Evidence that interval-counting neurons play a critical role in call recognition by Cope's gray treefrogs.

Species recognition is essential for reproductive isolation and plays a central role in the evolution of mating signals. In acoustically communicating species, temporal features of calls are critical for distinguishing conspecific from heterospecific signals. Anurans rely heavily on the precise timing of pulse trains for mate recognition. Females of Hyla chrysoscelis use the species-specific temporal structure of male advertisement (Adv) calls-specifically pulse rate (PR)-to select mates. For stimuli with the Adv call PR (40-60 pulses/s), females require at least ~ 6-7 pulses to approach a sound source, implicating interval-counting neurons (ICNs) in call recognition. To test this model and further investigate the neural basis of this temporal selectivity, we used behavioral and neurophysiological approaches. We lengthened interpulse intervals (IPIs) in pulse trains either at a single midpoint or in an alternating fashion while holding pulse number and, thus, stimulus energy constant. In phonotaxis assays, females showed sharply reduced responses when even one IPI was lengthened twofold or more, revealing high sensitivity to temporal irregularity. Single-unit in vivo extracellular recordings from the auditory midbrain revealed that ICNs exhibited a progressive decline in activity with increasing IPI length, closely mirroring behavioral trends. In contrast, long-interval neurons (LINs) responded more strongly to temporally irregular stimuli. These results support the hypothesis that ICNs mediate behavioral selectivity for conspecific Adv call temporal patterns, whereas LINs may contribute to processing other call types. Our study directly links a defined neuronal population to natural behavior, underscoring how midbrain temporal computations underlie species-specific recognition in Hyla chrysoscelis.