Abstract
Introduction Dietary nitrate lowers blood pressure and improves athletic performance in humans, yet data supporting observations that it may increase cerebral blood flow and improve cognitive performance are mixed. Here we tested the hypothesis that nitrate and nitrite treatment would improve indicators of learning and cognitive performance in a zebrafish (Danio rerio) model. We also explored the extent to which nitrate and nitrite treatment affected the brain metabolome in order to understand how nitrate and nitrite supplementation may affect indices of cognitive function.
Methods Fish were exposed to sodium nitrate (606.9 mg/L), sodium nitrite (19.5 mg/L), or control water for 2-4 weeks and free swim, startle response, innate predator avoidance, social cohesion, and shuttle box assays were performed.
Results Nitrate and nitrite treatment did not change fish weight, length, predator avoidance, or distance and velocity traveled in an unstressed environment. Nitrate- and nitrite-treated fish initially experienced more negative reinforcement and increased time to decision in the shuttle box assay, which is consistent with a decrease in associative learning or executive function however, over multiple trials, all treatment groups demonstrated behaviors associated with learning. Nitrate and nitrite treatment significantly increased anxiety-like behavior but did not alter epinephrine, norepinephrine or dopamine levels. Targeted LC-MS/MS analysis revealed no significant increase in brain nitrate or nitrite concentrations with treatment. An untargeted metabolomics analysis found 47 metabolites whose abundance was significantly altered in the brain with nitrate and nitrite treatment including an 18-19% reduction in the neurotransmitter γ-aminobutyric acid (GABA), and 17-22% reduction in its precursor, glutamine, which may contribute to the increased anxiety-like behavior.
Conclusion Nitrate and nitrite treatment did not adversely affect multiple parameters of zebrafish health but was associated with mild anxiety-like behavior, changes in the brain metabolome, and caused a short-term decrease in executive function or associative learning.
Footnotes
↵* Co-first authors
E-mail addresses: M Garcia-Jaramillo: manuel.g.jaramillo{at}oregonstate.edu, LM Beaver: Laura.Beaver{at}oregonstate.edu, L Truong: Lisa.Truong{at}oregonstate.edu, ER Axton: Elizabeth.Axton{at}jax.org, RM Keller: kellerro{at}oregonstate.edu, MC Prater: praterm{at}oregonstate.edu, KR Magnusson: kathy.magnusson{at}oregonstate.edu, RL Tanguay: Robyn.Tanguay{at}oregonstate.edu, J.F. Stevens fred.stevens{at}oregonstate.edu, NG Hord: Norman.Hord{at}oregonstate.edu
Funding Sources: This work was supported in part by Celia Strickland and G. Kenneth Austin III Endowment (NGH), the Oregon Agricultural Experimental Station and OSU College of Pharmacy Faculty Development Funds (JFS). It was also supported by National Institutes of Health grants 1S10RR027878-01 (JFS), and NIEHS Environmental Health Sciences P30 ES030287 (RLT). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.