Single-cell analysis of inhibitory efferent neurons of the zebrafish lateral line.

The zebrafish lateral line system is a sensory network made up of neuromasts, which contain hair cells to detect water flow. Neuromasts signal via sensory afferent neurons and their activity is modulated by efferent neurons. Inhibitory efferent neurons consist of REN, ROLE, and RELL cells and previous work has shown that neuromasts can be innervated by multiple efferent neurons, suggesting potential functional differences. To explore this, we performed single-cell RNA sequencing on REN, ROLE, and RELL neurons in 5-day-old zebrafish larvae. GO analysis across differentially expressed genes did not reveal pathways that suggest differences in cellular function. Comparing markers for neurotransmitter phenotype showed all inhibitory efferent neurons to be cholinergic, but also expressed genes related to other neurotransmitters. Expression of selected genes related to rhombomere location, axon guidance, or gap junctions was similar across efferent neurons. Expression of genes encoding proteins related to membrane potential suggest that REN neurons might be more sensitive to glutamate and may have different action potential dynamics, although functional validation remains to be done. In addition, we assessed neuromast innervation by ROLE and RELL neurons. We found that both ROLE and RELL neurons synapse to approximately 50% of hair cells within a neuromast, compared to approximately 75% innervation by all inhibitory efferent neurons combined. In addition, we did not observe flow polarity bias by innervating efferent axons. However, we did find that RELL neurons had a lower number of synaptic boutons compared to ROLE, which may reflect differences in synaptic output capacity. Taken that our transcriptional analysis did not reveal major intrinsic molecular differences, but we did observe differences in neuromast innervation, raises the possibility that functional differences, if present, may come from upstream inputs. Future work, such as retrograde tracing, could help map these input partners and clarify how different types of efferent neurons contribute to sensory modulation.