The adrenal stress response involves distinct dynamics of both cortisol and corticosterone in the axolotl salamander

lab animal Article https://doi.org/10.1038/s41684-026-01692-y The adrenal stress response involves distinct dynamics of both cortisol and corticosterone in the axolotl salamander Check for updates Anita Dittrich    1 , Sofie Amalie Andersson1, Emil A. B. Winkel    1,2, Aaron Savage Steven J. Blair3, Kelly E. Dooling    3, Alexandra C. Wagner3, Jessica L. Whited3, Catherine J. A. Williams    4,5,6,7 & Henrik Lauridsen    1,7    3, The axolotl is a popular model organism in regenerative biology owing to its ability to regenerate amputated limbs and internal organs. The role of injury-derived signals in initiating the regenerative response is still not well understood, but the potential involvement of the stress response is of interest, as injury and stress are temporally linked. The dominant glucocorticoid response to stress varies among species, with corticosterone generally considered dominant in most amphibians, whereas cortisol predominates in others. Here we characterize the adrenal stress response in the axolotl and describe methods to measure axolotl stress hormones to facilitate their inclusion in future research involving axolotl development and regeneration. We describe an intricate and unexpected axolotl stress response that involves cortisol and corticosterone, each being dominant under different conditions. Corticosterone is preferably activated by the classical hypothalamus–pituitary–interrenal axis pathway, with both arginine vasotocin and adrenocorticotropic hormone promoting its synthesis and release. Under manual stress and direct stimuli with acetylcholine, cortisol is more prominent, suggesting an alternative mechanism involving sympathetic nerve signaling. In response to an amputation injury, both cortisol and corticosterone are increased, with corticosterone being dominant, suggesting an injury-specific response. Finally, when administering glucocorticoids directly and measuring classical physiological effects of glucocorticoid signaling, cortisol is more potent. We propose a hypothesis that axolotls rely on cortisol as their dominant glucocorticoid, functioning in part as an extension of the catecholamine system. By contrast, corticosterone is mainly regulated classically via the hypothalamus–pituitary–interrenal axis. The axolotl salamander Ambystoma mexicanum (Shaw and Nodder, 1798) is a popular model organism in the fields of aging, development and, most prominently, tissue regeneration, owing to its ability to fully regenerate amputated limbs, as well as damaged or lost tissue in the heart, spinal cord, lung, skin and brain, among others, as extensively reviewed by Yun and Vieira et al.1,2. Regenerative research generally requires an initial injury, thereby linking regeneration and the stress response. Furthermore, the role of injury signals involving stress pathways in directly stimulating regenerative processes is not well understood despite being potentially central to the field. Nonetheless, the activation of stress pathways in the 1 axolotl is largely unexplored, and, in fact, the dominant glucocorticoid (GC) is not currently defined. Stress-response pathways are highly conserved among vertebrates3. Once an animal encounters and senses an environmental stressor, the sympathoadrenal system is activated. Here, the sympathetic nervous system triggers an acute release of catecholamines such as adrenaline from the adrenochromaffin cells in the adrenal tissue via acetylcholine (ACh) neurotransmitter release from preganglionic sympathetic neurons, preparing the animal to initiate a fight or flight response. The adrenal tissue of urodele amphibians (salamanders) is located as clusters of cells Comparative Medicine Lab, Department of Clinical Medicine, Aarhus University, Aarhus, Denmark. 2Department of Forensic Medicine, Aarhus University, Aarhus, Denmark. 3Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA. 4Zoophysiology, Department of Biology, Aarhus University, Aarhus, Denmark. 5Aarhus Institute of Advanced Studies, Aarhus University, Aarhus, Denmark. 6Department of Animal and Veterinary Science, Aarhus University, Aarhus, Denmark. 7These authors contributed equally: Catherine J. A. Williams, Henrik Lauridsen. e-mail: Lab Animal Article https://doi.org/10.1038/s41684-026-01692-y Axolotl kidney and adrenals Central artery Adrenal glands Sympathetic peripheral nerve AVT CRH Hypothalamus TRH Pituitary ACTH Kidney parenchyma TSH ACh Adrenal Thyroid Catecholamines α Corticosterone Cortisol T4 T3 β Adrenergic receptors Membrane GR Cytosolic GR T3 T4 Target tissues TF Promoter Target gene Fig. 1 | GC and catecholamine stress signaling pathways common to vertebrates. Key elements of the stress pathways involve the adrenal glands situated as disperse interrenal structures in salamanders as well as the hypothalamus and pituitary in the brain. The catecholamine signaling cascade is classically described to be initiated by sympathetic nerves that trigger the production and release of catecholamines (such as adrenaline) from the adrenal glands via ACh signaling. GC signaling (the HPI axis in amphibians) is classically described as being initiated by the release of CRH (in most species) and/or AVT (believed to be more prominent in amphibians), which then triggers the release of ACTH from the pituitary, which in turn stimulates the production and release of the GCs cortisol and/or corticosterone from the adrenal glands. Catecholamines mainly affect target tissues via adrenergic receptors, while GCs bind to extracellular and intracellular GRs, which then act as transcription factors (TFs). There is extensive crosstalk between the HPI and HPT axes, for instance through CRH-induced activation of thyrotropin-releasing hormone (TRH) and GC-driven promotion of the conversion of the inactive thyroid hormone thyroxine (T4) to its active form, triiodothyronine (T3). Figure created with BioRender.com. along the ventro-medial surface and central vessels of the kidneys4,5 (Fig. 1). These structures contain both chromaffin cells and clusters of corticosteroid-producing (steroidogenic) cells; thus, the hypothalamic–pituitary–adrenal (HPA) axis is referred to as the hypothalamic– pituitary–interrenal (HPI) axis in salamanders. This HPI hormonal cascade is activated by an initial release of corticotrophin-releasing hormone (CRH) and/or arginine vasotocin (AVT) from the hypothalamus in the brain6. This, in turn, stimulates the pituitary to release adrenocorticotropic hormone (ACTH) into the systemic circulation, which acts on the adrenal steroidogenic cells to synthesize and release GCs into the bloodstream (Fig. 1). GCs then trigger longer-lasting effects compared with, but complementary to, catecholamines, including adaptations in metabolism, cardiac output, blood pressure, muscle tone and the immune system7–11. In amphibians, the HPI axis serves important functions in response to environmental (...truncated)