Faculty Mentor: Ayako Yamaguchi (School of Biological Sciences, University of Utah)

African clawed frogs (Xenopus laevis) produce sex-specific vocalizations to coordinate reproduction. Male and female calls are composed of a series of fast and slow clicks, respectively. A previous study showed that administering testosterone to adult female X. laevis masculinizes their vocalizations, indicating vocal pathways reconfiguration in the brainstem [10]. This discovery provides us with an opportunity to examine how the mature nervous system can reorganize in response to hormones, the results of which potentially have high clinical relevance in reversing neurodegenerative diseases [11].

Neural plasticity is partly regulated by perineuronal nets (PNNs), a specialized extracellular matrix in the central nervous system of vertebrates. An increase in PNNs towards the end of development restricts synaptic plasticity, stabilizing neural networks [5, 8]. Disrupting PNNs can allow modifications in normally stable synaptic connections [2, 13]. Interestingly, PNNs surround GABAergic neurons that express parvalbumin (PV), a calcium-binding protein that allows fast spiking [1,7]. This suggests androgen may modulate synaptic formation on PV-positive neurons surrounded by PNNs in female Xenopus laevis (X. laevis).

We hypothesized that androgen-induced vocal masculinization is mediated by the loss of PNNs around PV-positive neurons in the vocal pathways. Testing this hypothesis required effective staining protocols for PV-positive neurons and PNNs in X. laevis brains—a challenging endeavor. The goal of the project was to develop immunohistological methods to effectively visualize PV-positive neurons and PNNs in X. laevis brains.

We succeeded in staining PV-positive neurons in frogs. In the vocal nuclei of female X. laevis, we found a significant number of PV-positive neurons, suggesting that these neurons are fastspiking. However, staining PNNs has proven more complex. Wisteria floribunda agglutinin (WFA), the most commonly used method to visualize PNNs, showed high variability in staining patterns and intensity across individuals [6, 12]. Given species differences in WFA efficacy, we explored four additional approaches targeting different components of the PNN structure: an antibody targeting chondroitin sulfate proteoglycan (CSPG) found in PNNs; an antibody against glycosaminoglycan (GAG) portion of the Aggrecan CSPG; an antibody against the core protein of Aggrecan (Cat-301); and biotinylated hyaluronic acid binding protein (HABP) which binds to the hyaluronan chains abundant in PNNs. The CSPG, GAG, and Cat-301 antibodies all produced inconsistent staining across individuals, while HABP showed no specific binding. These results suggest that visualizing PNNs in the brains of X. laevis may be particularly challenging.

We succeeded in staining PV-positive neurons in frogs. In the vocal nuclei of female X. laevis, we found a significant number of PV-positive neurons, suggesting that these neurons are fastspiking. However, staining PNNs has proven more complex. Wisteria floribunda agglutinin (WFA), the most commonly used method to visualize PNNs, showed high variability in staining patterns and intensity across individuals [6, 12]. Given species differences in WFA efficacy, we explored four additional approaches targeting different components of the PNN structure: an antibody targeting chondroitin sulfate proteoglycan (CSPG) found in PNNs; an antibody against glycosaminoglycan (GAG) portion of the Aggrecan CSPG; an antibody against the core protein of Aggrecan (Cat-301); and biotinylated hyaluronic acid binding protein (HABP) which binds to the hyaluronan chains abundant in PNNs. The CSPG, GAG, and Cat-301 antibodies all produced inconsistent staining across individuals, while HABP showed no specific binding. These results suggest that visualizing PNNs in the brains of X. laevis may be particularly challenging.

Bibliography

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[10] Potter, K., & Yamaguchi, A. (2005, July 01). Androgen-Induced Vocal Transformation in Adult Female African Clawed Frogs. Wikipedia. Retrieved April 8, 2024, from https://journals.physiology.org/doi/full/10.1152/jn.01279.2004

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