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.

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.

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

The goldfish (Carassius auratus) is a hardy exotic species that have established sporadically distributed feral populations throughout North America. In one shallow seasonally anoxic pond goldfish formed a large stunted population of 15-17,000 ind ha$ sp{-1}$, with 53% being small young of the year. Goldfish were predominantly benthic herbivores with little diet overlap with resident red-spotted newts (Notapthalmus v. viredescens). Thus in relatively simple systems lacking fish predators goldfish can be very successful. However in systems with a complex native fish community, goldfish have had less success in colonizing. This could be related to an inability of goldfish to cope with native predators. / Tests for assortative shoaling between brown and gold coloured morphs showed that gold coloured fish exhibited no colour based assortive shoaling, while brown fish showed slight but significant colour preferences for like-coloured fish. This level of shoaling preference did not improve after visual exposure or interaction with native predators, indicating that goldfish showed limited behaviourial responses to predators, and that they were unable to modify their response to a predation threat. Further trials allowing goldfish to interact with either pike (Esox lucius) or bass (Ambloplites rupestris), in both single species groups of predator-naive goldfish, and mixed species conditions of goldfish with predator-experience minnows, showed that goldfish did not alter their behaviour in the presence of minnows (Pimephales notatus) when the predators were not present. However, with the predators present goldfish altered their activities to a more minnow-like pattern and showed a significant improvement in anti-predator behaviour. This improved behaviour continued by goldfish when they were retested on their own, indicating that the goldfish were reacting to the predator and not the minnows. Goldfish colonization may therefore be limited not so much by predation or competition from native cyprinids, but more by the absence/presence of a suitable, native, predator-experienced fish from which to copy the appropriate anti-predator behaviours.

Although artificially selected goldfish exhibit swimming performance decrements, with the most derived morphotypes more affected, they can be utilized to explore functional design and movement pattern principles in aquatic vertebrates. Drag, steady swimming kinematics (tailbeat frequency, amplitude, stride length), energetics (standard and active metabolic rate), fast-start performance (average and maximum velocity and acceleration), stability in yaw and roll and propulsive muscle ultrastructural characteristics (mitochondrial volume density and spacing, myofibril diameter and capillary to fibre ratio in red and white muscle) were measured for four morphotypes: common, comet, fantail and eggfish, of comparable length (≈ 5 cm). A performance “pairing” (common and comet; fantail and eggfish) was a recurrent theme for most performance parameters. Vertebral numbers (30), segment lengths (≈ 0.85 mm) and standard metabolic rates (≈ 140 mg O2 kg-1 hr-1) are exceptions where values are the same. Fantail and eggfish drag and drag coefficients (referenced to frontally projected area ≈ 0.6 - 0.9) were higher (requiring more thrust at any given velocity) than those for the more streamlined common and comet (≈ 0.3 - 0.6; P < 0.05). This is reflected in kinematics; tailbeat frequency and stride length at any given velocity for the common and comet are lower and higher respectively than that of the fantail and eggfish (P < 0.05). Common and comet fatigue times are not significantly different from that of their ancestor, Crucian carp (P > 0.05), and are lower than those of the fantail and eggfish (P < 0.05). The cost of transport of the common and comet (≈ 0.6 mg O2 kg-1 m-1) is accurately predicted from the mass scaling relationship for fish (P > 0.05), but values for the fantail and eggfish (≈ 1.3 mg O2 kg-1 m-1) are not (P < 0.05). Eggfish steady swimming (dorsal fin absent) was characterized by rolling and yawing motions associated with significant energy losses. Common and comet fast-start performance (average velocity ≈ 0.45 m s-1, maximum velocity ≈ 1.2 m s-1, average acceleration ≈ 7.5 m s-2, maximum acceleration ≈ 35 m s-2) was similar to that of other locomotor generalists (e.g. trout). Eggfish maximum acceleration (≈ 5 m s-2) is poor due to the absence of inertial and lifting contributions to thrust from the dorsal fin and energy wasting rolling motions. Artificially selected fish can bear upon fitness related adaptations associated with form and movement, providing insights into the “performance envelope” of natural systems subject to ecological speciation.

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.

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.

Teleost retinal ganglion cells can regenerate severed axons following injury, something their mammalian counterparts cannot do. In the teleost, successful regeneration has been attributed in part to microglial cell activities including the phagocytosis of myelin. Although the regulation of microglial phagocytosis has been studied in mammals, in the teleost it is largely unexamined. The present study was designed to identify mediators of microglial phagocytosis released by injured goldfish optic nerve during the course of regeneration. We found that microglial phagocytosis was significantly enhanced in the presence of a 7 day regenerating nerve or medium conditioned by the nerve (CM). When either nerve or CM was incubated with microglia along with an antibody against tumour necrosis factor alpha (TNFalpha), this effect was neutralized. The L929 cell cytotoxicity assay further demonstrated TNFalpha activity in the CM. However, Western blot analysis did not confirm this result. Therefore, further work is necessary to clearly establish the presence of TNFalpha.

Aromatase is an enzyme that converts androgens to estrogens. In teleosts, brain aromatase, also known as aromatase B (cy19a1b), is only expressed in radial glial cells (RGCs). This is in contrast to aromatase A, which is expressed in gonads. Estrogens such as estradiol (E2) modulate neurogenesis in the adult teleost brain. Recent studies show that E2 also differentially regulates aromatase B expression in goldfish RGCs. As a result, teleost RGCs are suggested to be involved in regulating neurogenesis. In addition, aromatase B expression in goldfish RGC is under the control of dopamine suggesting that neurons and neurotransmitters can regulate RGC function. Interestingly, goldfish RGC transcriptome data shows the expression of one group of metabotropic glutamate receptors (mGluRs), group III mGluRs, which suggests that glutamate may affect RGC function. In this thesis, I present my findings regarding potential glutamatergic regulation of RGCs. Firstly, I investigated the distribution of glutamatergic synaptic vesicles and RGCs in the female goldfish forebrain. Double-staining immunohistochemistry shows that vesicular glutamate transporter (vGLUT) 1/2-labelled glutamatergic synaptic vesicles are in close anatomical proximity to aromatase B-labelled RGCs, which suggests potential regulation of RGCs by glutamate. Glutamatergic regulation of cyp19a1b, cyclin D1 (ccnd1), cyclin A2 (ccna2), mGluR6b (grm6b), mGluR7 (grm7), and mGluR8b (grm8b) expression in cultured adult female goldfish RGCs was also examined. Results from pharmacological manipulations and qPCR data analysis show that selective activation of group III mGluRs decreased cyp19a1b, ccnd1, and ccna2 mRNA via inhibition of cAMP/PKA signalling. Furthermore, grm7 mRNA is positively regulated by cAMP-dependent signalling. The glutamate analog L-glutamic acid decreased cyp19a1b mRNA and increased ccnd1 and grm6b mRNA in a dose-dependent manner. This suggests that ccnd1 and grm6b expression may be regulated by glutamate receptors other than group III mGluRs, for example, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are expressed in cultured goldfish RGCs. It was found that E2 upregulated cyp19a1b, ccnd1 and grm7 mRNA. However, selective activation of group III mGluRs decreases the stimulatory effect of E2 on ccnd1 expression. My findings show that glutamate finely regulates RGC neurogenic and steroidogenic genes, which may implicate glutamate in the regulation of RGC differentiation, RGC proliferation, and neurogenesis in surrounding cells.

Although artificially selected goldfish exhibit swimming performance decrements, with the most derived morphotypes more affected, they can be utilized to explore functional design and movement pattern principles in aquatic vertebrates. Drag, steady swimming kinematics (tailbeat frequency, amplitude, stride length), energetics (standard and active metabolic rate), fast-start performance (average and maximum velocity and acceleration), stability in yaw and roll and propulsive muscle ultrastructural characteristics (mitochondrial volume density and spacing, myofibril diameter and capillary to fibre ratio in red and white muscle) were measured for four morphotypes: common, comet, fantail and eggfish, of comparable length (≈ 5 cm). A performance “pairing” (common and comet; fantail and eggfish) was a recurrent theme for most performance parameters. Vertebral numbers (30), segment lengths (≈ 0.85 mm) and standard metabolic rates (≈ 140 mg O2 kg-1 hr-1) are exceptions where values are the same. Fantail and eggfish drag and drag coefficients (referenced to frontally projected area ≈ 0.6 - 0.9) were higher (requiring more thrust at any given velocity) than those for the more streamlined common and comet (≈ 0.3 - 0.6; P < 0.05). This is reflected in kinematics; tailbeat frequency and stride length at any given velocity for the common and comet are lower and higher respectively than that of the fantail and eggfish (P < 0.05). Common and comet fatigue times are not significantly different from that of their ancestor, Crucian carp (P > 0.05), and are lower than those of the fantail and eggfish (P < 0.05). The cost of transport of the common and comet (≈ 0.6 mg O2 kg-1 m-1) is accurately predicted from the mass scaling relationship for fish (P > 0.05), but values for the fantail and eggfish (≈ 1.3 mg O2 kg-1 m-1) are not (P < 0.05). Eggfish steady swimming (dorsal fin absent) was characterized by rolling and yawing motions associated with significant energy losses. Common and comet fast-start performance (average velocity ≈ 0.45 m s-1, maximum velocity ≈ 1.2 m s-1, average acceleration ≈ 7.5 m s-2, maximum acceleration ≈ 35 m s-2) was similar to that of other locomotor generalists (e.g. trout). Eggfish maximum acceleration (≈ 5 m s-2) is poor due to the absence of inertial and lifting contributions to thrust from the dorsal fin and energy wasting rolling motions. Artificially selected fish can bear upon fitness related adaptations associated with form and movement, providing insights into the “performance envelope” of natural systems subject to ecological speciation. / Science, Faculty of / Zoology, Department of / Graduate