Determining the Dominant Glucocorticoid in the Mexican Axolotl

Abstract

IntroductionThe Mexican axolotl salamander (Ambystoma mexicanum) is a popular animal model in regenerative research due to its remarkable regenerative capacity, and it is the oldest self‐sustained laboratory animal. However, despite the widespread laboratory use of the axolotl, little has been reported on stress hormones in this species. Glucocorticoids are commonly used as biomarkers for stress and affect many vital processes such as immune response, metabolism, and cardiovascular function. The stress associated with tissue damage, handling or other interventions could be important mechanisms for induction of inflammation and the initiation of regeneration. For humans, and most types of fish, cortisol is the dominating stress hormone while corticosterone usually dominates in rodents and many types of amphibians. The discovery of cortisol as the primary glucocorticoid in the fully aquatic, paedomorphic eastern hellbender salamander (Cryptobranchus alleganiensis) disputed the generalization that amphibians primarily utilize corticosterone. As such, this study aims to determine the dominant glucocorticoid in the axolotl to encourage the inclusion of this biomarker in future experiments. We hypothesize that the axolotl responds to stress in a similar way as the hellbender thus using cortisol as the dominant glucocorticoid.MethodsUsing different enzyme‐based assays, the circulating concentration of glucocorticoids as well as the tissue‐specific activation of the glucocorticoid receptor has been investigated. Plasma was isolated 1‐72 hours post stress induction to determine the time for maximal response to induced stress (chemically or manually induced). At the determined peak response, selected target tissues were harvested for later determination of the activation status of the glucocorticoid receptor in specific tissues. Lastly, the regulation of the glucocorticoid synthesis pathway will be investigated by quantification of glucocorticoid‐specific enzymes through quantitative PCR.ResultsAxolotls were found to be relatively unresponsive to manually and chemically induced stress using adrenocorticotropic hormone (an upstream regulator for both cortisol and corticosterone) though a significant stress response was induced using the synthetic analog Synacthen peaking at 1h post injection. Quantification of glucocorticoid receptor activation showed a tissue‐specific response to glucocorticoids.ConclusionPrevious studies have shown that simultaneous injections of T4 and dexamethasone, a synthetic glucocorticoid, can induce metamorphosis at submetamorphic doses. Additionally, stressing axolotls by altering their environmental conditions through lowering of the water levels have also been shown to induce metamorphosis. As such, it is possible that the weak response seen in this study is an indicator of strict regulation of the glucocorticoid response to retain neoteny. In future experiments it might therefore be interesting to measure the catecholamine response to see whether these hormones are also governed by strict regulation or could be used as biomarkers for stress.