My research program includes three distinct but related areas. The theme is fish physiology and endocrinology using the gulf toadfish, Opsanus beta, as a model organism. Within this theme, my main research areas are as follows:
The Regulation of Urea Transport in Teleost Fish
In terms of urea excretion, gulf toadfish, Opsanus beta, are extremely interesting. The gulf toadfish is a marine teleost fish that, like terrestrial organisms, has a fully functional ornithine-urea cycle (O-UC), an unusual characteristic in fish because, in general, fish are capable of excreting their nitrogenous wastes as ammonia instead of using energy to convert it into urea. When in its natural environment, toadfish are believed to excrete a combination of ammonia and urea. However, when stressed in the laboratory by exposure to environmental stressors, toadfish will switch to excreting predominantly urea. Most of the early work involving urea transport in toadfish was done by my Ph.D and postdoctoral supervisors, Dr. Chris M. Wood and Dr. Patrick J. Walsh, respectively. They determined that urea excretion occurs across the gill via a facilitated diffusion urea transporter, tUT. In association with NSF grant #IOS-0455944, research by my laboratory has shown that when toadfish are initially stressed the mRNA expression of important, rate-limiting O-UC enzymes and of the toadfish urea transporter, tUT, are upregulated (McDonald et al., 2009; Laberge et al., 2009). This results in an increase in urea production but surprisingly does not result in an increase in urea excretion, revealing a disconnection between tUT mRNA expression levels and tUT protein function (McDonald et al., 2009). Instead of urea being excreted continuously across the gill via tUT, tUT is believed to be only periodically activated, allowing for the excretion of urea only once or twice a day. At this time, cortisol levels in toadfish are cycling, periodically dropping to concentrations that are more characteristic of unstressed fish and then quickly returning to circulating levels that are typical of stressed toadfish. It is during these drops in cortisol that tUT is activated, as a pulse of urea will be excreted at this time. As a graduate student I determined that the drop of cortisol does not directly activate tUT, as exogenous infusion of cortisol in an attempt to prevent the pre-urea pulse drop does not reduce the frequency of urea pulses (McDonald et al., 2004). However, cortisol infusion does reduce the pulse size, suggesting that endogenous or exogenous elevations in cortisol are interfering with the activation of tUT (i.e., the more tUT are activated the bigger the urea pulse), which is a hypothesis that I have been investigating ever since (see below). The role of glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs) in the regulation of tUT activation was further investigated in my laboratory (Rodela et al., 2009a), confirming the role of GRs in mediating the response (McDonald et al., 2004). Furthermore, we have recently localized a cortisol-sensitive, facilitated diffusion urea transporter, likely tUT but it could be another urea transporter, to the basolateral membrane of the toadfish gill using an isolated basolateral membrane vesicle preparation (Rodela et al., 2009b).
Based on preliminary evidence supporting the neurotransmitter, serotonin (5-HT; 5-hydroxytryptamine) as the potential activator of tUT, as a postdoctoral researcher I established that arterial injection of the serotonin type 2 (5-HT2) receptor agonist, α-methyl 5-HT, results in urea pulses from toadfish within 5 minutes (McDonald and Walsh, 2004). Furthermore, this response is inhibited when fish are pre-injected with the 5-HT2A receptor antagonist, ketanserin, suggesting that this specific receptor subtype might be involved in tUT activation. The focus of my most current research (in association with NSF grant #IOS-0920547) is characterizing this receptor subtype in toadfish, in terms of its role in the regulation of urea excretion via tUT. I have recently cloned and sequenced the entire toadfish 5-HT2A receptor which shows > 70% sequence homology on the level of amino acids to other mammalian 5-HT2A receptors (GenBank Accession #FJ611960.1). I have also been able to express the toadfish 5-HT2A receptor in Xenopus laevis oocytes and show that oocytes that are injected with 5-HT2A cRNA will bind more [3H]-5-HT than oocytes that are injected with water, illustrating that the toadfish 5-HT2A receptor functions like the mammalian receptor. The hypothesis that I will be testing is that 5-HT2A receptors in the gill mediate tUT activation and it is these receptors that are sensitive to circulating cortisol levels. I hypothesize that 5-HT2A receptors become desensitized or downregulated when cortisol levels are high, resulting in a reduced activation of tUT. When cortisol levels drop before a pulse, 5-HT2A receptors become sensitive to 5-HT and will mediate activation of tUT, potentially by phosphorylation. Other hypotheses that I will test related to pulsatile urea excretion in the future is that the activation of tUT is preceded by an increase in the activity of serotonergic cells that are controlled by extrinsic nerve fibers. I do believe that the increase in neuronal firing results in a release of 5-HT, which ultimately activates tUT. Lastly, I hypothesize that the activation of tUT is preceded by an increase in cortisol catabolism and followed by an increase in cortisol secretion from the interrenal tissue.
The second aspect of my research program is looking at the role of 5-HT, and the 5-HT1A receptor, in the regulation of the hypothalamic-pituitary-interrenal (HPI) axis. The HPI axis is involved in mediating the stress response, namely cortisol release, in fish and is analogous to the mammalian hypothalamic-pituitary-adrenal (HPA) axis. This work on 5-HT1A is related to the urea project outlined above, as I believe that the fluctuations in circulating cortisol concentrations around the time of a pulse are mediated by 5-HT through 5-HT1A. However, this project also has direct human health implications. Major depressive disorder (MDD) represents one of the most prevalent clinical diagnoses globally and MDD and anxiety-related disorders are only predicted to rise in prevalence over the next 20 years. Dysfunction in the 5-HT1A receptor could account for the 5-HT deficiency and the hyperactivity of the stress response that make up the two predominant theories explaining the cause of depression. We believe that the gulf toadfish, Opsanus beta, may make an excellent non-mammalian model to study this human health issue.
Similar to findings in mammals, our work has shown the mammalian 5-HT1A receptor agonist, 8-hydroxy- 2-(di-n- propylamino) tetraline (8-OH-DPAT) causes a significant increase in circulating levels of cortisol within 0.25 h in the gulf toadfish, Opsanus beta (Medeiros et al., submitted). This pharmacological evidence was recently supported by molecular evidence; we have recently cloned and sequenced the full length toadfish 5-HT1A receptor (GenBank Accession #FJ769221), which shows 68% homology to the mammalian receptor. Using qPCR, we have determined that 5-HT1A receptors are found throughout tissues of the gulf toadfish, though predominantly in the brain and reproductive system, including the swim bladder, which is an important organ for communication during reproduction (Medeiros et al., submitted). Different brain regions express the receptor in varying amounts with 5-HT1A appearing to be most concentrated in the pituitary and the medulla, similar to the pattern of expression found in mammals. The toadfish 5-HT1A receptor has been expressed in Xenopus oocytes to determine toadfish 5-HT1A receptor binding kinetics and pharmacological specificity to different mammalian-based 5-HT1A receptor agonists and antagonists (Medeiros et al., submitted). Our future work includes testing the hypotheses that toadfish 5-HT1A is sensitive to fluctuations in circulating cortisol, that the location of 5-HT1A immunoreactivity and serotonergic innervation throughout toadfish brain is comparable to that of mammalian brain and that downregulation of 5-HT1A through chronic antidepressant administration has repercussions on the stress response.
The Pharmacodynamics of Selective Serotonin Reuptake Inhibitors in Aquatic Organisms
Fluoxetine is the active compound in Prozac&tm; and falls under the category of emerging environmental pollutants. It is a selective serotonin reuptake inhibitor (SSRI) that works to prevent the reuptake of serotonin (5-HT) by 5-HT specific transporters located in neurons, platelets, lymphocytes and the gastrointestinal tract. Fluoxetine is used most commonly for the treatment of illnesses such as depression and anxiety as well as panic and obsessive-compulsive disorders. After consumption, it enters wastewater treatment facilities in human waste and through the disposal of unused drug down the sink or toilet, and these treatment facilities are ill-equipped to remove these compounds. Consequently, low levels of fluoxetine have been continuously released into the environment (McDonald and Reimer, 2008), resulting in now measurable quantities in freshwater and marine coastal ecosystems. While the potential human health consequences through the consumption of tainted fish or drinking water are considered very low, the effects of chronic fluoxetine exposure on aquatic organisms are still not completely understood.
This third aspect of my research program is related to the previous two areas in terms of the action of fluoxetine. Treatment with or exposure to fluoxetine results in changes in synaptic and circulating levels of 5-HT, and as described above, 5-HT is involved in the regulation of many physiological and behavioral processes in fish, including nitrogen waste excretion and the stress response. Long-term exposure to fluoxetine also results in a downregulation of the 5-HT1A receptor in mammals. The goal of my research is to determine how treatment with fluoxetine or exposure to waterborne fluoxetine alters fish physiology. My most recent work in this area investigated the impact of fluoxetine treatment via intraperitoneal implants on two aspects of toadfish physiology: urea excretion and osmoregulation (Morando et al., 2009). We hypothesized that both processes would be affected by fluoxetine treatment, since both have been shown to be regulated by 5-HT. Our findings indicated that urea excretion and intestinal osmoregulation are both responsive to fluoxetine. In addition, the HPI axis was also stimulated in fish that were treated with the highest level of fluoxetine, an effect that had secondary implications on toadfish physiology. Further investigation is needed to determine the sensitivity of all these processes to chronic exposure to low levels of waterborne fluoxetine contamination. Looking at the effects of other SSRIs on fish physiology is another potential avenue of research.
I am also investigating the impact of fluoxetine treatment and waterborne fluoxetine exposure on toadfish behavior. This ongoing project has been funded by an NIEHS/MFBS Center pilot grant (2006) and a University of Miami General Research Support Award (2008). This work is in collaboration with Dr. Kath Sloman, a fish behavioralist from the University of Plymouth, UK. The project stems from the known affects of fluoxetine on mammalian behavior, on the dependency of toadfish pulsatile urea excretion on 5-HT (see above in i), the known sensitivity of this mechanism to fluoxetine treatment (Morando et al., 2009) and the belief that pulsatile urea excretion may be a social or chemical signal between toadfish (Sloman et al., 2005). Specifically, when toadfish are confined in individual chambers, urea pulses occur randomly over the course of the day. However, when two fish are placed in a tank together, they will pulse within one hour of one another, suggesting a role for the urea pulse in social communication and behavior (Sloman et al., 2005). Since pulsatile urea excretion is sensitive to fluoxetine (Morando et al., 2009), will fluoxetine treatment or exposure also result in changes in toadfish social interaction and behavior? Our results suggests that this may be the case: when toadfish are treated with fluoxetine via intraperitoneal implants, dominant fish show a significant increase in aggression. Interestingly, there is no change in the behavior of submissive fish (McDonald et al., submitted).
Other Research Interests
In addition to these three main projects, I have a couple of other research interests, one that began with my Ph.D. dissertation work (McDonald et al., 2000, 2002a, 2002b, 2003) and the other that has evolved with my most recent research. Of the former, I continue to be interested in the kidney function in the family of batrachoidid fishes, which includes the gulf toadfish (McDonald and Grosell, 2006) and also its Pacific relative, the plainfin midshipman (Porichthys notatus) (McDonald and Walsh, 2007). This family of fishes has an aglomerular kidney, which is highly evolved to survive in the marine environment. This has osmoregulatory implications if the fish moves to a more dilute environment (McDonald and Grosell, 2006; McDonald, 2007). My work investigates the transport processes within this kidney and focuses on how urea enters the kidney tubule. My second peripheral research interest is that of 5-HT receptor physiology, essentially determining the physiological mechanism(s) that is/are associated with different 5-HT receptor subtypes in fish. While molecular evidence exists for the presence of most of the 5-HT receptors that have been described in mammals, the physiological roles for most of these receptors have not been elucidated in fish. To date, I have determined that stimulation of the 5-HT7 receptor results in changes in intestinal transepithelial potential of toadfish, likely due to modulations in Cl- secretion, which has implications for osmoregulation in fish. I began studying the gastrointestinal tract (GIT) after realizing that the GIT of vertebrates contains > 80% of the serotonergic activity in the body, making it a very important organ with respect to 5-HT receptor physiology. THTI have also characterized roles for the 5-HT2A and 5-HT2B receptors in cardio-respiratory and vascular physiology, respectively (McDonald et al., 2010). This work came about after investigating whether the hypoxia response is involved in regulating pulsatile urea excretion in toadfish (McDonald et al., 2007).