STUDENT-FACULTY SUMMER RESEARCH

Current Summer Project Title:
No Oxygen, No Problem: Investigating the role of GABA in Vertebrate Anoxia Tolerance

Abstract/Summary of Project Proposal:

For most animals, even short periods of low oxygen levels can result in irreversible harm to critical organs, such as the heart and brain. Only a small number of vertebrates can survive extended periods without oxygen, and even fewer can prosper and recuperate from anoxic stress. The annual killifish (Austrofundulus limnaeus) resides in temporary pools within Venezuela's coastal deserts. What sets this fish apart is its embryo's exceptional endurance of anoxic conditions (i.e., no oxygen) that can persist for several months. The embryos of A. limnaeus react to anoxic conditions by producing large amounts of γ-aminobutyric acid (GABA). We previously discovered that GABA metabolism is crucial for survival during anoxia. This study aims to further understand the role of GABA in supporting the metabolic response to anoxia. To explore if we can increase anoxia tolerance, we will supplement cell media with GABA and observe anoxia tolerance in embryo-derived A. limnaeus cell lines. Additionally, we will precondition cells with lactate as well as brief exposures to anoxia to see if anoxia tolerance is affected. Results from this project will be disseminated at regional and national conferences as well as be part of peer-reviewed publications that I will co-author with my student researcher.

Current Student Researcher:

Devan Duey

Junior; Biochemistry and Molecular Biology Major

I grew up in Newberg, Oregon, with three older siblings and a mom and a dad. Despite years of begging, I only just recently got a pet dog, during our long lockdown months. Even more recently, I finally started to realize that I am an adult with free will and got myself a pet I have wanted for years; a ball python! His name is Percy and I'm not sure we get along all that well, but we have reached a happy medium and he's coming around, I just know it.

My love for science began when I was younger, it all started with dinosaurs. Prehistoric Planet became a staple in my daily schedule. From there, the sciences interested, almost any science that there was. Geology, the water cycle, photosynthesis, you name it. Up until about my junior year, I had my heart set on being a paleontologist. When college started to loom a little closer, I realized I might need to alter my plans to something more attainable. While still keeping my heart close to dinosaurs and the theory of evolution, I decided I wanted to be a researcher, specifically on genetics. I hope to do research on genetic diseases and maybe figure out a way to avoid them, or bring back the dinosaurs. Whichever comes first.

Research Project Background:

Model organisms are extensively used in biological research as they are accessible and convenient systems to study a particular area or question in biology. Traditionally, only a select number of organisms have been widely studied, but with so many advancements in modern technology and research tools, researchers are now able to extend beyond the status quo and explore understudied and unusual organisms. A recent paper by Goldstein and King (2016) argues that some of the biggest future discoveries in biology could come from the development and study of new and atypical model organisms. Goldstein and King (2016) emphasize the need for researchers to ask questions that they know their model organism is appropriate to answer, which was paramount to the development of my research program.

The annual killifish, Austrofundulus limnaeus, survives in ephemeral ponds in the coastal deserts of Venezuela (Podrabsky and Hand, 1999). They have a unique life history (see figure at bottom of page) that has allowed for the evolution of unique physiological mechanisms in their embryos (Podrabsky et al., 2016). Adult fish are abundant during the rainy seasons, constantly spawning and producing eggs which they deposit into the mud. While water is abundant, embryos are deposited in the sediment where high microbial activity creates a severely hypoxic (low oxygen) or anoxic (no oxygen) environment (Podrabsky et al., 1998). Thus, embryos face periods of little to no oxygen availability as part of their normal development. As the ponds dry up and the dry season begins, adult fish die and the embryos must endure the entirety of the dry season until water returns and they can hatch. Survival is attributed to the ability of embryos of A. limnaeus to enter drastic metabolic dormancy (diapause) as a part of their normal development (Podrabsky and Hand, 1999).

In most organisms, even brief episodes of low oxygen can cause irreparable damages to vital organs, such as the brain and heart (Larson et al., 2014). Few vertebrates are able to survive long bouts of anoxia (no oxygen), while even fewer are able to thrive and recover from anoxic stress. At their peak anoxia tolerance, embryos of A. limnaeus have the remarkable ability to tolerate anoxic conditions for months at 25°C – two orders of magnitude higher than any other vertebrate (Podrabsky et al., 1998; Podrabsky et al., 2012). This makes the annual killifish an ideal system to study anoxia tolerance.

When exposed to anoxia, embryos of A. limnaeus respond by producing large amounts (> 10 mmol l-1) of γ-aminobutyric acid (GABA) (Zajic and Podrabsky, 2020; Podrabsky et al., 2007). GABA is the primary inhibitory neurotransmitter in the adult central nervous system; however, it appears to function in an excitatory manner during early vertebrate development (Ben-Ari, 2002). The high levels of GABA produced in anoxic embryos of A. limnaeus suggest a role beyond inhibitory neurotransmission. We previously suggested multiple roles for GABA during anoxia and aerobic recovery in A. limnaeus embryos: as a neurotransmitter, as a source of energy, and as an antioxidant (Zajic and Podrabsky, 2020). In this paper, we showed that inhibition of GABA metabolism directly relates to survival time in anoxia. While at the same time, embryos accumulate toxic levels of the anaerobic metabolic end-product lactate during anoxia. Understanding how the most anoxia tolerant stages of embryos, composed mostly of cardiac tissue and neural tissue, utilize GABA and compartmentalize lactate may provide insight into how these tissues are able to survive long-term anoxia. The goals of this project are to shed light on how preconditioning (through brief anoxia and lactate exposure) and GABA supplementation affect anoxia tolerance in cells of A. limnaeus. The results of this project will increase our understanding of the physiological mechanisms that GABA and lactate play in survival during anoxia.

Works Cited:

Ben-Ari, Y. (2002). Excitatory actions of GABA during development: the nature of the nurture. Nature Reviews Neuroscience 3, 728-740.

Goldstein, B. and King, N. (2016). The Future of Cell Biology: Emerging Model Organisms. Trends in Cell Biology 26, 818-824.

Larson, J., Drew, K. L., Folkow, L. P., Milton, S. L. and Park, T. J. (2014). No oxygen? No problem! Intrinsic brain tolerance to hypoxia in vertebrates. Journal of Experimental Biology 217, 1024-1039.

Podrabsky, J. E. and Hand, S. C. (1999). The bioenergetics of embryonic diapause in an annual killifish, Austrofundulus limnaeus. Journal of Experimental Biology 202, 2567-2580.

Podrabsky, J., Riggs, C. and Wagner, J. (2016). Tolerance of Environmental Stress. In Annual Fishes. Life History Strategy, Diversity, and Evolution,  eds. N. Berois G. García and R. De Sá), pp. 159-184. Boca Raton, FL USA: CRC Press, Taylor & Francis.

Podrabsky, J. E., Hrbek, T. and Hand, S. C. (1998). Physical and chemical characteristics of ephemeral pond habitats in the Maracaibo basin and Llanos region of Venezuela. Hydrobiologia 362, 67-78.

Podrabsky, J. E., Riggs, C. L. and Duerr, J. M. (2012b). Anoxia Tolerance During Vertebrate Development - Insights from Studies on the Annual Killifish Austrofundulus limnaeus. In Anoxia,  (ed. P. Padilla), pp. 3-24. Rijeka, Croatia: InTech.

Podrabsky, J. E., Lopez, J. P., Fan, T. W. M., Higashi, R. and Somero, G. N. (2007). Extreme anoxia tolerance in embryos of the annual killifish Austrofundulus limnaeus: Insights from a metabolomics analysis. Journal of Experimental Biology 210, 2253-2266.

Riggs, C.L., Le, R., Kültz, D., Zajic, D., Summers, A., Alvarez, L., & Podrabsky, J.E. 2019. Establishment and characterization of an Anoxia-Tolerant Cell Line, PSU-AL-WS40NE, Derived from the Annual Killifish Austrofundulus limnaeus. Comparative Physiology and Biochemistry, Part B. 10.1016/j.cbpb.2019.02.008 Riggs et al 2019

Zajic, D.E. and Podrabsky, J.E. 2020. GABA metabolism is crucial for long-term survival of anoxia in annual killifish embryos. Journal of Experimental Biology. DOI: 10.1242/jeb.229716