Molecular Characterization of Parabrachial Neurons in Xenopus laevis and Silurana tropicalis : Evolutionary Conservation and Sex-Specific Differences

Clawed frogs communicate acoustically to coordinate reproduction, with males producing species-specific advertisement calls to attract females. In Xenopus laevis, males generate fast trills composed of clicks repeated at 60 Hz, a feature absent in Silurana tropicalis males and X. laevis females, whose calls consist of slower click rates (30 Hz and 7 Hz, respectively). In male X. laevis, fast trills are generated by premotor neurons in the parabrachial nucleus (PBN), known as fast trill neurons (FTNs). We hypothesized that FTNs are unique in male X. laevis, and absent or distinct in clawed frogs lacking fast trills. To test this, we used constellation pharmacology to profile receptor and ion channel expression in PBN neurons by measuring intracellular Ca²⁺ responses to pharmacological agents in male X. laevis, male S. tropicalis, and female X. laevis. Surprisingly, we found putative FTNs (pFTNs) in all three groups. Furthermore, a similar proportion of pFTNs across groups expressed fast-kinetic voltage-gated potassium channels known to support rapid firing, indicating the presence of these channels does not correlate with producing fast trills. Instead, some of these channels were more prevalent in males of both species than in female X. laevis, suggesting a sex-specific, non-vocal function. These results suggest FTNs may have an unrecognized function conserved across species but were repurposed for fast trills during X. laevis speciation; alternatively, they may differ in unresolved molecular or electrophysiological properties, or represent latent ancestral circuitry persisting in slow-trilling species. These findings provide new insight into neural circuit evolution across species and sexes.