Mapping the vocal circuitry of Alston’s singing mouse with pseudorabies virus

Abstract

Vocalizations, like many social displays, are often elaborate, rhythmically structured behaviors that are modulated by a complex combination of cues. Vocal motor patterns require close coordination of neural circuits governing the muscles of the larynx, jaw, and respiratory system. In the elaborate vocalization of Alstons singing mouse (Scotinomys teguina), for example, each note of its rapid, frequency-modulated trill is accompanied by equally rapid modulation of breath and gape. To elucidate the neural circuitry underlying this behavior, we introduced the polysynaptic retrograde neuronal tracer pseudorabies virus (PRV) into the cricothyroid and digastricus muscles, which control frequency modulation and jaw opening respectively. Each virus singly labels ipsilateral motoneurons (nucleus ambiguous for cricothyroid, and motor trigeminal nucleus for digastricus). We find that the two isogenic viruses heavily and bilaterally co-label neurons in the gigantocellular reticular formation, a putative central pattern generator. The viruses also show strong co-labeling in compartments of the midbrain including the ventrolateral periaqueductal grey and the parabrachial nucleus, two structures strongly implicated in vocalizations. In the forebrain, regions important to social cognition and energy balance both exhibit extensive co-labeling. This includes the paraventricular and arcuate nuclei of the hypothalamus, the lateral hypothalamus, preoptic area, extended amygdala, central amygdala, and the bed nucleus of the stria terminalis. Finally, we find doubly labeled neurons in M1 motor cortex previously described as laryngeal, as well as in the prelimbic cortex, which indicate these cortical regions play a role in vocal production. Although we observe some novel patterns of double-labelling, the progress of both viruses is broadly consistent with vertebrate-general patterns of vocal circuitry, as well as with circuit models derived from primate literature.

De novo assembly and annotation of the singing mouse genome

“…A quality transcriptome assembly and gene annotations allowed us to examine gene expression of single nuclei to identify specific brain cell types that may contribute to behavior (see Single-nuclei sequencing of the OMC). Single-nuclei sequencing of regions implicated in song production (31,85) can be paired with other approaches such as sequence-based interventions and epigenetic profiling to understand how the brain patterns vocal output.…”

“…The brain region we chose for single nuclei analysis is an important temporal regulator of the singing mouse advertisement song (31). By combining single-cell analysis of relevant brain regions with circuit-level studies (85), we can develop hypotheses about the role of each network node and the cellular mechanisms that underlie these functions. Together, these resources allow us to examine how the nervous system directs complex behavior.…”

“…A quality transcriptome assembly and gene annotations allowed us to examine gene expression of single nuclei to identify specific brain cell types that may contribute to behavior (see Single-nuclei sequencing of the OMC). Single-nuclei sequencing of regions implicated in song production (31,85) can be paired with other approaches such as sequence- The genome, transcriptome, and annotation GTF file can be accessed at the UCSC genome browser (https://bit.ly/3hfUiIy). We identified 14,989 genes that have at least 80% sequence similarity to reference genes.…”