Abstract
It is well established that serotonin (5-HT; 5-hydroxytryptamine) plays a role in regulating the mammalian hypothalamic-pituitary-adrenal (HPA) axis via the 5-HT receptor subtype 1A (5-HT1A). To date, there has not been a comprehensive investigation of the molecular, pharmacological and physiological aspects of the 5-HT1A receptor and its role in the activation of the hypothalamic-pituitary-interrenal (HPI) axis in a single species of teleost fish. The Gulf toadfish (Opsanus beta) 5-HT1A receptor was cloned and sequenced, showing 67.5% amino acid similarity to the human homologue. The 5-HT1A receptor was distributed throughout the brain, with the midbrain/diencephalon region containing significantly higher transcript levels than any other brain region. Substantial levels of transcript were also found in the pituitary, while very low levels were found in the kidney, which contains the interrenal cells. Xenopus oocyte binding experiments demonstrated that the pharmacology of the Gulf toadfish 5-HT1A receptor is similar to the mammalian form. Confirming these molecular and pharmacological findings, intravenous injection of 8-OH-DPAT, a mammalian 5-HT1A receptor agonist, stimulated the HPI axis to cause a 2-fold increase in circulating levels of cortisol. As the pharmacology and functionality of the toadfish 5-HT1A receptor is similar to the mammalian 5-HT1A receptor, it seemed likely that regulation of the receptor would also be subject to the negative feedback loop that exists in mammals whereby increased circulating levels of cortisol inhibit 5-HT1A receptor activity. To investigate the possibility of such a feedback mechanism in teleosts, plasma cortisol levels in Gulf toadfish (Opsanus beta) were manipulated and the role of cortisol and glucocorticoid receptors (GRs) in the control of 5-HT1A was evaluated. Initial investigations revealed that chronic elevation of plasma cortisol mediates changes in brain 5-HT1A receptor mRNA and protein levels via the GR; however, there appears to be a disconnect between brain levels of the receptor and activation of cortisol release from the interrenal tissue. Targeted investigations confirmed that the release of both corticotropin-releasing factor (CRF) and adrenocorticotropic hormone (ACTH) can be stimulated by activation of centrally located 5-HT1A receptors. Additionally, it was discovered that 8-OH-DPAT-stimulated release of CRF and ACTH was attenuated by crowding stress, and that treatment with RU486 returned secretion rates to control levels. However, while it appears that the GR is responsible for mediating the negative feedback of cortisol on 5-HT1A receptors located in the central nervous system, it does not appear to be responsible for mediating attenuation of cortisol secretion from the interrenal cells. In vitro experiments using isolated kidney tissue suggest that chronically elevated plasma cortisol attenuates ACTH- and 5-HT-stimulated cortisol secretion from the interrenal cells of toadfish. Furthermore, the 5-HT1A receptor is not a mediator of cortisol release at the level of the interrenal cells, explaining in part the disconnect between brain 5-HT1A levels and HPI axis function. The series of investigations outlined in this dissertation have provided key information about the functional aspects of the 5-HT1A receptor in a single species, providing a comprehensive examination of the role it plays in both activating and attenuating the stress response in the Gulf toadfish (Opsanus beta). These findings, combined with the fact that cortisol is the predominant corticosteroid in both humans and fish, provide support for considering the Gulf toadfish as a model for the human 5-HT1A receptor.