Developmental fluoxetine exposure in zebrafish reduces offspring basal cortisol concentration via life stage-dependent maternal transmission

Fluoxetine (FLX) is a pharmaceutical used to treat affective disorders in humans, but as environmental contaminant also affects inadvertently exposed fish in urban watersheds. In humans and fish, acute FLX treatment and exposure are linked to endocrine disruption, including effects on the reproductive and stress axes. Using the zebrafish model, we build on the recent finding that developmental FLX exposure reduced cortisol production across generations, to determine possible parental and/or life-stage-dependent (age and/or breeding experience) contributions to this phenotype. Specifically, we combined control and developmentally FLX-exposed animals of both sexes (F0) into four distinct breeding groups mated at 5 and 9 months, and measured offspring (F1) basal cortisol at 12 dpf. Basal cortisol was lower in F1 descended from developmentally FLX-exposed F0 females bred at 5, but not 9 months, revealing a maternal, life-stage dependent effect. To investigate potential molecular contributions to this phenotype, we profiled maternally deposited transcripts involved in endocrine stress axis development and regulation, epigenetic (de novo DNA methyltransferases) and post-transcriptional (miRNA pathway components and specific miRNAs) regulation of gene expression in unfertilized eggs. Maternal FLX exposure resulted in decreased transcript abundance of glucocorticoid receptor, dnmt3 paralogues and miRNA pathway components in eggs collected at 5 months, and increased transcript abundance of miRNA pathway components at 9 months. Specific miRNAs predicted to target stress axis transcripts decreased (miR-740) or increased (miR-26, miR-30d, miR-92a, miR-103) in eggs collected from FLX females at 5 months. Increased abundance of miRNA-30d and miRNA-92a persisted in eggs collected from FLX females at 9 months. Clustering and principal component analyses of egg transcript profiles separated eggs collected from FLX-females at 5 months from other groups, suggesting that oocyte molecular signatures, and miRNAs in particular, may serve as predictive tools for the offspring phenotype of reduced basal cortisol in response to maternal FLX exposure

Knock-out of vasotocin reduces reproductive success in female zebrafish, Danio rerio

The vertebrate nonapeptide vasotocin/vasopressin is evolutionarily highly conserved and acts as neuromodulator and endocrine/paracrine signaling molecule. Circumstantial and mechanistic evidence from pharmacological manipulations of the vasotocin system in several teleost fishes suggest sex- and species-specific reproductive roles of vasotocin. While effects of vasotocin on teleost reproductive physiology involve both courtship behaviors and the regulation of the hypothalamic-pituitary-gonadal (HPG) axes, comprehensive studies investigating behavioral and physiological reproductive consequences of genetic ablation of vasotocin in a genetically tractable fish model, such as the zebrafish, are currently lacking. Here, we report the generation of homozygous CRISPR/Cas9-based vasotocin gene knock-out zebrafish. Breeding pairs of vasotocin knock-out fish produce significantly fewer fertilized eggs per clutch compared to wildtype fish, an effect coincident with reduced female quivering courtship behavior. Crossbreeding experiments reveal that this reproductive phenotype is entirely female-dependent, as vasotocin-deficient males reproduce normally when paired with female wild-type fish. Histological analyses of vasotocin knock-out ovaries revealed an overall reduction in oocytes and differential distribution of oocyte maturation stages, demonstrating that the reproductive phenotype is linked to oocyte maturation and release. Ovarian hormone quantification and gene expression analysis in mutant fish indicated reduced synthesis of Prostaglandin F2α, a hormone involved in ovarian maturation, egg release and regulation of female courtship behavior in some cyprinids. However, acute injection of vasotocin did not rescue the female mutant reproductive phenotype, suggesting a contribution of organizational effects of vasotocin. Together, this study provides further support for emerging roles of vasotocin in female teleost reproduction in an important teleost model species

Defining Global Neuroendocrine Gene Expression Patterns Associated with Reproductive Seasonality in Fish

Many vertebrates, including the goldfish, exhibit seasonal reproductive rhythms, which are a result of interactions between external environmental stimuli and internal endocrine systems in the hypothalamo-pituitary-gonadal axis. While it is long believed that differential expression of neuroendocrine genes contributes to establishing seasonal reproductive rhythms, no systems-level investigation has yet been conducted. gamma2 receptor, calmodulin, and aromatase b by independent samplings of goldfish brains from six seasonal time points and real-time PCR assays.Using both theoretical and experimental strategies, we report for the first time global gene expression patterns throughout a breeding season which may account for dynamic neuroendocrine regulation of seasonal reproductive development

Epigenetics in teleost fish: from molecular mechanisms to physiological phenotypes

The final publication is available at Elsevier via https://doi.org/10.1016/j.cbpb.2018.01.006. © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/While the field of epigenetics is increasingly recognized to contribute to the emergence of phenotypes in mammalian research models across different developmental and generational timescales, the comparative biology of epigenetics in the large and physiologically diverse vertebrate infraclass of teleost fish remains comparatively understudied. The cypriniform zebrafish and the salmoniform rainbow trout and Atlantic salmon represent two especially important teleost orders, because they offer the unique possibility to comparatively investigate the role of epigenetic regulation in 3R and 4R duplicated genomes. In addition to their sequenced genomes, these teleost species are well-characterized model species for development and physiology, and therefore allow for an investigation of the role of epigenetic modifications in the emergence of physiological phenotypes during an organism's lifespan and in subsequent generations. This review aims firstly to describe the evolution of the repertoire of genes involved in key molecular epigenetic pathways including histone modifications, DNA methylation and microRNAs in zebrafish, rainbow trout, and Atlantic salmon, and secondly, to discuss recent advances in research highlighting a role for molecular epigenetics in shaping physiological phenotypes in these and other teleost models. Finally, by discussing themes and current limitations of the emerging field of teleost epigenetics from both theoretical and technical points of view, we will highlight future research needs and discuss how epigenetics will not only help address basic research questions in comparative teleost physiology, but also inform translational research including aquaculture, aquatic toxicology, and human disease

Differential predictors for alcohol use in adolescents as a function of familial risk

Abstract: Traditional models of future alcohol use in adolescents have used variable-centered approaches, predicting alcohol use from a set of variables across entire samples or populations. Following the proposition that predictive factors may vary in adolescents as a function of family history, we used a two-pronged approach by first defining clusters of familial risk, followed by prediction analyses within each cluster. Thus, for the first time in adolescents, we tested whether adolescents with a family history of drug abuse exhibit a set of predictors different from adolescents without a family history. We apply this approach to a genetic risk score and individual differences in personality, cognition, behavior (risk-taking and discounting) substance use behavior at age 14, life events, and functional brain imaging, to predict scores on the alcohol use disorders identification test (AUDIT) at age 14 and 16 in a sample of adolescents (N = 1659 at baseline, N = 1327 at follow-up) from the IMAGEN cohort, a longitudinal community-based cohort of adolescents. In the absence of familial risk (n = 616), individual differences in baseline drinking, personality measures (extraversion, negative thinking), discounting behaviors, life events, and ventral striatal activation during reward anticipation were significantly associated with future AUDIT scores, while the overall model explained 22% of the variance in future AUDIT. In the presence of familial risk (n = 711), drinking behavior at age 14, personality measures (extraversion, impulsivity), behavioral risk-taking, and life events were significantly associated with future AUDIT scores, explaining 20.1% of the overall variance. Results suggest that individual differences in personality, cognition, life events, brain function, and drinking behavior contribute differentially to the prediction of future alcohol misuse. This approach may inform more individualized preventive interventions

Differential predictors for alcohol use in adolescents as a function of familial risk

Abstract: Traditional models of future alcohol use in adolescents have used variable-centered approaches, predicting alcohol use from a set of variables across entire samples or populations. Following the proposition that predictive factors may vary in adolescents as a function of family history, we used a two-pronged approach by first defining clusters of familial risk, followed by prediction analyses within each cluster. Thus, for the first time in adolescents, we tested whether adolescents with a family history of drug abuse exhibit a set of predictors different from adolescents without a family history. We apply this approach to a genetic risk score and individual differences in personality, cognition, behavior (risk-taking and discounting) substance use behavior at age 14, life events, and functional brain imaging, to predict scores on the alcohol use disorders identification test (AUDIT) at age 14 and 16 in a sample of adolescents (N = 1659 at baseline, N = 1327 at follow-up) from the IMAGEN cohort, a longitudinal community-based cohort of adolescents. In the absence of familial risk (n = 616), individual differences in baseline drinking, personality measures (extraversion, negative thinking), discounting behaviors, life events, and ventral striatal activation during reward anticipation were significantly associated with future AUDIT scores, while the overall model explained 22% of the variance in future AUDIT. In the presence of familial risk (n = 711), drinking behavior at age 14, personality measures (extraversion, impulsivity), behavioral risk-taking, and life events were significantly associated with future AUDIT scores, explaining 20.1% of the overall variance. Results suggest that individual differences in personality, cognition, life events, brain function, and drinking behavior contribute differentially to the prediction of future alcohol misuse. This approach may inform more individualized preventive interventions

The Serotonergic System as a Target for Neuroendocrine Disruption in the Brain of Goldfish (Carassius auratus)

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

Hepatic microRNA profile in "glucose" intolerant rainbow trout and predicted consequences for the integration of endocrine signals

résumé poster résumé posterrésumé posterHepatic microRNA profile in "glucose" intolerant rainbow trout and predicted consequences for the integration of endocrine signals. 18. International Congress of Comparative Endocrinolog

Diet-induced hyperglycaemia alters the hepatic microRNA profile in rainbow trout, O. mykiss

résumé communication orale résumé communication oralerésumé communication oraleDiet-induced hyperglycaemia alters the hepatic microRNA profile in rainbow trout, O. mykiss. PWH Symposium (satellite symposium preceding the 56th annual meeting of the Canadian Society of Zoologists

Genetic ablation of bone marrow beta-adrenergic receptors in mice modulates miRNA-transcriptome networks of neuroinflammation in the paraventricular nucleus

Elucidating molecular pathways regulating neuroimmune communication is critical for therapeutic interventions in conditions characterized by overactive immune responses and dysfunctional autonomic nervous system. We generated a bone marrow-specific adrenergic beta 1 and beta 2 knockout mouse chimera (AdrB1.B2 KO) to determine how sympathetic drive to the bone affects transcripts and miRNAs in the hypothalamic paraventricular nucleus (PVN). This model has previously exhibited a dampened systemic immune response and decreased blood pressure compared with control animals. Reduced sympathetic responsiveness of the bone marrow hematopoietic cells of AdrB1.B2 KO chimera led to suppression of transcriptional networks that included leukocyte cell adhesion and migration and T cell-activation and recruitment. Transcriptome responses related to IL-17a signaling and the renin-angiotensin system were also suppressed in the PVN. Based on the transcriptome response, we next computationally predicted miRNAs in the PVN that may underscore the reduced sympathetic responsiveness of the bone marrow cells. These included miR-27b-3p, miR-150, miR-223-3p, and miR-326. Using real-time PCR, we measured a downregulation in the expression of miR-150-5p, miR-205-5p, miR-223-3p, miR-375-5p, miR-499a-5p, miR-27b-3p, let-7a-5p, and miR-21a-5p in the PVN of AdrB1.B2 KO chimera, confirming computational predictions that these miRNAs are associated with reduced neuro-immune responses and the loss of sympathetic responsiveness in the bone marrow. Intriguingly, directional responses of the miRNA corresponded to mRNAs, suggesting complex temporal or circuit-dependent posttranscriptional control of gene expression in the PVN. This study identifies molecular pathways involved in neural-immune interactions that may act as targets of therapeutic intervention for a dysfunctional autonomic nervous system.Peer reviewe