College of Science
71 Role of Perineuronal Nets in Androgen-Induced Masculinization of Female Vocal Pathways of African Clawed Frogs
Anuhya Yalavarty; Ayako Yamaguchi; Logan Klar; Cooper Gangi; Tayla Chiang; and Berlyn Prue
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
[1] Aronitz, E. M., Kamermans, B. A., & Duffy, K. R. (2021). Development of parvalbumin neurons and perineuronal nets in the visual cortex of normal and dark-exposed cats. The Journal of comparative neurology, 529(11), 2827–2841. https://doi.org/10.1002/cne.25127
[2] Collins, L., & Brunjes, P. (2019, September 30). Wiley Online Library | Scientific Research Articles, journals, books … Wiley Online Library. Retrieved October 19, 2022, from https://onlinelibrary.wiley.com/
[3] de Winter, F., Kwok, J. C. F., Fawcett, J. W., Vo, T. T., Carulli, D., & Verhaagen, J. (2016). The chemorepulsive protein Semaphorin 3A and perineuronal net-mediated plasticity. Neural plasticity. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4738953/
[4] Dick, G., Tan, C. L., Alves, J. N., Ehlert, E. M. E., Miller, G. M., Hsieh-Wilson, L. C., Sugahara, K., Oosterhof, A., van Kuppevelt, T. H., Verhaagen, J., Fawcett, J. W., & Kwok, J. C. F. (2013, September 20). Semaphorin 3A binds to the perineuronal nets via chondroitin sulfate type E motifs in rodent brains. The Journal of biological chemistry. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779733/
[5] Edwards, JA, Risch, M, Hoke, KL. (2020, October 16). Dynamics of perineuronal nets over amphibian metamorphosis. J Comp Neurol. Retrieved October 18, 2022 from https://doi.org/10.1002/cne.25055
[6] Gaál, B., Rácz, É., Juhász, T., Holló, K., & Matesz, C. (2014). Distribution of extracellular matrix macromolecules in the vestibular nuclei and cerebellum of the frog, Rana esculenta. Neuroscience, 258, 162–173. https://doi.org/10.1016/j.neuroscience.2013.10.080
[7] Hijazi, S., Smit, A., & Van Kesteren, R. (2023, July 7). Fast-spiking parvalbumin-positive interneurons in brain physiology and Alzheimer’s disease. Nature. Retrieved April 8, 2024, from https://www.nature.com/articles/s41380-023-02168-y
[8] Lledo, P.-M., Alonso, M., & Grubb, M. S. (2006, March 1). Adult neurogenesis and functional plasticity in neuronal circuits. Nature News. Retrieved October 18, 2022, from https://www.nature.com/articles/nrn1867
[9] Luo, F., Wang, J., Zhang, Z., You, Z., Bedolla, A., Okwubido-Williams, F., Huang, L. F., Silver, J., & Luo, Y. (2022). Inhibition of CSPG receptor PTPσ promotes migration of newly born neuroblasts, axonal sprouting, and recovery from stroke. Cell reports, 40(4), 111137. https://doi.org/10.1016/j.celrep.2022.111137
[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
[11] Reichelt, A. C. (2020, August 15). Is loss of perineuronal nets a critical pathological event in Alzheimer’s disease? eBioMedicine. Retrieved November 3, 2022, from https://www.thelancet.com/journals/ebiom/article/PIIS2352-3964(20)30322-4/fulltext
[12] Slaker, M. L., Harkness, J. H., & Sorg, B. A. (2016, December). A standardized and automated method of perineuronal net analysis using Wisteria floribunda agglutinin staining intensity. IBRO reports. Retrieved October 19, 2022, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507617/
[13] Tewari, B., Chaunsali, L., Prim, C., & Sontheimer, H. (2022, September 23). A glial perspective on the extracellular matrix and perineuronal net remodeling in the central nervous system. Frontiers. Retrieved April 8, 2024, from https://www.frontiersin.org/articles/10.3389/fncel.2022.1022754/full3/