single-cell quantitative PCR, and other experiments digging into mouse behaviors in response to the activation of specific sets of neurons.

In the process, the team identified 11 non-neuronal cell types, along with 15 clusters of glutamatergic neurons and 15 GABAergic neuron populations, clustered based on gene expression and the presence or absence of a handful of known pan-neuronal marker genes. The neuronal cell types typically contained more expressed genes and transcripts per cell, though cells from male and female mouse brains were scattered across the clusters, arguing against significant sex-dependent brain cell differences in the LHA.

"We found that neuronal clusters can be classified based on a confluence of markers that encode neurochemical phenotype, transcription factors, and synaptic proteins, among other gene categories," the authors reported. "These combinations of markers seem to specify the identity of transcriptionally distinct populations of LHA neurons, most likely reflecting a convergence of developmental lineage, neurochemistry, and functional connectivity."