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 both 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 either absent or molecularly distinct in clawed frogs that do not produce fast trills. To test this, we used constellation pharmacology to profile receptor expression of neurons via intracellular Ca²⁺ responses to pharmacological agents in PBN neurons from male X. laevis , male S. tropicalis , and female X. laevis . Surprisingly, we found putative FTNs in all three groups, including those that do not produce fast trills. Furthermore, a similar proportion of FTNs across groups expressed fast-kinetic voltage-gated potassium channels known to support rapid firing, indicating that the presence of these channels does not correlate with the ability to produce fast trills. Instead, some of these channels were more prevalent in males of both species compared to female X. laevis , suggesting a potential sex-specific, non-vocal function. The discovery of FTNs with similar molecular profiles in non-fast-trilling individuals suggests that these neurons are conserved across species and sexes, and may serve other functions. In male X. laevis , FTNs may have been repurposed for fast trill production during speciation. These findings provide new insight into understanding how neural circuits evolve and diversify across species and sexes.