Divergent NMJ Development Supports Female Vocal Suppression in the Zebra Finch Songbird (Taeniopygia guttata)

Songbirds have long been central to the study of vocal learning and production, and in most species, males and females sing. However, in the best-studied species, the zebra finch ( Taeniopygia guttata), only males sing. Most theories of this sexual dimorphism had suggested that male zebra finches have specializations, e.g. to promote song, that females do not. We recently found the opposite. At the synapses that connect motoneurons to muscle fibers (neuromuscular junctions; NMJs) of the avian voicebox (syrinx, analogous to larynx), we found female synaptic specializations that support song suppression: Female syrinx, and only female syrinx (not tongue, latissimus dorsi, or any male muscle measured) has specialized NMJs that could work to suppress singing ability in this species. Specifically, the female syrinx shows a high proportion of putatively weaker en grappe NMJs, biased towards smaller and weaker muscle fibers, which fail to form a motor endplate band (MEB). However, these results raise an unanswered question: are male and female syringes born differently, or do they have divergent developmental programs? We analyzed males and females (n=3) across critical ages - the robust onset of sensory song learning (post-hatch day P30), song-like vocalizations (P50), and the end of sensorimotor learning (P70). We find that NMJs in male and female syrinx muscles are similar early in development and diverge around the age that vocal behavior becomes sexually dimorphic, approaching their different adult phenotypes by the end of this learning phase. We find that outside the MEB, en grappe NMJs are pruned away in males, while en plaque NMJs are lost here in females. Additionally, these NMJ features are highly labile in development, as there are many examples of NMJs intermediate between the vertebrate-typical en plaque and the less well-described en grappe, implying some transitory nature of NMJ class. Preliminary work using developmental hormonal manipulations, which induces sex-reversal of vocal behavior (i.e., promoting female song, or suppressing male song), indicates the en grappe NMJs specifically outside the MEB are most directly tied to vocal ability. Interestingly, the songbird syrinx lacks the tonic muscle fibers on which en grappe NMJs are typically found in mammals, opening the question of the functional attributes of these NMJs. Future work aims to characterize the physiological properties of these putatively weaker synapses, as well as their functional consequences to vocal sexual dimorphism. NSF-GRFP awarded to JVG; Neuronex awarded to NBK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.