Author(s): Anuhya Yalavarty, Logan Klar, Cooper Gangi, Tayla Chiang, Berlyn Prue
Mentor(s): Ayako Yamaguchi
Institution U of U
African clawed frogs (Xenopus laevis) use sex-specific vocalizations—fast clicks by males and slow clicks by females—to facilitate reproduction. A previous study showed that administering testosterone to adult female frogs masculinizes their calls, indicating an androgen-induced reconfiguration of the neural vocal pathway. This finding opens avenues to explore how hormones can reorganize the mature nervous system, the results of which potentially have high clinical relevance in neurodegenerative disease reversal. Neural plasticity is partly regulated by perineuronal nets (PNNs), a specialized extracellular matrix in the vertebrate central nervous system that stabilizes neural networks by limiting synaptic plasticity after development. Disrupting PNNs can allow modifications in normally stable synaptic connections. Interestingly, PNNs surround neurons that express parvalbumin (PV), a calcium-binding protein that enables fast spiking. This suggests androgen may modulate synaptic formation on PV neurons surrounded by PNNs in female X. laevis. We hypothesized that androgen-induced vocal masculinization is mediated by the loss of PNNs around PV neurons in the vocal pathways. Testing this hypothesis required effective staining protocols for PV neurons and PNNs in X. laevis brains—a challenging endeavor. The present project aimed to develop immunohistological methods to visualize PV-positive neurons and PNNs in X. laevis brains effectively. We successfully stained PV neurons, revealing a large number of fast-spiking neurons in female X. laevis vocal nuclei. However, PNN staining posed difficulties. The standard approach using Wisteria floribunda agglutinin (WFA) showed high variability in staining patterns and intensity across individuals. Given the known variation in the efficacy of WFA staining across species, I tested four additional methods targeting different PNN components: antibodies for chondroitin sulfate proteoglycan (CSPG), glycosaminoglycan (GAG), and Cat-301, in addition to a biotinylated hyaluronic acid binding protein (HABP). None of the antibodies yielded consistent results, and HABP showed no specific binding. As an alternative, we began focusing on staining effector proteins bound to PNNs, such as Semaphorin 3A, which stabilizes neural circuits by repelling axon growth away from PV neurons and preventing new synaptic formation. Preliminary trials concurrently staining both Semaphorin 3A and PNN have been successful, showing the aggregation of Semaphoring 3A overlapping in the same location where PNN structures appear. These trials only used control female and male X. laevis samples. Progress on Semaphorin 3A labeling in testosterone-treated female X. laevis will be discussed.