Serotonin stimulates reproduction and inhibits feeding/growth in the neuroendocrine brain of goldfish. The objective of this thesis is to study the effects of selective serotonin reuptake inhibitor pharmaceuticals (SSRIs) on these systems, as SSRIs, such as fluoxetine, are detected in effluent and bioconcentrate in the brain of wild fish. Genes of the serotonin system were cloned to identify molecular conservation, seasonal expression, and tissue distribution. The serotonin transporter, the target molecule of fluoxetine, was highly conserved and ubiquitously expressed in goldfish. Seasonal changes of hypothalamic gene expression of the serotonin transporter support a role in the seasonal modulation of both processes. Fluoxetine injection experiments were used to assess effects on reproductive endpoints and to identify molecular mechanisms in the neuroendocrine brain. Fluoxetine inhibited serum estradiol concentrations in female goldfish and decreased isotocin mRNA abundance in the hypothalamus and telencephalon. Isotocin injections stimulated circulating estradiol concentrations, providing a causal link. Evidence for an involvement of serotonin in isotocin regulation was investigated using immunocytochemistry and 5-HT1A receptor agonists and antagonists. A close proximity of serotonin fibers and isotocin cell bodies and fibers was found in the telencephalon and pituitary,respectively. Injection of a 5-HT1A receptor antagonist inhibited isotocin mRNA expression in the telencephalon. Identified gene targets were investigated in waterborne fluoxetine exposures,including environmental concentrations. Waterborne fluoxetine led to a reduction in basal and pheromone-stimulated milt volume in male goldfish. Gene expression evidence indicated a central inhibitory effect of fluoxetine through the decrease in mRNA abundance of follicle-stimulating hormone in the pituitary and isotocin in the telencephalon. Feeding rate and weight decreased in fluoxetine-injected goldfish, indicating an anorexigenic effect. Fluoxetine induced changes in the gene expression of the feeding peptides neuropeptide Y, corticotropin-releasing factor, and cocaine- and amphetamine-regulated transcript-I in the hypothalamus and telencephalon. Waterborne exposure to fluoxetine validated the anorexigenic effect in goldfish and was correlated with increased expression of corticotropin-releasing factor mRNA, an anorectic peptide. The thesis provides evidence for disrupting effects of fluoxetine on neuroendocrine control of reproductive function and feeding/growth in goldfish, partially at environmental concentrations. The thesis provides the framework for the investigation of existing aquatic contaminants which modulate the serotonin system.

Fluoxetine, a selective serotonin re-uptake inhibitor (SSRI) and the active ingredient in Prozac®, is found in the environment and disrupts feeding and metabolism in exposed fish. The objective of this research was to investigate the mechanisms involved in the feeding and metabolism disruption in the model goldfish (Carassius auratus). Two short-term waterborne fluoxetine exposures (7- and 14-days) were performed using two environmentally relevant doses of fluoxetine (0.5 and 1 μg/L) and metabolic effects at the level of the brain, liver, serum and bile in goldfish were investigated. Abundances of mRNA transcripts coding for six feeding neuropeptides were examined to determine which may be involved in the initial neural changes associated with decreased appetite in goldfish. The 7-day fluoxetine exposure at 1 μg/L caused corticotropin-releasing factor (CRF) mRNA levels to increase by 2-fold in female hypothalamus and telencephalon, indicating that CRF may be one of the first of the feeding neuropeptides to be altered. Six hepatic miRNAs were also evaluated in the goldfish liver that were previously associated with fluoxetine exposure in zebrafish (Danio rerio). Following the 7-day exposure at 1 μg/L, miR-22b, miR-140, miR-210, miR-301a and miR-457b levels increased in the female goldfish liver by 4-6 fold. The 14-day fluoxetine exposure at 1 μg/L caused 2-fold increases in miR-210, miR-301a, miR-457b and let-7d in male goldfish liver. These miRNAs were associated with the down-regulation of anabolic metabolic pathways in zebrafish, indicating a conservation of miRNA and fluoxetine effect between fish species. Serum and bile metabolite profiles of fluoxetine exposed goldfish were evaluated using ultra performance liquid chromatography coupled to quadrupole time of flight mass spectrometry. Following the 14-day exposure at 1 μg/L, the bile metabolite profiles of male goldfish were significantly different from controls as detected by cluster analysis and fluoxetine was tentatively identified in the serum. No other discriminant metabolites were identified as of yet. The data presented suggest that fluoxetine causes metabolic disruption in goldfish at multiple organ levels. Because of the widespread detection of fluoxetine and other emerging SSRIs in the aquatic environment, future research is required to firmly establish this pharmaceutical class as a metabolic and endocrine disrupting chemical.

Studies on the survival of central nervous system of hypoxia-tolerant species under challenges of reduced energy availability have characterised adaptive mechanisms of brain at the cell and tissue level that lead to reduced excitability and protection. However, evidence of hypoxic suppression of retinal activity in these species has not been followed up with mechanistic studies. Microspectrofluorometric monitoring of intracellular free Ca2+ concentration ([Ca2+]i) is useful for identifying cellular mechanisms that may lead to adaptive strategies, as unregulated increases in [Ca2+]i cause toxicity. Horizontal cells (HCs) are second order retinal neurons that receive tonic excitatory input from photoreceptors, and possess voltage-gated Ca2+ conductances and other channels that can facilitate toxic increases in [Ca2+]i under conditions of reduced energy availability (modeled as oxygen-glucose deprivation, OGD). It was demonstrated that isolated HCs of the hypoxia-tolerant goldfish display spontaneous, transient [Ca2+]i activity (SA) which decreased in amplitude and area under the curve following OGD or glucose removal (20 min) without recovery. SA was shown to be dependent on extracellular Ca2+ influx through voltage-gated Ca2+ channels, though mechanisms of SA generation and regulation has yet to be determined. Additionally, glutamate-elicited peak increases in [Ca2+]i were reduced after 20 min of OGD. The removal of O2 during OGD insult seemed to be protective as an increase in baseline [Ca2+]i was seen during and following glucose removal under normoxic conditions. The mechanisms mediating these decreases in spontaneous and elicited [Ca2+]i activity are currently unknown, though candidate pathways are discussed. This thesis contributes a hint towards how HCs may tolerate conditions of low energy availability, which may also inform investigations on their role in situ during these insults.

Endogenously produced carbon monoxide (CO) is an important gaseous signalling molecule which regulates a variety of cardiorespiratory functions. CO is produced in cells by the heme oxygenase (HO) family of proteins by the breakdown of heme into equimolar amounts of CO, bilirubin and Fe2+. My thesis focuses on the hypoxia- and hyperoxia-inducible HO-1/CO system exclusively and aims to provide the first evidence that the HO-1/CO system is involved in cardiorespiratory control in the zebrafish (Danio rerio) and goldfish (Carassius auratus). Overall, I hypothesise that the HO-1/CO system acts as a negative regulator of cardiorespiratory function in fish. Using immunohistochemistry, I was able to characterise the distribution of HO-1 and thus reveal the potential for endogenous CO production (from heme breakdown) in branchial and skin neuroepithelial cells (NECs; putative O2 chemoreceptors) and associated innervation as well as the heart of the developing zebrafish larva. The presence of HO-1 in these structures suggests the likelihood of specific and localized production of CO in fish. To assess the functional significance of the HO-1/CO system in control of cardiorespiratory function, I used pharmacological and gene knock down approaches to diminish HO-1 activity, and presumably endogenous CO production, in adult and larval fish, respectively. The results from these experiments provided evidence that 1) CO has an inhibitory influence on ventilation in goldfish and zebrafish but that its function is temperature- and species-dependent and 2) showed that the HO-1/CO system tonically inhibits cardiac activity in larval zebrafish.

In the teleost brain, radial glial cells (RGCs) are the main macroglia and are stem- like progenitors that express key steroidogenic enzymes, including the estrogen- synthesizing enzyme, aromatase B (cyp19a1b). As a result, RGCs are integral to neurogenesis and neurosteroidogenesis in the brain, however little is known about the permissive factors and signaling mechanisms that control these functions. The aim of this thesis is to investigate if the secretogranin-derived neuropeptide secretoneurin (SN) can exert regulatory control over goldfish (Carassius auratus) RGCs. Immunohistochemistry revealed a close neuroanatomical relationship between RGCs and soma of SNa- immunoreactive magnocellular and parvocellular neurons in the preoptic nucleus in both goldfish and zebrafish (Danio rerio) models. Both intracerebroventricular injections of SNa into the third brain ventricle and SNa exposures of cultured goldfish RGCs in vitro show that SNa can reduce cyp19a1b expression, thus implicating SNa in the control of neuroestrogen production. RNA-sequencing was used to characterize the in vitro transcriptomic responses elicited by 1000 nM SNa in RGCs. These data revealed that gene networks related to central nervous system function (neurogenesis, glial cell development, synaptic plasticity) and immune function (immune system activation, leukocyte function, macrophage response) were increased by SNa. A dose-response study using quantitative proteomics indicates a low 10 nM dose of SNa increased expression of proteins involved in cell growth, proliferation, and migration whereas higher doses down- regulated proteins involved in these processes, indicating SNa has dose-dependent regulatory effects. Together, through these altered gene and protein networks, this thesis proposes SNa exerts trophic and immunogenic effects in RGCs. These datasets identified a total of 12,180 and 1,363 unique transcripts and proteins, respectively, and demonstrated that RGCs express a diverse receptor and signaling molecule profile. Therefore, RGCs can respond to and synthesize an array of hormones, peptides, cytokines, and growth factors, revealing a multiplicity of new functions critical to neuronal-glial interactions.

Serotonin (5-HT)-containing neuroepithelial cells (NECs) of the gill filament are believed to be the primary O2 chemosensors in fish. In the mammalian carotid body (CB), 5-HT is one of many neurotransmitters believed to play a role in transduction of hypoxic stimuli, with acetylcholine (ACh) being the primary fast-acting excitatory neurotransmitter. Immunohistochemistry and confocal microscopy was used to observe the presence of the vesicular acetylcholine transporter (VAChT), a marker for the presence of ACh, and its associated innervation in the gills of zebrafish. VAChT-positive cells were observed primarily along the afferent side of the filament, with some cells receiving extrabranchial innervation. No VAChT-positive cells were observed in the gills of goldfish; however, certain key morphological differences in the innervation of goldfish gills was observed, as compared to zebrafish. In addition, in zebrafish NECs, whole-cell current is dominated by an O2-sensitive background K+ current; however, this is just one of several currents observed in the mammalian CB. In zebrafish NECs and the CB, membrane depolarization in response to hypoxia, mediated by inhibition of the background K+ (KB) channels, is believed to lead to activation of voltage-gated Ca2+ (CaV) channels and Ca2+-dependent neurosecretion. Using patch-clamp electrophysiology, I discovered several ion channel types not previously observed in the gill chemosensors, including Ca2+-activated K+ (KCa), voltage-dependent K+ (KV), and voltage-activated Ca2+ (CaV) channels. Under whole-cell patch-clamp conditions, the goldfish NECs did not respond to hypoxia (PO2 ~ 11 mmHg). Employing ratiometric calcium imaging and an activity-dependent fluorescent vital dye, I observed that intact goldfish NECs respond to hypoxia (PO2 ~ 11 mmHg) with an increase in intracellular Ca2+ ([Ca2+]i) and increased synaptic vesicle activity. The results of these experiments demonstrate that (1) ACh appears to play a role in the zebrafish, but not goldfish gill, (2) goldfish NECs likely signal hypoxic stimuli primarily via the central nervous system (CNS), (3) goldfish NECs express a broad range of ion channels as compared to the NECs of zebrafish, and (4) goldfish NECs rely on some cytosolic factor(s) when responding to hypoxia (PO2 ~ 11 mmHg). This thesis represents a further step in the study of neurochemical and physiological adaptations to tolerance of extreme hypoxia.

Bottlenose dolphins (Tursiops truncatus) kept in zoos and dolphinarias rarely get an outlet for their echolocation abilities as their pool environment is often quite barren. Not much research has been carried out on enrichments promoting echolocation for dolphins in human care. In the present study a setup with live fish was compared to a setup with air-filled floats (providing strong sonar targets, similar to the swim bladders of large fish) and a control setup. A PCL (porpoise click logger) was used to record the echolocation click trains produced by the dolphins and aimed at the three setups. Behavioural data was also collected from video footage. Both the PCL data and all the behavioural observations indicated that the fish setup was more interesting than the float and the control setup, for the dolphins to echolocate towards. However, there were some contradictions with some parameters, where the floats and control seemed to be more interesting. This was probably due to the location of the PCL hydrophone in relation to the floats and fish, and not because the dolphins had a real bigger interest in these setups. To increase the possibility for dolphins to perform more echolocation in human care and increase their welfare, live fish can be recommended as echolocation enrichment.

No description available.

Development of techniques for observing variability at the major histocompatibility complex (MHC) of fishes could prove an important first step in understanding the genetic bases of disease resistance. In this study, using goldfish (Carassius auratus) as a model system, three approaches to generating antisera to putative MHC molecules and two methods for detecting antibody reactivities were evaluated. Seven full-sib families were produced, and red blood cells (RBCs) of goldfish family members were screened for reactivity with a panel of absorbed antisera. The antisera panel consisted of fish anti-fish, chicken anti-chicken, and chicken anti-fish antisera. The fish anti-fish antisera was produced by injecting RBCs from each parent into its mate, and the chicken anti-fish antisera was produced by injecting parental goldfish RBCs into chickens. The chicken anti-chicken antisera were obtained from a genetics laboratory where MHC-specific antisera had been prepared previously. The pattern of presence or absence of agglutination upon mixing with the respective reagents in this panel of antisera was regarded as the phenotype of the individual tested. Agglutinations observed macroscopically or microscopically were easily scored as positive or negative. Particular phenotypes were observed among individuals both within and between families. The large numbers of phenotypes observed may indicate: (1) the need for additional absorptions in the preparation of antisera, or (2) segregation of additional sets of phenotypic MHC haplotypes in the tetraploid goldfish. The utility of chicken anti-chicken reagents in serotyping of fish was demonstrated. Use of the traditional approach to conducting hemagglutination assays limited the number of assays executed because of the amount of blood required. In order to minimize the sample volumes required, antibody reactivities were evaluated by flow cytometry employing appropriate fluorescein labeled antibodies. Using this approach, scoring of positive and negative results was equivocal, and results did not always agree with those scored by hemagglutination assays. Results of this study strongly suggest that the development of immune allo- and xeno-antisera and use of hemagglutination assays can be used to characterize genetic variability of the MHC of fishes. Understanding of immunogenetic variability in fishes could be used to develop strains resistant to economically important fish pathogens. / Master of Science

The transmission of sex pheromone-mediated signals is essential for goldfish reproduction. However, the neural pathways underlying this reproductive signalling pathway in the goldfish brain is not well described. Lesioning experiments have shown previously that two brain areas, the preoptic area (POA) and the ventral telencephali pars ventralis (Vv) in particular, are important for reproduction. We used patch clamp electrophysiology to study the electrical activities of POA and Vv neurons. Based on the intrinsic properties of these neurons, we suggest there are five different functional classes of POA neurons and a single class of Vv neurons. In addition, by electrically stimulating the olfactory bulb (OB), we were able to show that this primary sensory structure makes monosynaptic glutamatergic connections with both POA and Vv neurons. While electrophysiology measures signalling events occurring at short time scales on the order of milliseconds to minutes, we were also interested in studying sex pheromone signalling in the goldfish brain over a long time scale. Thus, we describe changes in gene expression in male goldfish exposed to waterborne sex pheromones (17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha) over 6 hours. We perform cDNA microarrays on Prostaglandin-F2alpha-treated fish to study the rapid modulation of transcription and define the signalling pathways affected. Our microarrays showed that 71 genes were differentially regulated (67 up and 4 down). Through gene ontology enrichment analysis, we found that these genes were involved in various biological processes such as RNA processing, neurotransmission, neuronal development, apoptosis, cellular metabolism and sexual reproduction. RT-PCRs were performed to validate our microarrays and to facilitate direct comparisons of the effects of the two sex pheromones, 17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha. By combining electrophysiology and gene expression analyses, we were able to study sex-pheromone signalling on two different time scales. One short, occurring on the order of milliseconds to minutes, that involves electrical activities in the brain through the glutamatergic amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate receptors; and the other long occurring several hours later that involves changes in the gene expression levels of calmodulin and ependymin among other genes underlying neuroplasticity. Reproductive neuroplasticity in the goldfish may therefore require the activation of glutamatergic receptors which then activate downstream signals like calmodulin and ependymin to transform the sex pheromones-mediate signal into gene expression.