Cardiac parasympathetic tone mediates hypoxic bradycardia in fish, however the specific cholinergic mechanisms underlying this response have not been established. In Chapter 2, bradycardia in zebrafish (Danio rerio) larvae experiencing translational knockdown of the M2 muscarinic receptor was either prevented or limited at two different levels of hypoxia (PO2 = 30 or 40 Torr). Also, M2 receptor deficient fish exposed to exogenous procaterol (a presumed β2-adrenergic receptor agonist) had lower heart rates than similarly treated control fish, implying that the β2-adrenergic receptor may have a cardioinhibitory role in this species. Zebrafish have a single β1-adrenergic receptor (β1AR), but express two distinct β2-adrenergic receptor genes (β2aAR and β2bAR). Zebrafish β1AR deficient larvae described in Chapter 3 had lower resting heart rates than control larvae, which conforms to the stereotypical stimulatory nature of this receptor in the vertebrate heart. However, in larvae where loss of β2a/β2bAR and β1/β2bAR function was combined, heart rate was significantly increased. This confirmed my previous observation that the β2-adrenergic receptor has an inhibitory effect on heart rate in vivo. Fish release the catecholamines epinephrine and norepinephrine (the endogenous ligands of adrenergic receptors) into the circulation when exposed to hypoxia, if sufficiently severe. Zebrafish have two genes for tyrosine hydroxylase (TH1 and TH2), the rate limiting enzyme for catecholamine synthesis, which requires molecular oxygen as a cofactor. In Chapter 4, zebrafish larvae exposed to hypoxia for 4 days exhibited increased whole body epinephrine and norepinephrine content. TH2, but not TH1, mRNA expression decreased after 2 days of hypoxic exposure. The results of this thesis provide some of the first data on receptor-specific control of heart rate in fish under normal and hypoxic conditions. It also provides the first observations that catecholamine turnover and the mRNA expression of enzymes required for catecholamine synthesis in larvae are sensitive to hypoxia. Taken together, these data provide an interesting perspective on the balance of adrenergic and cholinergic control of heart rate in zebrafish larvae.

Abstract The Dlx genes play an important role in the differentiation and migration of gamma-aminobutyric acid (GABA) interneurons of mice. GABAergic interneurons are born in the proliferative zones of the ventral telencephalon and migrate to the cortex early during mouse development. Single Dlx mutant mice show only subtle phenotypes. However, the migration of immature interneurons is blocked in the ventral telencephalon of Dlx1/Dlx2 double mutant mice leading to reduction of GABAergic interneurons in the cortex. Also, Dlx5/Dlx6 expression is almost entirely absent in the forebrain, most probably due to cross-regulatory mechanisms. In zebrafish, the role of dlx genes in GABAergic interneuron development is unknown. By injecting Morpholino, we double knocked down dlx1 and dlx2 genes in wildtype zebrafish to investigate the function of the two genes in zebrafish GABAergic interneuron development. By comparing different subsets of GABAergic interneuron development in wildtype and dlx1/2 morphant zebrafish forebrain, we found out that at 3dpf, 4dpf and 7dpf, double knockdown of dlx1 and dlx2 genes in zebrafish remarkably reduced the number of Calbindin-, Somatostatin- and Parvalbumin-positive GABAergic neurons, whereas the development of Calretinin-positive neurons is slightly affected. These results suggest that in zebrafish, dlx1a and dlx2a genes are important for the development of certain subtypes of GABAergic interneurons (Calbindin-, Somatostatin- and Parvalbumin-positive neurons) and may have minor influence on Calretinin-positive neuron development. This also suggests that different regulatory mechanisms are involved in the development of the different subtypes of GABAergic interneurons.

SFWT Zebrafish were exposed to various concentrations of EtOH at 24 hours post-fertilization for a period of two hours. When fish reached maturity they were placed in individual tanks in a larger open field. A preliminary strain comparison was also conducted using control (EtOH untreated) SFWT and AB fish. The behaviour of fish was recorded for 24 hours during a normal light:dark cycle. Motor patterns and general activity were quantified and analyzed and several behaviors were found to change significantly throughout the daytime and nighttime period. Also, fish exposed to the highest concentration of alcohol were found to exhibit significantly reduced amount of thrashing towards other subjects as compared to fish in the control group confirming previous results that demonstrated reduction of shoaling after early embryonic alcohol exposure.

SFWT Zebrafish were exposed to various concentrations of EtOH at 24 hours post-fertilization for a period of two hours. When fish reached maturity they were placed in individual tanks in a larger open field. A preliminary strain comparison was also conducted using control (EtOH untreated) SFWT and AB fish. The behaviour of fish was recorded for 24 hours during a normal light:dark cycle. Motor patterns and general activity were quantified and analyzed and several behaviors were found to change significantly throughout the daytime and nighttime period. Also, fish exposed to the highest concentration of alcohol were found to exhibit significantly reduced amount of thrashing towards other subjects as compared to fish in the control group confirming previous results that demonstrated reduction of shoaling after early embryonic alcohol exposure.

Developmental exposure to aryl hydrocarbon receptor (AhR) agonists in fish causes severe defects in the cardiovascular system. However, the effects of acute AhR agonist exposure on the adult fish cardiovascular system as well as the genes mediating developmental AhR-induced deformities remain unclear. In this thesis, two studies were carried out to address these issues. Before experiments could begin, methods for quantitative real-time reverse transcriptase polymerase chain reaction (rtrt-PCR) as well as larval exposure and rearing were developed, validated, and optimized.<p> Following method development, a series of experiments was performed on adult zebrafish (<i>Danio rerio</i>) to assess how expression of cytochrome P450 (CYP) and cyclooxygenase (COX) enzyme mRNA in hepatic and vascular tissues is altered after intraperitoneal injection of AhR agonists benzo(a)pyrene (BaP) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alone and in combination with the purported AhR antagonists resveratrol (Res) or alpha-naphthoflavone (ANF). Both TCDD and BaP induced similar patterns of gene expression in arteries, although with different efficacies, and had slightly different effects in hepatic tissues. Resveratrol was generally without effect in all treatment groups and tissues with the exception of reducing TCDD-induced CYP1C2 in vascular tissues. In contrast, ANF antagonized TCDD- and BaP-induced changes, as well as reduced baseline gene expression in liver. However, in arteries, ANF alone acted as an agonist to increase expression of several of the genes investigated.<p> The second series of experiments involved zebrafish eggs aqueously exposed to BaP or TCDD alone and in combination with Res or ANF. Whole larvae CYP and COX isoform mRNA expression was quantified at 5 and 10 days post-fertilization (dpf), then correlated with developmental phenotype. Both TCDD and BaP caused concentration-dependent AhR-associated deformities with a significant increase in mortalities by 10 dpf and increased CYP1A mRNA expression, while TCDD alone decreased CYP1C2 expression. BaP/ANF co-exposure exhibited the highest rate of deformities and mortalities at both 5 and 10 dpf, caused marked alterations in cardiac and vascular morphology at 10 dpf, and increased CYP1A expression. Furthermore, ANF exhibited additive agonistic effects on gene expression with both BaP and TCDD. Correlation analyses revealed that gene expression at 5 dpf, but not 10 dpf, was strongly linked to abnormal cardiac and vascular phenotypes at 10 dpf with several genes related to cardiac development and one primary gene linked to vascular development.

Examination of the swimming capabilities of fish is increasingly being considered as an effective method for determining sublethal toxicity. Acute toxicant exposure is known to cause decreases in swim performance in fish but less is known about how developmental exposure can cause persistent effects that hinder swimming. In addition, little is known about how triglyceride levels fluctuate during fish swimming upon both acute and developmental exposure to toxicant. In this thesis, two studies, one acute and one developmental, were carried out using two different toxicants in order to address these issues.<p> In order to examine acute effects, adult zebrafish (Danio rerio) were exposed to ethanol vehicle or increasing concentrations of 2,4-dinitrophenol (DNP), a mitochondrial electron transport chain uncoupler, for a 24 h period. Following exposure, fish were placed in a swim tunnel for critical swimming speed (Ucrit) determination and swim motion analysis. Whole body triglyceride levels were then determined. Ucrit was decreased in a concentration dependent manner in both the 6 mg/L and 12 mg/L DNP exposure groups, with 6 mg/L DNP being considered sublethal and 12 mg/L approaching the LC50. A decrease in tail beat frequency was observed and is likely the main cause for the decrease in Ucrit in the DNP exposure groups. Triglyceride levels were elevated in a concentration dependent manner in the DNP exposure groups. This increase in triglyceride stores may be due to a behavioral adaption limiting swimming capabilities or due to a direct toxic action of DNP on lipid catabolism.<p> The second study examined whether developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure would cause persistent toxic effects. Zebrafish embryos were exposed to dimethyl sulfoxide control or increasing concentrations of TCDD between 2-4 days post fertilization (dpf). At 5 dpf, cytochrome P450 1A (CYP1A) activity was determined. Fish were raised to 90 dpf with mortalities and deformities being recorded at 5 dpf, 10 dpf, and 90 dpf. At 90 dpf, fish were placed in swim tunnel and Ucrit , swimming motion, and aerobic scope (oxygen consumption rate during exercise minus oxygen consumption rate during rest) were determined. Following swimming, some fish were used for whole body triglyceride analysis while others were used for histological examination. Ucrit was shown to be decreased in the two highest sublethal TCDD exposure groups (0.1 and 1 ng/L) but not in the lowest TCDD exposure group (0.01 ng/L). The exact cause of the decrease in Ucrit is not known, but may be linked to the observed decrease in dorsal aorta diameter, an inability to mobilize triglyceride stores, behavioral adaptations limiting swimming, decreased body length, or a combination of these factors. This TCDD related defect in swimming ability is not due to any increases in gross deformity or mortality rates, nor does it appear that CYP1A induction is required to mediate the toxic effects. Thus, it appears that examination of swim performance may serve as an effective measure of both sublethal acute and developmental toxicities.

Cardiac parasympathetic tone mediates hypoxic bradycardia in fish, however the specific cholinergic mechanisms underlying this response have not been established. In Chapter 2, bradycardia in zebrafish (Danio rerio) larvae experiencing translational knockdown of the M2 muscarinic receptor was either prevented or limited at two different levels of hypoxia (PO2 = 30 or 40 Torr). Also, M2 receptor deficient fish exposed to exogenous procaterol (a presumed β2-adrenergic receptor agonist) had lower heart rates than similarly treated control fish, implying that the β2-adrenergic receptor may have a cardioinhibitory role in this species. Zebrafish have a single β1-adrenergic receptor (β1AR), but express two distinct β2-adrenergic receptor genes (β2aAR and β2bAR). Zebrafish β1AR deficient larvae described in Chapter 3 had lower resting heart rates than control larvae, which conforms to the stereotypical stimulatory nature of this receptor in the vertebrate heart. However, in larvae where loss of β2a/β2bAR and β1/β2bAR function was combined, heart rate was significantly increased. This confirmed my previous observation that the β2-adrenergic receptor has an inhibitory effect on heart rate in vivo. Fish release the catecholamines epinephrine and norepinephrine (the endogenous ligands of adrenergic receptors) into the circulation when exposed to hypoxia, if sufficiently severe. Zebrafish have two genes for tyrosine hydroxylase (TH1 and TH2), the rate limiting enzyme for catecholamine synthesis, which requires molecular oxygen as a cofactor. In Chapter 4, zebrafish larvae exposed to hypoxia for 4 days exhibited increased whole body epinephrine and norepinephrine content. TH2, but not TH1, mRNA expression decreased after 2 days of hypoxic exposure. The results of this thesis provide some of the first data on receptor-specific control of heart rate in fish under normal and hypoxic conditions. It also provides the first observations that catecholamine turnover and the mRNA expression of enzymes required for catecholamine synthesis in larvae are sensitive to hypoxia. Taken together, these data provide an interesting perspective on the balance of adrenergic and cholinergic control of heart rate in zebrafish larvae.

Abstract The Dlx genes play an important role in the differentiation and migration of gamma-aminobutyric acid (GABA) interneurons of mice. GABAergic interneurons are born in the proliferative zones of the ventral telencephalon and migrate to the cortex early during mouse development. Single Dlx mutant mice show only subtle phenotypes. However, the migration of immature interneurons is blocked in the ventral telencephalon of Dlx1/Dlx2 double mutant mice leading to reduction of GABAergic interneurons in the cortex. Also, Dlx5/Dlx6 expression is almost entirely absent in the forebrain, most probably due to cross-regulatory mechanisms. In zebrafish, the role of dlx genes in GABAergic interneuron development is unknown. By injecting Morpholino, we double knocked down dlx1 and dlx2 genes in wildtype zebrafish to investigate the function of the two genes in zebrafish GABAergic interneuron development. By comparing different subsets of GABAergic interneuron development in wildtype and dlx1/2 morphant zebrafish forebrain, we found out that at 3dpf, 4dpf and 7dpf, double knockdown of dlx1 and dlx2 genes in zebrafish remarkably reduced the number of Calbindin-, Somatostatin- and Parvalbumin-positive GABAergic neurons, whereas the development of Calretinin-positive neurons is slightly affected. These results suggest that in zebrafish, dlx1a and dlx2a genes are important for the development of certain subtypes of GABAergic interneurons (Calbindin-, Somatostatin- and Parvalbumin-positive neurons) and may have minor influence on Calretinin-positive neuron development. This also suggests that different regulatory mechanisms are involved in the development of the different subtypes of GABAergic interneurons.

Developmental exposure to aryl hydrocarbon receptor (AhR) agonists in fish causes severe defects in the cardiovascular system. However, the effects of acute AhR agonist exposure on the adult fish cardiovascular system as well as the genes mediating developmental AhR-induced deformities remain unclear. In this thesis, two studies were carried out to address these issues. Before experiments could begin, methods for quantitative real-time reverse transcriptase polymerase chain reaction (rtrt-PCR) as well as larval exposure and rearing were developed, validated, and optimized.<p> Following method development, a series of experiments was performed on adult zebrafish (<i>Danio rerio</i>) to assess how expression of cytochrome P450 (CYP) and cyclooxygenase (COX) enzyme mRNA in hepatic and vascular tissues is altered after intraperitoneal injection of AhR agonists benzo(a)pyrene (BaP) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alone and in combination with the purported AhR antagonists resveratrol (Res) or alpha-naphthoflavone (ANF). Both TCDD and BaP induced similar patterns of gene expression in arteries, although with different efficacies, and had slightly different effects in hepatic tissues. Resveratrol was generally without effect in all treatment groups and tissues with the exception of reducing TCDD-induced CYP1C2 in vascular tissues. In contrast, ANF antagonized TCDD- and BaP-induced changes, as well as reduced baseline gene expression in liver. However, in arteries, ANF alone acted as an agonist to increase expression of several of the genes investigated.<p> The second series of experiments involved zebrafish eggs aqueously exposed to BaP or TCDD alone and in combination with Res or ANF. Whole larvae CYP and COX isoform mRNA expression was quantified at 5 and 10 days post-fertilization (dpf), then correlated with developmental phenotype. Both TCDD and BaP caused concentration-dependent AhR-associated deformities with a significant increase in mortalities by 10 dpf and increased CYP1A mRNA expression, while TCDD alone decreased CYP1C2 expression. BaP/ANF co-exposure exhibited the highest rate of deformities and mortalities at both 5 and 10 dpf, caused marked alterations in cardiac and vascular morphology at 10 dpf, and increased CYP1A expression. Furthermore, ANF exhibited additive agonistic effects on gene expression with both BaP and TCDD. Correlation analyses revealed that gene expression at 5 dpf, but not 10 dpf, was strongly linked to abnormal cardiac and vascular phenotypes at 10 dpf with several genes related to cardiac development and one primary gene linked to vascular development.

Examination of the swimming capabilities of fish is increasingly being considered as an effective method for determining sublethal toxicity. Acute toxicant exposure is known to cause decreases in swim performance in fish but less is known about how developmental exposure can cause persistent effects that hinder swimming. In addition, little is known about how triglyceride levels fluctuate during fish swimming upon both acute and developmental exposure to toxicant. In this thesis, two studies, one acute and one developmental, were carried out using two different toxicants in order to address these issues.<p> In order to examine acute effects, adult zebrafish (Danio rerio) were exposed to ethanol vehicle or increasing concentrations of 2,4-dinitrophenol (DNP), a mitochondrial electron transport chain uncoupler, for a 24 h period. Following exposure, fish were placed in a swim tunnel for critical swimming speed (Ucrit) determination and swim motion analysis. Whole body triglyceride levels were then determined. Ucrit was decreased in a concentration dependent manner in both the 6 mg/L and 12 mg/L DNP exposure groups, with 6 mg/L DNP being considered sublethal and 12 mg/L approaching the LC50. A decrease in tail beat frequency was observed and is likely the main cause for the decrease in Ucrit in the DNP exposure groups. Triglyceride levels were elevated in a concentration dependent manner in the DNP exposure groups. This increase in triglyceride stores may be due to a behavioral adaption limiting swimming capabilities or due to a direct toxic action of DNP on lipid catabolism.<p> The second study examined whether developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure would cause persistent toxic effects. Zebrafish embryos were exposed to dimethyl sulfoxide control or increasing concentrations of TCDD between 2-4 days post fertilization (dpf). At 5 dpf, cytochrome P450 1A (CYP1A) activity was determined. Fish were raised to 90 dpf with mortalities and deformities being recorded at 5 dpf, 10 dpf, and 90 dpf. At 90 dpf, fish were placed in swim tunnel and Ucrit , swimming motion, and aerobic scope (oxygen consumption rate during exercise minus oxygen consumption rate during rest) were determined. Following swimming, some fish were used for whole body triglyceride analysis while others were used for histological examination. Ucrit was shown to be decreased in the two highest sublethal TCDD exposure groups (0.1 and 1 ng/L) but not in the lowest TCDD exposure group (0.01 ng/L). The exact cause of the decrease in Ucrit is not known, but may be linked to the observed decrease in dorsal aorta diameter, an inability to mobilize triglyceride stores, behavioral adaptations limiting swimming, decreased body length, or a combination of these factors. This TCDD related defect in swimming ability is not due to any increases in gross deformity or mortality rates, nor does it appear that CYP1A induction is required to mediate the toxic effects. Thus, it appears that examination of swim performance may serve as an effective measure of both sublethal acute and developmental toxicities.