Comprehensive Expression Map of Transcription Regulators in the Adult Zebrafish Telencephalon Reveals Distinct Neurogenic Niches

PubMed Central

Diotel, Nicolas; Rodriguez Viales, Rebecca; Armant, Olivier; März, Martin; Ferg, Marco; Rastegar, Sepand; Strähle, Uwe

2015-01-01

The zebrafish has become a model to study adult vertebrate neurogenesis. In particular, the adult telencephalon has been an intensely studied structure in the zebrafish brain. Differential expression of transcriptional regulators (TRs) is a key feature of development and tissue homeostasis. Here we report an expression map of 1,202 TR genes in the telencephalon of adult zebrafish. Our results are summarized in a database with search and clustering functions to identify genes expressed in particular regions of the telencephalon. We classified 562 genes into 13 distinct patterns, including genes expressed in the proliferative zone. The remaining 640 genes displayed unique and complex patterns of expression and could thus not be grouped into distinct classes. The neurogenic ventricular regions express overlapping but distinct sets of TR genes, suggesting regional differences in the neurogenic niches in the telencephalon. In summary, the small telencephalon of the zebrafish shows a remarkable complexity in TR gene expression. The adult zebrafish telencephalon has become a model to study neurogenesis. We established the expression pattern of more than 1200 transcription regulators (TR) in the adult telencephalon. The neurogenic regions express overlapping but distinct sets of TR genes suggesting regional differences in the neurogenic potential. J. Comp. Neurol. 523:1202–1221, 2015. © 2015 Wiley Periodicals, Inc. PMID:25556858

Comprehensive expression map of transcription regulators in the adult zebrafish telencephalon reveals distinct neurogenic niches.

PubMed

Diotel, Nicolas; Rodriguez Viales, Rebecca; Armant, Olivier; März, Martin; Ferg, Marco; Rastegar, Sepand; Strähle, Uwe

2015-06-01

The zebrafish has become a model to study adult vertebrate neurogenesis. In particular, the adult telencephalon has been an intensely studied structure in the zebrafish brain. Differential expression of transcriptional regulators (TRs) is a key feature of development and tissue homeostasis. Here we report an expression map of 1,202 TR genes in the telencephalon of adult zebrafish. Our results are summarized in a database with search and clustering functions to identify genes expressed in particular regions of the telencephalon. We classified 562 genes into 13 distinct patterns, including genes expressed in the proliferative zone. The remaining 640 genes displayed unique and complex patterns of expression and could thus not be grouped into distinct classes. The neurogenic ventricular regions express overlapping but distinct sets of TR genes, suggesting regional differences in the neurogenic niches in the telencephalon. In summary, the small telencephalon of the zebrafish shows a remarkable complexity in TR gene expression. The adult zebrafish telencephalon has become a model to study neurogenesis. We established the expression pattern of more than 1200 transcription regulators (TR) in the adult telencephalon. The neurogenic regions express overlapping but distinct sets of TR genes suggesting regional differences in the neurogenic potential. © 2015 Wiley Periodicals, Inc.

Characterization of multiciliated ependymal cells that emerge in the neurogenic niche of the aged zebrafish brain.

PubMed

Ogino, Takashi; Sawada, Masato; Takase, Hiroshi; Nakai, Chiemi; Herranz-Pérez, Vicente; Cebrián-Silla, Arantxa; Kaneko, Naoko; García-Verdugo, José Manuel; Sawamoto, Kazunobu

2016-10-15

In mammals, ventricular walls of the developing brain maintain a neurogenic niche, in which radial glial cells act as neural stem cells (NSCs) and generate new neurons in the embryo. In the adult brain, the neurogenic niche is maintained in the ventricular-subventricular zone (V-SVZ) of the lateral wall of lateral ventricles and the hippocampal dentate gyrus. In the neonatal V-SVZ, radial glial cells transform into astrocytic postnatal NSCs and multiciliated ependymal cells. On the other hand, in zebrafish, radial glial cells continue to cover the surface of the adult telencephalic ventricle and maintain a higher neurogenic potential in the adult brain. However, the cell composition of the neurogenic niche of the aged zebrafish brain has not been investigated. Here we show that multiciliated ependymal cells emerge in the neurogenic niche of the aged zebrafish telencephalon. These multiciliated cells appear predominantly in the dorsal part of the ventral telencephalic ventricular zone, which also contains clusters of migrating new neurons. Scanning electron microscopy and live imaging analyses indicated that these multiple cilia beat coordinately and generate constant fluid flow within the ventral telencephalic ventricle. Analysis of the cell composition by transmission electron microscopy revealed that the neurogenic niche in the aged zebrafish contains different types of cells, with ultrastructures similar to those of ependymal cells, transit-amplifying cells, and migrating new neurons in postnatal mice. These data suggest that the transformation capacity of radial glial cells is conserved but that its timing is different between fish and mice. J. Comp. Neurol. 524:2982-2992, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

G-protein-coupled estrogen receptor 1 is involved in brain development during zebrafish (Danio rerio) embryogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Yanan; Liu, Xiaochun; Zhu, Pei

Highlights: •The Gper expression was detected in the developing brain of zebrafish. •Gper morpholino knockdown induced apoptosis of brain cells. •Gper morpholino knockdown reduced expression in neuron markers. •Zebrafish Gper may be involved in neuronal development. -- Abstract: G-protein-coupled estrogen receptor 1 (Gper, formerly known as GPR30) is found to be a trophic and protective factor in mediating action of estrogen in adult brain, while its role in developing brain remains to be elucidated. Here we present the expression pattern of Gper and its functions during embryogenesis in zebrafish. Both the mRNA and protein of Gper were detected throughout embryogenesis.more » Whole mount in situ hybridization (WISH) revealed a wide distribution of gper mRNAs in various regions of the developing brain. Gper knockdown by specific morpholinos resulted in growth retardation in embryos and morphological defects in the developing brain. In addition, induced apoptosis, decreased proliferation of the brain cells and maldevelopment of sensory and motor neurons were also found in the morphants. Our results provide novel insights into Gper functions in the developing brain, revealing that Gper can maintain the survival of the brain cells, and formation and/or differentiation of the sensory and motor neurons.« less

A novel perivascular cell population in the zebrafish brain.

PubMed

Venero Galanternik, Marina; Castranova, Daniel; Gore, Aniket V; Blewett, Nathan H; Jung, Hyun Min; Stratman, Amber N; Kirby, Martha R; Iben, James; Miller, Mayumi F; Kawakami, Koichi; Maraia, Richard J; Weinstein, Brant M

2017-04-11

The blood-brain barrier is essential for the proper homeostasis and function of the CNS, but its mechanism of function is poorly understood. Perivascular cells surrounding brain blood vessels are thought to be important for blood-brain barrier establishment, but their roles are not well defined. Here, we describe a novel perivascular cell population closely associated with blood vessels on the zebrafish brain. Based on similarities in their morphology, location, and scavenger behavior, these cells appear to be the zebrafish equivalent of cells variably characterized as Fluorescent Granular Perithelial cells (FGPs), perivascular macrophages, or 'Mato Cells' in mammals. Despite their macrophage-like morphology and perivascular location, zebrafish FGPs appear molecularly most similar to lymphatic endothelium, and our imaging studies suggest that these cells emerge by differentiation from endothelium of the optic choroidal vascular plexus. Our findings provide the first report of a perivascular cell population in the brain derived from vascular endothelium.

A novel perivascular cell population in the zebrafish brain

PubMed Central

Galanternik, Marina Venero; Castranova, Daniel; Gore, Aniket V; Blewett, Nathan H; Jung, Hyun Min; Stratman, Amber N; Kirby, Martha R; Iben, James; Miller, Mayumi F; Kawakami, Koichi; Maraia, Richard J; Weinstein, Brant M

2017-01-01

The blood-brain barrier is essential for the proper homeostasis and function of the CNS, but its mechanism of function is poorly understood. Perivascular cells surrounding brain blood vessels are thought to be important for blood-brain barrier establishment, but their roles are not well defined. Here, we describe a novel perivascular cell population closely associated with blood vessels on the zebrafish brain. Based on similarities in their morphology, location, and scavenger behavior, these cells appear to be the zebrafish equivalent of cells variably characterized as Fluorescent Granular Perithelial cells (FGPs), perivascular macrophages, or ‘Mato Cells’ in mammals. Despite their macrophage-like morphology and perivascular location, zebrafish FGPs appear molecularly most similar to lymphatic endothelium, and our imaging studies suggest that these cells emerge by differentiation from endothelium of the optic choroidal vascular plexus. Our findings provide the first report of a perivascular cell population in the brain derived from vascular endothelium. DOI: http://dx.doi.org/10.7554/eLife.24369.001 PMID:28395729

Regenerative response following stab injury in the adult zebrafish telencephalon.

PubMed

März, Martin; Schmidt, Rebecca; Rastegar, Sepand; Strähle, Uwe

2011-09-01

In contrast to mammals, the brain of the adult zebrafish has a remarkable ability to regenerate. In mammals, injuries induce proliferation of astrocytes and oligodendrocyte progenitors contributing to the formation of a glial scar. We analyzed the proliferation of glial cells and microglia in response to stab injury in the adult zebrafish telencephalon: Radial glial markers were up-regulated at the ventricle and co-expressed the proliferation nuclear antigen (PCNA). Microglia and oligodendrocyte progenitors accumulated transiently at the site of lesion. However, we could not find evidence of permanent scar formation. Parenchymal proliferation was almost negligible in comparison to the increase in proliferation at the ventricular zone. This suggests that most of the cellular material for regeneration is derived from regions of constitutive neurogenesis. Remarkably, the proliferative response is almost completely restricted to the lesioned hemisphere indicating that signals inducing regeneration remain mainly confined within the lesioned half of the telencephalon. Copyright © 2011 Wiley-Liss, Inc.

Enantio-alteration of gene transcription associated with bioconcentration in adult zebrafish (Danio rerio) exposed to chiral PCB149

NASA Astrophysics Data System (ADS)

Chai, Tingting; Cui, Feng; Mu, Pengqian; Yang, Yang; Xu, Nana; Yin, Zhiqiang; Jia, Qi; Yang, Shuming; Qiu, Jing; Wang, Chengju

2016-01-01

Enantioselective enrichment of chiral PCB149 (2,2’,3,4’,5’,6-hexachlorobiphenyl) was analysed in adult zebrafish (Danio rerio) exposed to the racemate, (-)-PCB149, and (+)-PCB149. Greater enrichment of (-)-PCB149 compared to (+) PCB149 was observed following 0.5 ng/L exposure; however, as the exposure time and concentration increased, racemic enrichment was observed in adult fish exposed to the racemate. No biotransformation between the two isomers was observed in fish exposed to single enantiomers. When zebrafish were exposed to different forms of chiral PCB149, enantioselective expression of genes associated with polychlorinated biphenyls (PCBs) was observed in brain and liver tissues and enantioselective correlations between bioconcentration and target gene expression levels were observed in brain and liver tissues. The strong positive correlations between expression levels of target genes (alox5a and alox12) and PCB149 bioconcentration suggest that prolonged exposure to the racemate of chiral PCB149 may result in inflammation-associated diseases. Prolonged exposure to (-)-PCB149 may also affect metabolic pathways such as dehydrogenation and methylation in the brain tissues of adult zebrafish. Hepatic expression levels of genes related to the antioxidant system were significantly negatively correlated with bioconcentration following exposure to (+)-PCB149.

A Novel Model of Traumatic Brain Injury in Adult Zebrafish Demonstrates Response to Injury and Treatment Comparable with Mammalian Models.

PubMed

McCutcheon, Victoria; Park, Eugene; Liu, Elaine; Sobhebidari, Pooya; Tavakkoli, Jahan; Wen, Xiao-Yan; Baker, Andrew J

2017-04-01

Traumatic brain injury (TBI) is a leading cause of death and morbidity in industrialized countries with considerable associated health care costs. The cost and time associated with pre-clinical development of TBI therapeutics is lengthy and expensive with a poor track record of successful translation to the clinic. The zebrafish is an emerging model organism in research with unique technical and genomic strengths in the study of disease and development. Its high degree of genetic homology and cell signaling pathways relative to mammalian species and amenability to high and medium throughput assays has potential to accelerate the rate of therapeutic drug identification. Accordingly, we developed a novel closed-head model of TBI in adult zebrafish using a targeted, pulsed, high-intensity focused ultrasound (pHIFU) to induce mechanical injury of the brain. Western blot results indicated altered microtubule and neurofilament expression as well as increased expression of cleaved caspase-3 and beta APP (β-APP; p < 0.05). We used automated behavioral tracking software to evaluate locomotor deficits 24 and 48 h post-injury. Significant behavioral impairment included decreased swim distance and velocity (p < 0.05), as well as heightened anxiety and altered group social dynamics. Responses to injury were pHIFU dose-dependent and modifiable with MK-801, MDL-28170, or temperature modulation. Together, results indicate that the zebrafish exhibits responses to injury and intervention similar to mammalian TBI pathophysiology and suggest the potential for use to rapidly evaluate therapeutic compounds with high efficiency.

An assay for lateral line regeneration in adult zebrafish.

PubMed

Pisano, Gina C; Mason, Samantha M; Dhliwayo, Nyembezi; Intine, Robert V; Sarras, Michael P

2014-04-08

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al. that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish

PubMed Central

Hui, Subhra Prakash; Nag, Tapas Chandra; Ghosh, Sukla

2015-01-01

Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2+ neural progenitor, A2B5+ astrocyte/ glial progenitor, NG2+ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration. PMID:26630262

A Dynamic Anesthesia System for Long-Term Imaging in Adult Zebrafish

PubMed Central

Wynd, Brenen M.; Watson, Claire J.; Patil, Karuna; Sanders, George E.

2017-01-01

Abstract Long-term in vivo imaging in adult zebrafish (i.e., 1–24 h) has been limited by the fact that regimens for long-term anesthesia in embryos and larvae are ineffective in adults. Here, we examined the potential for dynamic administration of benzocaine to enable long-term anesthesia in adult zebrafish. We developed a computer-controlled perfusion system comprised of programmable peristaltic pumps that enabled automatic exchange between anesthetic and system water. Continuous administration of benzocaine in adult zebrafish resulted in a mean time to respiratory arrest of 5.0 h and 8-h survival of 14.3%. We measured characteristic sedation and recovery times in response to benzocaine, and used them to devise an intermittent dosing regimen consisting of 14.5 min of benzocaine followed by 5.5 min of system water. Intermittent benzocaine administration in adult zebrafish resulted in a mean time to respiratory arrest of 7.6 h and 8-h survival of 71.4%. Finally, we performed a single 24-h trial and found that intermittent dosing maintained anesthesia in an adult zebrafish over the entire 24-h period. In summary, our studies demonstrate the potential for dynamic administration of benzocaine to enable prolonged anesthesia in adult zebrafish, expanding the potential for imaging in adult physiologies that unfold over 1–24 h. PMID:27409411

A Dynamic Anesthesia System for Long-Term Imaging in Adult Zebrafish.

PubMed

Wynd, Brenen M; Watson, Claire J; Patil, Karuna; Sanders, George E; Kwon, Ronald Y

2017-02-01

Long-term in vivo imaging in adult zebrafish (i.e., 1-24 h) has been limited by the fact that regimens for long-term anesthesia in embryos and larvae are ineffective in adults. Here, we examined the potential for dynamic administration of benzocaine to enable long-term anesthesia in adult zebrafish. We developed a computer-controlled perfusion system comprised of programmable peristaltic pumps that enabled automatic exchange between anesthetic and system water. Continuous administration of benzocaine in adult zebrafish resulted in a mean time to respiratory arrest of 5.0 h and 8-h survival of 14.3%. We measured characteristic sedation and recovery times in response to benzocaine, and used them to devise an intermittent dosing regimen consisting of 14.5 min of benzocaine followed by 5.5 min of system water. Intermittent benzocaine administration in adult zebrafish resulted in a mean time to respiratory arrest of 7.6 h and 8-h survival of 71.4%. Finally, we performed a single 24-h trial and found that intermittent dosing maintained anesthesia in an adult zebrafish over the entire 24-h period. In summary, our studies demonstrate the potential for dynamic administration of benzocaine to enable prolonged anesthesia in adult zebrafish, expanding the potential for imaging in adult physiologies that unfold over 1-24 h.

What is the Thalamus in Zebrafish?

PubMed Central

Mueller, Thomas

2012-01-01

Current research on the thalamus and related structures in the zebrafish diencephalon identifies an increasing number of both neurological structures and ontogenetic processes as evolutionary conserved between teleosts and mammals. The patterning processes, for example, which during the embryonic development of zebrafish form the thalamus proper appear largely conserved. Yet also striking differences between zebrafish and other vertebrates have been observed, particularly when we look at mature and histologically differentiated brains. A case in point is the migrated preglomerular complex of zebrafish which evolved only within the lineage of ray-finned fish and has no counterpart in mammals or tetrapod vertebrates. Based on its function as a sensory relay station with projections to pallial zones, the preglomerular complex has been compared to specific thalamic nuclei in mammals. However, no thalamic projections to the zebrafish dorsal pallium, which corresponds topologically to the mammalian isocortex, have been identified. Merely one teleostean thalamic nucleus proper, the auditory nucleus, projects to a part of the dorsal telencephalon, the pallial amygdala. Studies on patterning mechanisms identify a rostral and caudal domain in the embryonic thalamus proper. In both, teleosts and mammals, the rostral domain gives rise to GABAergic neurons, whereas glutamatergic neurons originate in the caudal domain of the zebrafish thalamus. The distribution of GABAergic derivatives in the adult zebrafish brain, furthermore, revealed previously overlooked thalamic nuclei and redefined already established ones. These findings require some reconsideration regarding the topological origin of these adult structures. In what follows, I discuss how evolutionary conserved and newly acquired features of the developing and adult zebrafish thalamus can be compared to the mammalian situation. PMID:22586363

Distinct structure and activity of monoamine oxidase in the brain of zebrafish (Danio rerio).

PubMed

Anichtchik, Oleg; Sallinen, Ville; Peitsaro, Nina; Panula, Pertti

2006-10-10

Monoamine oxidase (MAO) is a mitochondrial flavoprotein involved in the metabolism of, e.g., aminergic neurotransmitters and the parkinsonism-inducing neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). We have reported earlier MPTP-related alterations of brain catecholaminergic system in zebrafish (Danio rerio) brain. Here we describe the structural and functional properties of zebrafish MAO and the distribution of MAO mRNA and activity in zebrafish brain. The gene is located in chromosome 9 and consists of 15 exons. The amino acid composition of the active center resembles both human MAO-A and MAO-B. The enzyme displayed the highest substrate specificity for tyramine, followed by serotonin, phenylethylamine, MPTP, and dopamine; isoform-specific antagonists blocked the activity of the enzyme with equal potency. Zebrafish MAO mRNA, which was present in several tissues, and enzyme displayed differential distribution in the brain; dopaminergic cell clusters had low to moderate levels of MAO activity, whereas the highest levels of MAO activity were detected in noradrenergic and serotonergic cell groups and the habenulointerpeduncular pathway, including its caudal projection to the medial ventral rhombencephalon. The results of this study confirm the presence of functionally active MAO in zebrafish brain and other tissues and characterize the neural systems that express MAO and areas of intense activity in the brain. They also suggest that MPTP toxicity not related to MAO may affect the zebrafish brain.

Effects of EGCG and Chlorpyrifos on the Mortality, AChE and GSH of Adult Zebrafish: Independent and Combination

NASA Astrophysics Data System (ADS)

Zhang, Rong; Zhang, Jian; Gao, Qian; Guo, Nichun

2018-01-01

Chlorpyrifos is a neurotoxic agent and also causes oxidative stress in the body. EGCG is a typical strong antioxidant and has been reported to be neuroprotective. Our study investigated the mortality, the activity of acetylcholinesterase (AChE) in the brain and glutathione (GSH) in the liver of the adult Zebrafish in present of Chlorpyrifos and EGCG independent and combination. The results indicated that after the addition of EGCG, the mortality of zebrafish induced by Chlorpyrifos was reduced and the activity of AChE and glutathione (GSH) inhibited by Chlorpyrifos in zebrafish was significantly increased, which demonstrated that EGCG inhibited the toxicity Chlorpyrifos to zebrafish. The inhibition was dependent on the concentration of EGCG and Chlorpyrifos, which was not shown a gradual change trend but a complex situation.

4-dimensional functional profiling in the convulsant-treated larval zebrafish brain.

PubMed

Winter, Matthew J; Windell, Dylan; Metz, Jeremy; Matthews, Peter; Pinion, Joe; Brown, Jonathan T; Hetheridge, Malcolm J; Ball, Jonathan S; Owen, Stewart F; Redfern, Will S; Moger, Julian; Randall, Andrew D; Tyler, Charles R

2017-07-26

Functional neuroimaging, using genetically-encoded Ca 2+ sensors in larval zebrafish, offers a powerful combination of high spatiotemporal resolution and higher vertebrate relevance for quantitative neuropharmacological profiling. Here we use zebrafish larvae with pan-neuronal expression of GCaMP6s, combined with light sheet microscopy and a novel image processing pipeline, for the 4D profiling of chemoconvulsant action in multiple brain regions. In untreated larvae, regions associated with autonomic functionality, sensory processing and stress-responsiveness, consistently exhibited elevated spontaneous activity. The application of drugs targeting different convulsant mechanisms (4-Aminopyridine, Pentylenetetrazole, Pilocarpine and Strychnine) resulted in distinct spatiotemporal patterns of activity. These activity patterns showed some interesting parallels with what is known of the distribution of their respective molecular targets, but crucially also revealed system-wide neural circuit responses to stimulation or suppression. Drug concentration-response curves of neural activity were identified in a number of anatomically-defined zebrafish brain regions, and in vivo larval electrophysiology, also conducted in 4dpf larvae, provided additional measures of neural activity. Our quantification of network-wide chemoconvulsant drug activity in the whole zebrafish brain illustrates the power of this approach for neuropharmacological profiling in applications ranging from accelerating studies of drug safety and efficacy, to identifying pharmacologically-altered networks in zebrafish models of human neurological disorders.

High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

PubMed Central

Blackburn, Jessica S; Liu, Sali; Raimondi, Aubrey R; Ignatius, Myron S; Salthouse, Christopher D; Langenau, David M

2011-01-01

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis. PMID:21293462

Short-term developmental effects and potential mechanisms of azoxystrobin in larval and adult zebrafish (Danio rerio).

PubMed

Cao, Fangjie; Wu, Peizhuo; Huang, Lan; Li, Hui; Qian, Le; Pang, Sen; Qiu, Lihong

2018-05-01

Previous study indicated that azoxystrobin had high acute toxicity to zebrafish, and larval zebrafish were more sensitive to azoxystrobin than adult zebrafish. The objective of the present study was to investigate short-term developmental effects and potential mechanisms of azoxystrobin in larval and adult zebrafish. After zebrafish embryos and adults were exposed to 0.01, 0.05 and 0.20 mg/L azoxystrobin (equal to 25, 124 and 496 nM azoxystrobin, respectively) for 8 days, the lethal effect, physiological responses, liver histology, mitochondrial ultrastructure, and expression alteration of genes related to mitochondrial respiration, oxidative stress, cell apoptosis and innate immune response were determined. The results showed that there was no significant effect on larval and adult zebrafish after exposure to 0.01 mg/L azoxystrobin. However, increased ROS, MDA concentration and il1b in larval zebrafish, as well as increased il1b, il8 and cxcl-c1c in adult zebrafish were induced after exposure to 0.05 mg/L azoxystrobin. Reduced mitochondrial complex III activity and ATP concentration, increased SOD activity, ROS and MDA concentration, decreased cytb, as well as increased sod1, sod2, cat, il1b, il8 and cxcl-c1c were observed both in larval and adult zebrafish after exposure to 0.20 mg/L azoxystrobin; meanwhile, increased p53, bax, apaf1 and casp9, alteration of liver histology and mitochondrial ultrastructure in larval zebrafish, and alteration of mitochondrial ultrastructure in adult zebrafish were also induced. The results demonstrated that azoxytrobin induced short-term developmental effects on larval zebrafish and adult zebrafish, including mitochondrial dysfunction, oxidative stress, cell apoptosis and innate immune response. Statistical analysis indicated that azoxystrobin induced more negative effects on larval zebrafish, which might be the reason for the differences of developmental toxicity between larval and adult zebrafish caused by

Embryonic exposure to benzo(a)pyrene inhibits reproductive capability in adult female zebrafish and correlation with DNA methylation.

PubMed

Gao, Dongxu; Lin, Jing; Ou, Kunlin; Chen, Ying; Li, Hongbin; Dai, Qinhua; Yu, Zhenni; Zuo, Zhenghong; Wang, Chonggang

2018-05-09

This study was conducted to investigate the effects of embryonic short-term exposure to benzo(a)pyrene (BaP), a model polycyclic aromatic hydrocarbon, on ovarian development and reproductive capability in adult female zebrafish. In 1-year-old fish after embryonic exposure to BaP for 96 h, the gonadosomatic indices and the percentage of mature oocytes were significantly decreased in the 0.5, 5 and 50 nmol/L treatments. The spawned egg number, the fertilization rate and the hatching success were significantly reduced, while the malformation rate of the F1 unexposed larvae were increased. The mRNA levels of follicle-stimulating hormone, luteinizing hormone, ovarian cytochrome P450 aromatase cyp19a1a and cyp19b, estrogen receptor esr1 and esr2, and hepatic vitellogenin vtg1 and vtg2 genes, were down-regulated in adult female zebrafish that were exposed to BaP during embryonic stage. Both 17β-estradiol and testosterone levels were reduced in the ovary of adult females. The methylation levels of the gonadotropin releasing hormone (GnRH) gene gnrh3 were significantly increased in the adult zebrafish brain, and those of the GnRH receptor gene gnrhr3 were elevated both in the larvae exposed to BaP and in the adult brain, which might cause the down-regulation of the mRNA levels of gnrh3 and gnrhr3. This epigenetic change caused by embryonic exposure to BaP might be a reason for physiological changes along the brain-pituitary-gonad axis. These results suggest that short-term exposure in early life should be included and evaluated in any risk assessment of pollutant exposure to the reproductive health of fish. Copyright © 2018 Elsevier Ltd. All rights reserved.

Probiotic modulation of the microbiota-gut-brain axis and behaviour in zebrafish.

PubMed

Borrelli, Luca; Aceto, Serena; Agnisola, Claudio; De Paolo, Sofia; Dipineto, Ludovico; Stilling, Roman M; Dinan, Timothy G; Cryan, John F; Menna, Lucia F; Fioretti, Alessandro

2016-07-15

The gut microbiota plays a crucial role in the bi-directional gut-brain axis, a communication that integrates the gut and central nervous system (CNS) activities. Animal studies reveal that gut bacteria influence behaviour, Brain-Derived Neurotrophic Factor (BDNF) levels and serotonin metabolism. In the present study, we report for the first time an analysis of the microbiota-gut-brain axis in zebrafish (Danio rerio). After 28 days of dietary administration with the probiotic Lactobacillus rhamnosus IMC 501, we found differences in shoaling behaviour, brain expression levels of bdnf and of genes involved in serotonin signalling/metabolism between control and treated zebrafish group. In addition, in microbiota we found a significant increase of Firmicutes and a trending reduction of Proteobacteria. This study demonstrates that selected microbes can be used to modulate endogenous neuroactive molecules in zebrafish.

Expression and characterization of a brain-specific protein kinase BSK146 from zebrafish.

PubMed

Chou, Chih-Ming; Chen, Yi-Chung; Lee, Ming-Ting; Chen, Gen-Der; Lu, I-Ching; Chen, Shui-Tsung; Huang, Chang-Jen

2006-02-17

We have previously identified a novel protein kinase, pk146, in the brain of Tetraodon. In the present study, we cloned the homologous protein kinase gene encoding a protein of 385 amino acid residues from zebrafish. The overall amino acid sequence and the kinase domain of zebrafish BSK146 shows 48% and 69% identity to that of rat sbk, a SH3-containing serine/threonine protein kinase. By whole-mount in situ hybridization and RT-PCR, the expression of bsk146 mRNA was mainly in the brain. To explore the in vivo function of BSK146 during zebrafish development, we used morpholino knockdown approach and found that BSK146 morphants displayed enlarged hindbrain ventricle and smaller eyes. Whole-mount in situ hybridization was further performed to analyze the brain defects in BSK146-MO-injected embryos. The expression of brain-specific markers, such as otx2, pax2.1, and krox20, was found normal in morphant embryos at 24hpf, while expression of pax2.1 exerted changes in midbrain-hindbrain boundary and hindbrain in morphant embryos at 48hpf. These data suggest that BSK146 may play an important role in later ventricle expansion in zebrafish brain development. Although the recombinant BSK146 protein produced in insect cells was active and could phosphorylate both histone H1 and histone 2B, the endogenous substrate of BSK146 in the embryonic brain of zebrafish is not clear at the present time and needs further investigation.

Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development.

PubMed

van Lessen, Max; Shibata-Germanos, Shannon; van Impel, Andreas; Hawkins, Thomas A; Rihel, Jason; Schulte-Merker, Stefan

2017-05-12

The lymphatic system controls fluid homeostasis and the clearance of macromolecules from interstitial compartments. In mammals brain lymphatics were only recently discovered, with significant implications for physiology and disease. We examined zebrafish for the presence of brain lymphatics and found loosely connected endothelial cells with lymphatic molecular signature covering parts of the brain without forming endothelial tubular structures. These brain lymphatic endothelial cells (BLECs) derive from venous endothelium, are distinct from macrophages, and are sensitive to loss of Vegfc. BLECs endocytose macromolecules in a selective manner, which can be blocked by injection of mannose receptor ligands. This first report on brain lymphatic endothelial cells in a vertebrate embryo identifies cells with unique features, including the uptake of macromolecules at a single cell level. Future studies will address whether this represents an uptake mechanism that is conserved in mammals and how these cells affect functions of the embryonic and adult brain.

Whole-body and multispectral photoacoustic imaging of adult zebrafish

NASA Astrophysics Data System (ADS)

Huang, Na; Xi, Lei

2016-10-01

Zebrafish is a top vertebrate model to study developmental biology and genetics, and it is becoming increasingly popular for studying human diseases due to its high genome similarity to that of humans and the optical transparency in embryonic stages. However, it becomes difficult for pure optical imaging techniques to volumetric visualize the internal organs and structures of wild-type zebrafish in juvenile and adult stages with excellent resolution and penetration depth. Even with the establishment of mutant lines which remain transparent over the life cycle, it is still a challenge for pure optical imaging modalities to image the whole body of adult zebrafish with micro-scale resolution. However, the method called photoacoustic imaging that combines all the advantages of the optical imaging and ultrasonic imaging provides a new way to image the whole body of the zebrafish. In this work, we developed a non-invasive photoacoustic imaging system with optimized near-infrared illumination and cylindrical scanning to image the zebrafish. The lateral and axial resolution yield to 80 μm and 600 μm, respectively. Multispectral strategy with wavelengths from 690 nm to 930 nm was employed to image various organs inside the zebrafish. From the reconstructed images, most major organs and structures inside the body can be precisely imaged. Quantitative and statistical analysis of absorption for organs under illumination with different wavelengths were carried out.

Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development

PubMed Central

van Lessen, Max; Shibata-Germanos, Shannon; van Impel, Andreas; Hawkins, Thomas A; Rihel, Jason; Schulte-Merker, Stefan

2017-01-01

The lymphatic system controls fluid homeostasis and the clearance of macromolecules from interstitial compartments. In mammals brain lymphatics were only recently discovered, with significant implications for physiology and disease. We examined zebrafish for the presence of brain lymphatics and found loosely connected endothelial cells with lymphatic molecular signature covering parts of the brain without forming endothelial tubular structures. These brain lymphatic endothelial cells (BLECs) derive from venous endothelium, are distinct from macrophages, and are sensitive to loss of Vegfc. BLECs endocytose macromolecules in a selective manner, which can be blocked by injection of mannose receptor ligands. This first report on brain lymphatic endothelial cells in a vertebrate embryo identifies cells with unique features, including the uptake of macromolecules at a single cell level. Future studies will address whether this represents an uptake mechanism that is conserved in mammals and how these cells affect functions of the embryonic and adult brain. DOI: http://dx.doi.org/10.7554/eLife.25932.001 PMID:28498105

Methods for studying the zebrafish brain: past, present and future.

PubMed

Wyatt, Cameron; Bartoszek, Ewelina M; Yaksi, Emre

2015-07-01

The zebrafish (Danio rerio) is one of the most promising new model organisms. The increasing popularity of this amazing small vertebrate is evident from the exponentially growing numbers of research articles, funded projects and new discoveries associated with the use of zebrafish for studying development, brain function, human diseases and screening for new drugs. Thanks to the development of novel technologies, the range of zebrafish research is constantly expanding with new tools synergistically enhancing traditional techniques. In this review we will highlight the past and present techniques which have made, and continue to make, zebrafish an attractive model organism for various fields of biology, with a specific focus on neuroscience. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Estrogenic Effects of Several BPA Analogs in the Developing Zebrafish Brain

PubMed Central

Cano-Nicolau, Joel; Vaillant, Colette; Pellegrini, Elisabeth; Charlier, Thierry D.; Kah, Olivier; Coumailleau, Pascal

2016-01-01

Important set of studies have demonstrated the endocrine disrupting activity of Bisphenol A (BPA). The present work aimed at defining estrogenic-like activity of several BPA structural analogs, including BPS, BPF, BPAF, and BPAP, on 4- or 7-day post-fertilization (dpf) zebrafish larva as an in vivo model. We measured the induction level of the estrogen-sensitive marker cyp19a1b gene (Aromatase B), expressed in the brain, using three different in situ/in vivo strategies: (1) Quantification of cyp19a1b transcripts using RT-qPCR in wild type 7-dpf larva brains exposed to bisphenols; (2) Detection and distribution of cyp19a1b transcripts using in situ hybridization on 7-dpf brain sections (hypothalamus); and (3) Quantification of the cyp19a1b promoter activity in live cyp19a1b-GFP transgenic zebrafish (EASZY assay) at 4-dpf larval stage. These three different experimental approaches demonstrated that BPS, BPF, or BPAF exposure, similarly to BPA, significantly activates the expression of the estrogenic marker in the brain of developing zebrafish. In vitro experiments using both reporter gene assay in a glial cell context and competitive ligand binding assays strongly suggested that up-regulation of cyp19a1b is largely mediated by the zebrafish estrogen nuclear receptor alpha (zfERα). Importantly, and in contrast to other tested bisphenol A analogs, the bisphenol AP (BPAP) did not show estrogenic activity in our model. PMID:27047331

Recording the adult zebrafish cerebral field potential during pentylenetetrazole seizures

PubMed Central

Pineda, Ricardo; Beattie, Christine E.; Hall, Charles W.

2017-01-01

Although the zebrafish is increasingly used as a model organism to study epilepsy, no standard electrophysiological technique for recording electrographic seizures in adult fish exists. The purpose of this paper is to introduce a readily implementable technique for recording pentylenetetrazole seizures in the adult zebrafish. We find that we can consistently record a high quality field potential over the zebrafish cerebrum using an amplification of 5000 V/V and bandpass filtering at corner frequencies of 1.6 and 16 Hz. The cerebral field potential recordings show consistent features in the baseline, pre-seizure, seizure and post-seizure time periods that can be easily recognized by visual inspection as is the case with human and rodent electroencephalogram. Furthermore, numerical analysis of the field potential at the time of seizure onset reveals an increase in the total power, bandwidth and peak frequency in the power spectrum, as is also the case with human and rodent electroencephalogram. The techniques presented herein stand to advance the utility of the adult zebrafish in the study of epilepsy by affording an equivalent to the electroencephalogram used in mammalian models and human patients. PMID:21689682

Limb regeneration is impaired in an adult zebrafish model of diabetes mellitus.

PubMed

Olsen, Ansgar S; Sarras, Michael P; Intine, Robert V

2010-01-01

The zebrafish (Danio rerio) is an established model organism for the study of developmental processes, human disease, and tissue regeneration. We report that limb regeneration is severely impaired in our newly developed adult zebrafish model of type I diabetes mellitus. Intraperitoneal streptozocin injection of adult, wild-type zebrafish results in a sustained hyperglycemic state as determined by elevated fasting blood glucose values and increased glycation of serum protein. Serum insulin levels are also decreased and pancreas immunohistochemisty revealed a decreased amount of insulin signal in hyperglycemic fish. Additionally, the diabetic complications of retinal thinning and glomerular basement membrane thickening (early signs of retinopathy and nephropathy) resulting from the hyperglycemic state were evident in streptozocin-injected fish at 3 weeks. Most significantly, limb regeneration, following caudal fin amputation, is severely impaired in diabetic zebrafish and nonspecific toxic effects outside the pancreas were not found to contribute to impaired limb regeneration. This experimental system using adult zebrafish facilitates a broad spectrum of genetic and molecular approaches to study regeneration in the diabetic background. © 2010 by the Wound Healing Society.

Satellite-like cells contribute to pax7-dependent skeletal muscle repair in adult zebrafish

PubMed Central

Berberoglu, Michael A.; Gallagher, Thomas L.; Morrow, Zachary T.; Talbot, Jared C.; Hromowyk, Kimberly J.; Tenente, Inês M.; Langenau, David M.; Amacher, Sharon L.

2017-01-01

Satellite cells, also known as muscle stem cells, are responsible for skeletal muscle growth and repair in mammals. Pax7 and Pax3 transcription factors are established satellite cell markers required for muscle development and regeneration, and there is great interest in identifying additional factors that regulate satellite cell proliferation, differentiation, and/or skeletal muscle regeneration. Due to the powerful regenerative capacity of many zebrafish tissues, even in adults, we are exploring the regenerative potential of adult zebrafish skeletal muscle. Here, we show that adult zebrafish skeletal muscle contains cells similar to mammalian satellite cells. Adult zebrafish satellite-like cells have dense heterochromatin, express Pax7 and Pax3, proliferate in response to injury, and show peak myogenic responses 4–5 days post-injury (dpi). Furthermore, using a pax7a-driven GFP reporter, we present evidence implicating satellite-like cells as a possible source of new muscle. In lieu of central nucleation, which distinguishes regenerating myofibers in mammals, we describe several characteristics that robustly identify newly-forming myofibers from surrounding fibers in injured adult zebrafish muscle. These characteristics include partially overlapping expression in satellite cells and regenerating myofibers of two RNA-binding proteins Rbfox2 and Rbfoxl1, known to regulate embryonic muscle development and function. Finally, by analyzing pax7a; pax7b double mutant zebrafish, we show that Pax7 is required for adult skeletal muscle repair, as it is in the mouse. PMID:28279710

Rapid whole brain imaging of neural activity in freely behaving larval zebrafish (Danio rerio)

PubMed Central

Shang, Chunfeng; Yang, Wenbin; Bai, Lu; Du, Jiulin

2017-01-01

The internal brain dynamics that link sensation and action are arguably better studied during natural animal behaviors. Here, we report on a novel volume imaging and 3D tracking technique that monitors whole brain neural activity in freely swimming larval zebrafish (Danio rerio). We demonstrated the capability of our system through functional imaging of neural activity during visually evoked and prey capture behaviors in larval zebrafish. PMID:28930070

Lineage tracing of dlx1a/2a and dlx5a/6a expressing cells in the developing zebrafish brain.

PubMed

Solek, Cynthia M; Feng, Shengrui; Perin, Sofia; Weinschutz Mendes, Hellen; Ekker, Marc

2017-07-01

Lineage tracing of specific populations of progenitor cells provides crucial information about developmental programs. Four members of the Dlx homeobox gene family, Dlx1,2, 5 and 6, are involved in the specification of γ-aminobutyric acid (GABA)ergic neurons in the vertebrate forebrain. Orthologous genes in mammals and teleost show similarities in expression patterns and transcriptional regulation mechanisms. We have used lineage tracing to permanently label dlx-expressing cells in the zebrafish and have characterized the progeny of these cells in the larva and in the juvenile and adult brain. We have found that dlx1a/2a and dlx5a/6a expressing progenitors give rise, for the most part, to small populations of cells which constitute only a small proportion of GABAergic cells in the adult brain tissue. Moreover, some of the cells do not acquire a neuronal phenotype suggesting that, regardless of the time a cell expresses dlx genes in the brain, it can potentially give rise to cells other than neurons. In some instances, labeling larval dlx5a/6a-expressing cells, but not dlx1a/2a-expressing cells, results in massively expanding, widespread clonal expansion throughout the adult brain. Our data provide a detailed lineage analysis of the dlx1a/2a and dlx5a/6a expressing progenitors in the zebrafish brain and lays the foundation for further characterization of the role of these transcription factors beyond the specification of GABAergic neurons. Copyright © 2017 Elsevier Inc. All rights reserved.

Benomyl induction of brain aromatase and toxic effects in the zebrafish embryo.

PubMed

Kim, Dong-Jae; Seok, Seung-Hyeok; Baek, Min-Won; Lee, Hui-Young; Na, Yi-Rang; Park, Sung-Hoon; Lee, Hyun-Kyoung; Dutta, Noton Kumar; Kawakami, Koichi; Park, Jae-Hak

2009-05-01

Benomyl is a benzimidazole fungicide that has been widely used on a variety of food crops and ornamental plants. It is known to cause adverse effects on reproductive systems, including decreased testicular and epididymal weights and reduced epididymal sperm counts and fertility. The brain aromatase gene is up-regulated by estrogens and estrogen mimics and considered a target gene to screen estrogen mimics. This study was designed to test the estrogenic potential and toxic effects of benomyl in the zebrafish system, and validated this system as a model that may correspond to the effect of benomyl in rodents. Concentrations of 20 x 10(-6), 40 x 10(-6) and 80 x 10(-6) M of benomyl-treated embryos showed decreased survival, hatching and heart rates, and increased incidence of malformations, such as pericardial edema, spinal lordosis, elongated heart, head edema, eye lens protrusion and caudal fin disappearance. Benomyl induced enhanced green fluorescent protein (EGFP) expression in the mediobasal hypothalamus (MBH) in transient zebrafish embryos with a brain aromatase-based reporter gene. In this study, we determined that benomyl has estrogenic potential based on zebrafish brain aromatase gene induction, and that benomyl is toxic at 20 x 10(-6) M concentration and higher. These results demonstrate the usefulness of zebrafish embryos as an in vivo system to examine the estrogenic and developmental toxic potential of unknown compounds.

Nickel exposure alters behavioral parameters in larval and adult zebrafish.

PubMed

Nabinger, Débora Dreher; Altenhofen, Stefani; Bitencourt, Paula Eliete Rodrigues; Nery, Laura Roesler; Leite, Carlos Eduardo; Vianna, Mônica Ryff Moreira Roca; Bonan, Carla Denise

2018-05-15

Nickel is a heavy metal that, at high concentrations, leads to environmental contamination and causes health problems. We evaluated the effects of NiCl 2 exposure on cognition and behavior in larval and adult zebrafish. Larval and adult zebrafish were exposed to NiCl 2 concentrations (0.025, 2.0, 5.0, and 15.0mg/L) or water (control) in two treatment regimens: acute and subchronic. Larvae were exposed to NiCl 2 for 2h (acute treatment: 5-day-old larvae treated for 2h, tested after treatment) or 11days (subchronic treatment: 11-day-old larvae treated since fertilization, tested at 5, 8 and 11days post-fertilization, dpf). Adults were exposed for 12h (acute treatment) or 96h (subchronic treatment) and were tested after the treatment period. In both regimens, exposed zebrafish showed concentration-dependent increases in body nickel levels compared with controls. For larvae, delayed hatching, decreased heart rate and morphological alterations were observed in subchronically treated zebrafish. Larvae from subchronic treatment tested at 5dpf decrease distance and mean speed at a low concentration (0.025mg/L) and increased at higher concentrations (5.0 and 15.0mg/L). Subchronic treated larvae decrease locomotion at 15.0mg/L at 8 and 11dpf, whereas decreased escape responses to an aversive stimulus was observed at 2.0, 5.0 and 15.0mg/L in all developmental stages. For adults, the exploratory behavior test showed that subchronic nickel exposure induced anxiogenic-like behavior and decrease aggression, whereas impaired memory was observed in both treatments. These results indicate that exposure to nickel in early life stages of zebrafish leads to morphological alterations, avoidance response impairment and locomotor deficits whereas acute and subchronic exposure in adults resulst in anxiogenic effects, impaired memory and decreased aggressive behavior. These effects may be associated to neurotoxic actions of nickel and suggest this metal may influence animals' physiology in

Localization of BDNF expression in the developing brain of zebrafish

PubMed Central

De Felice, E; Porreca, I; Alleva, E; De Girolamo, P; Ambrosino, C; Ciriaco, E; Germanà, A; Sordino, P

2014-01-01

The brain-derived neurotrophic factor (BDNF) gene is expressed in differentiating and post-mitotic neurons of the zebrafish embryo, where it has been implicated in Huntington's disease. Little is known, however, about the full complement of neuronal cell types that express BDNF in this important vertebrate model. Here, we further explored the transcriptional profiles during the first week of development using real-time quantitative polymerase chain reaction (RT-qPCR) and whole-mount in situ hybridization (WISH). RT-qPCR results revealed a high level of maternal contribution followed by a steady increase of zygotic transcription, consistent with the notion of a prominent role of BDNF in neuronal maturation and maintenance. Based on WISH, we demonstrate for the first time that BDNF expression in the developing brain of zebrafish is structure specific. Anatomical criteria and co-staining with genetic markers (shh, pax2a, emx1, krox20, lhx2b and lhx9) visualized major topological domains of BDNF-positive cells in the pallium, hypothalamus, posterior tuberculum and optic tectum. Moreover, the relative timing of BDNF transcription in the eye and tectum may illustrate a mechanism for coordinated development of the retinotectal system. Taken together, our results are compatible with a local delivery and early role of BDNF in the developing brain of zebrafish, adding basic knowledge to the study of neurotrophin functions in neural development and disease. PMID:24588510

Long-term drug administration in the adult zebrafish using oral gavage for cancer preclinical studies

PubMed Central

Dang, Michelle; Henderson, Rachel E.; Garraway, Levi A.

2016-01-01

ABSTRACT Zebrafish are a major model for chemical genetics, and most studies use embryos when investigating small molecules that cause interesting phenotypes or that can rescue disease models. Limited studies have dosed adults with small molecules by means of water-borne exposure or injection techniques. Challenges in the form of drug delivery-related trauma and anesthesia-related toxicity have excluded the adult zebrafish from long-term drug efficacy studies. Here, we introduce a novel anesthetic combination of MS-222 and isoflurane to an oral gavage technique for a non-toxic, non-invasive and long-term drug administration platform. As a proof of principle, we established drug efficacy of the FDA-approved BRAFV600E inhibitor, Vemurafenib, in adult zebrafish harboring BRAFV600E melanoma tumors. In the model, adult casper zebrafish intraperitoneally transplanted with a zebrafish melanoma cell line (ZMEL1) and exposed to daily sub-lethal dosing at 100 mg/kg of Vemurafenib for 2 weeks via oral gavage resulted in an average 65% decrease in tumor burden and a 15% mortality rate. In contrast, Vemurafenib-resistant ZMEL1 cell lines, generated in culture from low-dose drug exposure for 4 months, did not respond to the oral gavage treatment regimen. Similarly, this drug treatment regimen can be applied for treatment of primary melanoma tumors in the zebrafish. Taken together, we developed an effective long-term drug treatment system that will allow the adult zebrafish to be used to identify more effective anti-melanoma combination therapies and opens up possibilities for treating adult models of other diseases. PMID:27482819

Rhodopsin expression in the zebrafish pineal gland from larval to adult stage.

PubMed

Magnoli, Domenico; Zichichi, Rosalia; Laurà, Rosaria; Guerrera, Maria Cristina; Campo, Salvatore; de Carlos, Felix; Suárez, Alberto Álvarez; Abbate, Francesco; Ciriaco, Emilia; Vega, Jose Antonio; Germanà, Antonino

2012-03-09

The zebrafish pineal gland plays an important role in different physiological functions including the regulation of the circadian clock. In the fish pineal gland the pinealocytes are made up of different segments: outer segment, inner segment and basal pole. Particularly, in the outer segment the rhodopsin participates in the external environment light reception that represents the first biochemical step in the melatonin production. It is well known that the rhodopsin in the adult zebrafish is well expressed in the pineal gland but both the expression and the cellular localization of this protein during development remain still unclear. In this study using qRT-PCR, sequencing and immunohistochemistry the expression as well as the protein localization of the rhodopsin in the zebrafish from larval (10 dpf) to adult stage (90 dpf) were demonstrated. The rhodopsin mRNA expression presents a peak of expression at 10 dpf, a further reduction to 50 dpf before increasing again in the adult stage. Moreover, the cellular localization of the rhodopsin-like protein was always localized in the pinealocyte at all ages examined. Our results demonstrated the involvement of the rhodopsin in the zebrafish pineal gland physiology particularly in the light capture during the zebrafish lifespan. Copyright © 2012 Elsevier B.V. All rights reserved.

Advanced Echocardiography in Adult Zebrafish Reveals Delayed Recovery of Heart Function after Myocardial Cryoinjury

PubMed Central

Kossack, Mandy; Juergensen, Lonny; Fuchs, Dieter; Katus, Hugo A.; Hassel, David

2015-01-01

Translucent zebrafish larvae represent an established model to analyze genetics of cardiac development and human cardiac disease. More recently adult zebrafish are utilized to evaluate mechanisms of cardiac regeneration and by benefiting from recent genome editing technologies, including TALEN and CRISPR, adult zebrafish are emerging as a valuable in vivo model to evaluate novel disease genes and specifically validate disease causing mutations and their underlying pathomechanisms. However, methods to sensitively and non-invasively assess cardiac morphology and performance in adult zebrafish are still limited. We here present a standardized examination protocol to broadly assess cardiac performance in adult zebrafish by advancing conventional echocardiography with modern speckle-tracking analyses. This allows accurate detection of changes in cardiac performance and further enables highly sensitive assessment of regional myocardial motion and deformation in high spatio-temporal resolution. Combining conventional echocardiography measurements with radial and longitudinal velocity, displacement, strain, strain rate and myocardial wall delay rates after myocardial cryoinjury permitted to non-invasively determine injury dimensions and to longitudinally follow functional recovery during cardiac regeneration. We show that functional recovery of cryoinjured hearts occurs in three distinct phases. Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi). The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage. PMID:25853735

Production of zebrafish cardiospheres and cardiac progenitor cells in vitro and three-dimensional culture of adult zebrafish cardiac tissue in scaffolds.

PubMed

Zeng, Wendy R; Beh, Siew-Joo; Bryson-Richardson, Robert J; Doran, Pauline M

2017-09-01

The hearts of adult zebrafish (Danio rerio) are capable of complete regeneration in vivo even after major injury, making this species of particular interest for understanding the growth and differentiation processes required for cardiac tissue engineering. To date, little research has been carried out on in vitro culture of adult zebrafish cardiac cells. In this work, progenitor-rich cardiospheres suitable for cardiomyocyte differentiation and myocardial regeneration were produced from adult zebrafish hearts. The cardiospheres contained a mixed population of c-kit + and Mef2c + cells; proliferative peripheral cells of possible mesenchymal lineage were also observed. Cellular outgrowth from cardiac explants and cardiospheres was enhanced significantly using conditioned medium harvested from cultures of a rainbow trout cell line, suggesting that fish-specific trophic factors are required for zebrafish cardiac cell expansion. Three-dimensional culture of zebrafish heart cells in fibrous polyglycolic acid (PGA) scaffolds was carried out under dynamic fluid flow conditions. High levels of cell viability and cardiomyocyte differentiation were maintained within the scaffolds. Expression of cardiac troponin T, a marker of differentiated cardiomyocytes, increased during the first 7 days of scaffold culture; after 15 days, premature disintegration of the biodegradable scaffolds led to cell detachment and a decline in differentiation status. This work expands our technical capabilities for three-dimensional zebrafish cardiac cell culture with potential applications in tissue engineering, drug and toxicology screening, and ontogeny research. Biotechnol. Bioeng. 2017;114: 2142-2148. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Developmental origins of neurotransmitter and transcriptome alterations in adult female zebrafish exposed to atrazine during embryogenesis.

PubMed

Wirbisky, Sara E; Weber, Gregory J; Sepúlveda, Maria S; Xiao, Changhe; Cannon, Jason R; Freeman, Jennifer L

2015-07-03

Atrazine is an herbicide applied to agricultural crops and is indicated to be an endocrine disruptor. Atrazine is frequently found to contaminate potable water supplies above the maximum contaminant level of 3μg/L as defined by the U.S. Environmental Protection Agency. The developmental origin of adult disease hypothesis suggests that toxicant exposure during development can increase the risk of certain diseases during adulthood. However, the molecular mechanisms underlying disease progression are still unknown. In this study, zebrafish embryos were exposed to 0, 0.3, 3, or 30μg/L atrazine throughout embryogenesis. Larvae were then allowed to mature under normal laboratory conditions with no further chemical treatment until 7 days post fertilization (dpf) or adulthood and neurotransmitter analysis completed. No significant alterations in neurotransmitter levels was observed at 7dpf or in adult males, but a significant decrease in 5-hydroxyindoleacetic acid (5-HIAA) and serotonin turnover was seen in adult female brain tissue. Transcriptomic analysis was completed on adult female brain tissue to identify molecular pathways underlying the observed neurological alterations. Altered expression of 1928, 89, and 435 genes in the females exposed to 0.3, 3, or 30μg/L atrazine during embryogenesis were identified, respectively. There was a high level of overlap between the biological processes and molecular pathways in which the altered genes were associated. Moreover, a subset of genes was down regulated throughout the serotonergic pathway. These results provide support of the developmental origins of neurological alterations observed in adult female zebrafish exposed to atrazine during embryogenesis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Expression and activity profiling of the steroidogenic enzymes of glucocorticoid biosynthesis and the fdx1 co-factors in zebrafish.

PubMed

Weger, M; Diotel, N; Weger, B D; Beil, T; Zaucker, A; Eachus, H L; Oakes, J A; do Rego, J L; Storbeck, K-H; Gut, P; Strähle, U; Rastegar, S; Müller, F; Krone, N

2018-04-01

The spatial and temporal expression of steroidogenic genes in zebrafish has not been fully characterised. Because zebrafish are increasingly employed in endocrine and stress research, a better characterisation of steroidogenic pathways is required to target specific steps in the biosynthetic pathways. In the present study, we have systematically defined the temporal and spatial expression of steroidogenic enzymes involved in glucocorticoid biosynthesis (cyp21a2, cyp11c1, cyp11a1, cyp11a2, cyp17a1, cyp17a2, hsd3b1, hsd3b2), as well as the mitochondrial electron-providing ferredoxin co-factors (fdx1, fdx1b), during zebrafish development. Our studies showed an early expression of all these genes during embryogenesis. In larvae, expression of cyp11a2, cyp11c1, cyp17a2, cyp21a2, hsd3b1 and fdx1b can be detected in the interrenal gland, which is the zebrafish counterpart of the mammalian adrenal gland, whereas the fdx1 transcript is mainly found in the digestive system. Gene expression studies using quantitative reverse transcriptase-PCR and whole-mount in situ hybridisation in the adult zebrafish brain revealed a wide expression of these genes throughout the encephalon, including neurogenic regions. Using ultra-high-performance liquid chromatography tandem mass spectrometry, we were able to demonstrate the presence of the glucocorticoid cortisol in the adult zebrafish brain. Moreover, we demonstrate de novo biosynthesis of cortisol and the neurosteroid tetrahydrodeoxycorticosterone in the adult zebrafish brain from radiolabelled pregnenolone. Taken together, the present study comprises a comprehensive characterisation of the steroidogenic genes and the fdx co-factors facilitating glucocorticoid biosynthesis in zebrafish. Furthermore, we provide additional evidence of de novo neurosteroid biosynthesising in the brain of adult zebrafish facilitated by enzymes involved in glucocorticoid biosynthesis. Our study provides a valuable source for establishing the zebrafish as a

Oral dosing in adult zebrafish: proof-of-concept using pharmacokinetics and pharmacological evaluation of carbamazepine.

PubMed

Kulkarni, Pushkar; Chaudhari, Girish Hari; Sripuram, Vijaykumar; Banote, Rakesh Kumar; Kirla, Krishna Tulasi; Sultana, Razia; Rao, Pallavi; Oruganti, Srinivas; Chatti, Kiranam

2014-02-01

We describe a method for obtaining pharmacokinetics (PK) and pharmacology data from adult zebrafish in terms of mg/kg using a novel method of oral administration. Using carbamazepine (CBZ) as a test drug, we employed dried blood spot (DBS) cards to enable drug quantification for PK; and we evaluated the pharmacological anxiolytic effect using novel tank test. The PK study confirmed the presence of CBZ in both blood and brain and the behavioural study showed dose dependent anxiolytic effect. The reproducibility of oral dosing was confirmed by the fact that the results obtained in both the experiments had negligible errors. This report enables a novel approach for optimizing the utility of zebrafish in drug discovery and drug delivery research. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Localization of BDNF expression in the developing brain of zebrafish.

PubMed

De Felice, E; Porreca, I; Alleva, E; De Girolamo, P; Ambrosino, C; Ciriaco, E; Germanà, A; Sordino, P

2014-05-01

The brain-derived neurotrophic factor (BDNF) gene is expressed in differentiating and post-mitotic neurons of the zebrafish embryo, where it has been implicated in Huntington's disease. Little is known, however, about the full complement of neuronal cell types that express BDNF in this important vertebrate model. Here, we further explored the transcriptional profiles during the first week of development using real-time quantitative polymerase chain reaction (RT-qPCR) and whole-mount in situ hybridization (WISH). RT-qPCR results revealed a high level of maternal contribution followed by a steady increase of zygotic transcription, consistent with the notion of a prominent role of BDNF in neuronal maturation and maintenance. Based on WISH, we demonstrate for the first time that BDNF expression in the developing brain of zebrafish is structure specific. Anatomical criteria and co-staining with genetic markers (shh, pax2a, emx1, krox20, lhx2b and lhx9) visualized major topological domains of BDNF-positive cells in the pallium, hypothalamus, posterior tuberculum and optic tectum. Moreover, the relative timing of BDNF transcription in the eye and tectum may illustrate a mechanism for coordinated development of the retinotectal system. Taken together, our results are compatible with a local delivery and early role of BDNF in the developing brain of zebrafish, adding basic knowledge to the study of neurotrophin functions in neural development and disease. © 2014 Anatomical Society.

Whole-brain activity mapping onto a zebrafish brain atlas.

PubMed

Randlett, Owen; Wee, Caroline L; Naumann, Eva A; Nnaemeka, Onyeka; Schoppik, David; Fitzgerald, James E; Portugues, Ruben; Lacoste, Alix M B; Riegler, Clemens; Engert, Florian; Schier, Alexander F

2015-11-01

In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system. Using brain registration across hundreds of larval zebrafish, we have built an expandable open-source atlas containing molecular labels and definitions of anatomical regions, the Z-Brain. Using this platform and immunohistochemical detection of phosphorylated extracellular signal–regulated kinase (ERK) as a readout of neural activity, we have developed a system to create and contextualize whole-brain maps of stimulus- and behavior-dependent neural activity. This mitogen-activated protein kinase (MAP)-mapping assay is technically simple, and data analysis is completely automated. Because MAP-mapping is performed on freely swimming fish, it is applicable to studies of nearly any stimulus or behavior. Here we demonstrate our high-throughput approach using pharmacological, visual and noxious stimuli, as well as hunting and feeding. The resultant maps outline hundreds of areas associated with behaviors.

Whole-brain activity mapping onto a zebrafish brain atlas

PubMed Central

Randlett, Owen; Wee, Caroline L.; Naumann, Eva A.; Nnaemeka, Onyeka; Schoppik, David; Fitzgerald, James E.; Portugues, Ruben; Lacoste, Alix M.B.; Riegler, Clemens; Engert, Florian; Schier, Alexander F.

2015-01-01

In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system. Using brain registration across hundreds of larval zebrafish, we have built an expandable open source atlas containing molecular labels and anatomical region definitions, the Z-Brain. Using this platform and immunohistochemical detection of phosphorylated-Extracellular signal-regulated kinase (ERK/MAPK) as a readout of neural activity, we have developed a system to create and contextualize whole brain maps of stimulus- and behavior-dependent neural activity. This MAP-Mapping (Mitogen Activated Protein kinase – Mapping) assay is technically simple, fast, inexpensive, and data analysis is completely automated. Since MAP-Mapping is performed on fish that are freely swimming, it is applicable to nearly any stimulus or behavior. We demonstrate the utility of our high-throughput approach using hunting/feeding, pharmacological, visual and noxious stimuli. The resultant maps outline hundreds of areas associated with behaviors. PMID:26778924

Breaking symmetry: the zebrafish as a model for understanding left-right asymmetry in the developing brain.

PubMed

Roussigne, Myriam; Blader, Patrick; Wilson, Stephen W

2012-03-01

How does left-right asymmetry develop in the brain and how does the resultant asymmetric circuitry impact on brain function and lateralized behaviors? By enabling scientists to address these questions at the levels of genes, neurons, circuitry and behavior,the zebrafish model system provides a route to resolve the complexity of brain lateralization. In this review, we present the progress made towards characterizing the nature of the gene networks and the sequence of morphogenetic events involved in the asymmetric development of zebrafish epithalamus. In an attempt to integrate the recent extensive knowledge into a working model and to identify the future challenges,we discuss how insights gained at a cellular/developmental level can be linked to the data obtained at a molecular/genetic level. Finally, we present some evolutionary thoughts and discuss how significant discoveries made in zebrafish should provide entry points to better understand the evolutionary origins of brain lateralization.

Increased cell proliferation and neural activity by physostigmine in the telencephalon of adult zebrafish.

PubMed

Lee, Yunkyoung; Lee, Bongkyu; Jeong, Sumin; Park, Ji-Won; Han, Inn-Oc; Lee, Chang-Joong

2016-08-26

Physostigmine, an acetylcholinesterase inhibitor, is known to affect the brain function in various aspects. This study was conducted to test whether physostigmine affects cell proliferation in the telencephalon of zebrafish. BrdU-labeled cells was prominently observed in the ventral zone of the ventral telencephalon of zebrafish. The increased number of BrdU- and proliferating cell nuclear antigen-labeled cells were shown in zebrafish treated with 200μM physostigmine, which was inhibited by pretreatment with 200μM scopolamine. iNOS mRNA expression was increased in the brain of zebrafish treated with 200μM physostigmine. Consistently, aminoguanidine, an iNOS inhibitor, attenuated the increase in the number of BrdU-labeled cells by physostigmine treatment. Zebrafish also showed seizure-like locomotor activity characterized by a rapid and abrupt movement during a 30min treatment with 200μM physostigmine. Neural activity in response to an electrical stimulus was increased in the isolated telencephalon of zebrafish continuously perfused with 200μM physostigmine. None of the number of BrdU-labeled cells, neural activity, or locomotor activity was affected by treatment with 20μM physostigmine. These results suggest that 200μM physostigmine increased neural activity and induced cell proliferation via nitric oxide production in zebrafish. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis.

PubMed

Baumgart, Emily Violette; Barbosa, Joana S; Bally-Cuif, Laure; Götz, Magdalena; Ninkovic, Jovica

2012-03-01

Reactive glia, including astroglia and oligodendrocyte progenitors (OPCs) are at the core of the reaction to injury in the mammalian brain with initially beneficial and later partially adverse functions such as scar formation. Given the different glial composition in the adult zebrafish brain with radial ependymoglia but no parenchymal astrocytes, we examined the glial response to an invasive stab wound injury model in the adult zebrafish telencephalon. Strikingly, already a few days after injury the wound was closed without any scar tissue. Similar to mammals, microglia cells reacted first and accumulated close to the injury site, while neither GFAP+ radial ependymoglia nor adult OPCs were recruited to the injury site. Moreover, OPCs failed to increase their proliferation after this injury, while the number of proliferating GFAP+ glia was increased until 7 days after injury. Importantly, neurogenesis was also increased after injury, generating additional neurons recruited to the parenchyma which survived for several months. Thus, these data suggest that the specific glial environment in the adult zebrafish telencephalon is not only permissive for long-term neuronal survival, but avoids scar formation. Invasive injury in the adult zebrafish telencephalon may therefore provide a useful model to untangle the molecular mechanisms involved in these beneficial glial reactions. Copyright © 2011 Wiley Periodicals, Inc.

Gene expression changes in female zebrafish (Danio rerio) brain in response to acute exposure to methylmercury

USGS Publications Warehouse

Richter, Catherine A.; Garcia-Reyero, Natàlia; Martyniuk, Chris; Knoebl, Iris; Pope, Marie; Wright-Osment, Maureen K.; Denslow, Nancy D.; Tillitt, Donald E.

2011-01-01

Methylmercury (MeHg) is a potent neurotoxicant and endocrine disruptor that accumulates in aquatic systems. Previous studies have shown suppression of hormone levels in both male and female fish, suggesting effects on gonadotropin regulation in the brain. The gene expression profile in adult female zebrafish whole brain induced by acute (96 h) MeHg exposure was investigated. Fish were exposed by injection to 0 or 0.5(mu or u)g MeHg/g. Gene expression changes in the brain were examined using a 22,000-feature zebrafish microarray. At a significance level of pbrain. Future studies will compare the gene expression profile induced in response to MeHg with that induced by other toxicants and will investigate responsive genes as potential biomarkers of MeHg exposure.

Gross and fine dissection of inner ear sensory epithelia in adult zebrafish (Danio rerio).

PubMed

Liang, Jin; Burgess, Shawn M

2009-05-08

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss. Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Phylostratigraphic Profiles in Zebrafish Uncover Chordate Origins of the Vertebrate Brain

PubMed Central

Šestak, Martin Sebastijan; Domazet-Lošo, Tomislav

2015-01-01

An elaborated tripartite brain is considered one of the important innovations of vertebrates. Other extant chordate groups have a more basic brain organization. For instance, cephalochordates possess a relatively simple brain possibly homologous to the vertebrate forebrain and hindbrain, whereas tunicates display the tripartite organization, but without the specialized brain centers. The difference in anatomical complexity is even more pronounced if one compares chordates with other deuterostomes that have only a diffuse nerve net or alternatively a rather simple central nervous system. To gain a new perspective on the evolutionary roots of the complex vertebrate brain, we made here a phylostratigraphic analysis of gene expression patterns in the developing zebrafish (Danio rerio). The recovered adaptive landscape revealed three important periods in the evolutionary history of the zebrafish brain. The oldest period corresponds to preadaptive events in the first metazoans and the emergence of the nervous system at the metazoan–eumetazoan transition. The origin of chordates marks the next phase, where we found the overall strongest adaptive imprint in almost all analyzed brain regions. This finding supports the idea that the vertebrate brain evolved independently of the brains within the protostome lineage. Finally, at the origin of vertebrates we detected a pronounced signal coming from the dorsal telencephalon, in agreement with classical theories that consider this part of the cerebrum a genuine vertebrate innovation. Taken together, these results reveal a stepwise adaptive history of the vertebrate brain where most of its extant organization was already present in the chordate ancestor. PMID:25415965

Optimal Anesthetic Regime for Motionless Three-Dimensional Image Acquisition During Longitudinal Studies of Adult Nonpigmented Zebrafish.

PubMed

Lockwood, Nicola; Parker, Jennifer; Wilson, Carole; Frankel, Paul

2017-04-01

With many live imaging techniques, it is crucial that a deep level of anesthesia is reached and maintained throughout image acquisition without reducing zebrafish viability. This is particularly true for three-dimensional tomographic imaging modalities. Currently, the most commonly used anesthetic in the zebrafish community, MS-222 (tricaine methanesulfonate), does not allow this. We show, using a combination of both MS-222 and isoflurane, that we can significantly improve the anesthetic regime required for motionless image acquisition of live adult zebrafish. We have benchmarked this against the requirements of our novel quantitative imaging platform, compressive sensing optical projection tomography. Using nonpigmented transgenic zebrafish, we show that a combination of 175 ppm of both anesthetics improves the maintenance of deep anesthesia for prolonged periods of time and it can be used repeatedly to enable longitudinal imaging. Importantly, it does not affect the health or viability of the adult zebrafish. We also show that nonpigmented fish, with a mutated form of the gene transparent, took significantly longer to reach deep anesthesia. The anesthetic regime presented in this study should lead to significant improvements in accuracy and information achievable from imaging live adult zebrafish and in its application to longitudinal studies.

Copper at low levels impairs memory of adult zebrafish (Danio rerio) and affects swimming performance of larvae.

PubMed

Acosta, Daiane da Silva; Danielle, Naissa Maria; Altenhofen, Stefani; Luzardo, Milene Dornelles; Costa, Patrícia Gomes; Bianchini, Adalto; Bonan, Carla Denise; da Silva, Rosane Souza; Dafre, Alcir Luiz

2016-01-01

Metal contamination at low levels is an important issue because it usually produces health and environmental effects, either positive or deleterious. Contamination of surface waters with copper (Cu) is a worldwide event, usually originated by mining, agricultural, industrial, commercial, and residential activities. Water quality criteria for Cu are variable among countries but allowed limits are generally in the μg/L range, which can disrupt several functions in the early life-stages of fish species. Behavioral and biochemical alterations after Cu exposure have also been described at concentrations close to the allowed limits. Aiming to search for the effects of Cu in the range of the allowed limits, larvae and adult zebrafish (Danio rerio) were exposed to different concentrations of dissolved Cu (nominally: 0, 5, 9, 20 and 60μg/L; measured: 0.4, 5.7, 7.2 16.6 and 42.3μg/L, respectively) for 96h. Larvae swimming and body length, and adult behavior and biochemical biomarkers (activity of glutathione-related enzymes in gills, muscle, and brain) were assessed after Cu exposure. Several effects were observed in fish exposed to 9μg/L nominal Cu, including increased larvae swimming distance and velocity, abolishment of adult inhibitory avoidance memory, and decreased glutathione S-transferase (GST) activity in gills of adult fish. At the highest Cu concentration tested (nominally: 60μg/L), body length of larvae, spatial memory of adults, and gill GST activity were decreased. Social behavior (aggressiveness and conspecific interaction), and glutathione reductase (GR) activity were not affected in adult zebrafish. Exposure to Cu, at concentrations close to the water quality criteria for this metal in fresh water, was able to alter larvae swimming performance and to induce detrimental effects on the behavior of adult zebrafish, thus indicating the need for further studies to reevaluate the currently allowed limits for Cu in fresh water. Copyright © 2016 Elsevier Inc

Amigo adhesion protein regulates development of neural circuits in zebrafish brain.

PubMed

Zhao, Xiang; Kuja-Panula, Juha; Sundvik, Maria; Chen, Yu-Chia; Aho, Vilma; Peltola, Marjaana A; Porkka-Heiskanen, Tarja; Panula, Pertti; Rauvala, Heikki

2014-07-18

The Amigo protein family consists of three transmembrane proteins characterized by six leucine-rich repeat domains and one immunoglobulin-like domain in their extracellular moieties. Previous in vitro studies have suggested a role as homophilic adhesion molecules in brain neurons, but the in vivo functions remain unknown. Here we have cloned all three zebrafish amigos and show that amigo1 is the predominant family member expressed during nervous system development in zebrafish. Knockdown of amigo1 expression using morpholino oligonucleotides impairs the formation of fasciculated tracts in early fiber scaffolds of brain. A similar defect in fiber tract development is caused by mRNA-mediated expression of the Amigo1 ectodomain that inhibits adhesion mediated by the full-length protein. Analysis of differentiated neural circuits reveals defects in the catecholaminergic system. At the behavioral level, the disturbed formation of neural circuitry is reflected in enhanced locomotor activity and in the inability of the larvae to perform normal escape responses. We suggest that Amigo1 is essential for the development of neural circuits of zebrafish, where its mechanism involves homophilic interactions within the developing fiber tracts and regulation of the Kv2.1 potassium channel to form functional neural circuitry that controls locomotion. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Phylostratigraphic profiles in zebrafish uncover chordate origins of the vertebrate brain.

PubMed

Šestak, Martin Sebastijan; Domazet-Lošo, Tomislav

2015-02-01

An elaborated tripartite brain is considered one of the important innovations of vertebrates. Other extant chordate groups have a more basic brain organization. For instance, cephalochordates possess a relatively simple brain possibly homologous to the vertebrate forebrain and hindbrain, whereas tunicates display the tripartite organization, but without the specialized brain centers. The difference in anatomical complexity is even more pronounced if one compares chordates with other deuterostomes that have only a diffuse nerve net or alternatively a rather simple central nervous system. To gain a new perspective on the evolutionary roots of the complex vertebrate brain, we made here a phylostratigraphic analysis of gene expression patterns in the developing zebrafish (Danio rerio). The recovered adaptive landscape revealed three important periods in the evolutionary history of the zebrafish brain. The oldest period corresponds to preadaptive events in the first metazoans and the emergence of the nervous system at the metazoan-eumetazoan transition. The origin of chordates marks the next phase, where we found the overall strongest adaptive imprint in almost all analyzed brain regions. This finding supports the idea that the vertebrate brain evolved independently of the brains within the protostome lineage. Finally, at the origin of vertebrates we detected a pronounced signal coming from the dorsal telencephalon, in agreement with classical theories that consider this part of the cerebrum a genuine vertebrate innovation. Taken together, these results reveal a stepwise adaptive history of the vertebrate brain where most of its extant organization was already present in the chordate ancestor. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Husbandry stress exacerbates mycobacterial infections in adult zebrafish, Danio rerio (Hamilton)

USGS Publications Warehouse

Ramsay, J.M.; Watral, Virginia G.; Schreck, C.B.; Kent, M.L.

2009-01-01

Mycobacteria are significant pathogens of laboratory zebrafish, Danio rerio (Hamilton). Stress is often implicated in clinical disease and morbidity associated with mycobacterial infections but has yet to be examined with zebrafish. The aim of this study was to examine the effects of husbandry stressors on zebrafish infected with mycobacteria. Adult zebrafish were exposed to Mycobacterium marinum or Mycobacterium chelonae, two species that have been associated with disease in zebrafish. Infected fish and controls were then subjected to chronic crowding and handling stressors and examined over an 8-week period. Whole-body cortisol was significantly elevated in stressed fish compared to non-stressed fish. Fish infected with M. marinum ATCC 927 and subjected to husbandry stressors had 14% cumulative mortality while no mortality occurred among infected fish not subjected to husbandry stressors. Stressed fish, infected with M. chelonae H1E2 from zebrafish, were 15-fold more likely to be infected than non-stressed fish at week 8 post-injection. Sub-acute, diffuse infections were more common among stressed fish infected with M. marinum or M. chelonae than non-stressed fish. This is the first study to demonstrate an effect of stress and elevated cortisol on the morbidity, prevalence, clinical disease and histological presentation associated with mycobacterial infections in zebrafish. Minimizing husbandry stress may be effective at reducing the severity of outbreaks of clinical mycobacteriosis in zebrafish facilities. ?? 2009 Blackwell Publishing Ltd.

The photoreceptive cells of the pineal gland in adult zebrafish (Danio rerio).

PubMed

Laurà, Rosaria; Magnoli, Domenico; Zichichi, Rosalia; Guerrera, Maria Cristina; De Carlos, Felix; Suárez, Alberto Álvarez; Abbate, Francesco; Ciriaco, Emilia; Vega, Jose Antonio; Germanà, Antonino

2012-03-01

The zebrafish pineal gland plays a fundamental role in the regulation of the circadian rhythm through the melatonin secretion. The pinealocytes, also called photoreceptive cells, are considered the morphofunctional unit of pineal gland. In literature, the anatomical features, the cellular characteristics, and the pinealocytes morphology of zebrafish pineal gland have not been previously described in detail. Therefore, this study was undertaken to analyze the structure and ultrastructure, as well as the immunohistochemical profile of the zebrafish pineal gland with particular reference to the pinealocytes. Here, we demonstrated, using RT-PCR, immunohistochemistry and transmission electron microscopy, the expression of the mRNA for rhodopsin in the pineal gland of zebrafish, as well as its cellular localization exclusively in the pinealocytes of adult zebrafish. Moreover, the ultrastructural observations demonstrated that the pinealocytes were constituted by an outer segment with numerous lamellar membranes, an inner segment with many mitochondria, and a basal pole with the synapses. Our results taken together demonstrated a central role of zebrafish pinealocytes in the control of pineal gland functions. Copyright © 2011 Wiley Periodicals, Inc.

ScreenCube: A 3D Printed System for Rapid and Cost-Effective Chemical Screening in Adult Zebrafish.

PubMed

Monstad-Rios, Adrian T; Watson, Claire J; Kwon, Ronald Y

2018-02-01

Phenotype-based small molecule screens in zebrafish embryos and larvae have been successful in accelerating pathway and therapeutic discovery for diverse biological processes. Yet, the application of chemical screens to adult physiologies has been relatively limited due to additional demands on cost, space, and labor associated with screens in adult animals. In this study, we present a 3D printed system and methods for intermittent drug dosing that enable rapid and cost-effective chemical administration in adult zebrafish. Using prefilled screening plates, the system enables dosing of 96 fish in ∼3 min, with a 10-fold reduction in drug quantity compared to that used in previous chemical screens in adult zebrafish. We characterize water quality kinetics during immersion in the system and use these kinetics to rationally design intermittent dosing regimens that result in 100% fish survival. As a demonstration of system fidelity, we show the potential to identify two known chemical inhibitors of adult tail fin regeneration, cyclopamine and dorsomorphin. By developing methods for rapid and cost-effective chemical administration in adult zebrafish, this study expands the potential for small molecule discovery in postembryonic models of development, disease, and regeneration.

Characterization and classification of zebrafish brain morphology mutants

PubMed Central

Lowery, Laura Anne; De Rienzo, Gianluca; Gutzman, Jennifer H.; Sive, Hazel

2010-01-01

The mechanisms by which the vertebrate brain achieves its three-dimensional structure are clearly complex, requiring the functions of many genes. Using the zebrafish as a model, we have begun to define genes required for brain morphogenesis, including brain ventricle formation, by studying 16 mutants previously identified as having embryonic brain morphology defects. We report the phenotypic characterization of these mutants at several time-points, using brain ventricle dye injection, imaging, and immunohistochemistry with neuronal markers. Most of these mutants display early phenotypes, affecting initial brain shaping, while others show later phenotypes, affecting brain ventricle expansion. In the early phenotype group, we further define four phenotypic classes and corresponding functions required for brain morphogenesis. Although we did not use known genotypes for this classification, basing it solely on phenotypes, many mutants with defects in functionally related genes clustered in a single class. In particular, class 1 mutants show midline separation defects, corresponding to epithelial junction defects; class 2 mutants show reduced brain ventricle size; class 3 mutants show midbrain-hindbrain abnormalities, corresponding to basement membrane defects; and class 4 mutants show absence of ventricle lumen inflation, corresponding to defective ion pumping. Later brain ventricle expansion requires the extracellular matrix, cardiovascular circulation, and transcription/splicing-dependent events. We suggest that these mutants define processes likely to be used during brain morphogenesis throughout the vertebrates. PMID:19051268

Morphometric analysis and neuroanatomical mapping of the zebrafish brain.

PubMed

Gupta, Tripti; Marquart, Gregory D; Horstick, Eric J; Tabor, Kathryn M; Pajevic, Sinisa; Burgess, Harold A

2018-06-21

Large-scale genomic studies have recently identified genetic variants causative for major neurodevelopmental disorders, such as intellectual disability and autism. However, determining how underlying developmental processes are affected by these mutations remains a significant challenge in the field. Zebrafish is an established model system in developmental neurogenetics that may be useful in uncovering the mechanisms of these mutations. Here we describe the use of voxel-intensity, deformation field, and volume-based morphometric techniques for the systematic and unbiased analysis of gene knock-down and environmental exposure-induced phenotypes in zebrafish. We first present a computational method for brain segmentation based on transgene expression patterns to create a comprehensive neuroanatomical map. This map allowed us to disclose statistically significant changes in brain microstructure and composition in neurodevelopmental models. We demonstrate the effectiveness of morphometric techniques in measuring changes in the relative size of neuroanatomical subdivisions in atoh7 morphant larvae and in identifying phenotypes in larvae treated with valproic acid, a chemical demonstrated to increase the risk of autism in humans. These tools enable rigorous evaluation of the effects of gene mutations and environmental exposures on neural development, providing an entry point for cellular and molecular analysis of basic developmental processes as well as neurodevelopmental and neurodegenerative disorders. Published by Elsevier Inc.

GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish.

PubMed

Marín-Juez, Rubén; Rovira, Mireia; Crespo, Diego; van der Vaart, Michiel; Spaink, Herman P; Planas, Josep V

2015-01-01

Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing.

Understanding spatio-temporal strategies of adult zebrafish exploration in the open field test.

PubMed

Stewart, Adam Michael; Gaikwad, Siddharth; Kyzar, Evan; Kalueff, Allan V

2012-04-27

Zebrafish (Danio rerio) are emerging as a useful model organism for neuroscience research. Mounting evidence suggests that various traditional rodent paradigms may be adapted for testing zebrafish behavior. The open field test is a popular rodent test of novelty exploration, recently applied to zebrafish research. To better understand fish novelty behavior, we exposed adult zebrafish to two different open field arenas for 30 min, assessing the amount and temporal patterning of their exploration. While (similar to rodents) zebrafish scale their locomotory activity depending on the size of the tank, the temporal patterning of their activity was independent of arena size. These observations strikingly parallel similar rodent behaviors, suggesting that spatio-temporal strategies of animal exploration may be evolutionarily conserved across vertebrate species. In addition, we found interesting oscillations in zebrafish exploration, with the per-minute distribution of their horizontal activity demonstrating sinusoidal-like patterns. While such patterning is not reported for rodents and other higher vertebrates, a nonlinear regression analysis confirmed the oscillation patterning of all assessed zebrafish behavioral endpoints in both open field arenas, revealing a potentially important aspect of novelty exploration in lower vertebrates. Copyright © 2012 Elsevier B.V. All rights reserved.

Reproductive toxicity of azoxystrobin to adult zebrafish (Danio rerio).

PubMed

Cao, Fangjie; Zhu, Lizhen; Li, Hui; Yu, Song; Wang, Chengju; Qiu, Lihong

2016-12-01

In the past few decades, extensive application of azoxystrobin has led to great concern regarding its adverse effects on aquatic organisms. The objective of the present study was to evaluate the reproductive toxicity of azoxystrobin to zebrafish. After adult zebrafish of both sexes were exposed to 2, 20 and 200 μg/L azoxystrobin for 21 days, egg production, the fertilization rate, the gonadosomatic index (GSI) and hepatosomatic index (HSI), 17β-estradiol (E2), testosterone (T) and vitellogenin (Vtg) concentrations, and histological alterations in the gonads and livers were measured. Meanwhile, expression alterations of genes encoding gonadotropins and gonadotropin receptors (fshb, lhb, fshr and lhr), steroid hormone receptors (era, er2b and ar), steroidogenic enzymes (cyp11a, cyp11b, cyp17, cyp19a, cyp19b, hsd3b and hsd17b) in the hypothalamic-pituitary-gonad (HPG) axis and vitellogenin (vtg1 and vtg2) in the livers were also investigated. The results showed that reduced egg production and fertilization rates were observed at 200 μg/L azoxystrobin. In female zebrafish, reduced E2 and Vtg concentrations, decreased GSI, increased T concentrations, and histological alterations in the ovaries and livers were observed at 200 μg/L azoxystrobin, along with significant down-regulation of lhb, cyp19b, lhr, cyp19a, vtg1 and vtg2, and up-regulation of cyp17, hsd3b and hsd17b. In male zebrafish, increased E2 and Vtg concentrations, reduced T concentration and GSI, and histological alterations in the testes and livers were observed after exposure to 20 and 200 μg/L azoxystrobin, along with significant up-regulations of cyp19b, cyp11a, cyp17, cyp19a, hsd3b and hsd17b, vtg1 and vtg2. Moreover, cyp11a, hsd3b, cyp19a, vtg1 and vtg2 in male zebrafish were significantly up-regulated after treatment with 2 μg/L azoxystrobin. The results of the present study indicate that azoxystrobin led to reproductive toxicity in zebrafish and male zebrafish were more sensitive to

Differential expression of neuroligin genes in the nervous system of zebrafish.

PubMed

Davey, Crystal; Tallafuss, Alexandra; Washbourne, Philip

2010-02-01

The establishment and maturation of appropriate synaptic connections is crucial in the development of neuronal circuits. Cellular adhesion is believed to play a central role in this process. Neuroligins are neuronal cell adhesion molecules that are hypothesized to act in the initial formation and maturation of synaptic connections. In order to establish the zebrafish as a model to investigate the in vivo role of Neuroligin proteins in nervous system development, we identified the zebrafish orthologs of neuroligin family members and characterized their expression. Zebrafish possess seven neuroligin genes. Synteny analysis and sequence comparisons show that NLGN2, NLGN3, and NLGN4X are duplicated in zebrafish, but NLGN1 has a single zebrafish ortholog. All seven zebrafish neuroligins are expressed in complex patterns in the developing nervous system and in the adult brain. The spatial and temporal expression patterns of these genes suggest that they occupy a role in nervous system development and maintenance.

Stable multilineage xenogeneic replacement of definitive hematopoiesis in adult zebrafish.

PubMed

Hess, Isabell; Boehm, Thomas

2016-01-18

Bony fishes are the most numerous and phenotypically diverse group of vertebrates inhabiting our planet, making them an ideal target for identifying general principles of tissue development and function. However, lack of suitable experimental platforms prevents the exploitation of this rich source of natural phenotypic variation. Here, we use a zebrafish strain lacking definitive hematopoiesis for interspecific analysis of hematopoietic cell development. Without conditioning prior to transplantation, hematopoietic progenitor cells from goldfish stably engraft in adult zebrafish homozygous for the c-myb(I181N) mutation. However, in competitive repopulation experiments, zebrafish hematopoietic cells exhibit an advantage over their goldfish counterparts, possibly owing to subtle species-specific functional differences in hematopoietic microenvironments resulting from over 100 million years of independent evolution. Thus, our unique animal model provides an unprecedented opportunity to genetically and functionally disentangle universal and species-specific contributions of the microenvironment to hematopoietic progenitor cell maintenance and development.

Exercise quantity-dependent muscle hypertrophy in adult zebrafish (Danio rerio).

PubMed

Hasumura, Takahiro; Meguro, Shinichi

2016-07-01

Exercise is very important for maintaining and increasing skeletal muscle mass, and is particularly important to prevent and care for sarcopenia and muscle disuse atrophy. However, the dose-response relationship between exercise quantity, duration/day, and overall duration and muscle mass is poorly understood. Therefore, we investigated the effect of exercise duration on skeletal muscle to reveal the relationship between exercise quantity and muscle hypertrophy in zebrafish forced to exercise. Adult male zebrafish were exercised 6 h/day for 4 weeks, 6 h/day for 2 weeks, or 3 h/day for 2 weeks. Flow velocity was adjusted to maximum velocity during continual swimming (initial 43 cm/s). High-speed consecutive photographs revealed that zebrafish mainly drove the caudal part. Additionally, X-ray micro computed tomography measurements indicated muscle hypertrophy of the mid-caudal half compared with the mid-cranial half part. The cross-sectional analysis of the mid-caudal half muscle revealed that skeletal muscle (red, white, or total) mass increased with increasing exercise quantity, whereas that of white muscle and total muscle increased only under the maximum exercise load condition of 6 h/day for 4 weeks. Additionally, the muscle fiver size distributions of exercised fish were larger than those from non-exercised fish. We revealed that exercise quantity, duration/day, and overall duration were correlated with skeletal muscle hypertrophy. The forced exercise model enabled us to investigate the relationship between exercise quantity and skeletal muscle mass. These results open up the possibility for further investigations on the effects of exercise on skeletal muscle in adult zebrafish.

Comparison of the In Vivo Biotransformation of Two Emerging Estrogenic Contaminants, BP2 and BPS, in Zebrafish Embryos and Adults

PubMed Central

Le Fol, Vincent; Brion, François; Hillenweck, Anne; Perdu, Elisabeth; Bruel, Sandrine; Aït-Aïssa, Selim; Cravedi, Jean-Pierre; Zalko, Daniel

2017-01-01

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using 3H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances. PMID:28346357

Comparison of the In Vivo Biotransformation of Two Emerging Estrogenic Contaminants, BP2 and BPS, in Zebrafish Embryos and Adults.

PubMed

Le Fol, Vincent; Brion, François; Hillenweck, Anne; Perdu, Elisabeth; Bruel, Sandrine; Aït-Aïssa, Selim; Cravedi, Jean-Pierre; Zalko, Daniel

2017-03-25

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using ³H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances.

Heterogeneity and Fgf dependence of adult neural progenitors in the zebrafish telencephalon.

PubMed

Ganz, Julia; Kaslin, Jan; Hochmann, Sarah; Freudenreich, Dorian; Brand, Michael

2010-08-15

Adult telencephalic neurogenesis is a conserved trait of all vertebrates studied. It has been investigated in detail in rodents, but very little is known about the composition of neurogenic niches and the cellular nature of progenitors in nonmammalian vertebrates. To understand the components of the progenitor zones in the adult zebrafish telencephalon and the link between glial characteristics and progenitor state, we examined whether canonical glial markers are colocalized with proliferation markers. In the adult zebrafish telencephalon, we identify heterogeneous progenitors that reside in two distinct glial domains. We find that the glial composition of the progenitor zone is linked to its proliferative behavior. Analyzing both fast-cycling proliferating cells as well as slowly cycling progenitors, we find four distinct progenitor types characterized by differential expression of glial markers. Importantly, a significant proportion of progenitors do not display typical radial glia characteristics. By blocking or activating Fgf signaling by misexpression of a dominant negative Fgf-receptor 1 or Fgf8a, respectively, we find that ventral and dorsal progenitors in the telencephalon also differ in their requirement for Fgf signaling. Together with data on the expression of Fgf signaling components in the ventricular zone of the telencephalon, this suggests that Fgf signaling directly regulates proliferation of specific subsets of adult telencephalic progenitors in vivo. Taken together our results show that adult neural progenitor cells are heterogeneous with their respect to distribution into two distinct glial domains and their dependence upon Fgf signaling as a proliferative cue in the zebrafish telencephalon.

Effects of piracetam on behavior and memory in adult zebrafish.

PubMed

Grossman, Leah; Stewart, Adam; Gaikwad, Siddharth; Utterback, Eli; Wu, Nadine; Dileo, John; Frank, Kevin; Hart, Peter; Howard, Harry; Kalueff, Allan V

2011-04-25

Piracetam, a derivative of γ-aminobutyric acid, exerts memory-enhancing and mild anxiolytic effects in human and rodent studies. To examine the drug's behavioral profile further, we assessed its effects on behavioral and endocrine (cortisol) responses of adult zebrafish (Danio rerio)--a novel model species rapidly gaining popularity in neurobehavioral research. Overall, acute piracetam did not affect zebrafish novel tank and light-dark box behavior at mild doses (25-400mg/L), but produced nonspecific behavioral inhibition at 700mg/L. No effects on cortisol levels or inter-/intra-session habituation in the novel tank test were observed for acute or chronic mild non-sedative dose of 200mg/L. In contrast, fish exposed to chronic piracetam at this dose performed significantly better in the cued learning plus-maze test. This observation parallels clinical and rodent literature on the behavioral profile of piracetam, supporting the utility of zebrafish paradigms for testing nootropic agents. Copyright © 2011 Elsevier Inc. All rights reserved.

Short-term memory in zebrafish (Danio rerio).

PubMed

Jia, Jason; Fernandes, Yohaan; Gerlai, Robert

2014-08-15

Learning and memory represent perhaps the most complex behavioral phenomena. Although their underlying mechanisms have been extensively analyzed, only a fraction of the potential molecular components have been identified. The zebrafish has been proposed as a screening tool with which mechanisms of complex brain functions may be systematically uncovered. However, as a relative newcomer in behavioral neuroscience, the zebrafish has not been well characterized for its cognitive and mnemonic features, thus learning and/or memory screens with adults have not been feasible. Here we study short-term memory of adult zebrafish. We show animated images of conspecifics (the stimulus) to the experimental subject during 1 min intervals on ten occasions separated by different (2, 4, 8 or 16 min long) inter-stimulus intervals (ISI), a between subject experimental design. We quantify the distance of the subject from the image presentation screen during each stimulus presentation interval, during each of the 1-min post-stimulus intervals immediately following the stimulus presentations and during each of the 1-min intervals furthest away from the last stimulus presentation interval and just before the next interval (pre-stimulus interval), respectively. Our results demonstrate significant retention of short-term memory even in the longest ISI group but suggest no acquisition of reference memory. Because in the employed paradigm both stimulus presentation and behavioral response quantification is computer automated, we argue that high-throughput screening for drugs or mutations that alter short-term memory performance of adult zebrafish is now becoming feasible. Copyright © 2014 Elsevier B.V. All rights reserved.

Nestin is essential for zebrafish brain and eye development through control of progenitor cell apoptosis.

PubMed

Chen, Hua-Ling; Yuh, Chiou-Hwa; Wu, Kenneth K

2010-02-19

Nestin is expressed in neural progenitor cells (NPC) of developing brain. Despite its wide use as an NPC marker, the function of nestin in embryo development is unclear. As nestin is conserved in zebrafish and its predicted sequence is clustered with the mammalian nestin orthologue, we used zebrafish as a model to investigate its role in embryogenesis. Injection of nestin morpholino (MO) into fertilized eggs induced time- and dose-dependent brain and eye developmental defects. Nestin morphants exhibited characteristic morphological changes including small head, small eyes and hydrocephalus. Histological examinations show reduced hind- and mid-brain size, dilated ventricle, poorly organized retina and underdeveloped lens. Injection of control nestin MO did not induce brain or eye changes. Nestin MO injection reduced expression of ascl1b (achaete-scute complex-like 1b), a marker of NPCs, without affecting its distribution. Nestin MO did not influence Elavl3/4 (Embryonic lethal, abnormal vision, Drosophila-like 3/4) (a neuronal marker), or otx2 (a midbrain neuronal marker), but severely perturbed cranial motor nerve development and axon distribution. To determine whether the developmental defects are due to excessive NPC apoptosis and/or reduced NPC proliferation, we analyzed apoptosis by TUNEL assay and acridine orange staining and proliferation by BrdU incorporation, pcna and mcm5 expressions. Excessive apoptosis was noted in hindbrain and midbrain cells. Apoptotic signals were colocalized with ascl1b. Proliferation markers were not significantly altered by nestin MO. These results suggest that nestin is essential for zebrafish brain and eye development probably through control of progenitor cell apoptosis.

In vivo cell tracking and quantification method in adult zebrafish

NASA Astrophysics Data System (ADS)

Zhang, Li; Alt, Clemens; Li, Pulin; White, Richard M.; Zon, Leonard I.; Wei, Xunbin; Lin, Charles P.

2012-03-01

Zebrafish have become a powerful vertebrate model organism for drug discovery, cancer and stem cell research. A recently developed transparent adult zebrafish using double pigmentation mutant, called casper, provide unparalleled imaging power in in vivo longitudinal analysis of biological processes at an anatomic resolution not readily achievable in murine or other systems. In this paper we introduce an optical method for simultaneous visualization and cell quantification, which combines the laser scanning confocal microscopy (LSCM) and the in vivo flow cytometry (IVFC). The system is designed specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish casper, under physiological conditions in the same fish over time. The confocal imaging part in this system serves the dual purposes of imaging fish tissue microstructure and a 3D navigation tool to locate a suitable vessel for circulating cell counting. The multi-color, multi-channel instrument allows the detection of multiple cell populations or different tissues or organs simultaneously. We demonstrate initial testing of this novel instrument by imaging vasculature and tracking circulating cells in CD41: GFP/Gata1: DsRed transgenic casper fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. Circulating fluorescent cell incidents were recorded and counted repeatedly over time and in different types of vessels. Great application opportunities in cancer and stem cell researches are discussed.

Craniofacial skeletal defects of adult zebrafish glypican 4 (knypek) mutants

PubMed Central

LeClair, Elizabeth E.; Mui, Stephanie R.; Huang, Angela; Topczewska, Jolanta M.; Topczewski, Jacek

2010-01-01

The heparan sulfate proteoglycan Glypican 4 (Gpc4) is part of the Wnt/planar cell polarity pathway, which is required for convergence and extension during zebrafish gastrulation. To observe Glypican 4-deficient phenotypes at later stages, we rescued gpc4−/− (knypek) homozygotes and raised them for more than one year. Adult mutants showed diverse cranial malformations of both dermal and endochondral bones, ranging from shortening of the rostral-most skull to loss of the symplectic. Additionally, the adult palatoquadrate cartilage was disorganized, with abnormal chondrocyte orientation. To understand how the palatoquadrate cartilage normally develops, we examined a juvenile series of wild type and mutant specimens. This identified two novel domains of elongated chondrocytes in the larval palatoquadrate, which normally form prior to endochondral ossification. In contrast, gpc4−/− larvae never form these domains, suggesting a failure of chondrocyte orientation, though not differentiation. Our findings implicate Gpc4 in the regulation of zebrafish cartilage and bone morphogenesis. PMID:19777561

GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish

PubMed Central

Marín-Juez, Rubén; Rovira, Mireia; Crespo, Diego; van der Vaart, Michiel; Spaink, Herman P; Planas, Josep V

2015-01-01

Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing. PMID:25294126

Differences in Acute Alcohol-Induced Behavioral Responses Among Zebrafish Populations

PubMed Central

Gerlai, Robert; Ahmad, Fahad; Prajapati, Sonal

2009-01-01

Background With the arsenal of genetic tools available for zebrafish, this species has been successfully used to investigate the genetic aspects of human diseases from developmental disorders to cancer. Interest in the behavior and brain function of zebrafish is also increasing as CNS disorders may be modeled and studied with this species. Alcoholism and alcohol abuse are among the most devastating and costliest diseases. However, the mechanisms of these diseases are not fully understood. Zebrafish has been proposed as a model organism to study such mechanisms. Characterization of alcohol’s effects on zebrafish is a necessary step in this research. Methods Here, we compare the effects of acute alcohol (EtOH) administration on the behavior of zebrafish from 4 distinct laboratory-bred populations using automated as well as observation based behavioral quantification methods. Results Alcohol treatment resulted in significant dose-dependent behavioral changes but the dose–response trajectories differed among zebrafish populations. Conclusions The results demonstrate for the first time a genetic component in alcohol responses in adult zebrafish and also show the feasibility of high throughput behavioral screening. We discuss the exploration and exploitation of the genetic differences found. PMID:18652595

Sex differences in DNA methylation and expression in zebrafish brain: a test of an extended 'male sex drive' hypothesis.

PubMed

Chatterjee, Aniruddha; Lagisz, Malgorzata; Rodger, Euan J; Zhen, Li; Stockwell, Peter A; Duncan, Elizabeth J; Horsfield, Julia A; Jeyakani, Justin; Mathavan, Sinnakaruppan; Ozaki, Yuichi; Nakagawa, Shinichi

2016-09-30

The sex drive hypothesis predicts that stronger selection on male traits has resulted in masculinization of the genome. Here we test whether such masculinizing effects can be detected at the level of the transcriptome and methylome in the adult zebrafish brain. Although methylation is globally similar, we identified 914 specific differentially methylated CpGs (DMCs) between males and females (435 were hypermethylated and 479 were hypomethylated in males compared to females). These DMCs were prevalent in gene body, intergenic regions and CpG island shores. We also discovered 15 distinct CpG clusters with striking sex-specific DNA methylation differences. In contrast, at transcriptome level, more female-biased genes than male-biased genes were expressed, giving little support for the male sex drive hypothesis. Our study provides genome-wide methylome and transcriptome assessment and sheds light on sex-specific epigenetic patterns and in zebrafish for the first time. Copyright © 2016 Elsevier B.V. All rights reserved.

Zebrafish: a model animal for analyzing the impact of environmental pollutants on muscle and brain mitochondrial bioenergetics.

PubMed

Bourdineaud, Jean-Paul; Rossignol, R; Brèthes, D

2013-01-01

Mercury, anthropogenic release of uranium (U), and nanoparticles constitute hazardous environmental pollutants able to accumulate along the aquatic food chain with severe risk for animal and human health. The impact of such pollutants on living organisms has been up to now approached by classical toxicology in which huge doses of toxic compounds, environmentally irrelevant, are displayed through routes that never occur in the lifespan of organisms (for instance injecting a bolus of mercury to an animal although the main route is through prey and fish eating). We wanted to address the effect of such pollutants on the muscle and brain mitochondrial bioenergetics under realistic conditions, at unprecedented low doses, using an aquatic model animal, the zebrafish Danio rerio. We developed an original method to measure brain mitochondrial respiration: a single brain was put in 1.5 mL conical tube containing a respiratory buffer. Brains were gently homogenized by 13 strokes with a conical plastic pestle, and the homogenates were immediately used for respiration measurements. Skinned muscle fibers were prepared by saponin permeabilization. Zebrafish were contaminated with food containing 13 μg of methylmercury (MeHg)/g, an environmentally relevant dose. In permeabilized muscle fibers, we observed a strong inhibition of both state 3 mitochondrial respiration and cytochrome c oxidase activity after 49 days of MeHg exposure. We measured a dramatic decrease in the rate of ATP release by skinned muscle fibers. Contrarily to muscles, brain mitochondrial respiration was not modified by MeHg exposure although brain accumulated twice as much MeHg than muscles. When zebrafish were exposed to 30 μg/L of waterborne U, the basal mitochondrial respiratory control ratio was decreased in muscles after 28 days of exposure. This was due to an increase of the inner mitochondrial membrane permeability. The impact of a daily ration of food containing gold nanoparticles of two sizes (12 and

Long-term (30 days) toxicity of NiO nanoparticles for adult zebrafish Danio rerio.

PubMed

Kovrižnych, Jevgenij A; Sotníková, Ružena; Zeljenková, Dagmar; Rollerová, Eva; Szabová, Elena

2014-03-01

Nickel oxide in the form of nanoparticles (NiO NPs) is extensively used in different industrial branches. In a test on adult zebrafish, the acute toxicity of NiO NPs was shown to be low, however longlasting contact with this compound can lead to its accumulation in the tissues and to increased toxicity. In this work we determined the 30-day toxicity of NiO NPs using a static test for zebrafish Danio rerio. We found the 30-day LC50 value to be 45.0 mg/L, LC100 (minimum concentration causing 100% mortality) was 100.0 mg/L, and LC0 (maximum concentration causing no mortality) was 6.25 mg/L for adult individuals of zebrafish. Considering a broad use of Ni in the industry, NiO NPs chronic toxicity may have a negative impact on the population of aquatic organisms and on food web dynamics in aquatic systems.

Identification of a brain center whose activity discriminates a choice behavior in zebrafish

PubMed Central

Lau, Billy Y. B.; Mathur, Priya; Gould, Georgianna G.; Guo, Su

2011-01-01

The ability to make choices and carry out appropriate actions is critical for individual survival and well-being. Choice behaviors, from hard-wired to experience-dependent, have been observed across the animal kingdom. Although differential engagement of sensory neuronal pathways is a known mechanism, neurobiological substrates in the brain that underlie choice making downstream of sensory perception are not well understood. Here, we report a behavioral paradigm in zebrafish in which a half-light/half-dark visual image evokes an innate choice behavior, light avoidance. Neuronal activity mapping using the immediate early gene c-fos reveals the engagement of distinct brain regions, including the medial zone of the dorsal telencephalic region (Dm) and the dorsal nucleus of the ventral telencephalic area (Vd), the teleost anatomical homologs of the mammalian amygdala and striatum, respectively. In animals that were subjected to the identical sensory stimulus but displayed little or no avoidance, strikingly, the Dm and Vd were not engaged, despite similar levels of activation in the brain nuclei involved in visual processing. Based on these findings and previous connectivity data, we propose a neural circuitry model in which the Dm serves as a brain center, the activity of which predicates this choice behavior in zebrafish. PMID:21262817

Emotions and motivated behavior converge on an amygdala-like structure in the zebrafish

PubMed Central

von Trotha, Jakob William; Vernier, Philippe; Bally-Cuif, Laure

2014-01-01

The brain reward circuitry plays a key role in emotional and motivational behaviors, and its dysfunction underlies neuropsychiatric disorders such as schizophrenia, depression and drug addiction. Here, we characterized the neuronal activity pattern induced by acute amphetamine administration and during drug-seeking behavior in the zebrafish, and demonstrate the existence of conserved underlying brain circuitry. Combining quantitative analyses of cfos expression with neuronal subtype-specific markers at single-cell resolution, we show that acute d-amphetamine administration leads to both increased neuronal activation and the recruitment of neurons in the medial (Dm) and the lateral (Dl) domains of the adult zebrafish pallium, which contain homologous structures to the mammalian amygdala and hippocampus, respectively. Calbindin-positive and glutamatergic neurons are recruited in Dm, and glutamatergic and γ-aminobutyric acid (GABAergic) neurons in Dl. The drug-activated neurons in Dm and Dl are born at juvenile stage rather than in the embryo or during adulthood. Furthermore, the same territory in Dm is activated during both drug-seeking approach and light avoidance behavior, while these behaviors do not elicit activation in Dl. These data identify the pallial territories involved in acute psychostimulant response and reward formation in the adult zebrafish. They further suggest an evolutionarily conserved function of amygdala-like structures in positive emotions and motivated behavior in zebrafish and mammals. PMID:25145867

Gene expression changes in a zebrafish model of drug dependency suggest conservation of neuro-adaptation pathways.

PubMed

Kily, Layla J M; Cowe, Yuka C M; Hussain, Osman; Patel, Salma; McElwaine, Suzanne; Cotter, Finbarr E; Brennan, Caroline H

2008-05-01

Addiction is a complex psychiatric disorder considered to be a disease of the brain's natural reward reinforcement system. Repeated stimulation of the 'reward' pathway leads to adaptive changes in gene expression and synaptic organization that reinforce drug taking and underlie long-term changes in behaviour. The primitive nature of reward reinforcement pathways and the near universal ability of abused drugs to target the same system allow drug-associated reward and reinforcement to be studied in non-mammalian species. Zebrafish have proved to be a valuable model system for the study of vertebrate development and disease. Here we demonstrate that adult zebrafish show a dose-dependent acute conditioned place preference (CPP) reinforcement response to ethanol or nicotine. Repeated exposure of adult zebrafish to either nicotine or ethanol leads to a robust CPP response that persists following 3 weeks of abstinence and in the face of adverse stimuli, a behavioural indicator of the establishment of dependence. Microarray analysis using whole brain samples from drug-treated and control zebrafish identified 1362 genes that show a significant change in expression between control and treated individuals. Of these genes, 153 are common to both ethanol- and nicotine-treated animals. These genes include members of pathways and processes implicated in drug dependence in mammalian models, revealing conservation of neuro-adaptation pathways between zebrafish and mammals.

Delivery of Small Interfering RNA to Inhibit Vascular Endothelial Growth Factor in Zebrafish Using Natural Brain Endothelia Cell-Secreted Exosome Nanovesicles for the Treatment of Brain Cancer.

PubMed

Yang, Tianzhi; Fogarty, Brittany; LaForge, Bret; Aziz, Salma; Pham, Thuy; Lai, Leanne; Bai, Shuhua

2017-03-01

Although small interfering RNA (siRNA) holds great therapeutic promise, its delivery to the disease site remains a paramount obstacle. In this study, we tested whether brain endothelial cell-derived exosomes could deliver siRNA across the blood-brain barrier (BBB) in zebrafish. Natural exosomes were isolated from brain endothelial bEND.3 cell culture media and vascular endothelial growth factor (VEGF) siRNA was loaded in exosomes with the assistance of a transfection reagent. While fluorescence-activated cell flow cytometry and immunocytochemistry staining studies indicated that wild-type exosomes significantly increased the uptake of fluorescence-labeled siRNA in the autologous brain endothelial cells, decreased fluorescence intensity was observed in the cells treated with the tetraspanin CD63 antibody-blocked exosome-delivered formulation (p < 0.05). In the transport study, exosomes also enhanced the permeability of rhodamine 123 in a co-cultured monolayer of brain endothelial bEND.3 cell and astrocyte. Inhibition at the expression of VEGF RNA and protein levels was observed in glioblastoma-astrocytoma U-87 MG cells treated with exosome-delivered siRNAs. Imaging results showed that exosome delivered more siRNAs across the BBB in Tg(fli1:GFP) zebrafish. In a xenotransplanted brain tumor model, exosome-delivered VEGF siRNAs decreased the fluorescence intensity of labeled cancer cells in the brain of zebrafish. Brain endothelial cell-derived exosomes could be potentially used as a natural carrier for the brain delivery of exogenous siRNA.

Long-term (30 days) toxicity of NiO nanoparticles for adult zebrafish Danio rerio

PubMed Central

Kovrižnych, Jevgenij A.; Zeljenková, Dagmar; Rollerová, Eva; Szabová, Elena

2014-01-01

Nickel oxide in the form of nanoparticles (NiO NPs) is extensively used in different industrial branches. In a test on adult zebrafish, the acute toxicity of NiO NPs was shown to be low, however longlasting contact with this compound can lead to its accumulation in the tissues and to increased toxicity. In this work we determined the 30-day toxicity of NiO NPs using a static test for zebrafish Danio rerio. We found the 30-day LC50 value to be 45.0 mg/L, LC100 (minimum concentration causing 100% mortality) was 100.0 mg/L, and LC0 (maximum concentration causing no mortality) was 6.25 mg/L for adult individuals of zebrafish. Considering a broad use of Ni in the industry, NiO NPs chronic toxicity may have a negative impact on the population of aquatic organisms and on food web dynamics in aquatic systems. PMID:26038672

Effect of social isolation on anxiety-related behaviors, cortisol, and monoamines in adult zebrafish.

PubMed

Shams, Soaleha; Seguin, Diane; Facciol, Amanda; Chatterjee, Diptendu; Gerlai, Robert

2017-12-01

Social isolation can be used to study behavioral, neural, and hormonal mechanisms that regulate interactions in social animals. Although isolation effects have been reported in social mammals and various fish species, systematic studies with isolated zebrafish are rare. Here, the authors examined behavior (social and nonsocial), physiological stress (whole-body cortisol levels), and neurochemicals (serotonin, dopamine, and their metabolites), following acute and chronic social isolation in adult zebrafish. To observe how isolated fish respond behaviorally to social stimuli, they exposed zebrafish to live conspecifics or animated images after acute (24 hr) or chronic (6 months) social isolation. The authors observed that isolation did not affect locomotor activity, but acute isolation had weak nonsignificant anxiogenic effects in adult zebrafish. They also found that all isolated fish responded to both live and animated social stimuli, and the stress hormone, cortisol was lower in chronically isolated fish. Finally, neurochemical analyses showed that serotonin levels increased when fish were exposed to social stimulus after acute isolation, but its metabolite 5HIAA decreased in response to social stimulus following both acute and chronic isolation. Levels of both dopamine and its metabolite DOPAC were also reduced in fish exposed to social stimulus after acute and chronic isolation. Overall, these results show that isolation in zebrafish is an effective tool to study fundamental mechanisms controlling social interaction at behavioral and physiological levels. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Quantitative in vivo optical tomography of cancer progression & vasculature development in adult zebrafish

PubMed Central

Kumar, Sunil; Lockwood, Nicola; Ramel, Marie-Christine; Correia, Teresa; Ellis, Matthew; Alexandrov, Yuriy; Andrews, Natalie; Patel, Rachel; Bugeon, Laurence; Dallman, Margaret J.; Brandner, Sebastian; Arridge, Simon; Katan, Matilda; McGinty, James; Frankel, Paul; French, Paul M.W.

2016-01-01

We describe a novel approach to study tumour progression and vasculature development in vivo via global 3-D fluorescence imaging of live non-pigmented adult zebrafish utilising angularly multiplexed optical projection tomography with compressive sensing (CS-OPT). This “mesoscopic” imaging method bridges a gap between established ~μm resolution 3-D fluorescence microscopy techniques and ~mm-resolved whole body planar imaging and diffuse tomography. Implementing angular multiplexing with CS-OPT, we demonstrate the in vivo global imaging of an inducible fluorescently labelled genetic model of liver cancer in adult non-pigmented zebrafish that also present fluorescently labelled vasculature. In this disease model, addition of a chemical inducer (doxycycline) drives expression of eGFP tagged oncogenic K-RASV12 in the liver of immune competent animals. We show that our novel in vivo global imaging methodology enables non-invasive quantitative imaging of the development of tumour and vasculature throughout the progression of the disease, which we have validated against established methods of pathology including immunohistochemistry. We have also demonstrated its potential for longitudinal imaging through a study of vascular development in the same zebrafish from early embryo to adulthood. We believe that this instrument, together with its associated analysis and data management tools, constitute a new platform for in vivo cancer studies and drug discovery in zebrafish disease models. PMID:27259259

The gonadotropin-inhibitory hormone (Lpxrfa) system's regulation of reproduction in the brain-pituitary axis of the zebrafish (Danio rerio).

PubMed

Spicer, Olivia Smith; Zmora, Nilli; Wong, Ten-Tsao; Golan, Matan; Levavi-Sivan, Berta; Gothilf, Yoav; Zohar, Yonathan

2017-05-01

Gonadotropin-inhibitory hormone (GNIH) was discovered in quail with the ability to reduce gonadotropin expression/secretion in the pituitary. There have been few studies on GNIH orthologs in teleosts (LPXRFamide (Lpxrfa) peptides), which have provided inconsistent results. Therefore, the goal of this study was to determine the roles and modes of action by which Lpxrfa exerts its functions in the brain-pituitary axis of zebrafish (Danio rerio). We localized Lpxrfa soma to the ventral hypothalamus, with fibers extending throughout the brain and to the pituitary. In the preoptic area, Lpxrfa fibers interact with gonadotropin-releasing hormone 3 (Gnrh3) soma. In pituitary explants, zebrafish peptide Lpxrfa-3 downregulated luteinizing hormone beta subunit and common alpha subunit expression. In addition, Lpxrfa-3 reduced gnrh3 expression in brain slices, offering another pathway for Lpxrfa to exert its effects on reproduction. Receptor activation studies, in a heterologous cell-based system, revealed that all three zebrafish Lpxrfa peptides activate Lpxrf-R2 and Lpxrf-R3 via the PKA/cAMP pathway. Receptor activation studies demonstrated that, in addition to activating Lpxrf receptors, zebrafish Lpxrfa-2 and Lpxrfa-3 antagonize Kisspeptin-2 (Kiss2) activation of Kisspeptin receptor-1a (Kiss1ra). The fact that kiss1ra-expressing neurons in the preoptic area are innervated by Lpxrfa-ir fibers suggests an additional pathway for Lpxrfa action. Therefore, our results suggest that Lpxrfa may act as a reproductive inhibitory neuropeptide in the zebrafish that interacts with Gnrh3 neurons in the brain and with gonadotropes in the pituitary, while also potentially utilizing the Kiss2/Kiss1ra pathway. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Fgf Signaling is Required for Photoreceptor Maintenance in the Adult Zebrafish Retina

PubMed Central

Hochmann, Sarah; Kaslin, Jan; Hans, Stefan; Weber, Anke; Machate, Anja; Geffarth, Michaela; Funk, Richard H. W.; Brand, Michael

2012-01-01

Fibroblast growth factors (Fgf) are secreted signaling molecules that have mitogenic, patterning, neurotrophic and angiogenic properties. Their importance during embryonic development in patterning and morphogenesis of the vertebrate eye is well known, but less is known about the role of Fgfs in the adult vertebrate retina. To address Fgf function in adult retina, we determined the spatial distribution of components of the Fgf signaling pathway in the adult zebrafish retina. We detected differential expression of Fgf receptors, ligands and downstream Fgf targets within specific retinal layers. Furthermore, we blocked Fgf signaling in the retina, by expressing a dominant negative variant of Fgf receptor 1 conditionally in transgenic animals. After blocking Fgf signaling we observe a fast and progressive photoreceptor degeneration and disorganization of retinal tissue, coupled with cell death in the outer nuclear layer. Following the degeneration of photoreceptors, a profound regeneration response is triggered that starts with proliferation in the inner nuclear layer. Ultimately, rod and cone photoreceptors are regenerated completely. Our study reveals the requirement of Fgf signaling to maintain photoreceptors and for proliferation during regeneration in the adult zebrafish retina. PMID:22291943

Primary Spinal OPC Culture System from Adult Zebrafish to Study Oligodendrocyte Differentiation In Vitro.

PubMed

Kroehne, Volker; Tsata, Vasiliki; Marrone, Lara; Froeb, Claudia; Reinhardt, Susanne; Gompf, Anne; Dahl, Andreas; Sterneckert, Jared; Reimer, Michell M

2017-01-01

Endogenous oligodendrocyte progenitor cells (OPCs) are a promising target to improve functional recovery after spinal cord injury (SCI) by remyelinating denuded, and therefore vulnerable, axons. Demyelination is the result of a primary insult and secondary injury, leading to conduction blocks and long-term degeneration of the axons, which subsequently can lead to the loss of their neurons. In response to SCI, dormant OPCs can be activated and subsequently start to proliferate and differentiate into mature myelinating oligodendrocytes (OLs). Therefore, researchers strive to control OPC responses, and utilize small molecule screening approaches in order to identify mechanisms of OPC activation, proliferation, migration and differentiation. In zebrafish, OPCs remyelinate axons of the optic tract after lysophosphatidylcholine (LPC)-induced demyelination back to full thickness myelin sheaths. In contrast to zebrafish, mammalian OPCs are highly vulnerable to excitotoxic stress, a cause of secondary injury, and remyelination remains insufficient. Generally, injury induced remyelination leads to shorter internodes and thinner myelin sheaths in mammals. In this study, we show that myelin sheaths are lost early after a complete spinal transection injury, but are re-established within 14 days after lesion. We introduce a novel, easy-to-use, inexpensive and highly reproducible OPC culture system based on dormant spinal OPCs from adult zebrafish that enables in vitro analysis. Zebrafish OPCs are robust, can easily be purified with high viability and taken into cell culture. This method enables to examine why zebrafish OPCs remyelinate better than their mammalian counterparts, identify cell intrinsic responses, which could lead to pro-proliferating or pro-differentiating strategies, and to test small molecule approaches. In this methodology paper, we show efficient isolation of OPCs from adult zebrafish spinal cord and describe culture conditions that enable analysis up to 10

Zebrafish models for translational neuroscience research: from tank to bedside

PubMed Central

Stewart, Adam Michael; Braubach, Oliver; Spitsbergen, Jan; Gerlai, Robert; Kalueff, Allan V.

2014-01-01

The zebrafish (Danio rerio) is emerging as a new important species for studying mechanisms of brain function and dysfunction. Focusing on selected central nervous system (CNS) disorders (brain cancer, epilepsy, and anxiety) and using them as examples, we discuss the value of zebrafish models in translational neuroscience. We further evaluate the contribution of zebrafish to neuroimaging, circuit level, and drug discovery research. Outlining the role of zebrafish in modeling a wide range of human brain disorders, we also summarize recent applications and existing challenges in this field. Finally, we emphasize the potential of zebrafish models in behavioral phenomics and high-throughput genetic/small molecule screening, which is critical for CNS drug discovery and identifying novel candidate genes. PMID:24726051

Increased radial glia quiescence, decreased reactivation upon injury and unaltered neuroblast behavior underlie decreased neurogenesis in the aging zebrafish telencephalon.

PubMed

Edelmann, Kathrin; Glashauser, Lena; Sprungala, Susanne; Hesl, Birgit; Fritschle, Maike; Ninkovic, Jovica; Godinho, Leanne; Chapouton, Prisca

2013-09-01

The zebrafish has recently become a source of new data on the mechanisms of neural stem cell (NSC) maintenance and ongoing neurogenesis in adult brains. In this vertebrate, neurogenesis occurs at high levels in all ventricular regions of the brain, and brain injuries recover successfully, owing to the recruitment of radial glia, which function as NSCs. This new vertebrate model of adult neurogenesis is thus advancing our knowledge of the molecular cues in use for the activation of NSCs and fate of their progeny. Because the regenerative potential of somatic stem cells generally weakens with increasing age, it is important to assess the extent to which zebrafish NSC potential decreases or remains unaltered with age. We found that neurogenesis in the ventricular zone, in the olfactory bulb, and in a newly identified parenchymal zone of the telencephalon indeed declines as the fish ages and that oligodendrogenesis also declines. In the ventricular zone, the radial glial cell population remains largely unaltered morphologically but enters less frequently into the cell cycle and hence produces fewer neuroblasts. The neuroblasts themselves do not change their behavior with age and produce the same number of postmitotic neurons. Thus, decreased neurogenesis in the physiologically aging zebrafish brain is correlated with an increasing quiescence of radial glia. After injuries, radial glia in aged brains are reactivated, and the percentage of cell cycle entry is increased in the radial glia population. However, this reaction is far less pronounced than in younger animals, pointing to irreversible changes in aging zebrafish radial glia. Copyright © 2013 Wiley Periodicals, Inc.

Differential requirement for irf8 in formation of embryonic and adult macrophages in zebrafish

DOE PAGES

Shiau, Celia E.; Kaufman, Zoe; Meireles, Ana M.; ...

2015-01-23

Interferon regulatory factor 8 (Irf8) is critical for mammalian macrophage development and innate immunity, but its role in teleost myelopoiesis remains incompletely understood. Specifically, genetic tools to analyze the role of irf8 in zebrafish macrophage development at larval and adult stages are lacking. In this study, we generated irf8 null mutants in zebrafish using TALEN-mediated targeting. Our analysis defines different requirements for irf8 at different stages. irf8 is required for formation of all macrophages during primitive and transient definitive hematopoiesis, but not during adult-phase definitive hematopoiesis starting at 5-6 days postfertilization. At early stages, irf8 mutants have excess neutrophils andmore » excess cell death in pu.1-expressing myeloid cells. Macrophage fates were recovered in irf8 mutants after wildtype irf8 expression in neutrophil and macrophage lineages, suggesting that irf8 regulates macrophage specification and survival. In juvenile irf8 mutant fish, mature macrophages are present, but at numbers significantly reduced compared to wildtype, indicating an ongoing requirement for irf8 after embryogenesis. As development progresses, tissue macrophages become apparent in zebrafish irf8 mutants, with the possible exception of microglia. Our study defines distinct requirement for irf8 in myelopoiesis before and after transition to the adult hematopoietic system.« less

Zebrafish is a predictive model for identifying compounds that protect against brain toxicity in severe acute organophosphorus intoxication.

PubMed

Faria, Melissa; Prats, Eva; Padrós, Francesc; Soares, Amadeu M V M; Raldúa, Demetrio

2017-04-01

Acute organophosphorus (OP) intoxication is a worldwide clinical and public health problem. In addition to cholinergic crisis, neurodegeneration and brain damage are hallmarks of the severe form of this toxidrome. Recently, we generated a chemical model of severe acute OP intoxication in zebrafish that is characterized by altered head morphology and brain degeneration. The pathophysiological pathways resulting in brain toxicity in this model are similar to those described in humans. The aim of this study was to assess the predictive power of this zebrafish model by testing the effect of a panel of drugs that provide protection in mammalian models. The selected drugs included "standard therapy" drugs (atropine and pralidoxime), reversible acetylcholinesterase inhibitors (huperzine A, galantamine, physostigmine and pyridostigmine), N-methyl-D-aspartate (NMDA) receptor antagonists (MK-801 and memantine), dual-function NMDA receptor and acetylcholine receptor antagonists (caramiphen and benactyzine) and anti-inflammatory drugs (dexamethasone and ibuprofen). The effects of these drugs on zebrafish survival and the prevalence of abnormal head morphology in the larvae exposed to 4 µM chlorpyrifos oxon [1 × median lethal concentration (LC 50 )] were determined. Moreover, the neuroprotective effects of pralidoxime, memantine, caramiphen and dexamethasone at the gross morphological level were confirmed by histopathological and transcriptional analyses. Our results demonstrated that the zebrafish model for severe acute OP intoxication has a high predictive value and can be used to identify new compounds that provide neuroprotection against severe acute OP intoxication.

Neurodevelopmental Low-dose Bisphenol A Exposure Leads to Early Life-stage Hyperactivity and Learning Deficits in Adult Zebrafish

PubMed Central

Saili, Katerine S.; Corvi, Margaret M.; Weber, Daniel N.; Patel, Ami U.; Das, Siba R.; Przybyla, Jennifer; Anderson, Kim A.; Tanguay, Robert L.

2011-01-01

Developmental bisphenol A (BPA) exposure has been implicated in adverse behavior and learning deficits. The mode of action underlying these effects is unclear. The zebrafish model was employed to investigate the neurobehavioral effects of developmental bisphenol A (BPA) exposure. The objectives of this study were to identify whether low-dose, developmental BPA exposure affects larval zebrafish locomotor behavior and whether learning deficits occur in adults exposed during development. Two control compounds, 17β-estradiol (an estrogen receptor ligand) and GSK4716 (a synthetic estrogen related receptor gamma ligand), were included. Larval toxicity assays were used to determine appropriate BPA, 17β-estradiol, and GSK4716 concentrations for behavior testing. BPA tissue uptake was analyzed using HPLC and lower doses were extrapolated using a linear regression analysis. Larval behavior tests were conducted using a ViewPoint Zebrabox. Adult learning tests were conducted using a custom-built T-maze. BPA exposure to ≤30 μM was nonteratogenic in zebrafish. Neurodevelopmental BPA exposure to 0.01, 0.1, or 1 μM led to larval hyperactivity or learning deficits in adult zebrafish. Exposure to 0.1 μM 17β-estradiol or GSK4716 also led to larval hyperactivity. This study demonstrates the efficacy of using the larval zebrafish model for studying the neurobehavioral effects of low-dose developmental BPA exposure. PMID:22108044

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain.

PubMed

Kuipers, Jeroen; Kalicharan, Ruby D; Wolters, Anouk H G; van Ham, Tjakko J; Giepmans, Ben N G

2016-05-25

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae(1-7). Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture(1-5). Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)(8) on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

PubMed Central

Kuipers, Jeroen; Kalicharan, Ruby D.; Wolters, Anouk H. G.

2016-01-01

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner. PMID:27285162

Brain tumor - primary - adults

MedlinePlus

... Vestibular schwannoma (acoustic neuroma) - adults; Meningioma - adults; Cancer - brain tumor (adults) ... Primary brain tumors include any tumor that starts in the brain. Primary brain tumors can start from brain cells, ...

Novel approaches to determine contractile function of the isolated adult zebrafish ventricular cardiac myocyte.

PubMed

Dvornikov, Alexey V; Dewan, Sukriti; Alekhina, Olga V; Pickett, F Bryan; de Tombe, Pieter P

2014-05-01

The zebrafish (Danio rerio) has been used extensively in cardiovascular biology, but mainly in the study of heart development. The relative ease of its genetic manipulation may indicate the suitability of this species as a cost-effective model system for the study of cardiac contractile biology. However, whether the zebrafish heart is an appropriate model system for investigations pertaining to mammalian cardiac contractile structure-function relationships remains to be resolved. Myocytes were isolated from adult zebrafish hearts by enzymatic digestion, attached to carbon rods, and twitch force and intracellular Ca(2+) were measured. We observed the modulation of twitch force, but not of intracellular Ca(2+), by both extracellular [Ca(2+)] and sarcomere length. In permeabilized cells/myofibrils, we found robust myofilament length-dependent activation. Moreover, modulation of myofilament activation-relaxation and force redevelopment kinetics by varied Ca(2+) activation levels resembled that found previously in mammalian myofilaments. We conclude that the zebrafish is a valid model system for the study of cardiac contractile structure-function relationships.

Cardiac and Metabolic Effects of Dietary Selenomethionine Exposure in Adult Zebrafish.

PubMed

Pettem, Connor M; Weber, Lynn P; Janz, David M

2017-10-01

Selenium (Se) is an essential micronutrient involved in important metabolic functions for all vertebrate species. As Se is reported to have a narrow margin between essentiality and toxicity, there is growing concern surrounding the adverse effects of elevated Se exposure caused by anthropogenic activities. Recent studies have reported that elevated dietary exposure of fish to selenomethionine (Se-Met) can alter aerobic metabolic capacity, energetics and swimming performance. This study aims to further investigate mechanisms of sublethal Se-Met toxicity, particularly potential underlying cardiovascular implications of chronic exposure to environmentally relevant concentrations of dietary Se-Met in adult zebrafish (Danio rerio). Adult zebrafish were fed either control food (1.1 μg Se/g dry mass [d.m.]) or Se-Met spiked food (10.3 or 28.8 μg Se/g d.m.) for 90 d at 5% body weight per day. Following exposure, ultrahigh resolution B-mode and Doppler ultrasound was used to characterize cardiac function. Chronic dietary exposure to elevated Se-Met significantly reduced ventricular contractile rate, stroke volume, and cardiac output. Exposure to Se-Met significantly decreased mRNA expression of methionine adenosyltransferase 1 alpha and glutathione-S-transferase pi class in liver, and a key cardiac remodelling enzyme, matrix metalloproteinase 2, in adult zebrafish heart. Se-Met significantly increased echodensity at the junction between atrium and ventricle, and these results combined with increased matrix metalloproteinase 2 expression are consistent with cardiac remodelling and fibrosis. The results of this study suggest that chronic exposure to dietary Se-Met can negatively impact cardiac function, and such physiological consequences could reduce the aerobic capacity and survivability of fish. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Exercise-Induced Hypertrophic and Oxidative Signaling Pathways and Myokine Expression in Fast Muscle of Adult Zebrafish

PubMed Central

Rovira, Mireia; Arrey, Gerard; Planas, Josep V.

2017-01-01

Skeletal muscle is a plastic tissue that undergoes cellular and metabolic adaptations under conditions of increased contractile activity such as exercise. Using adult zebrafish as an exercise model, we previously demonstrated that swimming training stimulates hypertrophy and vascularization of fast muscle fibers, consistent with the known muscle growth-promoting effects of exercise and with the resulting increased aerobic capacity of this tissue. Here we investigated the potential involvement of factors and signaling mechanisms that could be responsible for exercise-induced fast muscle remodeling in adult zebrafish. By subjecting zebrafish to swimming-induced exercise, we observed an increase in the activity of mammalian target of rapamycin (mTOR) and Mef2 protein levels in fast muscle. We also observed an increase in the protein levels of the mitotic marker phosphorylated histone H3 that correlated with an increase in the protein expression levels of Pax7, a satellite-like cell marker. Furthermore, the activity of AMP-activated protein kinase (AMPK) was also increased by exercise, in parallel with an increase in the mRNA expression levels of pgc1α and also of pparda, a β-oxidation marker. Changes in the mRNA expression levels of slow and fast myosin markers further supported the notion of an exercise-induced aerobic phenotype in zebrafish fast muscle. The mRNA expression levels of il6, il6r, apln, aplnra and aplnrb, sparc, decorin and igf1, myokines known in mammals to be produced in response to exercise and to signal through mTOR/AMPK pathways, among others, were increased in fast muscle of exercised zebrafish. These results support the notion that exercise increases skeletal muscle growth and myogenesis in adult zebrafish through the coordinated activation of the mTOR-MEF2 and AMPK-PGC1α signaling pathways. These results, coupled with altered expression of markers for oxidative metabolism and fast-to-slow fiber-type switch, also suggest improved aerobic

Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy.

PubMed

Panier, Thomas; Romano, Sebastián A; Olive, Raphaël; Pietri, Thomas; Sumbre, Germán; Candelier, Raphaël; Debrégeas, Georges

2013-01-01

The optical transparency and the small dimensions of zebrafish at the larval stage make it a vertebrate model of choice for brain-wide in-vivo functional imaging. However, current point-scanning imaging techniques, such as two-photon or confocal microscopy, impose a strong limit on acquisition speed which in turn sets the number of neurons that can be simultaneously recorded. At 5 Hz, this number is of the order of one thousand, i.e., approximately 1-2% of the brain. Here we demonstrate that this limitation can be greatly overcome by using Selective-plane Illumination Microscopy (SPIM). Zebrafish larvae expressing the genetically encoded calcium indicator GCaMP3 were illuminated with a scanned laser sheet and imaged with a camera whose optical axis was oriented orthogonally to the illumination plane. This optical sectioning approach was shown to permit functional imaging of a very large fraction of the brain volume of 5-9-day-old larvae with single- or near single-cell resolution. The spontaneous activity of up to 5,000 neurons was recorded at 20 Hz for 20-60 min. By rapidly scanning the specimen in the axial direction, the activity of 25,000 individual neurons from 5 different z-planes (approximately 30% of the entire brain) could be simultaneously monitored at 4 Hz. Compared to point-scanning techniques, this imaging strategy thus yields a ≃20-fold increase in data throughput (number of recorded neurons times acquisition rate) without compromising the signal-to-noise ratio (SNR). The extended field of view offered by the SPIM method allowed us to directly identify large scale ensembles of neurons, spanning several brain regions, that displayed correlated activity and were thus likely to participate in common neural processes. The benefits and limitations of SPIM for functional imaging in zebrafish as well as future developments are briefly discussed.

From Whole-Brain Data to Functional Circuit Models: The Zebrafish Optomotor Response.

PubMed

Naumann, Eva A; Fitzgerald, James E; Dunn, Timothy W; Rihel, Jason; Sompolinsky, Haim; Engert, Florian

2016-11-03

Detailed descriptions of brain-scale sensorimotor circuits underlying vertebrate behavior remain elusive. Recent advances in zebrafish neuroscience offer new opportunities to dissect such circuits via whole-brain imaging, behavioral analysis, functional perturbations, and network modeling. Here, we harness these tools to generate a brain-scale circuit model of the optomotor response, an orienting behavior evoked by visual motion. We show that such motion is processed by diverse neural response types distributed across multiple brain regions. To transform sensory input into action, these regions sequentially integrate eye- and direction-specific sensory streams, refine representations via interhemispheric inhibition, and demix locomotor instructions to independently drive turning and forward swimming. While experiments revealed many neural response types throughout the brain, modeling identified the dimensions of functional connectivity most critical for the behavior. We thus reveal how distributed neurons collaborate to generate behavior and illustrate a paradigm for distilling functional circuit models from whole-brain data. Copyright © 2016 Elsevier Inc. All rights reserved.

Brain-wide neuronal dynamics during motor adaptation in zebrafish.

PubMed

Ahrens, Misha B; Li, Jennifer M; Orger, Michael B; Robson, Drew N; Schier, Alexander F; Engert, Florian; Portugues, Ruben

2012-05-09

A fundamental question in neuroscience is how entire neural circuits generate behaviour and adapt it to changes in sensory feedback. Here we use two-photon calcium imaging to record the activity of large populations of neurons at the cellular level, throughout the brain of larval zebrafish expressing a genetically encoded calcium sensor, while the paralysed animals interact fictively with a virtual environment and rapidly adapt their motor output to changes in visual feedback. We decompose the network dynamics involved in adaptive locomotion into four types of neuronal response properties, and provide anatomical maps of the corresponding sites. A subset of these signals occurred during behavioural adjustments and are candidates for the functional elements that drive motor learning. Lesions to the inferior olive indicate a specific functional role for olivocerebellar circuitry in adaptive locomotion. This study enables the analysis of brain-wide dynamics at single-cell resolution during behaviour.

Brain-wide neuronal dynamics during motor adaptation in zebrafish

PubMed Central

Ahrens, Misha B; Li, Jennifer M; Orger, Michael B; Robson, Drew N; Schier, Alexander F; Engert, Florian; Portugues, Ruben

2013-01-01

A fundamental question in neuroscience is how entire neural circuits generate behavior and adapt it to changes in sensory feedback. Here we use two-photon calcium imaging to record activity of large populations of neurons at the cellular level throughout the brain of larval zebrafish expressing a genetically-encoded calcium sensor, while the paralyzed animals interact fictively with a virtual environment and rapidly adapt their motor output to changes in visual feedback. We decompose the network dynamics involved in adaptive locomotion into four types of neural response properties, and provide anatomical maps of the corresponding sites. A subset of these signals occurred during behavioral adjustments and are candidates for the functional elements that drive motor learning. Lesions to the inferior olive indicate a specific functional role for olivocerebellar circuitry in adaptive locomotion. This study enables the analysis of brain-wide dynamics at single-cell resolution during behavior. PMID:22622571

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hinfray, N., E-mail: nathalie.hinfray@ineris.fr

Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase genemore » under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures. - Highlights: • Combined effects of EE2 and LNG were assessed on ER-dependent cyp19a1b expression. • EE2 and LNG alone induced brain aromatase in zebrafish specific bioassays. • Experimental ray design allowed complete concentration-response surfaces modeling. �

Valerenic acid and Valeriana officinalis extracts delay onset of Pentylenetetrazole (PTZ)-Induced seizures in adult Danio rerio (Zebrafish).

PubMed

Torres-Hernández, Bianca A; Del Valle-Mojica, Lisa M; Ortíz, José G

2015-07-14

Anticonvulsant properties have been attributed to extracts of the herbal medicine Valeriana officinalis. Our aims were to examine the anticonvulsant properties of valerenic acid and valerian extracts and to determine whether valerian preparations interact with the activity of other anti-epileptic drugs (phenytoin or clonazepam). To achieve these goals, we validated the adult zebrafish, Danio rerio, as an animal model for studying anticonvulsant drugs. All drug treatments were administered by immersion in water containing the drug. For assays of anticonvulsant activity, zebrafish were pretreated with: anti-epileptic drugs, valerenic acid, aqueous or ethanolic valerian extracts, or mixtures (phenytoin or clonazepam with valerenic acid or valerian extracts). Seizures were then induced with pentylenetetrazole (PTZ). A behavioral scale was developed for scoring PTZ-induced seizures in adult zebrafish. The seizure latency was evaluated for all pretreatments and control, untreated fish. Valerenic acid and both aqueous and ethanolic extracts of valerian root were also evaluated for their ability to improve survival after pentylenetetrazole-challenge. The assay was validated by comparison with well-studied anticonvulsant drugs (phenytoin, clonazepam, gabapentin and valproate). One-way ANOVA followed by Tukey post-hoc test was performed, using a p < 0.05 level of significance. All treatments were compared with the untreated animals and with the other pretreatments. After exposure to pentylenetetrazole, zebrafish exhibited a series of stereotypical behaviors prior to the appearance of clonic-like movements--convulsions. Both valerenic acid and valerian extracts (aqueous and ethanolic) significantly extended the latency period to the onset of seizure (convulsion) in adult zebrafish. The ethanolic valerian extract was a more potent anticonvulsant than the aqueous extract. Valerenic acid and both valerian extracts interacted synergistically with clonazepam to extended the

Manganese(II) Chloride Alters Nucleotide and Nucleoside Catabolism in Zebrafish (Danio rerio) Adult Brain.

PubMed

Altenhofen, Stefani; Nabinger, Débora Dreher; Pereira, Talita Carneiro Brandão; Leite, Carlos Eduardo; Bogo, Maurício Reis; Bonan, Carla Denise

2018-05-01

ATP and adenosine, the main signaling molecules of purinergic system, are involved in toxicological effects induced by metals. The manganese (Mn) exposure induces several cellular changes, which could interfere with signaling pathways, such as the purinergic system. In this study, we evaluated the effects of exposure to manganese(II) chloride (MnCl 2 ) during 96 h on nucleoside triphosphate diphosphohydrolase (NTPDase), ecto-5'-nucleotidase, and adenosine deaminase (ADA) activities, followed by analyzing the gene expression patterns of NTPDases (entpd1, entpd2a.1, entpd2a.2, entpd2-like, entpd3) and ADA (ADA 1 , ADA 2.1 , ADA 2.2 , ADAasi, ADAL) families in zebrafish brain. In addition, the brain metabolism of nucleotides and nucleosides was evaluated after MnCl 2 exposure. The results showed that MnCl 2 exposure during 96 h inhibited the NTPDase (1.0 and 1.5 mM) and ecto-ADA (0.5, 1.0, and 1.5 mM) activities, further decreasing ADA2.1 expression at all MnCl 2 concentrations analyzed. Purine metabolism was also altered by the action of MnCl 2 . An increased amount of ADP appeared at all MnCl 2 concentrations analyzed; however, AMP and adenosine levels are decreased at the concentrations of 1.0 and 1.5 mM MnCl 2 , whereas decreased inosine (INO) levels were observed at all concentrations tested. The findings of this study demonstrated that MnCl 2 may inhibit NTPDase and ecto-ADA activities, consequently modulating nucleotide and nucleoside levels, which may contribute for the toxicological effects induced by this metal.

A novel TRIM family member, Trim69, regulates zebrafish development through p53-mediated apoptosis.

PubMed

Han, Ruiqin; Zhao, Qing; Zong, Shudong; Miao, Shiying; Song, Wei; Wang, Linfang

2016-05-01

Trim69 contains the hallmark domains of a tripartite motif (TRIM) protein, including a Ring-finger domain, B-box domain, and coiled-coil domain. Trim69 is structurally and evolutionarily conserved in zebrafish, mouse, rat, human, and chimpanzee. The role of this protein is unclear, however, so we investigated its function in zebrafish development. Trim69 is extensively expressed in zebrafish adults and developing embryos-particularly in the testis, brain, ovary, and heart-and its expression decreases in a time- and stage-dependent manner. Loss of trim69 in zebrafish induces apoptosis and activates apoptosis-related processes; indeed, the tp53 pathway was up-regulated in response to the knockdown. Expression of human trim69 rescued the apoptotic phenotype, while overexpression of trim69 does not increase cellular apoptosis. Taken together, our results suggest that trim69 participates in tp53-mediated apoptosis during zebrafish development. Mol. Reprod. Dev. 83: 442-454, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Normal anatomy and histology of the adult zebrafish.

PubMed

Menke, Aswin L; Spitsbergen, Jan M; Wolterbeek, Andre P M; Woutersen, Ruud A

2011-08-01

The zebrafish has been shown to be an excellent vertebrate model for studying the roles of specific genes and signaling pathways. The sequencing of its genome and the relative ease with which gene modifications can be performed have led to the creation of numerous human disease models that can be used for testing the potential and the toxicity of new pharmaceutical compounds. Many pharmaceutical companies already use the zebrafish for prescreening purposes. So far, the focus has been on ecotoxicity and the effects on embryonic development, but there is a trend to expand the use of the zebrafish with acute, subchronic, and chronic toxicity studies that are currently still carried out with the more conventional test animals such as rodents. However, before we can fully realize the potential of the zebrafish as an animal model for understanding human development, disease, and toxicology, we must first greatly advance our knowledge of normal zebrafish physiology, anatomy, and histology. To further this knowledge, we describe, in the present article, location and histology of the major zebrafish organ systems with a brief description of their function.

Zebrafish Whole-Adult-Organism Chemogenomics for Large-Scale Predictive and Discovery Chemical Biology

PubMed Central

Lam, Siew Hong; Mathavan, Sinnakarupan; Tong, Yan; Li, Haixia; Karuturi, R. Krishna Murthy; Wu, Yilian; Vega, Vinsensius B.; Liu, Edison T.; Gong, Zhiyuan

2008-01-01

The ability to perform large-scale, expression-based chemogenomics on whole adult organisms, as in invertebrate models (worm and fly), is highly desirable for a vertebrate model but its feasibility and potential has not been demonstrated. We performed expression-based chemogenomics on the whole adult organism of a vertebrate model, the zebrafish, and demonstrated its potential for large-scale predictive and discovery chemical biology. Focusing on two classes of compounds with wide implications to human health, polycyclic (halogenated) aromatic hydrocarbons [P(H)AHs] and estrogenic compounds (ECs), we generated robust prediction models that can discriminate compounds of the same class from those of different classes in two large independent experiments. The robust expression signatures led to the identification of biomarkers for potent aryl hydrocarbon receptor (AHR) and estrogen receptor (ER) agonists, respectively, and were validated in multiple targeted tissues. Knowledge-based data mining of human homologs of zebrafish genes revealed highly conserved chemical-induced biological responses/effects, health risks, and novel biological insights associated with AHR and ER that could be inferred to humans. Thus, our study presents an effective, high-throughput strategy of capturing molecular snapshots of chemical-induced biological states of a whole adult vertebrate that provides information on biomarkers of effects, deregulated signaling pathways, and possible affected biological functions, perturbed physiological systems, and increased health risks. These findings place zebrafish in a strategic position to bridge the wide gap between cell-based and rodent models in chemogenomics research and applications, especially in preclinical drug discovery and toxicology. PMID:18618001

Treatment with Myf5-morpholino results in somite patterning and brain formation defects in zebrafish.

PubMed

Chen, Yau-Hung; Tsai, Huai-Jen

2002-10-01

Myf-5 is a stage-dependent transcription factor associated with somitogenesis. To study its biological functions in zebrafish, we injected the Myf5-morpholinos ZMF-MO (antisense nucleotides 28 to 52) and ZMF-OTHER (antisense nucleotides 3 to 27) into zebrafish embryos to establish a myf-5 gene knockdown. No phenotypic abnormalities were observed following injection with 0.2 ng of ZMF-MO, but defects were displayed in 2 of 118 (1.7%) surviving embryos injected with 1 ng ZMF-MO. Morphological defects became more severe with increased dosages: 105 of 270 (38.9%) surviving embryos injected with 4.5 ng of ZMF-MO displayed such abnormalities as the absence of eyes or brains in addition to the following low-dosage defects in 24 hpf embryos: longitudinal yolk sacs, incomplete epiboly coverage, abnormal and suspended tail buds, diffused somite boundaries, and head shrinkage. Similar results were observed in the 4.5 ng ZMF-OTHER injection group. However, when fish were co-injected with 4.5 ng ZMF-MO and 4.5 ng myf-5 mRNA, abnormality rates decreased from 49.6% to 5.5%. Our results show that the brain krox20 gene was down-regulated at rhombomere 3; the pax2.1 gene was completely down-regulated; myoD was expressed normally; myogenin was substantially down-regulated in whole somites; and desmin was partly inhibited in newly forming somites. Our conclusion is that zebrafish Myf-5 may play important roles in brain formation and in the convergence and extension of shield epiblasts and tail buds during early embryogenesis, in addition to its well-understood role as a muscle regulatory factor in somites.

Novel function of vitamin E in regulation of zebrafish (Danio rerio) brain lysophospholipids discovered using lipidomics

PubMed Central

Choi, Jaewoo; Leonard, Scott W.; Kasper, Katherine; McDougall, Melissa; Stevens, Jan F.; Tanguay, Robert L.; Traber, Maret G.

2015-01-01

We hypothesized that brains from vitamin E-deficient (E−) zebrafish (Danio rerio) would undergo increased lipid peroxidation because they contain highly polyunsaturated fatty acids, thus susceptible lipids could be identified. Brains from zebrafish fed for 9 months defined diets without (E−) or with (E+) added vitamin E (500 mg RRR-α-tocopheryl acetate per kilogram diet) were studied. Using an untargeted approach, 1-hexadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine [DHA-PC 38:6, PC 16:0/22:6]was the lipid that showed the most significant and greatest fold-differences between groups. DHA-PC concentrations were approximately 1/3 lower in E− (4.3 ± 0.6 mg/g) compared with E+ brains (6.5 ± 0.9 mg/g, mean ± SEM, n = 10 per group, P = 0.04). Using lipidomics, 155 lipids in brain extracts were identified. Only four phospholipids (PLs) were different (P < 0.05) between groups; they were lower in E− brains and contained DHA with DHA-PC 38:6 at the highest abundances. Moreover, hydroxy-DHA-PC 38:6 was increased in E− brains (P = 0.0341) supporting the hypothesis of DHA peroxidation. More striking was the depletion in E− brains of nearly 60% of 19 different lysophospholipids (lysoPLs) (combined P = 0.0003), which are critical for membrane PL remodeling. Thus, E− brains contained fewer DHA-PLs, more hydroxy-DHA-PCs, and fewer lysoPLs, suggesting that lipid peroxidation depletes membrane DHA-PC and homeostatic mechanisms to repair the damage resulting in lysoPL depletion. PMID:25855633

[Effect of rhynchophylline on behaviors of methamphetamine-dependent zebrafish and the mechanism].

PubMed

Chen, Yi-Fei; Peng, Ju; Fang, Miao; Liu, Yi; Nie, Ling-Hui; Mo, Zhi-Xian; Zhu, Ling-Ling

2016-11-20

To observe the effect of rhynchophylline on methamphetamine-dependent zebrafish and explore the possible mechanism. Zebrafish were divided into control group, amphetamine group, low- (50 mg/kg) and high (100 mg/kg)-dose rhynchophylline groups, and ketamine (150 mg/kg) group. Conditioned place preference (CPP) was induced in zebrafish with methamphetamine, and the staying time in the drug box and the tracking map of the zebrafish were observed with Noldus Ethovision XT system. The protein expressions of TH, NR2B and GLUR2 in the brain of zebrafish with CPP were detected with Western blotting. Compared with the control group, zebrafish in methamphetamine group showed significant variations in the staying time and swimming distance in the drug box after conditioning (P<0.05) with obvious alterations of NR2B, TH and GLUR2 expressions in the brain (P<0.05). Treatment of methamphetamine-dependent zebrafish with high-dose rhynchophylline significantly reduced the variations in the staying time and swimming distance in the drug box (P<0.05) and in the expressions of NR2B, TH and GLUR2 in the brain (P<0.05). Rhynchophylline can inhibit methamphetamine dependence in zebrafish, the mechanism of which may involve the expressions of TH, NR2B and GLUR2 proteins in the brain.

Comparison of molecular marker expression in early zebrafish brain development following chronic ethanol or morpholino treatment.

PubMed

Zhang, Chengjin; Boa-Amponsem, Oswald; Cole, Gregory J

2017-08-01

This study was undertaken to ascertain whether defined markers of early zebrafish brain development are affected by chronic ethanol exposure or morpholino knockdown of agrin, sonic hedgehog, retinoic acid, and fibroblast growth factors, four signaling molecules that are suggested to be ethanol sensitive. Zebrafish embryos were exposed to 2% ethanol from 6 to 24 hpf or injected with agrin, shha, aldh1a3, or fgf8a morpholinos. In situ hybridization was employed to analyze otx2, pax6a, epha4a, krx20, pax2a, fgf8a, wnt1, and eng2b expression during early brain development. Our results showed that pax6a mRNA expression was decreased in eye, forebrain, and hindbrain of both chronic ethanol exposed and select MO treatments. Epha4a expression in rhombomere R1 boundary was decreased in chronic ethanol exposure and aldh1a3 morphants, lost in fgf8a morphants, but largely unaffected in agrin and shha morphants. Ectopic pax6a and epha4a expression in midbrain was only found in fgf8a morphants. These results suggest that while chronic ethanol induces obvious morphological change in brain architecture, many molecular markers of these brain structures are relatively unaffected by ethanol exposure.

A novel method for automated tracking and quantification of adult zebrafish behaviour during anxiety.

PubMed

Nema, Shubham; Hasan, Whidul; Bhargava, Anamika; Bhargava, Yogesh

2016-09-15

Behavioural neuroscience relies on software driven methods for behavioural assessment, but the field lacks cost-effective, robust, open source software for behavioural analysis. Here we propose a novel method which we called as ZebraTrack. It includes cost-effective imaging setup for distraction-free behavioural acquisition, automated tracking using open-source ImageJ software and workflow for extraction of behavioural endpoints. Our ImageJ algorithm is capable of providing control to users at key steps while maintaining automation in tracking without the need for the installation of external plugins. We have validated this method by testing novelty induced anxiety behaviour in adult zebrafish. Our results, in agreement with established findings, showed that during state-anxiety, zebrafish showed reduced distance travelled, increased thigmotaxis and freezing events. Furthermore, we proposed a method to represent both spatial and temporal distribution of choice-based behaviour which is currently not possible to represent using simple videograms. ZebraTrack method is simple and economical, yet robust enough to give results comparable with those obtained from costly proprietary software like Ethovision XT. We have developed and validated a novel cost-effective method for behavioural analysis of adult zebrafish using open-source ImageJ software. Copyright © 2016 Elsevier B.V. All rights reserved.

Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing.

PubMed

Tang, Qin; Iyer, Sowmya; Lobbardi, Riadh; Moore, John C; Chen, Huidong; Lareau, Caleb; Hebert, Christine; Shaw, McKenzie L; Neftel, Cyril; Suva, Mario L; Ceol, Craig J; Bernards, Andre; Aryee, Martin; Pinello, Luca; Drummond, Iain A; Langenau, David M

2017-10-02

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA-protein kinase catalytic subunit ( prkdc ), interleukin-2 receptor γ a ( il2rga ), and double-homozygous-mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish. © 2017 Tang et al.

Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing

PubMed Central

Iyer, Sowmya; Lobbardi, Riadh; Chen, Huidong; Hebert, Christine; Shaw, McKenzie L.; Neftel, Cyril; Suva, Mario L.; Bernards, Andre; Aryee, Martin; Drummond, Iain A.

2017-01-01

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA–protein kinase catalytic subunit (prkdc), interleukin-2 receptor γ a (il2rga), and double-homozygous–mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish. PMID:28878000

Whole-brain serial-section electron microscopy in larval zebrafish.

PubMed

Hildebrand, David Grant Colburn; Cicconet, Marcelo; Torres, Russel Miguel; Choi, Woohyuk; Quan, Tran Minh; Moon, Jungmin; Wetzel, Arthur Willis; Scott Champion, Andrew; Graham, Brett Jesse; Randlett, Owen; Plummer, George Scott; Portugues, Ruben; Bianco, Isaac Henry; Saalfeld, Stephan; Baden, Alexander David; Lillaney, Kunal; Burns, Randal; Vogelstein, Joshua Tzvi; Schier, Alexander Franz; Lee, Wei-Chung Allen; Jeong, Won-Ki; Lichtman, Jeff William; Engert, Florian

2017-05-18

High-resolution serial-section electron microscopy (ssEM) makes it possible to investigate the dense meshwork of axons, dendrites, and synapses that form neuronal circuits. However, the imaging scale required to comprehensively reconstruct these structures is more than ten orders of magnitude smaller than the spatial extents occupied by networks of interconnected neurons, some of which span nearly the entire brain. Difficulties in generating and handling data for large volumes at nanoscale resolution have thus restricted vertebrate studies to fragments of circuits. These efforts were recently transformed by advances in computing, sample handling, and imaging techniques, but high-resolution examination of entire brains remains a challenge. Here, we present ssEM data for the complete brain of a larval zebrafish (Danio rerio) at 5.5 days post-fertilization. Our approach utilizes multiple rounds of targeted imaging at different scales to reduce acquisition time and data management requirements. The resulting dataset can be analysed to reconstruct neuronal processes, permitting us to survey all myelinated axons (the projectome). These reconstructions enable precise investigations of neuronal morphology, which reveal remarkable bilateral symmetry in myelinated reticulospinal and lateral line afferent axons. We further set the stage for whole-brain structure-function comparisons by co-registering functional reference atlases and in vivo two-photon fluorescence microscopy data from the same specimen. All obtained images and reconstructions are provided as an open-access resource.

Whole-brain serial-section electron microscopy in larval zebrafish

NASA Astrophysics Data System (ADS)

Hildebrand, David Grant Colburn; Cicconet, Marcelo; Torres, Russel Miguel; Choi, Woohyuk; Quan, Tran Minh; Moon, Jungmin; Wetzel, Arthur Willis; Scott Champion, Andrew; Graham, Brett Jesse; Randlett, Owen; Plummer, George Scott; Portugues, Ruben; Bianco, Isaac Henry; Saalfeld, Stephan; Baden, Alexander David; Lillaney, Kunal; Burns, Randal; Vogelstein, Joshua Tzvi; Schier, Alexander Franz; Lee, Wei-Chung Allen; Jeong, Won-Ki; Lichtman, Jeff William; Engert, Florian

2017-05-01

High-resolution serial-section electron microscopy (ssEM) makes it possible to investigate the dense meshwork of axons, dendrites, and synapses that form neuronal circuits. However, the imaging scale required to comprehensively reconstruct these structures is more than ten orders of magnitude smaller than the spatial extents occupied by networks of interconnected neurons, some of which span nearly the entire brain. Difficulties in generating and handling data for large volumes at nanoscale resolution have thus restricted vertebrate studies to fragments of circuits. These efforts were recently transformed by advances in computing, sample handling, and imaging techniques, but high-resolution examination of entire brains remains a challenge. Here, we present ssEM data for the complete brain of a larval zebrafish (Danio rerio) at 5.5 days post-fertilization. Our approach utilizes multiple rounds of targeted imaging at different scales to reduce acquisition time and data management requirements. The resulting dataset can be analysed to reconstruct neuronal processes, permitting us to survey all myelinated axons (the projectome). These reconstructions enable precise investigations of neuronal morphology, which reveal remarkable bilateral symmetry in myelinated reticulospinal and lateral line afferent axons. We further set the stage for whole-brain structure-function comparisons by co-registering functional reference atlases and in vivo two-photon fluorescence microscopy data from the same specimen. All obtained images and reconstructions are provided as an open-access resource.

Nicotine increases fear responses and brain acetylcholinesterase activity in a context-dependent manner in zebrafish.

PubMed

Ziani, Paola R; Müller, Talise E; Stefanello, Flavia V; Fontana, Barbara D; Duarte, Tâmie; Canzian, Julia; Rosemberg, Denis B

2018-07-01

Nicotine is an alkaloid with positive effects on learning and memory processes. Exposure to conspecific alarm substance (CAS) elicits fear responses in zebrafish, but the effects of nicotine on aversive behaviors and associative learning in this species remain unclear. Here, we evaluated whether nicotine enhances contextual fear responses in zebrafish and investigated a putative involvement of brain acetylcholinesterase (AChE) in associative learning. Fish were exposed to 1 mg/L nicotine for 3 min and then kept in non-chlorinated water for 20 min. Later, animals were transferred to experimental tanks in the absence or presence of 3.5 mL/L CAS for 5 min (training session). After 24 h, fish were tested in tanks with similar or altered context in the absence of CAS (post-training session) and brain AChE activity was further assessed. At training, CAS increased freezing, erratic movements, and decreased the time spent in top area, while nicotine abolished the effects of CAS on erratic movements. Nicotine/CAS group tested in a similar context showed exacerbated freezing and reduced transitions to top area. Moreover, a decrease in distance traveled was observed in control, nicotine, and nicotine/CAS groups at post-training. Nicotine also stimulated brain AChE activity in CAS-exposed animals reintroduced in tanks with similar context. Although freezing bouts and time spent in top could serve as behavioral endpoints that reflect CAS-induced sensitization, the effects of nicotine occurred in a context-dependent manner. Collectively, our data suggest an involvement of cholinergic signaling in aversive learning, reinforcing the growing utility of zebrafish models to explore the neurobehavioral effects of nicotine in vertebrates. Copyright © 2018 Elsevier Inc. All rights reserved.

A review of monoaminergic neuropsychopharmacology in zebrafish.

PubMed

Maximino, Caio; Herculano, Anderson Manoel

2010-12-01

Monoamine neurotransmitters are the major regulatory mechanisms in the vertebrate brain, involved in the adjustment of motivation, emotion, and cognition. The chemical anatomy of these systems is thought to be highly conserved in the brain of all vertebrates, including zebrafish. Recently, the development of behavioral assays in zebrafish allowed the neuropsychopharmacological investigation of these circuits and its functions. Here we review neuroanatomical, genetic, neurochemical, and psychopharmacological evidence regarding the roles of histaminergic, dopaminergic, noradrenergic, serotonergic, and melatonergic systems in this species. We conclude that, in spite of species differences, zebrafish are suitable for the investigation of neuropsychopharmacology of drugs that affect theses systems; nonetheless, more thorough validation of behavioral methods is still needed.

Startle response memory and hippocampal changes in adult zebrafish pharmacologically-induced to exhibit anxiety/depression-like behaviors.

PubMed

Pittman, Julian T; Lott, Chad S

2014-01-17

Zebrafish (Danio rerio) are rapidly becoming a popular animal model for neurobehavioral and psychopharmacological research. While startle testing is a well-established assay to investigate anxiety-like behaviors in different species, screening of the startle response and its habituation in zebrafish is a new direction of translational biomedical research. This study focuses on a novel behavioral protocol to assess a tapping-induced startle response and its habituation in adult zebrafish that have been pharmacologically-induced to exhibit anxiety/depression-like behaviors. We demonstrated that zebrafish exhibit robust learning performance in a task adapted from the mammalian literature, a modified plus maze, and showed that ethanol and fluoxetine impair memory performance in this maze when administered after training at a dose that does not impair motor function, however, leads to significant upregulation of hippocampal serotoninergic neurons. These results suggest that the maze associative learning paradigm has face and construct validity and that zebrafish may become a translationally relevant study species for the analysis of the mechanisms of learning and memory changes associated with psychopharmacological treatment of anxiety/depression. © 2013.

Mitragynine Attenuates Withdrawal Syndrome in Morphine-Withdrawn Zebrafish

PubMed Central

Khor, Beng-Siang; Amar Jamil, Mohd Fadzly; Adenan, Mohamad Ilham; Chong Shu-Chien, Alexander

2011-01-01

A major obstacle in treating drug addiction is the severity of opiate withdrawal syndrome, which can lead to unwanted relapse. Mitragynine is the major alkaloid compound found in leaves of Mitragyna speciosa, a plant widely used by opiate addicts to mitigate the harshness of drug withdrawal. A series of experiments was conducted to investigate the effect of mitragynine on anxiety behavior, cortisol level and expression of stress pathway related genes in zebrafish undergoing morphine withdrawal phase. Adult zebrafish were subjected to two weeks chronic morphine exposure at 1.5 mg/L, followed by withdrawal for 24 hours prior to tests. Using the novel tank diving tests, we first showed that morphine-withdrawn zebrafish display anxiety-related swimming behaviors such as decreased exploratory behavior and increased erratic movement. Morphine withdrawal also elevated whole-body cortisol levels, which confirms the phenotypic stress-like behaviors. Exposing morphine-withdrawn fish to mitragynine however attenuates majority of the stress-related swimming behaviors and concomitantly lower whole-body cortisol level. Using real-time PCR gene expression analysis, we also showed that mitragynine reduces the mRNA expression of corticotropin releasing factor receptors and prodynorphin in zebrafish brain during morphine withdrawal phase, revealing for the first time a possible link between mitragynine's ability to attenuate anxiety during opiate withdrawal with the stress-related corticotropin pathway. PMID:22205946

Automated processing of zebrafish imaging data: a survey.

PubMed

Mikut, Ralf; Dickmeis, Thomas; Driever, Wolfgang; Geurts, Pierre; Hamprecht, Fred A; Kausler, Bernhard X; Ledesma-Carbayo, María J; Marée, Raphaël; Mikula, Karol; Pantazis, Periklis; Ronneberger, Olaf; Santos, Andres; Stotzka, Rainer; Strähle, Uwe; Peyriéras, Nadine

2013-09-01

Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines.

Automated Processing of Zebrafish Imaging Data: A Survey

PubMed Central

Dickmeis, Thomas; Driever, Wolfgang; Geurts, Pierre; Hamprecht, Fred A.; Kausler, Bernhard X.; Ledesma-Carbayo, María J.; Marée, Raphaël; Mikula, Karol; Pantazis, Periklis; Ronneberger, Olaf; Santos, Andres; Stotzka, Rainer; Strähle, Uwe; Peyriéras, Nadine

2013-01-01

Abstract Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines. PMID:23758125

Automatic multiple zebrafish larvae tracking in unconstrained microscopic video conditions.

PubMed

Wang, Xiaoying; Cheng, Eva; Burnett, Ian S; Huang, Yushi; Wlodkowic, Donald

2017-12-14

The accurate tracking of zebrafish larvae movement is fundamental to research in many biomedical, pharmaceutical, and behavioral science applications. However, the locomotive characteristics of zebrafish larvae are significantly different from adult zebrafish, where existing adult zebrafish tracking systems cannot reliably track zebrafish larvae. Further, the far smaller size differentiation between larvae and the container render the detection of water impurities inevitable, which further affects the tracking of zebrafish larvae or require very strict video imaging conditions that typically result in unreliable tracking results for realistic experimental conditions. This paper investigates the adaptation of advanced computer vision segmentation techniques and multiple object tracking algorithms to develop an accurate, efficient and reliable multiple zebrafish larvae tracking system. The proposed system has been tested on a set of single and multiple adult and larvae zebrafish videos in a wide variety of (complex) video conditions, including shadowing, labels, water bubbles and background artifacts. Compared with existing state-of-the-art and commercial multiple organism tracking systems, the proposed system improves the tracking accuracy by up to 31.57% in unconstrained video imaging conditions. To facilitate the evaluation on zebrafish segmentation and tracking research, a dataset with annotated ground truth is also presented. The software is also publicly accessible.

Single-cell in vivo imaging of adult neural stem cells in the zebrafish telencephalon.

PubMed

Barbosa, Joana S; Di Giaimo, Rossella; Götz, Magdalena; Ninkovic, Jovica

2016-08-01

Adult neural stem cells (aNSCs) in zebrafish produce mature neurons throughout their entire life span in both the intact and regenerating brain. An understanding of the behavior of aNSCs in their intact niche and during regeneration in vivo should facilitate the identification of the molecular mechanisms controlling regeneration-specific cellular events. A greater understanding of the process in regeneration-competent species may enable regeneration to be achieved in regeneration-incompetent species, including humans. Here we describe a protocol for labeling and repetitive imaging of aNSCs in vivo. We label single aNSCs, allowing nonambiguous re-identification of single cells in repetitive imaging sessions using electroporation of a red-reporter plasmid in Tg(gfap:GFP)mi2001 transgenic fish expressing GFP in aNSCs. We image using two-photon microscopy through the thinned skull of anesthetized and immobilized fish. Our protocol allows imaging every 2 d for a period of up to 1 month. This methodology allowed the visualization of aNSC behavior in vivo in their natural niche, in contrast to previously available technologies, which rely on the imaging of either dissociated cells or tissue slices. We used this protocol to follow the mode of aNSC division, fate changes and cell death in both the intact and injured zebrafish telencephalon. This experimental setup can be widely used, with minimal prior experience, to assess key factors for processes that modulate aNSC behavior. A typical experiment with data analysis takes up to 1.5 months.

The utility of zebrafish to study the mechanisms by which ethanol affects social behavior and anxiety during early brain development.

PubMed

Parker, Matthew O; Annan, Leonette V; Kanellopoulos, Alexandros H; Brock, Alistair J; Combe, Fraser J; Baiamonte, Matteo; Teh, Muy-Teck; Brennan, Caroline H

2014-12-03

Exposure to moderate levels of ethanol during brain development has a number of effects on social behavior but the molecular mechanisms that mediate this are not well understood. Gaining a better understanding of these factors may help to develop therapeutic interventions in the future. Zebrafish offer a potentially useful model in this regard. Here, we introduce a zebrafish model of moderate prenatal ethanol exposure. Embryos were exposed to 20mM ethanol for seven days (48hpf-9dpf) and tested as adults for individual social behavior and shoaling. We also tested their basal anxiety with the novel tank diving test. We found that the ethanol-exposed fish displayed reductions in social approach and shoaling, and an increase in anxiety in the novel tank test. These behavioral differences corresponded to differences in hrt1aa, slc6a4 and oxtr expression. Namely, acute ethanol caused a spike in oxtr and ht1aa mRNA expression, which was followed by down-regulation at 7dpf, and an up-regulation in slc6a4 at 72hpf. This study confirms the utility of zebrafish as a model system for studying the molecular basis of developmental ethanol exposure. Furthermore, it proposes a putative developmental mechanism characterized by ethanol-induced OT inhibition leading to suppression of 5-HT and up-regulation of 5-HT1A, which leads, in turn, to possible homeostatic up-regulation of 5-HTT at 72hpf and subsequent imbalance of the 5-HT system. Copyright © 2014 Elsevier Inc. All rights reserved.

The neurogenetic frontier--lessons from misbehaving zebrafish.

PubMed

Burgess, Harold A; Granato, Michael

2008-11-01

One of the central questions in neuroscience is how refined patterns of connectivity in the brain generate and monitor behavior. Genetic mutations can influence neural circuits by disrupting differentiation or maintenance of component neuronal cells or by altering functional patterns of nervous system connectivity. Mutagenesis screens therefore have the potential to reveal not only the molecular underpinnings of brain development and function, but to illuminate the cellular basis of behavior. Practical considerations make the zebrafish an organism of choice for undertaking forward genetic analysis of behavior. The powerful array of experimental tools at the disposal of the zebrafish researcher makes it possible to link molecular function to neuronal properties that underlie behavior. This review focuses on specific challenges to isolating and analyzing behavioral mutants in zebrafish.

Culture of adult transgenic zebrafish retinal explants for live-cell imaging by multiphoton microscopy

PubMed Central

Lahne, Manuela; Gorsuch, Ryne A; Nelson, Craig M; Hyde, David R

2017-01-01

An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina i.e. they migrate between the basal inner nuclear layer (INL) and the outer nuclear layer (ONL), respectively, in a process described as interkinetic nuclear migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM. PMID:28287581

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy.

PubMed

Lahne, Manuela; Gorsuch, Ryne A; Nelson, Craig M; Hyde, David R

2017-02-24

An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP] mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

In silico predicted reproductive endocrine transcriptional regulatory networks during zebrafish (Danio rerio) development.

PubMed

Hala, D

2017-03-21

The interconnected topology of transcriptional regulatory networks (TRNs) readily lends to mathematical (or in silico) representation and analysis as a stoichiometric matrix. Such a matrix can be 'solved' using the mathematical method of extreme pathway (ExPa) analysis, which identifies uniquely activated genes subject to transcription factor (TF) availability. In this manuscript, in silico multi-tissue TRN models of brain, liver and gonad were used to study reproductive endocrine developmental programming in zebrafish (Danio rerio) from 0.25h post fertilization (hpf; zygote) to 90 days post fertilization (dpf; adult life stage). First, properties of TRN models were studied by sequentially activating all genes in multi-tissue models. This analysis showed the brain to exhibit lowest proportion of co-regulated genes (19%) relative to liver (23%) and gonad (32%). This was surprising given that the brain comprised 75% and 25% more TFs than liver and gonad respectively. Such 'hierarchy' of co-regulatory capability (brainbrain, alluding to its role as 'master controller' of endocrine function. Second, TRN models were constrained with varying TF availabilities during zebrafish development. Normalized numbers of genes active during development showed concomitant activations between brain and gonad from 10 to 12 hpf (embryonic life stage) up to 30-90 dpf (adult life stage). This indicated a putative 'syncing' between the brain and gonad, and initiation of an early reproductive endocrine developmental program. Finally, comparison of in vivo active genes with those predicted in silico showed relatively good agreement for brain (49%), liver (27%) and gonad (32%). The multi-tissue TRN models presented can lend diagnostic insights into the effects of changing environmental and/or genetic constraints on reproductive endocrine function. Copyright © 2017 Elsevier Ltd. All rights reserved.

Contextual Fear Conditioning in Zebrafish

ERIC Educational Resources Information Center

Kenney, Justin W.; Scott, Ian C.; Josselyn, Sheena A.; Frankland, Paul W.

2017-01-01

Zebrafish are a genetically tractable vertebrate that hold considerable promise for elucidating the molecular basis of behavior. Although numerous recent advances have been made in the ability to precisely manipulate the zebrafish genome, much less is known about many aspects of learning and memory in adult fish. Here, we describe the development…

Re-epithelialization of cutaneous wounds in adult zebrafish combines mechanisms of wound closure in embryonic and adult mammals.

PubMed

Richardson, Rebecca; Metzger, Manuel; Knyphausen, Philipp; Ramezani, Thomas; Slanchev, Krasimir; Kraus, Christopher; Schmelzer, Elmon; Hammerschmidt, Matthias

2016-06-15

Re-epithelialization of cutaneous wounds in adult mammals takes days to complete and relies on numerous signalling cues and multiple overlapping cellular processes that take place both within the epidermis and in other participating tissues. Re-epithelialization of partial- or full-thickness skin wounds of adult zebrafish, however, is extremely rapid and largely independent of the other processes of wound healing. Live imaging after treatment with transgene-encoded or chemical inhibitors reveals that re-epithelializing keratinocytes repopulate wounds by TGF-β- and integrin-dependent lamellipodial crawling at the leading edges of the epidermal tongue. In addition, re-epithelialization requires long-range epithelial rearrangements, involving radial intercalations, flattening and directed elongation of cells - processes that are dependent on Rho kinase, JNK and, to some extent, planar cell polarity within the epidermis. These rearrangements lead to a massive recruitment of keratinocytes from the adjacent epidermis and make re-epithelialization independent of keratinocyte proliferation and the mitogenic effect of FGF signalling, which are only required after wound closure, allowing the epidermis outside the wound to re-establish its normal thickness. Together, these results demonstrate that the adult zebrafish is a valuable in vivo model for studying and visualizing the processes involved in cutaneous wound closure, facilitating the dissection of direct from indirect and motogenic from mitogenic effects of genes and molecules affecting wound re-epithelialization. © 2016. Published by The Company of Biologists Ltd.

The neurogenetic frontier—lessons from misbehaving zebrafish

PubMed Central

Granato, Michael

2008-01-01

One of the central questions in neuroscience is how refined patterns of connectivity in the brain generate and monitor behavior. Genetic mutations can influence neural circuits by disrupting differentiation or maintenance of component neuronal cells or by altering functional patterns of nervous system connectivity. Mutagenesis screens therefore have the potential to reveal not only the molecular underpinnings of brain development and function, but to illuminate the cellular basis of behavior. Practical considerations make the zebrafish an organism of choice for undertaking forward genetic analysis of behavior. The powerful array of experimental tools at the disposal of the zebrafish researcher makes it possible to link molecular function to neuronal properties that underlie behavior. This review focuses on specific challenges to isolating and analyzing behavioral mutants in zebrafish. PMID:18836206

Neurobehavioural Changes and Brain Oxidative Stress Induced by Acute Exposure to GSM900 Mobile Phone Radiations in Zebrafish (Danio rerio)

PubMed Central

Nirwane, Abhijit; Sridhar, Vinay; Majumdar, Anuradha

2016-01-01

The impact of mobile phone (MP) radiation on the brain is of specific interest to the scientific community and warrants investigations, as MP is held close to the head. Studies on humans and rodents revealed hazards MP radiation associated such as brain tumors, impairment in cognition, hearing etc. Melatonin (MT) is an important modulator of CNS functioning and is a neural antioxidant hormone. Zebrafish has emerged as a popular model organism for CNS studies. Herein, we evaluated the impact of GSM900MP (GSM900MP) radiation exposure daily for 1 hr for 14 days with the SAR of 1.34W/Kg on neurobehavioral and oxidative stress parameters in zebrafish. Our study revealed that, GSM900MP radiation exposure, significantly decreased time spent near social stimulus zone and increased total distance travelled, in social interaction test. In the novel tank dive test, the GSM900MP radiation exposure elicited anxiety as revealed by significantly increased time spent in bottom half; freezing bouts and duration and decreased distance travelled, average velocity, and number of entries to upper half of the tank. Exposed zebrafish spent less time in the novel arm of the Y-Maze, corroborating significant impairment in learning as compared to the control group. Exposure decreased superoxide dismutase (SOD), catalase (CAT) activities whereas, increased levels of reduced glutathione (GSH) and lipid peroxidation (LPO) was encountered showing compromised antioxidant defense. Treatment with MT significantly reversed the above neurobehavioral and oxidative derangements induced by GSM900MP radiation exposure. This study traced GSM900MP radiation exposure induced neurobehavioral aberrations and alterations in brain oxidative status. Furthermore, MT proved to be a promising therapeutic candidate in ameliorating such outcomes in zebrafish. PMID:27123163

Characterization of heme oxygenase and biliverdin reductase gene expression in zebrafish (Danio rerio): Basal expression and response to pro-oxidant exposures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holowiecki, Andrew

While heme is an important cofactor for numerous proteins, it is highly toxic in its unbound form and can perpetuate the formation of reactive oxygen species. Heme oxygenase enzymes (HMOX1 and HMOX2) degrade heme into biliverdin and carbon monoxide, with biliverdin subsequently being converted to bilirubin by biliverdin reductase (BVRa or BVRb). As a result of the teleost-specific genome duplication event, zebrafish have paralogs of hmox1 (hmox1a and hmox1b) and hmox2 (hmox2a and hmox2b). Expression of all four hmox paralogs and two bvr isoforms were measured in adult tissues (gill, brain and liver) and sexually dimorphic differences were observed, mostmore » notably in the basal expression of hmox1a, hmox2a, hmox2b and bvrb in liver samples. hmox1a, hmox2a and hmox2b were significantly induced in male liver tissues in response to 96 h cadmium exposure (20 μM). hmox2a and hmox2b were significantly induced in male brain samples, but only hmox2a was significantly reduced in male gill samples in response to the 96 h cadmium exposure. hmox paralogs displayed significantly different levels of basal expression in most adult tissues, as well as during zebrafish development (24 to 120 hpf). Furthermore, hmox1a, hmox1b and bvrb were significantly induced in zebrafish eleutheroembryos in response to multiple pro-oxidants (cadmium, hemin and tert-butylhydroquinone). Knockdown of Nrf2a, a transcriptional regulator of hmox1a, was demonstrated to inhibit the Cd-mediated induction of hmox1b and bvrb. These results demonstrate distinct mechanisms of hmox and bvr transcriptional regulation in zebrafish, providing initial evidence of the partitioning of function of the hmox paralogs. - Highlights: • hmox1a, hmox2a, hmox2b and bvrb are sexually dimorphic in expression. • hmox paralogs were induced in adult tissues by cadmium exposure. • hmox1a, hmox1b and bvrb were induced by multiple pro-oxidants zebrafish embryos. • Differential expression of zebrafish hmox paralogs

Single Low-Dose Ionizing Radiation Induces Genotoxicity in Adult Zebrafish and its Non-Irradiated Progeny.

PubMed

Lemos, J; Neuparth, T; Trigo, M; Costa, P; Vieira, D; Cunha, L; Ponte, F; Costa, P S; Metello, L F; Carvalho, A P

2017-02-01

This study investigated to what extent a single exposure to low doses of ionizing radiation can induce genotoxic damage in irradiated adult zebrafish (Danio rerio) and its non-irradiated F1 progeny. Four groups of adult zebrafish were irradiated with a single dose of X-rays at 0 (control), 100, 500 and 1000 mGy, respectively, and couples of each group were allowed to reproduce following irradiation. Blood of parental fish and whole-body offspring were analysed by the comet assay for detection of DNA damage. The level of DNA damage in irradiated parental fish increased in a radiation dose-dependent manner at day 1 post-irradiation, but returned to the control level thereafter. The level of DNA damage in the progeny was directly correlated with the parental irradiation dose. Results highlight the genotoxic risk of a single exposure to low-dose ionizing radiation in irradiated individuals and also in its non-irradiated progeny.

Recent advancements in understanding endogenous heart regeneration—insights from adult zebrafish and neonatal mice

PubMed Central

Rubin, Nicole; Harrison, Michael R.; Krainock, Michael; Kim, Richard; Lien, Ching-Ling

2016-01-01

Enhancing the endogenous regenerative capacity of the mammalian heart is a promising strategy that can lead to potential treatment of injured cardiac tissues. Studies on heart regeneration in zebrafish and neonatal mice have shown that cardiomyocyte proliferation is essential for replenishing myocardium. We will review recent advancements that have demonstrated the importance of Neuregulin 1/ErbB2 and innervation in regulating cardiomyocyte proliferation using both adult zebrafish and neonatal mouse heart regeneration models. Emerging findings suggest that different populations of macrophages and inflammation might contribute to regenerative versus fibrotic responses. Finally, we will discuss variation in the severity of the cardiac injury and size of the wound, which may explain the range of outcomes observed in different injury models. PMID:27132022

Subchronic atrazine exposure changes defensive behaviour profile and disrupts brain acetylcholinesterase activity of zebrafish.

PubMed

Schmidel, Ademir J; Assmann, Karla L; Werlang, Chariane C; Bertoncello, Kanandra T; Francescon, Francini; Rambo, Cassiano L; Beltrame, Gabriela M; Calegari, Daiane; Batista, Cibele B; Blaser, Rachel E; Roman Júnior, Walter A; Conterato, Greicy M M; Piato, Angelo L; Zanatta, Leila; Magro, Jacir Dal; Rosemberg, Denis B

2014-01-01

Animal behaviour is the interaction between environment and an individual organism, which also can be influenced by its neighbours. Variations in environmental conditions, as those caused by contaminants, may lead to neurochemical impairments altering the pattern of the behavioural repertoire of the species. Atrazine (ATZ) is an herbicide widely used in agriculture that is frequently detected in surface water, affecting non-target species. The zebrafish is a valuable model organism to assess behavioural and neurochemical effects of different contaminants since it presents a robust behavioural repertoire and also all major neurotransmitter systems described for mammalian species. The goal of this study was to evaluate the effects of subchronic ATZ exposure in defensive behaviours of zebrafish (shoaling, thigmotaxis, and depth preference) using the split depth tank. Furthermore, to investigate a putative role of cholinergic signalling on ATZ-mediated effects, we tested whether this herbicide alters acetylcholinesterase (AChE) activity in brain and muscle preparations. Fish were exposed to ATZ for 14days and the following groups were tested: control (0.2% acetone) and ATZ (10 and 1000μg/L). The behaviour of four animals in the same tank was recorded for 6min and biological samples were prepared. Our results showed that 1000μg/L ATZ significantly increased the inter-fish distance, as well as the nearest and farthest neighbour distances. This group also presented an increase in the shoal area with decreased social interaction. No significant differences were detected for the number of animals in the shallow area, latency to enter the shallow and time spent in shallow and deep areas of the apparatus, but the ATZ 1000 group spent significantly more time near the walls. Although ATZ did not affect muscular AChE, it significantly reduced AChE activity in brain. Exposure to 10μg/L ATZ did not affect behaviour or AChE activity. These data suggest that ATZ impairs defensive

Integrated toxic evaluation of sulfamethazine on zebrafish: Including two lifespan stages (embryo-larval and adult) and three exposure periods (exposure, post-exposure and re-exposure).

PubMed

Yan, Zhengyu; Yang, Qiulian; Jiang, Weili; Lu, Jilai; Xiang, Zhongrun; Guo, Ruixin; Chen, Jianqiu

2018-03-01

Persistence of antibiotics in aquatic environment may pose a risk to the non-target aquatic organisms. This study provided an integrated evaluation to analyze the toxic stress of sulfamethazine (SMZ) on zebrafish in two lifespan stages (embryo-larval and adult) and three exposure periods (exposure, post-exposure and re-exposure). Zebrafish embryos and adult zebrafish were exposed to SMZ at 0.2, 20 and 2000 μg/L, respectively. The results showed that SMZ at any given concentration inhibited the hatching of embryos at 58-96 hpf (hours post-fertilization). Our result also indicated that two major kinds of the malformation, which was induced by the antibiotic, were edema and spinal curvature. Additionally, the antibiotic stimulated the heartbeat while reduced the body length of the embryo at 72 hpf. Superoxide dismutase (SOD) activities and malondialdehyde (MDA) contents significantly increased at 120 hpf when the embryos were exposed to the lowest concentration (0.2 μg/L) of the antibiotic. On the other hand, the antibiotic induced SOD activities and MDA contents in adult zebrafish in the exposure and re-exposure periods. The MDA contents could recover while SOD activities still increased in 2 d after the exposure. Both SOD activities and MDA contents could recover in 7 d after the exposure. Levels of SOD and MDA in the re-exposure were higher than those in the first exposure. Our results suggested that SMZ had toxic effects on both embryos and adult zebrafish, and provided an integrated evaluation of the toxic effects of SMZ on zebrafish at a new perspective. Copyright © 2017 Elsevier Ltd. All rights reserved.

Reproductive impacts and physiological adaptations of zebrafish to elevated dietary nickel.

PubMed

Alsop, Derek; Lall, Santosh P; Wood, Chris M

2014-09-01

Nickel (Ni) concentrations in the environment can rise due to human industrial activities. The toxicity of waterborne Ni to aquatic animals has been examined in a number of previous studies; however, little is known about the impacts of elevated dietary Ni. In the present study, zebrafish were chronically fed diets containing two concentrations of Ni [3.7 (control) and 116 μg Ni/g diet]. Ni-exposed males, but not females, were significantly smaller (26%) compared to controls at 80 days. In addition, total egg production was decreased by 65% in the Ni treatment at 75-78 days of the experiment. Ni was ubiquitously distributed in control animals (similar to previous studies), and concentrations varied between tissues by 15-fold. Ni exposure resulted in modest but significant Ni accumulation in some tissues (increases were highest in brain, vertebrae and gut; 44%, 34% and 25%, respectively), an effect observed only at 80 days. The limited Ni accumulation may be due to (1) the lack of an acidified stomach in zebrafish and/or (2) the efficient upregulation of Ni transport and excretion mechanisms, as indicated by the 4.5-fold increase in waterborne (63)Ni uptake by Ni-exposed fish. Eggs from Ni-exposed adults had Ni concentrations that were 5.2-fold higher than controls. However, by 4 days post fertilization, larvae had similar Ni concentrations as controls, demonstrating a capacity for rapid Ni depuration. Larvae from Ni-exposed adults were also more resistant to waterborne Ni (35% increase in the 96-h LC50 over controls). In conclusion, elevated dietary Ni significantly affected zebrafish reproduction despite only modest tissue Ni accumulation. There were also indications of adaptation, including increased Ni uptake rates and increased Ni tolerance of offspring from Ni-exposed adults. Ni concentrations were particularly elevated in the brain with exposure; possible relations to growth and reproductive impacts require further study. Copyright © 2014 Elsevier Inc. All

Environmental estrogen(s) induced swimming behavioural alterations in adult zebrafish (Danio rerio).

PubMed

Goundadkar, Basavaraj B; Katti, Pancharatna

2017-09-01

The present study is an attempt to investigate the effects of long-term (75days) exposure to environmental estrogens (EE) on the swimming behaviour of zebrafish (Danio rerio). Adult zebrafish were exposed semi-statically to media containing commonly detected estrogenic water contaminants (EE2, DES and BPA) at a concentration (5ng/L) much lower than environmentally recorded levels. Time spent in swimming, surface preference, patterns and path of swimming were recorded (6mins) for each fish using two video cameras on day 15, 30 60 and 75. Video clips were analysed using a software program. Results indicate that chronic exposure to EE leads to increased body weight and size of females, reduced (P<0.05) swimming time, delay in latency, increased (P<0.05) immobility, erratic movements and freezing episodes. We conclude that estrogenic contamination of natural aquatic systems induces alterations in locomotor behaviour and associated physiological disturbances in inhabitant fish fauna. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficient Cargo Delivery into Adult Brain Tissue Using Short Cell-Penetrating Peptides.

PubMed

Kizil, Caghan; Iltzsche, Anne; Thomas, Alvin Kuriakose; Bhattarai, Prabesh; Zhang, Yixin; Brand, Michael

2015-01-01

Zebrafish brains can regenerate lost neurons upon neurogenic activity of the radial glial progenitor cells (RGCs) that reside at the ventricular region. Understanding the molecular events underlying this ability is of great interest for translational studies of regenerative medicine. Therefore, functional analyses of gene function in RGCs and neurons are essential. Using cerebroventricular microinjection (CVMI), RGCs can be targeted efficiently but the penetration capacity of the injected molecules reduces dramatically in deeper parts of the brain tissue, such as the parenchymal regions that contain the neurons. In this report, we tested the penetration efficiency of five known cell-penetrating peptides (CPPs) and identified two- polyR and Trans - that efficiently penetrate the brain tissue without overt toxicity in a dose-dependent manner as determined by TUNEL staining and L-Plastin immunohistochemistry. We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael's addition and azide-mediated copper-free click reaction. Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues.

Response mechanisms to joint exposure of triclosan and its chlorinated derivatives on zebrafish (Danio rerio) behavior.

PubMed

Liu, Jinfeng; Sun, Limei; Zhang, Hongqin; Shi, Mengru; Dahlgren, Randy A; Wang, Xuedong; Wang, Huili

2018-02-01

Triclosan (TCS), 2,4,6-trichlorophenol (2,4,6-TCP) and 2,4-dichlorophenol (2,4-DCP) frequently co-exist in real-world aquatic environments; the latter two contaminants contributing to TCS photolytic products or chlorinated derivatives. There is a paucity of information regarding their joint toxicity to aquatic organisms leading us to study their effects on the swimming behavior of zebrafish (Danio rerio). Herein, we reported that 0.28 mg/L TDT exposure (mixtures of TCS, 2,4,6-TCP and 2,4-DCP) enhanced 24-hpf embryonic spontaneous movement frequency, 96-hpf larval activity; however, the 0.56 and 1.12 mg/L TDT treatments decreased all of these behavioral endpoints. All adult behavioral tests demonstrated that chronic TDT exposure (0.14 mg/L) led to hyperactivity and restlessness in adult zebrafish. A 0.14 mg/L TD DATE /@ "M/d/yyyy" 11/21/2017T treatment led to anxiety-like behavior in a bottom dwelling test and excessive panic and low hedging capacity in a conditioned place preference test. Social interaction test demonstrated that zebrafish preferred quiet and isolated space in response to TDT stress. Zebrafish memory was significantly decreased in a T-maze experiment. Whole mount in situ hybridization of pax2a and bcl2l11 genes revealed that their differential expression in the brain and skeleton were related to the corresponding phenotypic behavioral abnormality. A series of biomarker and estrogen receptor assays demonstrated that TDT acute exposure caused abnormal energy metabolism and neurological diseases. AO staining revealed that TDT exposure produced vascular ablation in the head, as well as the occurrence of massive apoptosis in the brain. TEM observation showed pyknosis of nucleus following TDT exposure. These results allow assessment of mechanisms for zebrafish abnormal behavior in response to TDT exposure, and are useful for early intervention and gene therapy of contaminant-induced diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments.

PubMed

Giarmarco, Michelle M; Cleghorn, Whitney M; Hurley, James B; Brockerhoff, Susan E

2018-05-09

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method's versatility. This protocol was developed for imaging Ca 2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca 2+ or metabolites in Müller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca 2+ , and energy homeostasis.

DNA methylation regulates gabrb2 mRNA expression: developmental variations and disruptions in l-methionine-induced zebrafish with schizophrenia-like symptoms.

PubMed

Wang, L; Jiang, W; Lin, Q; Zhang, Y; Zhao, C

2016-11-01

Single nucleotide polymorphisms (SNPs) in the human type A gamma-aminobutyric acid (GABA) receptor β 2 subunit gene (GABRB2) have been associated with schizophrenia and quantitatively correlated with mRNA expression in the postmortem brain tissue of patients with schizophrenia. l-Methionine (MET) administration has been reported to cause a recrudescence of psychotic symptoms in patients with schizophrenia, and similar symptoms have been generated in MET-induced mice. In this study, a zebrafish animal model was used to evaluate the relationship between the gabrb2 mRNA expression and its promoter DNA methylation in developmental and MET-induced schizophrenia-like zebrafish. The results indicated developmental increases in global DNA methylation and decreases in gabrb2 promoter methylation in zebrafish. A significant increase in gabrb2 mRNA levels was observed after GABA was synthesized. Additionally, the MET-triggered schizophrenia-like symptoms in adult zebrafish, involving social withdrawal and cognitive dysfunction analyzed with social interaction and T-maze behavioral tests, were accompanied by significantly increased DNA methylation levels in the global genome and the gabrb2 promoter. Furthermore, the significant correlation between gabrb2 mRNA expression and gabrb2 promoter methylation observed in the developmental stages became non-significant in MET-triggered adult zebrafish. These findings demonstrate that gabrb2 mRNA expression is associated with DNA methylation varies by developmental stage and show that these epigenetic association mechanisms are disrupted in MET-triggered adult zebrafish with schizophrenia-like symptoms. In conclusion, these results provide plausible epigenetic evidence of the GABA A receptor β 2 subunit involvement in the schizophrenia-like behaviors and demonstrate the potential use of zebrafish models in neuropsychiatric research. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Left Brain/Right Brain Learning for Adult Education.

ERIC Educational Resources Information Center

Garvin, Barbara

1986-01-01

Contrasts and compares the theory and practice of adult education as it relates to the issue of right brain/left brain learning. The author stresses the need for a whole-brain approach to teaching and suggests that adult educators, given their philosophical directions, are the perfect potential users of this integrated system. (Editor/CT)

Behavioral Changes Over Time Following Ayahuasca Exposure in Zebrafish

PubMed Central

Savoldi, Robson; Polari, Daniel; Pinheiro-da-Silva, Jaquelinne; Silva, Priscila F.; Lobao-Soares, Bruno; Yonamine, Mauricio; Freire, Fulvio A. M.; Luchiari, Ana C.

2017-01-01

The combined infusion of Banisteriopsis caapi stem and Psychotria viridis leaves, known as ayahuasca, has been used for centuries by indigenous tribes. The infusion is rich in N, N-dimethyltryptamine (DMT) and monoamine oxidase inhibitors, with properties similar to those of serotonin. Despite substantial progress in the development of new drugs to treat anxiety and depression, current treatments have several limitations. Alternative drugs, such as ayahuasca, may shed light on these disorders. Here, we present time-course behavioral changes induced by ayahuasca in zebrafish, as first step toward establishing an ideal concentration for pre-clinical evaluations. We exposed adult zebrafish to five concentrations of the ayahuasca infusion: 0 (control), 0.1, 0.5, 1, and 3 ml/L (n = 14 each group), and behavior was recorded for 60 min. We evaluated swimming speed, distance traveled, freezing and bottom dwelling every min for 60 min. Swimming speed and distance traveled decreased with an increase in ayahuasca concentration while freezing increased with 1 and 3 ml/L. Bottom dwelling increased with 1 and 3 ml/L, but declined with 0.1 ml/L. Our data suggest that small amounts of ayahuasca do not affect locomotion and reduce anxiety-like behavior in zebrafish, while increased doses of the drug lead to crescent anxiogenic effects. We conclude that the temporal analysis of zebrafish behavior is a sensitive method for the study of ayahuasca-induced functional changes in the vertebrate brain. PMID:28804451

Behavioral Changes Over Time Following Ayahuasca Exposure in Zebrafish.

PubMed

Savoldi, Robson; Polari, Daniel; Pinheiro-da-Silva, Jaquelinne; Silva, Priscila F; Lobao-Soares, Bruno; Yonamine, Mauricio; Freire, Fulvio A M; Luchiari, Ana C

2017-01-01

The combined infusion of Banisteriopsis caapi stem and Psychotria viridis leaves, known as ayahuasca, has been used for centuries by indigenous tribes. The infusion is rich in N , N -dimethyltryptamine (DMT) and monoamine oxidase inhibitors, with properties similar to those of serotonin. Despite substantial progress in the development of new drugs to treat anxiety and depression, current treatments have several limitations. Alternative drugs, such as ayahuasca, may shed light on these disorders. Here, we present time-course behavioral changes induced by ayahuasca in zebrafish, as first step toward establishing an ideal concentration for pre-clinical evaluations. We exposed adult zebrafish to five concentrations of the ayahuasca infusion: 0 (control), 0.1, 0.5, 1, and 3 ml/L ( n = 14 each group), and behavior was recorded for 60 min. We evaluated swimming speed, distance traveled, freezing and bottom dwelling every min for 60 min. Swimming speed and distance traveled decreased with an increase in ayahuasca concentration while freezing increased with 1 and 3 ml/L. Bottom dwelling increased with 1 and 3 ml/L, but declined with 0.1 ml/L. Our data suggest that small amounts of ayahuasca do not affect locomotion and reduce anxiety-like behavior in zebrafish, while increased doses of the drug lead to crescent anxiogenic effects. We conclude that the temporal analysis of zebrafish behavior is a sensitive method for the study of ayahuasca-induced functional changes in the vertebrate brain.

Lactobacillus plantarum attenuates anxiety-related behavior and protects against stress-induced dysbiosis in adult zebrafish.

PubMed

Davis, Daniel J; Doerr, Holly M; Grzelak, Agata K; Busi, Susheel B; Jasarevic, Eldin; Ericsson, Aaron C; Bryda, Elizabeth C

2016-09-19

The consumption of probiotics has become increasingly popular as a means to try to improve health and well-being. Not only are probiotics considered beneficial to digestive health, but increasing evidence suggests direct and indirect interactions between gut microbiota (GM) and the central nervous system (CNS). Here, adult zebrafish were supplemented with Lactobacillus plantarum to determine the effects of probiotic treatment on structural and functional changes of the GM, as well as host neurological and behavioral changes. L. plantarum administration altered the β-diversity of the GM while leaving the major core architecture intact. These minor structural changes were accompanied by significant enrichment of several predicted metabolic pathways. In addition to GM modifications, L. plantarum treatment also significantly reduced anxiety-related behavior and altered GABAergic and serotonergic signaling in the brain. Lastly, L. plantarum supplementation provided protection against stress-induced dysbiosis of the GM. These results underscore the influence commensal microbes have on physiological function in the host, and demonstrate bidirectional communication between the GM and the host.

Neural stem cell quiescence and stemness are molecularly distinct outputs of the Notch3 signalling cascade in the vertebrate adult brain

PubMed Central

Than-Trong, Emmanuel; Ortica-Gatti, Sara; Mella, Sébastien; Nepal, Chirag; Alunni, Alessandro

2018-01-01

ABSTRACT Neural stem cells (NSCs) in the adult vertebrate brain are found in a quiescent state and can preserve long-lasting progenitor potential (stemness). Whether and how these two properties are linked, and to what extent they can be independently controlled by NSC maintenance pathways, is unresolved. We have previously identified Notch3 signalling as a major quiescence-promoting pathway in adult NSCs of the zebrafish pallium. We now show that Notch3 also controls NSC stemness. Using parallel transcriptomic characterizations of notch3 mutant NSCs and adult NSC physiological states, we demonstrate that a set of potentially direct Notch3 target genes distinguishes quiescence and stemness control. As a proof of principle, we focus on one ‘stemness’ target, encoding the bHLH transcription factor Hey1, that has not yet been analysed in adult NSCs. We show that abrogation of Hey1 function in adult pallial NSCs in vivo, including quiescent NSCs, leads to their differentiation without affecting their proliferation state. These results demonstrate that quiescence and stemness are molecularly distinct outputs of Notch3 signalling, and identify Hey1 as a major Notch3 effector controlling NSC stemness in the vertebrate adult brain. PMID:29695612

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays.

PubMed

Hinfray, N; Tebby, C; Garoche, C; Piccini, B; Bourgine, G; Aït-Aïssa, S; Kah, O; Pakdel, F; Brion, F

2016-09-15

Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase gene under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures. Copyright © 2016 Elsevier Inc. All rights reserved.

Zebrafish neurobehavioral phenomics for aquatic neuropharmacology and toxicology research.

PubMed

Kalueff, Allan V; Echevarria, David J; Homechaudhuri, Sumit; Stewart, Adam Michael; Collier, Adam D; Kaluyeva, Aleksandra A; Li, Shaomin; Liu, Yingcong; Chen, Peirong; Wang, JiaJia; Yang, Lei; Mitra, Anisa; Pal, Subharthi; Chaudhuri, Adwitiya; Roy, Anwesha; Biswas, Missidona; Roy, Dola; Podder, Anupam; Poudel, Manoj K; Katare, Deepshikha P; Mani, Ruchi J; Kyzar, Evan J; Gaikwad, Siddharth; Nguyen, Michael; Song, Cai

2016-01-01

Zebrafish (Danio rerio) are rapidly emerging as an important model organism for aquatic neuropharmacology and toxicology research. The behavioral/phenotypic complexity of zebrafish allows for thorough dissection of complex human brain disorders and drug-evoked pathological states. As numerous zebrafish models become available with a wide spectrum of behavioral, genetic, and environmental methods to test novel drugs, here we discuss recent zebrafish phenomics methods to facilitate drug discovery, particularly in the field of biological psychiatry. Additionally, behavioral, neurological, and endocrine endpoints are becoming increasingly well-characterized in zebrafish, making them an inexpensive, robust and effective model for toxicology research and pharmacological screening. We also discuss zebrafish behavioral phenotypes, experimental considerations, pharmacological candidates and relevance of zebrafish neurophenomics to other 'omics' (e.g., genomic, proteomic) approaches. Finally, we critically evaluate the limitations of utilizing this model organism, and outline future strategies of research in the field of zebrafish phenomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Polystyrene microplastics induce microbiota dysbiosis and inflammation in the gut of adult zebrafish.

PubMed

Jin, Yuanxiang; Xia, Jizhou; Pan, Zihong; Yang, Jiajing; Wang, Wenchao; Fu, Zhengwei

2018-04-01

Microplastic (MP) are environmental pollutants and have the potential to cause varying degrees of aquatic toxicity. In this study, the effects on gut microbiota of adult male zebrafish exposed for 14 days to 100 and 1000 μg/L of two sizes of polystyrene MP were evaluated. Both 0.5 and 50 μm-diameter spherical polystyrene MP increased the volume of mucus in the gut at a concentration of 1000 μg/L (about 1.456 × 10 10 particles/L for 0.5 μm and 1.456 × 10 4 particles/L for 50 μm). At the phylum level, the abundance of Bacteroidetes and Proteobacteria decreased significantly and the abundance of Firmicutes increased significantly in the gut after 14-day exposure to 1000 μg/L of both sizes of polystyrene MP. In addition, high throughput sequencing of the 16S rRNA gene V3-V4 region revealed a significant change in the richness and diversity of microbiota in the gut of polystyrene MP-exposed zebrafish. A more in depth analysis, at the genus level, revealed that a total of 29 gut microbes identified by operational taxonomic unit (OTU) analysis were significantly changed in both 0.5 and 50 μm-diameter polystyrene MP-treated groups. Moreover, it was observed that 0.5 μm polystyrene MP not only increased mRNA levels of IL1α, IL1β and IFN but also their protein levels in the gut, indicating that inflammation occurred after polystyrene MP exposure. Our findings suggest that polystyrene MP could induce microbiota dysbiosis and inflammation in the gut of adult zebrafish. Copyright © 2017 Elsevier Ltd. All rights reserved.

High-frequency dual mode pulsed wave Doppler imaging for monitoring the functional regeneration of adult zebrafish hearts

PubMed Central

Kang, Bong Jin; Park, Jinhyoung; Kim, Jieun; Kim, Hyung Ham; Lee, Changyang; Hwang, Jae Youn; Lien, Ching-Ling; Shung, K. Kirk

2015-01-01

Adult zebrafish is a well-known small animal model for studying heart regeneration. Although the regeneration of scars made by resecting the ventricular apex has been visualized with histological methods, there is no adequate imaging tool for tracking the functional recovery of the damaged heart. For this reason, high-frequency Doppler echocardiography using dual mode pulsed wave Doppler, which provides both tissue Doppler (TD) and Doppler flow in a same cardiac cycle, is developed with a 30 MHz high-frequency array ultrasound imaging system. Phantom studies show that the Doppler flow mode of the dual mode is capable of measuring the flow velocity from 0.1 to 15 cm s−1 with high accuracy (p-value = 0.974 > 0.05). In the in vivo study of zebrafish, both TD and Doppler flow signals were simultaneously obtained from the zebrafish heart for the first time, and the synchronized valve motions with the blood flow signals were identified. In the longitudinal study on the zebrafish heart regeneration, the parameters for diagnosing the diastolic dysfunction, for example, E/Em < 10, E/A < 0.14 for wild-type zebrafish, were measured, and the type of diastolic dysfunction caused by the amputation was found to be similar to the restrictive filling. The diastolic function was fully recovered within four weeks post-amputation. PMID:25505135

Characterization of glutathione-S-transferases in zebrafish (Danio rerio).

PubMed

Glisic, Branka; Mihaljevic, Ivan; Popovic, Marta; Zaja, Roko; Loncar, Jovica; Fent, Karl; Kovacevic, Radmila; Smital, Tvrtko

2015-01-01

Glutathione-S-transferases (GSTs) are one of the key enzymes that mediate phase II of cellular detoxification. The aim of our study was a comprehensive characterization of GSTs in zebrafish (Danio rerio) as an important vertebrate model species frequently used in environmental research. A detailed phylogenetic analysis of GST superfamily revealed 27 zebrafish gst genes. Further insights into the orthology relationships between human and zebrafish GSTs/Gsts were obtained by the conserved synteny analysis. Expression of gst genes in six tissues (liver, kidney, gills, intestine, brain and gonads) of adult male and female zebrafish was determined using qRT-PCR. Functional characterization was performed on 9 cytosolic Gst enzymes after overexpression in E. coli and subsequent protein purification. Enzyme kinetics was measured for GSH and a series of model substrates. Our data revealed ubiquitously high expression of gstp, gstm (except in liver), gstr1, mgst3a and mgst3b, high expression of gsto2 in gills and ovaries, gsta in intestine and testes, gstt1a in liver, and gstz1 in liver, kidney and brain. All zebrafish Gsts catalyzed the conjugation of GSH to model GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and monochlorobimane (MCB), apart from Gsto2 and Gstz1 that catalyzed GSH conjugation to dehydroascorbate (DHA) and dichloroacetic acid (DCA), respectively. Affinity toward CDNB varied from 0.28 mM (Gstp2) to 3.69 mM (Gstm3), while affinity toward MCB was in the range of 5 μM (Gstt1a) to 250 μM (Gstp1). Affinity toward GSH varied from 0.27 mM (Gstz1) to 4.45 mM (Gstt1a). Turnover number for CDNB varied from 5.25s(-1) (Gstt1a) to 112s(-1) (Gstp2). Only Gst Pi enzymes utilized ethacrynic acid (ETA). We suggest that Gstp1, Gstp2, Gstt1a, Gstz1, Gstr1, Mgst3a and Mgst3b have important role in the biotransformation of xenobiotics, while Gst Alpha, Mu, Pi, Zeta and Rho classes are involved in the crucial physiological processes. In summary, this study provides the

The common neural parasite Pseudoloma neurophilia is associated with altered startle response habituation in adult zebrafish (Danio rerio): Implications for the zebrafish as a model organism.

PubMed

Spagnoli, Sean; Xue, Lan; Kent, Michael L

2015-09-15

The zebrafish's potential as a model for human neurobehavioral research appears nearly limitless despite its relatively recent emergence as an experimental organism. Since the zebrafish has only been part of the research community for a handful of decades, pathogens from its commercial origins continue to plague laboratory stocks. One such pathogen is Pseudoloma neurophilia, a common microparasite in zebrafish laboratories world-wide that generally produces subclinical infections. Given its high prevalence, its predilection for the host's brain and spinal cord, and the delicate nature of neurobehavioral research, the behavioral consequences of subclinical P. neurophilia infection must be explored. Fish infected via cohabitation were tested for startle response habituation in parallel with controls in a device that administered ten taps over 10 min along with taps at 18 and 60 min to evaluate habituation extinction. After testing, fish were euthanized and evaluated for infection via histopathology. Infected fish had a significantly smaller reduction in startle velocity during habituation compared to uninfected tankmates and controls. Habituation was eliminated in infected and control fish at 18 min, whereas exposed negative fish retained partial habituation at 18 min. Infection was also associated with enhanced capture evasion: Despite the absence of external symptoms, infected fish tended to be caught later than uninfected fish netted from the same tank. The combination of decreased overall habituation, early extinction of habituation compared to uninfected cohorts, and enhanced netting evasion indicates that P. neurophilia infection is associated with a behavioral phenotype distinct from that of controls and uninfected cohorts. Because of its prevalence in zebrafish facilities, P. neurophilia has the potential to insidiously influence a wide range of neurobehavioral studies if these associations are causative. Rigorous health screening is therefore vital to the

Evaluation of anaesthetic protocols for laboratory adult zebrafish (Danio rerio).

PubMed

Martins, Tânia; Diniz, Enoque; Félix, Luís M; Antunes, Luís

2018-01-01

In the last decades, the use of zebrafish (Danio rerio) in biomedical research has increased. Anaesthesia is daily used in fish during experimental procedures to avoid discomfort, stress or pain. Also, fish welfare and the reliability of results can be compromised if an unsuitable anaesthetic protocol is used. Therefore, we aimed to refine anaesthetic protocols to be used in adult zebrafish by evaluating the efficacy of different anaesthetics, used alone or in combination. For that, zebrafish were randomly assigned to 8 different groups: 100 μg/mLMS-222 (MS); 0.2 μg/mL etomidate (E); 0.2 μg/mL etomidate + 100 μg/mL lidocaine (E+L); 1.25 μg/mL propofol (P); 1.25 μg/mL propofol + 100 μg/mL lidocaine (P+L); 100 μg/mL ketamine (K); 100 μg/mL ketamine + 1.25 μg/mL medetomidine (K+M); and 100 μg/mL ketamine + 1.25 μg/mL medetomidine/3.125 μg/mL atipamezole (K+M/A). The animals were placed in an anaesthetic water bath, then, the following parameters were registered: time for equilibrium loss and anaesthesia induction, loss of sensitivity to soft and painful stimuli, respiratory rate, recovery time, and activity after recovery. The combined forms of E+L, P+L and K+M were the fastest to induce a surgical anaesthetic stage. Nevertheless, E+L induced respiratory depression, while K+M was shown to have the longer recovery time compared to MS-222, even when atipamezole was added. In conclusion, the P+L combination was shown to provide good anaesthesia with analgesia, without causing a major respiratory depression, providing as well a quick recovery, similar to MS-222.

Axonal regeneration in zebrafish spinal cord

PubMed Central

Hui, Subhra Prakash

2018-01-01

Abstract In the present review we discuss two interrelated events—axonal damage and repair—known to occur after spinal cord injury (SCI) in the zebrafish. Adult zebrafish are capable of regenerating axonal tracts and can restore full functionality after SCI. Unlike fish, axon regeneration in the adult mammalian central nervous system is extremely limited. As a consequence of an injury there is very little repair of disengaged axons and therefore functional deficit persists after SCI in adult mammals. In contrast, peripheral nervous system axons readily regenerate following injury and hence allow functional recovery both in mammals and fish. A better mechanistic understanding of these three scenarios could provide a more comprehensive insight into the success or failure of axonal regeneration after SCI. This review summarizes the present understanding of the cellular and molecular basis of axonal regeneration, in both the peripheral nervous system and the central nervous system, and large scale gene expression analysis is used to focus on different events during regeneration. The discovery and identification of genes involved in zebrafish spinal cord regeneration and subsequent functional experimentation will provide more insight into the endogenous mechanism of myelination and remyelination. Furthermore, precise knowledge of the mechanism underlying the extraordinary axonal regeneration process in zebrafish will also allow us to unravel the potential therapeutic strategies to be implemented for enhancing regrowth and remyelination of axons in mammals. PMID:29721326

Axonal regeneration in zebrafish spinal cord.

PubMed

Ghosh, Sukla; Hui, Subhra Prakash

2018-03-01

In the present review we discuss two interrelated events-axonal damage and repair-known to occur after spinal cord injury (SCI) in the zebrafish. Adult zebrafish are capable of regenerating axonal tracts and can restore full functionality after SCI. Unlike fish, axon regeneration in the adult mammalian central nervous system is extremely limited. As a consequence of an injury there is very little repair of disengaged axons and therefore functional deficit persists after SCI in adult mammals. In contrast, peripheral nervous system axons readily regenerate following injury and hence allow functional recovery both in mammals and fish. A better mechanistic understanding of these three scenarios could provide a more comprehensive insight into the success or failure of axonal regeneration after SCI. This review summarizes the present understanding of the cellular and molecular basis of axonal regeneration, in both the peripheral nervous system and the central nervous system, and large scale gene expression analysis is used to focus on different events during regeneration. The discovery and identification of genes involved in zebrafish spinal cord regeneration and subsequent functional experimentation will provide more insight into the endogenous mechanism of myelination and remyelination. Furthermore, precise knowledge of the mechanism underlying the extraordinary axonal regeneration process in zebrafish will also allow us to unravel the potential therapeutic strategies to be implemented for enhancing regrowth and remyelination of axons in mammals.

Effects of two strobilurins (azoxystrobin and picoxystrobin) on embryonic development and enzyme activities in juveniles and adult fish livers of zebrafish (Danio rerio).

PubMed

Jia, Wei; Mao, Liangang; Zhang, Lan; Zhang, Yanning; Jiang, Hongyun

2018-09-01

Azoxystrobin and picoxystrobin are two primary strobilurin fungicides used worldwide. This study was conducted to test their effects on embryonic development and the activity of several enzyme in the zebrafish (Danio rerio). After fish eggs were separately exposed to azoxystrobin and picoxystrobin from 24 to 144 h post fertilization (hpf), the mortality, hatching, and teratogenetic rates were measured. Additionally, effects of azoxystrobin and picoxystrobin on activities of three important antioxidant enzymes [catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD)] and two primary detoxification enzymes [carboxylesterase (CarE) and glutathione S-transferase (GST)] and malondialdehyde (MDA) content in zebrafish larvae (96 h) and livers of adult zebrafish of both sexes were also assessed for potential toxicity mechanisms. Based on the embryonic development test results, the mortality, hatching, and teratogenetic rates of eggs treated with azoxystrobin and picoxystrobin all showed significant dose- and time-dependent effects, and the 144-h LC 50 values of azoxystrobin and picoxystrobin were 1174.9 and 213.8 μg L -1 , respectively. In the larval zebrafish (96 h) test, activities of CAT, POD, CarE, and GST and MDA content in azoxystrobin and picoxystrobin-treated zebrafish larvae increased significantly with concentrations of the pesticides compared with those in the control. We further revealed that azoxystrobin and picoxystrobin exposure both caused significant oxidative stress in adult fish livers and the changes differed between the sexes. Our results indicated that picoxystrobin led to higher embryonic development toxicity and oxidative stress than azoxystrobin in zebrafish and the male zebrafish liver had stronger ability to detoxify than that of the females. Copyright © 2018 Elsevier Ltd. All rights reserved.

Two knockdown models of the autism genes SYNGAP1 and SHANK3 in zebrafish produce similar behavioral phenotypes associated with embryonic disruptions of brain morphogenesis

PubMed Central

Kozol, Robert A.; Cukier, Holly N.; Zou, Bing; Mayo, Vera; De Rubeis, Silvia; Cai, Guiqing; Griswold, Anthony J.; Whitehead, Patrice L.; Haines, Jonathan L.; Gilbert, John R.; Cuccaro, Michael L.; Martin, Eden R.; Baker, James D.; Buxbaum, Joseph D.; Pericak-Vance, Margaret A.; Dallman, Julia E.

2015-01-01

Despite significant progress in the genetics of autism spectrum disorder (ASD), how genetic mutations translate to the behavioral changes characteristic of ASD remains largely unknown. ASD affects 1–2% of children and adults, and is characterized by deficits in verbal and non-verbal communication, and social interactions, as well as the presence of repetitive behaviors and/or stereotyped interests. ASD is clinically and etiologically heterogeneous, with a strong genetic component. Here, we present functional data from syngap1 and shank3 zebrafish loss-of-function models of ASD. SYNGAP1, a synaptic Ras GTPase activating protein, and SHANK3, a synaptic scaffolding protein, were chosen because of mounting evidence that haploinsufficiency in these genes is highly penetrant for ASD and intellectual disability (ID). Orthologs of both SYNGAP1 and SHANK3 are duplicated in the zebrafish genome and we find that all four transcripts (syngap1a, syngap1b, shank3a and shank3b) are expressed at the earliest stages of nervous system development with pronounced expression in the larval brain. Consistent with early expression of these genes, knockdown of syngap1b or shank3a cause common embryonic phenotypes including delayed mid- and hindbrain development, disruptions in motor behaviors that manifest as unproductive swim attempts, and spontaneous, seizure-like behaviors. Our findings indicate that both syngap1b and shank3a play novel roles in morphogenesis resulting in common brain and behavioral phenotypes. PMID:25882707

Zebrafish models in neuropsychopharmacology and CNS drug discovery.

PubMed

Khan, Kanza M; Collier, Adam D; Meshalkina, Darya A; Kysil, Elana V; Khatsko, Sergey L; Kolesnikova, Tatyana; Morzherin, Yury Yu; Warnick, Jason E; Kalueff, Allan V; Echevarria, David J

2017-07-01

Despite the high prevalence of neuropsychiatric disorders, their aetiology and molecular mechanisms remain poorly understood. The zebrafish (Danio rerio) is increasingly utilized as a powerful animal model in neuropharmacology research and in vivo drug screening. Collectively, this makes zebrafish a useful tool for drug discovery and the identification of disordered molecular pathways. Here, we discuss zebrafish models of selected human neuropsychiatric disorders and drug-induced phenotypes. As well as covering a broad range of brain disorders (from anxiety and psychoses to neurodegeneration), we also summarize recent developments in zebrafish genetics and small molecule screening, which markedly enhance the disease modelling and the discovery of novel drug targets. © 2017 The British Pharmacological Society.

The UV-absorber benzophenone-4 alters transcripts of genes involved in hormonal pathways in zebrafish (Danio rerio) eleuthero-embryos and adult males

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zucchi, Sara; Bluethgen, Nancy; University of Basel, Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, Klingelbergstrasse 50, CH-4056 Basel

Benzophenone-4 (BP-4) is frequently used as UV-absorber in cosmetics and materials protection. Despite its frequent detection in the aquatic environment potential effects on aquatic life are unknown. In this study, we evaluate the effects of BP-4 in eleuthero-embryos and in the liver, testis and brain of adult male fish on the transcriptional level by focusing on target genes involved in hormonal pathways to provide a more complete toxicological profile of this important UV-absorber. Eleuthero-embryos and males of zebrafish were exposed up to 3 days after hatching and for 14 days, respectively, to BP-4 concentrations between 30 and 3000 {mu}g/L. Inmore » eleuthero-embryos transcripts of vtg1, vtg3, esr1, esr2b, hsd17ss3, cyp19b cyp19a, hhex and pax8 were induced at 3000 {mu}g/L BP-4, which points to a low estrogenic activity and interference with early thyroid development, respectively. In adult males BP-4 displayed multiple effects on gene expression in different tissues. In the liver vtg1, vtg3, esr1 and esr2b were down-regulated, while in the brain, vtg1, vtg3 and cyp19b transcripts were up-regulated. In conclusion, the transcription profile revealed that BP-4 interferes with the expression of genes involved in hormonal pathways and steroidogenesis. The effects of BP-4 differ in life stages and adult tissues and point to an estrogenic activity in eleuthero-embryos and adult brain, and an antiestrogenic activity in the liver. The results indicate that BP-4 interferes with the sex hormone system of fish, which is important for the risk assessment of this UV-absorber.« less

Behavioral, morphometric, and gene expression effects in adult zebrafish (Danio rerio) embryonically exposed to PFOA, PFOS, and PFNA.

PubMed

Jantzen, Carrie E; Annunziato, Kate M; Cooper, Keith R

2016-11-01

Perfluoroalkylated substances (PFAS) are a class of persistent anthropogenic chemicals that have been detected worldwide. PFASs consist of fluorinated carbon chains of varying length, terminal groups, and have a number of industrial uses. A previous zebrafish study from our laboratory showed that acute (3-120h post fertilization, 0.02-2.0μM), waterborne embryonic exposure to these chemicals resulted in chemical specific alterations at 5days post fertilization (dpf), and some effects persisted up to 14 dpf. Using a gene battery consisting of 100 transcripts identified several genes that were up or down regulated. This current study looks at the long-term impacts of PFASs in adult zebrafish using the same exposure regimen. It was hypothesized that sub-lethal exposure of perfluorooctane sulfonate (PFOS), perfluorononanoic acid (PFNA), or perfluorooctane sulfonate (PFOA) in embryonic zebrafish (3-120 hpf) would result in permanent morphometric, gene expression, and behavioral changes in adult fish similar to those observed at 5 and 14 dpf. Zebrafish were exposed to PFOS, PFOA, and PFNA (Control 0μM, 2.0μM) for the first five days post fertilization. At six months post fertilization, no PFAS treatment resulted in a significant change in total body length or weight. In terms of behavior, PFNA males showed a reduction in total distance traveled and time of immobility, and an increase in thigmotaxis behavior, aggressive attacks, and preference for the bright section of the tank. PFOS treated males had a reduced aggression behavior, and PFOA females preferred the dark section of the tank. Gene expression of slco2b1, slco1d1, and tgfb1a were analyzed because these transcripts were previously found to be affected by PFAS exposure in 5dpf and 14 dpf zebrafish and resulted in: significant decrease in expression of slco2b1 for both sexes in PFNA and PFOS treated groups, significant decrease of slco1d1 in all treatment groups for females and PFOS and PFOA exposed males

Identification of oocyte progenitor cells in the zebrafish ovary.

PubMed

Draper, Bruce W

2012-01-01

Zebrafish breed year round and females are capable of producing thousands of eggs during their lifetime. This amazing fecundity is due to the fact that the adult ovary, contains premeiotic oocyte progenitor cells, called oogonia, which produce a continuous supply of new oocytes throughout adult life. Oocyte progenitor cells can be easily identified based on their expression of Vasa, and their characteristic nuclear morphology. Thus, the zebrafish ovary provides a unique and powerful system to study the genetic regulation of oocyte production in a vertebrate animal. A method is presented here for identifying oocyte progenitor cells in the zebrafish ovary using whole-mount confocal immunofluorescence that is simple and accurate.

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

PubMed Central

Maaswinkel, Hans; Zhu, Liqun; Weng, Wei

2013-01-01

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals. PMID:24336189

The effects of rearing light level and duration differences on the optic nerve, brain, and associated structures in developing zebrafish larvae: a light and transmission electron microscope study.

PubMed

Chapman, George B; Tarboush, Rania; Connaughton, Victoria P

2012-03-01

The ultrastructure of the optic nerve, brain, and some associated structures of larval zebrafish, grown under three different light regimens were studied. Fish grown under cyclic light (control), constant dark (CD), and constant light (CL) were studied for 4 and 8 days postfertilization (dpf). We also studied the control and CD fish at 15 dpf. The brains of the control and CL fish were larger at 4 dpf than at 8 dpf. In all 4 dpf fish, the brain occupied the entire expanse between the two retinas and the optic nerve extended the shortest distance between the retina and the brain. The 15 dpf zebrafish had the smallest brain size. Groups of skeletal muscle cells associated with the optic nerves became visible in all older larvae. In the 15 dpf larvae, bulges and dilations in the optic nerve occurred as it reached the brain and optic chiasms occurred proximal to the brain. Electron microscopy yielded information about myelinated and unmyelinated axons in the optic nerve, the dimensions of neurotubules, neurofilaments, and myofilaments, including a unique variation in actin myofilaments, and a confirmation of reported myosin myofilament changes (but with dimensions). We also describe the ultrastructure of a sheath-like structure that is confluent over the optic nerve and the brain, which has not been described before in zebrafish. Also presented are images of associated fibroblasts, epithelial cells lining the mouth, cartilage plates, blood vessels, nerve bundles, and skeletal muscle cells, most of which have not been previously described in the literature. Copyright © 2012 Wiley Periodicals, Inc.

Glyphosate and Roundup® alter morphology and behavior in zebrafish.

PubMed

Bridi, Daiane; Altenhofen, Stefani; Gonzalez, Jonas Brum; Reolon, Gustavo Kellermann; Bonan, Carla Denise

2017-12-01

Glyphosate has become the most widely used herbicide in the world, due to the wide scale adoption of transgenic glyphosate resistant crops after its introduction in 1996. Glyphosate may be used alone, but it is commonly applied as an active ingredient of the herbicide Roundup ® . This pesticide contains several adjuvants, which may promote an unknown toxicity. The indiscriminate application poses numerous problems, both for the health of the applicators and consumers, and for the environment, contaminating the soil, water and leading to the death of plants and animals. Zebrafish (Danio rerio) is quickly gaining popularity in behavioral research, because of physiological similarity to mammals, sensitivity to pharmacological factors, robust performance, low cost, short spawning intervals, external fertilization, transparency of embryos through larval stages, and rapid development. The aim of this study was evaluate the effects of glyphosate and Roundup ® on behavioral and morphological parameters in zebrafish larvae and adults. Zebrafish larvae at 3days post-fertilization and adults were exposed to glyphosate (0.01, 0.065, and 0.5mg/L) or Roundup ® (0.01, 0.065, and 0.5mg/L) for 96h. Immediately after the exposure, we performed the analysis of locomotor activity, aversive behavior, and morphology for the larvae and exploratory behavior, aggression and inhibitory avoidance memory for adult zebrafish. In zebrafish larvae, there were significant differences in the locomotor activity and aversive behavior after glyphosate or Roundup ® exposure when compared to the control group. Our findings demonstrated that exposure to glyphosate at the concentration of 0.5mg/L, Roundup ® at 0.065 or 0.5mg/L reduced the distance traveled, the mean speed and the line crossings in adult zebrafish. A decreased ocular distance was observed for larvae exposed at 0.5mg/L of glyphosate. We verified that at 0.5mg/L of Roundup ® -treated adult zebrafish demonstrated a significant

Zebrafish antipredatory responses: A future for translational research?

PubMed Central

Gerlai, Robert

2011-01-01

Human neuropsychiatric conditions associated with abnormally exaggerated or misdirected fear (anxiety disorders and phobias) still represent a large unmet medical need because the biological mechanisms underlying these diseases are not well understood. Animal models have been proposed to facilitate this research. Here I review the literature with a focus on zebrafish, an upcoming laboratory organism in behavioral brain research. I argue that abnormal human fear responses are likely the result of the malfunction of neurobiological mechanisms (brain areas, circuits and/or molecular mechanisms) that originally evolved to support avoidance of predators or other harm in nature. I also argue that the understanding of the normal as well as pathological functioning of such mechanisms may be best achieved if one utilizes naturalistic experimental approaches. In case of laboratory model organisms, this may entail presenting stimuli associated with predators and measuring species-specific antipredatory responses. Although zebrafish is a relatively new subject of such inquiry, I review the recently rapidly increasing number of zebrafish studies in this area, and conclude that zebrafish is a promising research tool for the analysis of the neurobiology and genetics of vertebrate fear responses. PMID:19836422

Development of the zebrafish mesonephros.

PubMed

Diep, Cuong Q; Peng, Zhenzhen; Ukah, Tobechukwu K; Kelly, Paul M; Daigle, Renee V; Davidson, Alan J

2015-01-01

The vertebrate kidney plays an essential role in removing metabolic waste and balancing water and salt. This is carried out by nephrons, which comprise a blood filter attached to an epithelial tubule with proximal and distal segments. In zebrafish, two nephrons are first formed as part of the embryonic kidney (pronephros) and hundreds are formed later to make up the adult kidney (mesonephros). Previous studies have focused on the development of the pronephros while considerably less is known about how the mesonephros is formed. Here, we characterize mesonephros development in zebrafish and examine the nephrons that form during larval metamorphosis. These nephrons, arising from proliferating progenitor cells that express the renal transcription factor genes wt1b, pax2a, and lhx1a, form on top of the pronephric tubules and develop a segmentation pattern similar to pronephric nephrons. We find that the pronephros acts as a scaffold for the mesonephros, where new nephrons fuse with the distal segments of the pronephric tubules to form the final branching network that characterizes the adult zebrafish kidney. © 2015 Wiley Periodicals, Inc.

Development of the zebrafish mesonephros

PubMed Central

Diep, Cuong Q.; Peng, Zhenzhen; Ukah, Tobechukwu K.; Kelly, Paul M.; Daigle, Renee V.; Davidson, Alan J.

2015-01-01

The vertebrate kidney plays an essential role in removing metabolic waste and balancing water and salt. This is carried out by nephrons, which comprise a blood filter attached to an epithelial tubule with proximal and distal segments. In zebrafish, two nephrons are first formed as part of the embryonic kidney (pronephros) and hundreds are formed later to make up the adult kidney (mesonephros). Previous studies have focused on the development of the pronephros while considerably less is known about how the mesonephros is formed. Here, we characterize mesonephros development in zebrafish and examine the nephrons that form during larval metamorphosis. These nephrons, arising from proliferating progenitor cells that express the renal transcription factor genes wt1b, pax2a, and lhx1a, form on top of the pronephric tubules and develop a segmentation pattern similar to pronephric nephrons. We find that the pronephros acts as a scaffold for the mesonephros, where new nephrons fuse with the distal segments of the pronephric tubules to form the final branching network that characterizes the adult zebrafish kidney. PMID:25677367

A rapid throughput approach identifies cognitive deficits in adult zebrafish from developmental exposure to polybrominated flame retardants.

PubMed

Truong, Lisa; Mandrell, David; Mandrell, Rick; Simonich, Michael; Tanguay, Robert L

2014-07-01

A substantial body of evidence has correlated the human body burdens of some polybrominated diphenyl ether (PBDE) flame retardants with cognitive and other behavioral deficits. Adult zebrafish exhibit testable learning and memory, making them an increasingly attractive model for neurotoxicology. Our goal was to develop a rapid throughput means of identifying the cognitive impact of developmental exposure to flame retardants in the zebrafish model. We exposed embryos from 6h post fertilization to 5 days post fertilization to either PBDE 47 (0.1μM), PBDE 99 (0.1μM) or PBDE 153 (0.1μM), vehicle (0.1% DMSO), or embryo medium (EM). The larvae were grown to adulthood and evaluated for the rate at which they learned an active-avoidance response in an automated shuttle box array. Zebrafish developmentally exposed to PBDE 47 learned the active avoidance paradigm significantly faster than the 0.1% DMSO control fish (P<0.0001), but exhibited significantly poorer performance when retested suggestive of impaired memory retention or altered neuromotor activity. Learning in the PBDE 153 group was not significantly different from the DMSO group. Developmental exposure to 0.1% DMSO impaired adult active avoidance learning relative to the sham group (n=39; P<0.0001). PBDE 99 prevented the DMSO effect, yielding a learning rate not significantly different from the sham group (n=36; P>0.9). Our results underscore the importance of vehicle choice in accurately assessing chemical effects on behavior. Active avoidance response in zebrafish is an effective model of learning that, combined with automated shuttle box testing, will provide a highly efficient platform for evaluating persistent neurotoxic hazard from many chemicals. Copyright © 2014 Elsevier Inc. All rights reserved.

MK-801 increases locomotor activity in a context-dependent manner in zebrafish.

PubMed

Tran, Steven; Muraleetharan, Arrujyan; Fulcher, Niveen; Chatterjee, Diptendu; Gerlai, Robert

2016-01-01

Zebrafish have become a popular animal model for behavioral neuroscience with an increasing number of studies examining the effects of pharmacological compounds targeting the brain. Exposure to MK-801, a non-competitive N-methyl-d-aspartate receptor antagonist has been shown to increase locomotor activity in zebrafish. However, others have failed to replicate this finding as several contradicting studies report no changes in locomotor activity following exposure to similar doses. In the current study we reconcile these behavioral reports by demonstrating that zebrafish do not exhibit changes in locomotor activity during exposure to non-sedative doses of MK-801. Interestingly, zebrafish do exhibit significant increases in locomotion if pre-treated with MK-801 followed by subsequent testing in a novel environment, which suggests the effects of MK-801 are context-dependent. In addition, we examine the potential role of the dopaminergic system in mediating MK-801's locomotor stimulant effect by quantifying the levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the brains of zebrafish following a 30 min exposure to 10 μM of MK-801 (the dose found to induce the largest increase in locomotor activity). Our findings indicate that the MK-801-induced increase in locomotor activity is not accompanied by changes in whole-brain levels of dopamine or DOPAC. Overall, our results suggest that MK-801's context-dependent locomotor stimulant effect may be independent of whole-brain dopaminergic activation. Copyright © 2015 Elsevier B.V. All rights reserved.

Neural stem cell quiescence and stemness are molecularly distinct outputs of the Notch3 signalling cascade in the vertebrate adult brain.

PubMed

Than-Trong, Emmanuel; Ortica-Gatti, Sara; Mella, Sébastien; Nepal, Chirag; Alunni, Alessandro; Bally-Cuif, Laure

2018-05-15

Neural stem cells (NSCs) in the adult vertebrate brain are found in a quiescent state and can preserve long-lasting progenitor potential (stemness). Whether and how these two properties are linked, and to what extent they can be independently controlled by NSC maintenance pathways, is unresolved. We have previously identified Notch3 signalling as a major quiescence-promoting pathway in adult NSCs of the zebrafish pallium. We now show that Notch3 also controls NSC stemness. Using parallel transcriptomic characterizations of notch3 mutant NSCs and adult NSC physiological states, we demonstrate that a set of potentially direct Notch3 target genes distinguishes quiescence and stemness control. As a proof of principle, we focus on one 'stemness' target, encoding the bHLH transcription factor Hey1, that has not yet been analysed in adult NSCs. We show that abrogation of Hey1 function in adult pallial NSCs in vivo , including quiescent NSCs, leads to their differentiation without affecting their proliferation state. These results demonstrate that quiescence and stemness are molecularly distinct outputs of Notch3 signalling, and identify Hey1 as a major Notch3 effector controlling NSC stemness in the vertebrate adult brain. © 2018. Published by The Company of Biologists Ltd.

Efficacy and Safety of 5 Anesthetics in Adult Zebrafish (Danio rerio)

PubMed Central

Collymore, Chereen; Tolwani, Angela; Lieggi, Christine; Rasmussen, Skye

2014-01-01

Although the safety and efficacy of tricaine methanesulfonate (MS222) for anesthesia of fish are well established, other anesthetics used less commonly in fish have been less extensively evaluated. Therefore, we compared gradual cooling, lidocaine hydrochloride (300, 325, and 350 mg/L), metomidate hydrochloride (2, 4, 6, 8, and 10 mg/L), and isoflurane (0.5 mL/L) with MS222 (150 mg/L) for anesthesia of adult zebrafish. The efficacy and safety of each agent was evaluated by observing loss of equilibrium, slowing of opercular movement, response to tail-fin pinch, recovery time, and anesthesia-associated mortality rates. At 15 min after anesthetic recovery, we used a novel-tank test to evaluate whether anesthetic exposure influenced short-term anxiety-like behavior. Behavioral parameters measured included latency to enter and number of transitions to the upper half of the tank, number of erratic movements, and number of freezing bouts. Behavior after anesthesia was unaltered regardless of the anesthetic used. Efficacy and safety differed among the anesthetics evaluated. Gradual cooling was useful for short procedures requiring immobilization only, but all instrumentation and surfaces that come in contact with fish must be maintained at approximately 10 °C. MS222 and lidocaine hydrochloride at 325 mg/L were effective as anesthetic agents for surgical procedures in adult zebrafish, but isoflurane and high-dose lidocaine hydrochloride were unsuitable as sole anesthetic agents due to high (30%) mortality rates. Although MS222 remains the best choice for generating a surgical plane of anesthesia, metomidate hydrochloride and gradual cooling were useful for sedation and immobilization for nonpainful procedures. PMID:24602548

V-ATPase proton pumping activity is required for adult zebrafish appendage regeneration.

PubMed

Monteiro, Joana; Aires, Rita; Becker, Jörg D; Jacinto, António; Certal, Ana C; Rodríguez-León, Joaquín

2014-01-01

The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration.

V-ATPase Proton Pumping Activity Is Required for Adult Zebrafish Appendage Regeneration

PubMed Central

Monteiro, Joana; Aires, Rita; Becker, Jörg D.; Jacinto, António; Certal, Ana C.; Rodríguez-León, Joaquín

2014-01-01

The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration. PMID:24671205

The ontogeny of sleep-wake cycles in zebrafish: a comparison to humans

PubMed Central

Sorribes, Amanda; Þorsteinsson, Haraldur; Arnardóttir, Hrönn; Jóhannesdóttir, Ingibjörg Þ.; Sigurgeirsson, Benjamín; de Polavieja, Gonzalo G.; Karlsson, Karl Æ.

2013-01-01

Zebrafish (Danio rerio) are used extensively in sleep research; both to further understanding of sleep in general and also as a model of human sleep. To date, sleep studies have been performed in larval and adult zebrafish but no efforts have been made to document the ontogeny of zebrafish sleep–wake cycles. Because sleep differs across phylogeny and ontogeny it is important to validate the use of zebrafish in elucidating the neural substrates of sleep. Here we describe the development of sleep and wake across the zebrafish lifespan and how it compares to humans. We find power-law distributions to best fit wake bout data but demonstrate that exponential distributions, previously used to describe sleep bout distributions, fail to adequately account for the data in either species. Regardless, the data reveal remarkable similarities in the ontogeny of sleep cycles in zebrafish and humans. Moreover, as seen in other organisms, zebrafish sleep levels are highest early in ontogeny and sleep and wake bouts gradually consolidate to form the adult sleep pattern. Finally, sleep percentage, bout duration, bout number, and sleep fragmentation are shown to allow for meaningful comparisons between zebrafish and human sleep. PMID:24312015

In vivo and in vitro biophysical properties of hair cells from the lateral line and inner ear of developing and adult zebrafish.

PubMed

Olt, Jennifer; Johnson, Stuart L; Marcotti, Walter

2014-05-15

Hair cells detect and process sound and movement information, and transmit this with remarkable precision and efficiency to afferent neurons via specialized ribbon synapses. The zebrafish is emerging as a powerful model for genetic analysis of hair cell development and function both in vitro and in vivo. However, the full exploitation of the zebrafish is currently limited by the difficulty in obtaining systematic electrophysiological recordings from hair cells under physiological recording conditions. Thus, the biophysical properties of developing and adult zebrafish hair cells are largely unknown. We investigated potassium and calcium currents, voltage responses and synaptic activity in hair cells from the lateral line and inner ear in vivo and using near-physiological in vitro recordings. We found that the basolateral current profile of hair cells from the lateral line becomes more segregated with age, and that cells positioned in the centre of the neuromast show more mature characteristics and those towards the edge retain a more immature phenotype. The proportion of mature-like hair cells within a given neuromast increased with zebrafish development. Hair cells from the inner ear showed a developmental change in current profile between the juvenile and adult stages. In lateral line hair cells from juvenile zebrafish, exocytosis also became more efficient and required less calcium for vesicle fusion. In hair cells from mature zebrafish, the biophysical characteristics of ion channels and exocytosis resembled those of hair cells from other lower vertebrates and, to some extent, those in the immature mammalian vestibular and auditory systems. We show that although the zebrafish provides a suitable animal model for studies on hair cell physiology, it is advisable to consider that the age at which the majority of hair cells acquire a mature-type configuration is reached only in the juvenile lateral line and in the inner ear from >2 months after hatching. © 2014 The

In vitro and in vivo estrogenic activity of BPA, BPF and BPS in zebrafish-specific assays.

PubMed

Le Fol, Vincent; Aït-Aïssa, Selim; Sonavane, Manoj; Porcher, Jean-Marc; Balaguer, Patrick; Cravedi, Jean-Pierre; Zalko, Daniel; Brion, François

2017-08-01

Bisphenol A (BPA) is a widely used chemical that has been extensively studied as an endocrine-disrupting chemical (EDC). Other bisphenols sharing close structural features with BPA, are increasingly being used as alternatives, increasing the need to assess associated hazards to the endocrine system. In the present study, the estrogenic activity of BPA, bisphenol S (BPS) and bisphenol F (BPF) was assessed by using a combination of zebrafish-specific mechanism-based in vitro and in vivo assays. The three bisphenols were found to efficiently transactivate all zebrafish estrogen receptor (zfER) subtypes in zebrafish hepatic reporter cell lines (ZELH-zfERs). BPA was selective for zfERα while BPS and BPF were slightly more potent on zfERβ subtypes. We further documented the estrogenic effect in vivo by quantifying the expression of brain aromatase using a transgenic cyp19a1b-GFP zebrafish embryo assay. All three bisphenols induced GFP in a concentration-dependent manner. BPS only partially induced brain aromatase at the highest tested concentrations (>30µM) while BPA and BPF strongly induced GFP, in an ER-dependent manner, at 1-10µM. Furthermore, we show that BPF strongly induced vitellogenin synthesis in adult male zebrafish. Overall, this study demonstrates the estrogenic activity of BPA, BPF and BPS in different cell- and tissue-contexts and at different stages of development. Differences between in vitro and in vivo responses are discussed in light of selective ER activation and the fate of the compounds in the models. This study confirms the relevance of combining cellular and whole-organism bioassays in a unique model species for the hazard assessment of candidate EDCs. Copyright © 2017 Elsevier Inc. All rights reserved.

Impact of low-dose chronic exposure to Bisphenol A (BPA) on adult male zebrafish adaption to the environmental complexity: Disturbing the color preference patterns and reliving the anxiety behavior.

PubMed

Li, Xiang; Sun, Ming-Zhu; Li, Xu; Zhang, Shu-Hui; Dai, Liang-Ti; Liu, Xing-Yu; Zhao, Xin; Chen, Dong-Yan; Feng, Xi-Zeng

2017-11-01

The extensive usage of xenobiotic endocrine disrupting chemicals (XEDCs), such as Bisphenol A (BPA), has created obvious threat to aquatic ecosystems worldwide. Although a comprehensive understanding of the adverse effect of BPA on behaviors and physiology have been proven, the potential impact of low-dose BPA on altering the basic ability of aquatic organism in adapting to the surrounded complex environment still remains elusive. In this research, we report that treatment of adult male zebrafish with chronic (7 weeks) low-dose (0.22 nM-2.2 nM) BPA, altered the ability in adapting the complex environment by disturbing the natural color preference patterns. In addition, chronic 50 ng/L (0.22 nM) BPA exposure alleviated the anxiety behavior of male zebrafish confronted with the novel environment by enhancing the preference towards light in the light/dark preference test. This phenotype was associated with less expression of serotonin (5-TH) in the hypothalamus and the down-regulation of tyrosine hydroxylase (TH) in brain tissues. As such, our results show that low-dose BPA remnant in surface waters altered zebrafish behavior that are known to have ecological and evolutionary consequences. Here we reported that the impact of chronic low-dose BPA exposure on the basic capability of zebrafish to adapt to the environmental complexity. Specifically, BPA at low concentration, under the environmental safety level and 3000-fold lower than the accepted human daily exposure, interfered with the ability to discriminate color and alleviate anxiety induced by the novel environment, which finally altered the capability of male zebrafish to adapt to the environmental complexity. These findings revealed the ecological effect of low-dose BPA and regular BPA concentration standard are not necessarily safe. The result also provided the consideration of retuning the hazard concentration level of BPA. Copyright © 2017 Elsevier Ltd. All rights reserved.

Developmental Neurotoxicity of Methamidophos in the Embryo-Larval Stages of Zebrafish.

PubMed

He, Xiaowei; Gao, Jiawei; Dong, Tianyu; Chen, Minjian; Zhou, Kun; Chang, Chunxin; Luo, Jia; Wang, Chao; Wang, Shoulin; Chen, Daozhen; Zhou, Zuomin; Tian, Ying; Xia, Yankai; Wang, Xinru

2016-12-28

Methamidophos is a representative organophosphate insecticide. The knowledge of its developmental neurotoxicity is limited, especially for zebrafish in the early stages of their life. Four hour post-fertilization (hpf) zebrafish embryos were exposed to several environmentally relevant concentrations of methamidophos (0, 25, and 500 μg/L) for up to 72 hpf. Locomotor behavior was then studied in the zebrafish larvae at this timepoint. Acridine orange (AO) staining was carried out in the zebrafish larvae, and the mRNA levels of genes associated with neural development ( mbp and syn2a ) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The number of escape responders for mechanical stimulation was significantly decreased in exposed groups. AO staining showed noticeable signs of apoptosis mainly in the brain. In addition, the mRNA levels of mbp and syn2a were both significantly down-regulated in exposed groups. Our study provides the first evidence that methamidophos exposure can cause developmental neurotoxicity in the early stages of zebrafish life, which may be caused by the effect of methamidophos on neurodevelopmental genes and the activation of cell apoptosis in the brain.

Can zebrafish be used as animal model to study Alzheimer's disease?

PubMed Central

Santana, Soraya; Rico, Eduardo P; Burgos, Javier S

2012-01-01

Zebrafish is rapidly emerging as a promising model organism to study various central nervous system (CNS) disorders, including Alzheimer’s disease (AD). AD is the main cause of dementia in the human population and there is an urgency to understand the causes of this neurodegenerative disease. In this respect, the development of new animal models to study the underlying neurodegenerative mechanisms of AD is an urgent need. In this review we analyze the current situation in the use of zebrafish as a model for AD, discussing the reasons to use this experimental paradigm in CNS investigation and analyzing the several strategies adopted to induce an AD-like pathology in zebrafish. We discuss the strategies of performing interventions to cause damage in the zebrafish brain by altering the major neurotransmitter systems (such as cholinergic, glutamatergic or GABAergic circuits). We also analyze the several transgenic zebrafish constructed for the AD study, discussing both the familial-AD models based on APP processing pathway (APP and presenilins) and in the TAU hyperphosphorylation, together with the genes involved in sporadic-AD, as apolipoprotein E. We conclude that zebrafish is in a preliminary stage of development in the AD field, and that the transgenic animals must be improved to use this fish as an optimal model for AD research. Furthermore, a deeper knowledge of the zebrafish brain and a better characterization of the injury caused by alterations in the major neurotransmitter systems are needed. PMID:23383380

Forkhead transcription factor foxe1 regulates chondrogenesis in zebrafish.

PubMed

Nakada, Chisako; Iida, Atsumi; Tabata, Yoko; Watanabe, Sumiko

2009-12-15

Forkhead transcription factor (Fox) e1 is a causative gene for Bamforth-Lazarus syndrome, which is characterized by hypothyroidism and cleft palate. Applying degenerate polymerase chain reaction using primers specific for the conserved forkhead domain, we identified zebrafish foxe1 (foxe1). Foxe1 is expressed in the thyroid, pharynx, and pharyngeal skeleton during development; strongly expressed in the gill and weakly expressed in the brain, eye, and heart in adult zebrafish. A loss of function of foxe1 by morpholino antisense oligo (MO) exhibited abnormal craniofacial development, shortening of Meckel's cartilage and the ceratohyals, and suppressed chondrycytic proliferation. However, at 27 hr post fertilization, the foxe1 MO-injected embryos showed normal dlx2, hoxa2, and hoxb2 expression, suggesting that the initial steps of pharyngeal skeletal development, including neural crest migration and specification of the pharyngeal arch occurred normally. In contrast, at 2 dpf, a severe reduction in the expression of sox9a, colIIaI, and runx2b, which play roles in chondrocytic proliferation and differentiation, was observed. Interestingly, fgfr2 was strongly upregulated in the branchial arches of the foxe1 MO-injected embryos. Unlike Foxe1-null mice, normal thyroid development in terms of morphology and thyroid-specific marker expression was observed in foxe1 MO-injected zebrafish embryos. Taken together, our results indicate that Foxe1 plays an important role in chondrogenesis during development of the pharyngeal skeleton in zebrafish, probably through regulation of fgfr2 expression. Furthermore, the roles reported for FOXE1 in mammalian thyroid development may have been acquired during evolution. (c) 2009 Wiley-Liss, Inc.

The Fanconi anemia/BRCA gene network in zebrafish: embryonic expression and comparative genomics.

PubMed

Titus, Tom A; Yan, Yi-Lin; Wilson, Catherine; Starks, Amber M; Frohnmayer, Jonathan D; Bremiller, Ruth A; Cañestro, Cristian; Rodriguez-Mari, Adriana; He, Xinjun; Postlethwait, John H

2009-07-31

Fanconi anemia (FA) is a genetic disease resulting in bone marrow failure, high cancer risks, and infertility, and developmental anomalies including microphthalmia, microcephaly, hypoplastic radius and thumb. Here we present cDNA sequences, genetic mapping, and genomic analyses for the four previously undescribed zebrafish FA genes (fanci, fancj, fancm, and fancn), and show that they reverted to single copy after the teleost genome duplication. We tested the hypothesis that FA genes are expressed during embryonic development in tissues that are disrupted in human patients by investigating fanc gene expression patterns. We found fanc gene maternal message, which can provide Fanc proteins to repair DNA damage encountered in rapid cleavage divisions. Zygotic expression was broad but especially strong in eyes, central nervous system and hematopoietic tissues. In the pectoral fin bud at hatching, fanc genes were expressed specifically in the apical ectodermal ridge, a signaling center for fin/limb development that may be relevant to the radius/thumb anomaly of FA patients. Hatching embryos expressed fanc genes strongly in the oral epithelium, a site of squamous cell carcinomas in FA patients. Larval and adult zebrafish expressed fanc genes in proliferative regions of the brain, which may be related to microcephaly in FA. Mature ovaries and testes expressed fanc genes in specific stages of oocyte and spermatocyte development, which may be related to DNA repair during homologous recombination in meiosis and to infertility in human patients. The intestine strongly expressed some fanc genes specifically in proliferative zones. Our results show that zebrafish has a complete complement of fanc genes in single copy and that these genes are expressed in zebrafish embryos and adults in proliferative tissues that are often affected in FA patients. These results support the notion that zebrafish offers an attractive experimental system to help unravel mechanisms relevant not only

The Fanconi anemia/BRCA gene network in zebrafish: Embryonic expression and comparative genomics

PubMed Central

Titus, Tom A.; Yan, Yi-Lin; Wilson, Catherine; Starks, Amber M.; Frohnmayer, Jonathan D.; Canestro, Cristian; Rodriguez-Mari, Adriana; He, Xinjun; Postlethwait, John H.

2008-01-01

Fanconi anemia (FA) is a genic disease resulting in bone marrow failure, high cancer risks, and infertility, and developmental anomalies including microphthalmia, microcephaly, hypoplastic radius and thumb. Here we present cDNA sequences, genetic mapping, and genomic analyses for the four previously undescribed zebrafish FA genes (fanci, fancj, fancm, and fancn, and show that they reverted to single copy after the teleost genome duplication. We tested the hypothesis that FA genes are expressed during embryonic development in tissues that are disrupted in human patients by investigating fanc gene expression patterns. We found fanc gene maternal message, which can provide Fanc proteins to repair DNA damage encountered in rapid cleavage divisions. Zygotic expression was broad but especially strong in eyes, central nervous system and hematopoietic tissues. In the pectoral fin bud at hatching, fanc genes were expressed specifically in the apical ectodermal ridge, a signaling center for fin/limb development that may be relevant to the radius/thumb anomaly of FA patients. Hatching embryos expressed fanc genes strongly in the oral epithelium, a site of squamous cell carcinomas in FA patients. Larval and adult zebrafish expressed fanc genes in proliferative regions of the brain, which may be related to microcephaly in FA. Mature ovaries and testes expressed fanc genes in specific stages of oocyte and spermatocyte development, which may be related to DNA repair during homologous recombination in meiosis and to infertility in human patients. The intestine strongly expressed some fanc genes specifically in proliferative zones. Our results show that zebrafish has a complete complement of fanc genes in single copy and that these genes are expressed in zebrafish embryos and adults in proliferative tissues that are often affected in FA patients. These results support the notion that zebrafish offers an attractive experimental system to help unravel mechanisms relevant not only to

Development of sensory systems in zebrafish (Danio rerio)

NASA Technical Reports Server (NTRS)

Moorman, S. J.

2001-01-01

Zebrafish possess all of the classic sensory modalities: taste, tactile, smell, balance, vision, and hearing. For each sensory system, this article provides a brief overview of the system in the adult zebrafish followed by a more detailed overview of the development of the system. By far the majority of studies performed in each of the sensory systems of the zebrafish have involved some aspect of molecular biology or genetics. Although molecular biology and genetics are not major foci of the paper, brief discussions of some of the mutant strains of zebrafish that have developmental defects in each specific sensory system are included. The development of the sensory systems is only a small sampling of the work being done using zebrafish and provides a mere glimpse of the potential of this model for the study of vertebrate development, physiology, and human disease.

Heart-specific expression of laminopathic mutations in transgenic zebrafish.

PubMed

Verma, Ajay D; Parnaik, Veena K

2017-07-01

Lamins are key determinants of nuclear organization and function in the metazoan nucleus. Mutations in human lamin A cause a spectrum of genetic diseases that affect cardiac muscle and skeletal muscle as well as other tissues. A few laminopathies have been modeled using the mouse. As zebrafish is a well established model for the study of cardiac development and disease, we have investigated the effects of heart-specific lamin A mutations in transgenic zebrafish. We have developed transgenic lines of zebrafish expressing conserved lamin A mutations that cause cardiac dysfunction in humans. Expression of zlamin A mutations Q291P and M368K in the heart was driven by the zebrafish cardiac troponin T2 promoter. Homozygous mutant embryos displayed nuclear abnormalities in cardiomyocyte nuclei. Expression analysis showed the upregulation of genes involved in heart regeneration in transgenic mutant embryos and a cell proliferation marker was increased in adult heart tissue. At the physiological level, there was deviation of up to 20% from normal heart rate in transgenic embryos expressing mutant lamins. Adult homozygous zebrafish were fertile and did not show signs of early mortality. Our results suggest that transgenic zebrafish models of heart-specific laminopathies show cardiac regeneration and moderate deviations in heart rate during embryonic development. © 2017 International Federation for Cell Biology.

Zebrafish: A Versatile Animal Model for Fertility Research.

PubMed

Hoo, Jing Ying; Kumari, Yatinesh; Shaikh, Mohd Farooq; Hue, Seow Mun; Goh, Bey Hing

2016-01-01

The utilization of zebrafish in biomedical research is very common in the research world nowadays. Today, it has emerged as a favored vertebrate organism for the research in science of reproduction. There is a significant growth in amount numbers of scientific literature pertaining to research discoveries in reproductive sciences in zebrafish. It has implied the importance of zebrafish in this particular field of research. In essence, the current available literature has covered from the very specific brain region or neurons of zebrafish, which are responsible for reproductive regulation, until the gonadal level of the animal. The discoveries and findings have proven that this small animal is sharing a very close/similar reproductive system with mammals. More interestingly, the behavioral characteristics and along with the establishment of animal courtship behavior categorization in zebrafish have laid an even stronger foundation and firmer reason on the suitability of zebrafish utilization in research of reproductive sciences. In view of the immense importance of this small animal for the development of reproductive sciences, this review aimed at compiling and describing the proximate close similarity of reproductive regulation on zebrafish and human along with factors contributing to the infertility, showing its versatility and its potential usage for fertility research.

Zebrafish: A Versatile Animal Model for Fertility Research

PubMed Central

Hoo, Jing Ying; Kumari, Yatinesh; Shaikh, Mohd Farooq; Hue, Seow Mun

2016-01-01

The utilization of zebrafish in biomedical research is very common in the research world nowadays. Today, it has emerged as a favored vertebrate organism for the research in science of reproduction. There is a significant growth in amount numbers of scientific literature pertaining to research discoveries in reproductive sciences in zebrafish. It has implied the importance of zebrafish in this particular field of research. In essence, the current available literature has covered from the very specific brain region or neurons of zebrafish, which are responsible for reproductive regulation, until the gonadal level of the animal. The discoveries and findings have proven that this small animal is sharing a very close/similar reproductive system with mammals. More interestingly, the behavioral characteristics and along with the establishment of animal courtship behavior categorization in zebrafish have laid an even stronger foundation and firmer reason on the suitability of zebrafish utilization in research of reproductive sciences. In view of the immense importance of this small animal for the development of reproductive sciences, this review aimed at compiling and describing the proximate close similarity of reproductive regulation on zebrafish and human along with factors contributing to the infertility, showing its versatility and its potential usage for fertility research. PMID:27556045

Biomarkers as a tool to assess effects of chromium (VI): comparison of responses in zebrafish early life stages and adults.

PubMed

Domingues, Inês; Oliveira, Rhaul; Lourenço, Joana; Grisolia, Cesar Koppe; Mendo, Sónia; Soares, A M V M

2010-09-01

The present work aims to compare the sensitivity of embryos and adult zebrafish to chromium (VI) (as potassium dichromate) focusing on biomarkers (cholinesterase, glutathione S-transferase and lactate dehydrogenase) as endpoints. Zebrafish eggs showed less sensitivity to Cr (VI) (96 h-LC50=145.7 mg/L) than adults (96 h-LC50=39.4 mg/L) probably due to the protective action of the chorion. However, biomarkers were much more responsive in larvae than in adults and gave clear indications about Cr (VI) mode of action: it seems to be neurotoxic (inhibited cholinesterase), to inhibit glutathione S-transferase activity and to interfere with cellular metabolic activity (changes in lactate dehydrogenase activity) in larvae. In adults, only glutathione S-transferase was responsive, showing a clear inhibition. The responsiveness of the analyzed biomarkers in larvae reinforces the idea of the usefulness of early life stage assays in the assessment of chemicals effects. Moreover, early life stage assays also contributed with relevant information regarding anomalies in larvae development and behavior. Further research should focus on the use of biomarkers to assess long term effects which are ecologically more relevant. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Behavioral and biochemical adjustments of the zebrafish Danio rerio exposed to the β-blocker propranolol.

PubMed

Mitchell, Kimberly M; Moon, Thomas W

2016-09-01

Propranolol (PROP) is a β-blocker prescribed mainly to treat human cardiovascular diseases and as a result of its wide usage and persistence, it is reported in aquatic environments. This study examined whether PROP alters developmental patterns and catecholamine (CA)-regulated processes in the zebrafish (Danio rerio) and if exposure during early life alters the stress response and behaviors of adults. The calculated 48h larva LC50 was 21.6mg/L, well above reported environmental levels (0.01-0.59μg/L). Stressed and PROP-exposed adult zebrafish had reduced testosterone and estradiol levels and exhibited behaviors indicating less anxiety than control fish. Furthermore, adults previously PROP-exposed as embryos/larvae had decreased growth in terms of body length and mass. Finally, these adults showed increased cholesterol and a dose-dependent decrease in testosterone levels compared with unexposed zebrafish. Thus PROP-exposure of zebrafish embryos/larvae alters developmental patterns and CA-regulated processes that may affect normal behaviors and responses to stressors, and at least some of these changes persist in the adult zebrafish. Copyright © 2015 Elsevier Inc. All rights reserved.

Embryological exposure to valproic acid induces social interaction deficits in zebrafish (Danio rerio): A developmental behavior analysis.

PubMed

Zimmermann, Fernanda Francine; Gaspary, Karina Vidarte; Leite, Carlos Eduardo; De Paula Cognato, Giana; Bonan, Carla Denise

2015-01-01

Changes in social behavior are associated with brain disorders, including mood disorders, stress, schizophrenia, Alzheimer's disease, and autism spectrum disorders (ASD). Autism is a complex neurodevelopmental disorder characterized by deficits in social interaction, impaired communication, anxiety, hyperactivity, and the presence of restricted interests. Zebrafish is one of the most social vertebrates used as a model in biomedical research, contributing to an understanding of the mechanisms that underlie social behavior. Valproic acid (VPA) is used as an anti-epileptic drug and mood stabilizer; however, prenatal VPA exposure in humans has been associated with an increased incidence of autism and it can also affect fetal brain development. Therefore, we conducted a behavioral screening at different periods of zebrafish development at 6, 30, 70, and 120dpf (days postfertilization) after VPA exposure in the early development stage to investigate social behavior, locomotion, aggression, and anxiety. VPA (48μM) exposure during the first 48hpf (hours postfertilization) did not promote changes on survival, morphology, and hatching rate at 24hpf, 48hpf, and 72hpf. The behavioral patterns suggest that VPA exposure induces changes in locomotor activity and anxiety at different developmental periods in zebrafish. Furthermore, a social interaction deficit is present at 70dpf and 120dpf. VPA exposure did not affect aggression in the adult stage at 70dpf and 120dpf. This is the first study that demonstrated zebrafish exposed to VPA during the first 48h of development exhibit deficits in social interaction, anxiety, and hyperactivity at different developmental periods. Copyright © 2015 Elsevier Inc. All rights reserved.

The common neural parasite Pseudoloma neurophilia is associated with altered startle response habituation in adult zebrafish (Danio rerio): Implications for the zebrafish as a model organism

PubMed Central

Spagnoli, Sean; Xue, Lan; Kent, Michael L.

2015-01-01

The zebrafish’s potential as a model for human neurobehavioral research appears nearly limitless despite its relatively recent emergence as an experimental organism. Since the zebrafish has only been part of the research community for a handful of decades, pathogens from its commercial origins continue to plague laboratory stocks. One such pathogen is Pseudoloma neurophilia, a common microparasite in zebrafish laboratories world-wide that generally produces subclinical infections. Given its high prevalence, its predilection for the host’s brain and spinal cord, and the delicate nature of neurobehavioral research, the behavioral consequences of subclinical P. neurophilia infection must be explored. Fish infected via cohabitation were tested for startle response habituation in parallel with controls in a device that administered ten taps over ten minutes along with taps at 18 and 60 minutes to evaluate habituation extinction. After testing, fish were euthanized and evaluated for infection via histopathology. Infected fish had a significantly smaller reduction in startle velocity during habituation compared to uninfected tankmates and controls. Habituation was eliminated in infected and control fish at 18 minutes, whereas exposed negative fish retained partial habituation at 18 minutes. Infection was also associated with enhanced capture evasion: Despite the absence of external symptoms, infected fish tended to be caught later than uninfected fish netted from the same tank. The combination of decreased overall habituation, early extinction of habituation compared to uninfected cohorts, and enhanced netting evasion indicates that P. neurophilia infection is associated with a behavioral phenotype distinct from that of controls and uninfected cohorts. Because of its prevalence in zebrafish facilities, P. neurophilia has the potential to insidiously influence a wide range of neurobehavioral studies if these associations are causative. Rigorous health screening is

Disruption of Epithalamic Left-Right Asymmetry Increases Anxiety in Zebrafish.

PubMed

Facchin, Lucilla; Duboué, Erik R; Halpern, Marnie E

2015-12-02

Differences between the left and right sides of the brain are found throughout the animal kingdom, but the consequences of altered neural asymmetry are not well understood. In the zebrafish epithalamus, the parapineal is located on the left side of the brain where it influences development of the adjacent dorsal habenular (dHb) nucleus, causing the left and right dHb to differ in their organization, gene expression, and connectivity. Left-right (L-R) reversal of parapineal position and dHb asymmetry occurs spontaneously in a small percentage of the population, whereas the dHb develop symmetrically following experimental ablation of the parapineal. The habenular region was previously implicated in modulating fear in both mice and zebrafish, but the relevance of its L-R asymmetry is unclear. We now demonstrate that disrupting directionality of the zebrafish epithalamus causes reduced exploratory behavior and increased cortisol levels, indicative of enhanced anxiety. Accordingly, exposure to buspirone, an anxiolytic agent, significantly suppresses atypical behavior. Axonal projections from the parapineal to the dHb are more variable when it is located on the right side of the brain, revealing that L-R reversals do not necessarily represent a neuroanatomical mirror image. The results highlight the importance of directional asymmetry of the epithalamus in the regulation of stress responses in zebrafish. Copyright © 2015 the authors 0270-6474/15/3515847-13$15.00/0.

Chondroitin sulfate and keratan sulfate are the major glycosaminoglycans present in the adult zebrafish Danio rerio (Chordata-Cyprinidae).

PubMed

Souza, Aline R C; Kozlowski, Eliene O; Cerqueira, Vinicius R; Castelo-Branco, Morgana T L; Costa, Manoel L; Pavão, Mauro S G

2007-12-01

The zebrafish Danio rerio (Chordata-Cyprinidae) is a model organism frequently used to study the functions of proteoglycans and their glycosaminoglycan (GAG) chains. Although several studies clearly demonstrate the participation of these polymers in different biological and cellular events that take place during embryonic development, little is known about the GAGs in adult zebrafish. In the present study, the total GAGs were extracted from the whole fish by proteolytic digestion, purified by anion-exchange chromatography and characterized by electrophoresis after degradation with specific enzymes and/or by high-performance liquid chromatography (HPLC) analysis of the disaccharides. Two GAGs were identified: a low-molecular-weight chondroitin sulfate (CS) and keratan sulfate (KS), corresponding to approximately 80% and 20% of the total GAGs, respectively. In the fish eye, KS represents approximately 80% of total GAGs. Surprisingly, no heparinoid was detected, but may be present in the fish at concentrations lower than the limit of the method used. HPLC of the disaccharides formed after chondroitin AC or ABC lyase degradation revealed that the zebrafish CS is composed by DeltaUA-1-->3-GalNAc(4SO4) (59.4%), DeltaUA-1-->3-GalNAc(6SO4) (23.1%), and DeltaUA-1-->3-GalNAc (17.5%) disaccharide units. No disulfated disaccharides were detected. Immunolocalization on sections from zebrafish retina using monoclonal antibodies against CS4- or 6-sulfate showed that in the retina these GAGs are restricted to the outer and inner plexiform layers. This is the first report showing the presence of KS in zebrafish eye, and the structural characterization of CS and its localization in the zebrafish retina. Detailed information about the structure and tissue localization of GAGs is important to understand the functions of these polymers in this model organism.

Inhibiting effects of rhynchophylline on zebrafish methamphetamine dependence are associated with amelioration of neurotransmitters content and down-regulation of TH and NR2B expression.

PubMed

Jiang, Mingjin; Chen, Yifei; Li, Chan; Peng, Qiuxian; Fang, Miao; Liu, Wei; Kang, Qunzhao; Lin, Yingbo; Yung, Ken Kin Lam; Mo, Zhixian

2016-07-04

Others and we have reported that rhynchophylline reverses amphetamine-induced conditioned place preference (CPP) effect which may be partly mediated by amelioration of central neurotransmitters and N-methyl-d-aspartate receptor 2B (NR2B) levels in the rat brains. The current study investigated the inhibiting effects of rhynchophylline on methamphetamine-induced (METH-induced) CPP in adult zebrafish and METH-induced locomotor activity in tyrosine hydroxylase-green fluorescent protein (TH-GFP) transgenic zebrafish larvae and attempted to confirm the hypothesis that these effects were mediated via regulation of neurotransmitters and dopaminergic and glutamatergic systems. After baseline preference test (on days 1-3), zebrafish were injected intraperitoneally METH (on days 4, 6 and 8) or the same volume of fish physiological saline (on days 5 and 7) and were immediately conditioned. Rhynchophylline was administered at 12h after injection of METH. On day 9, zebrafish were tested for METH-induced CPP. Results revealed that rhynchophylline (100mg/kg) significantly inhibited the acquisition of METH-induced CPP, reduced the content of dopamine and glutamate and down-regulated the expression of TH and NR2B in the CPP zebrafish brains. Furthermore, the influence of rhynchophylline on METH-induced locomotor activity was also observed in TH-GFP transgenic zebrafish larvae. Results showed that rhynchophylline (50mg/L) treatment led to a significant reduction on the locomotor activity and TH expression in TH-GFP transgenic zebrafish larvae. Taken together, these data indicate that the inhibition of the formation of METH dependence by rhynchophylline in zebrafish is associated with amelioration of the neurotransmitters dopamine and glutamate content and down-regulation of TH and NR2B expression. Copyright © 2016 Elsevier Inc. All rights reserved.

Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio.

PubMed

Yang, Tianzhi; Martin, Paige; Fogarty, Brittany; Brown, Alison; Schurman, Kayla; Phipps, Roger; Yin, Viravuth P; Lockman, Paul; Bai, Shuhua

2015-06-01

The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model. Four types of exosomes were isolated from brain cell culture media and characterized by particle size, morphology, total protein, and transmembrane protein markers. Transport mechanism, cell uptake, and cytotoxicity of optimized exosome delivery system were tested. Brain distribution of exosome delivered anticancer drugs was evaluated using transgenic zebrafish TG (fli1: GFP) embryos and efficacies of optimized formations were examined in a xenotransplanted zebrafish model of brain cancer model. Four exosomes in 30-100 diameters showed different morphologies and exosomes derived from brain endothelial cells expressed more CD63 tetraspanins transmembrane proteins. Optimized exosomes increased the uptake of fluorescent marker via receptor mediated endocytosis and cytotoxicity of anticancer drugs in cancer cells. Images of the zebrafish showed exosome delivered anticancer drugs crossed the BBB and entered into the brain. In the brain cancer model, exosome delivered anticancer drugs significantly decreased fluorescent intensity of xenotransplanted cancer cells and tumor growth marker. Brain endothelial cell derived exosomes could be potentially used as a carrier for brain delivery of anticancer drug for the treatment of brain cancer.

Triclosan Exposure Is Associated with Rapid Restructuring of the Microbiome in Adult Zebrafish

PubMed Central

Barton, Carrie L.; Proffitt, Sarah; Tanguay, Robert L.; Sharpton, Thomas J.

2016-01-01

Growing evidence indicates that disrupting the microbial community that comprises the intestinal tract, known as the gut microbiome, can contribute to the development or severity of disease. As a result, it is important to discern the agents responsible for microbiome disruption. While animals are frequently exposed to a diverse array of environmental chemicals, little is known about their effects on gut microbiome stability and structure. Here, we demonstrate how zebrafish can be used to glean insight into the effects of environmental chemical exposure on the structure and ecological dynamics of the gut microbiome. Specifically, we exposed forty-five adult zebrafish to triclosan-laden food for four or seven days or a control diet, and analyzed their microbial communities using 16S rRNA amplicon sequencing. Triclosan exposure was associated with rapid shifts in microbiome structure and diversity. We find evidence that several operational taxonomic units (OTUs) associated with the family Enterobacteriaceae appear to be susceptible to triclosan exposure, while OTUs associated with the genus Pseudomonas appeared to be more resilient and resistant to exposure. We also found that triclosan exposure is associated with topological alterations to microbial interaction networks and results in an overall increase in the number of negative interactions per microbe in these networks. Together these data indicate that triclosan exposure results in altered composition and ecological dynamics of microbial communities in the gut. Our work demonstrates that because zebrafish afford rapid and inexpensive interrogation of a large number of individuals, it is a useful experimental system for the discovery of the gut microbiome’s interaction with environmental chemicals. PMID:27191725

Using Zebrafish to Study Podocyte Genesis During Kidney Development and Regeneration

PubMed Central

Kroeger, Paul T.; Wingert, Rebecca A.

2014-01-01

SUMMARY During development, vertebrates form a progression of up to three different kidneys that are comprised of functional units termed nephrons. Nephron composition is highly conserved across species, and an increasing appreciation of the similarities between zebrafish and mammalian nephron cell types has positioned the zebrafish as a relevant genetic system for nephrogenesis studies. A key component of the nephron blood filter is a specialized epithelial cell known as the podocyte. Podocyte research is of the utmost importance as a vast majority of renal diseases initiate with the dysfunction or loss of podocytes, resulting in a condition known as proteinuria that causes nephron degeneration and eventually leads to kidney failure. Understanding how podocytes develop during organogenesis may elucidate new ways to promote nephron health by stimulating podocyte replacement in kidney disease patients. In this review, we discuss how the zebrafish model can be used to study kidney development, and how zebrafish research has provided new insights into podocyte lineage specification and differentiation. Further, we discuss the recent discovery of podocyte regeneration in adult zebrafish, and explore how continued basic research using zebrafish can provide important knowledge about podocyte genesis in embryonic and adult environments. PMID:24920186

Exposure to benzidine caused apoptosis and malformation of telencephalon region in zebrafish.

PubMed

Chen, Mark Hung-Chih; Hsu, Li-Chi; Wu, Jia-Lun; Yeh, Chi-Wei; Tsai, Jen-Ning; Hseu, You-Cheng; Hsu, Li-Sung

2014-12-01

Exposure to benzidine has been known to induce human cancers, particularly bladder carcinomas. In this study, the zebrafish model was used to investigate the developmental toxicity of benzidine. Embryos at 6 h postfertilization (hpf) that were exposed to benzidine exhibited embryonic death in a dose- and time-dependent manner. Benzidine induced malformations in zebrafish, such as small brain development, shorter axes, and a slight pericardial edema. High concentrations (50, 100, and 200 µM) of benzidine triggered widespread apoptosis in the brain and dorsal neurons, as evidenced by acridine orange and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays. Real-time polymerase chain reaction analysis also showed that benzidine treatment affected p53, bax, and noxa expression. Decreases in specific brain markers, such as emx1 in the telencephalon, ngn1 in differentiated neurons, and otx2 in the midbrain, were observed in benzidine-treated embryos at 24 hpf. Conversely, no overt changes to pax2.1 expression in the midbrain-hindbrain boundary were found. Moreover, the use of Tg(HuC:GFP) zebrafish showed that benzidine caused a malformation of the telencephalon region. Our findings show that benzidine exposure triggers widespread apoptosis in the zebrafish brain and dorsal neurons, resulting in the development of an abnormal telencephalon. © 2013 Wiley Periodicals, Inc.

Zebrafish Get Connected: Investigating Neurotransmission Targets and Alterations in Chemical Toxicity

PubMed Central

Horzmann, Katharine A.; Freeman, Jennifer L.

2016-01-01

Neurotransmission is the basis of neuronal communication and is critical for normal brain development, behavior, learning, and memory. Exposure to drugs and chemicals can alter neurotransmission, often through unknown pathways and mechanisms. The zebrafish (Danio rerio) model system is increasingly being used to study the brain and chemical neurotoxicity. In this review, the major neurotransmitter systems, including glutamate, GABA, dopamine, norepinephrine, serotonin, acetylcholine, histamine, and glutamate are surveyed and pathways of synthesis, transport, metabolism, and action are examined. Differences between human and zebrafish neurochemical pathways are highlighted. We also review techniques for evaluating neurological function, including the measurement of neurotransmitter levels, assessment of gene expression through transcriptomic analysis, and the recording of neurobehavior. Finally examples of chemical toxicity studies evaluating alterations in neurotransmitter systems in the zebrafish model are reviewed. PMID:28730152

Enhanced bio-concentration of tris(1,3-dichloro-2-propyl) phosphate in the presence of nano-TiO2 can lead to adverse reproductive outcomes in zebrafish.

PubMed

Ren, Xin; Zhao, Xuesong; Duan, Xiaoyue; Fang, Ziwei

2018-02-01

Interactions between organic toxicants and nano-particles in the aquatic environment may modify toxicant bioavailability and consequently the toxicant's fate and toxicity. To evaluate the potential impact of nano-titanium dioxide (TiO 2 ) on the bio-concentration and reproductive endocrine disruption of tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) in fish, a comparative bioaccumulation study was conducted on zebrafish (Danio rerio, AB strain) treated with 0, 5.74, 23.6, or 90.7 μg L -1 TDCIPP alone or co-exposed to TDCIPP and 0.09 mg L -1 nano-TiO 2 for 21 days. Nano-TiO 2 can absorb TDCIPP and nano-TiO 2 is taken up into zebrafish. Chemical measurements showed that TDCIPP was bio-concentrated in zebrafish, and the highest level was detected in the liver, followed by the brain and gonads. Compared with TDCIPP treatment, increased tissue burdens of both TDCIPP were observed in the liver, brain, and gonads suggesting that nano-TiO 2 adsorbed TDCIPP and acted as a carrier facilitating the uptake and translocation of TDCIPP in tissues. Higher bio-concentration in the presence of nano-TiO 2 resulted in a significant decrease in the hepatic-somatic index, gonad-somatic index and brain-somatic index in F0 females but not F0 males. Moreover, a further gender-dependent reduction in testosterone (T), estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and induction of plasma vitellogenin (VTG) concentrations in adults were observed following co-exposure. Co-exposure also inhibited egg production and caused significant developmental toxicity in F1 larvae. The results obtained using this multi-marker approach suggested that nano-TiO 2 is a carrier of TDCIPP and accelerated its bio-concentration in adult zebrafish, resulting in adverse reproduction outcomes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Impacts of 17beta-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-, juvenile- and adult-life stages in zebrafish (Danio rerio).

PubMed

Brion, F; Tyler, C R; Palazzi, X; Laillet, B; Porcher, J M; Garric, J; Flammarion, P

2004-06-24

Zebrafish (Danio rerio) were exposed for 3 weeks to low concentrations of estradiol including environmentally relevant concentrations (5, 25 and 100 ng/l), encompassing either their embryo-larvae (from fertilization to 21 day post-fertilization (dpf)), juvenile (from 21 to 42 dpf) or adult life stages (>200 dpf) with a view to investigating the most sensitive life stage of the zebrafish to 17beta-estradiol (E2). At all sampling points, whole-body vitellogenin concentrations and gonadal development were analyzed in order to investigate the effects of estrogen exposure on these endpoint in the zebrafish. In the adult stage, additional endpoints were measured including secondary sexual characteristics (manifestation of the uro-genital papillae (UGP) in males), gonadal growth (the gonado-somatic index (GSI)) and sex ratio. For all the different life stage exposures, reproductive performance of the F0 generation was assessed (egg production) and survival and development of the F1 embryo-larvae. Exposure to low concentrations of E2 resulted in vitellogenin induction whatever the life stage exposed but these effects were reversible after depuration. The effective concentration for vitellogenin induction in zebrafish early life stages was 100 ng E2/l, and in adult male zebrafish the effective concentration for vitellogenin induction (between 5 and 25 ng/l) was lower than for the early life stage fish. Exposure to E2 prior to (from fertilization to 21 dpf) and during the time of sex differentiation (from 21 to 42 dpf) also caused disruptions in the process of sexual differentiation (resulting in formation of a retrogonadal cavity in presumptive male, germ cell development and leading to a significant change of the sex ratio towards the female sex at the dose of 100 ng E2/l for the fish exposure as embryo-larvae) and altered patterns of egg production in the subsequent adults. Exposure of adult fish to E2 resulted in a modification of the secondary sexual characteristic in

The zebrafish as a model for complex tissue regeneration

PubMed Central

Gemberling, Matthew; Bailey, Travis J.; Hyde, David R.; Poss, Kenneth D.

2013-01-01

For centuries, philosophers and scientists have been fascinated by the principles and implications of regeneration in lower vertebrate species. Two features have made zebrafish an informative model system for determining mechanisms of regenerative events. First, they are highly regenerative, able to regrow amputated fins, as well as a lesioned brain, retina, spinal cord, heart, and other tissues. Second, they are amenable to both forward and reverse genetic approaches, with a research toolset regularly updated by an expanding community of zebrafish researchers. Zebrafish studies have helped identify new mechanistic underpinnings of regeneration in multiple tissues, and in some cases have served as a guide for contemplating regenerative strategies in mammals. Here, we review the recent history of zebrafish as a genetic model system for understanding how and why tissue regeneration occurs. PMID:23927865

Generation and characterization of Kctd15 mutations in zebrafish

PubMed Central

Heffer, Alison; Marquart, Gregory D.; Aquilina-Beck, Allisan; Saleem, Nabil; Burgess, Harold A.

2017-01-01

Potassium channel tetramerization domain containing 15 (Kctd15) was previously found to have a role in early neural crest (NC) patterning, specifically delimiting the region where NC markers are expressed via repression of transcription factor AP-2a and inhibition of Wnt signaling. We used transcription activator-like effector nucleases (TALENs) to generate null mutations in zebrafish kctd15a and kctd15b paralogs to study the in vivo role of Kctd15. We found that while deletions producing frame-shift mutations in each paralog showed no apparent phenotype, kctd15a/b double mutant zebrafish are smaller in size and show several phenotypes including some affecting the NC, such as expansion of the early NC domain, increased pigmentation, and craniofacial defects. Both melanophore and xanthophore pigment cell numbers and early markers are up-regulated in the double mutants. While we find no embryonic craniofacial defects, adult mutants have a deformed maxillary segment and missing barbels. By confocal imaging of mutant larval brains we found that the torus lateralis (TLa), a region implicated in gustatory networks in other fish, is absent. Ablation of this brain tissue in wild type larvae mimics some aspects of the mutant growth phenotype. Thus kctd15 mutants show deficits in the development of both neural crest derivatives, and specific regions within the central nervous system, leading to a strong reduction in normal growth rates. PMID:29216270

UPLC/MS MS data of testosterone metabolites in human and zebrafish liver microsomes and whole zebrafish larval microsomes.

PubMed

Saad, Moayad; Bijttebier, Sebastiaan; Matheeussen, An; Verbueken, Evy; Pype, Casper; Casteleyn, Christophe; Van Ginneken, Chris; Maes, Louis; Cos, Paul; Van Cruchten, Steven

2018-02-01

This article represents data regarding a study published in Toxicology in vitro entitled " in vitro CYP-mediated drug metabolism in the zebrafish (embryo) using human reference compounds" (Saad et al., 2017) [1]. Data were acquired with ultra-performance liquid chromatography - accurate mass mass spectrometry (UPLC-amMS). A full spectrum scan was conducted for the testosterone (TST) metabolites from the microsomal stability assay in zebrafish and humans. The microsomal proteins were extracted from adult zebrafish male (MLM) and female (FLM) livers, whole body homogenates of 96 h post fertilization larvae (EM) and a pool of human liver microsomes from 50 donors (HLM). Data are expressed as the abundance from the extracted ion chromatogram of the metabolites.

Ontogeny of classical and operant learning behaviors in zebrafish.

PubMed

Valente, André; Huang, Kuo-Hua; Portugues, Ruben; Engert, Florian

2012-03-20

The performance of developing zebrafish in both classical and operant conditioning assays was tested with a particular focus on the emergence of these learning behaviors during development. Strategically positioned visual cues paired with electroshocks were used in two fully automated assays to investigate both learning paradigms. These allow the evaluation of the behavioral performance of zebrafish continuously throughout development, from larva to adult. We found that learning improves throughout development, starts reliably around week 3, and reaches adult performance levels at week 6. Adult fish quickly learned to perform perfectly, and the expression of the learned behavior is manifestly controlled by vision. The memory is behaviorally expressed in adults for at least 6 h and retrievable for at least 12 h.

Ontogeny of classical and operant learning behaviors in zebrafish

PubMed Central

Valente, André; Huang, Kuo-Hua; Portugues, Ruben; Engert, Florian

2012-01-01

The performance of developing zebrafish in both classical and operant conditioning assays was tested with a particular focus on the emergence of these learning behaviors during development. Strategically positioned visual cues paired with electroshocks were used in two fully automated assays to investigate both learning paradigms. These allow the evaluation of the behavioral performance of zebrafish continuously throughout development, from larva to adult. We found that learning improves throughout development, starts reliably around week 3, and reaches adult performance levels at week 6. Adult fish quickly learned to perform perfectly, and the expression of the learned behavior is manifestly controlled by vision. The memory is behaviorally expressed in adults for at least 6 h and retrievable for at least 12 h. PMID:22434824

Malformation of certain brain blood vessels caused by TCDD activation of Ahr2/Arnt1 signaling in developing zebrafish.

PubMed

Teraoka, Hiroki; Ogawa, Akira; Kubota, Akira; Stegeman, John J; Peterson, Richard E; Hiraga, Takeo

2010-08-15

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes various signs of toxicity in early life stages of vertebrates through activation of the aryl hydrocarbon receptor (AHR). The AHR also plays important roles in normal development in mice, and AHR(-/-) mice show abnormal development of vascular structures in various blood vessels. Our previous studies revealed that Ahr type 2 (Ahr2) activation by TCDD and beta-naphthoflavone (BNF) caused a significant decrease in blood flow in the dorsal midbrain of zebrafish embryos. Here we report effects of TCDD exposure on the morphology of some blood vessels in the head of developing zebrafish. TCDD caused concentration-dependent anatomical rearrangements in the shape of the prosencephalic artery in zebrafish larvae. In contrast, no major vascular defects were recognized in the trunk and tail regions following exposure to TCDD at least at the concentrations used. Essentially, the same observations were also confirmed in BNF-exposed larvae. Knock-down of either Ahr2 or Ahr nuclear translocator type 1 (Arnt1) by morpholino oligonucleotides (MOs) protected larvae against abnormal shape of the prosencephalic artery caused by TCDD and BNF. On the other hand, knock-down of Ahr2 or Arnt1 in vehicle-exposed zebrafish larvae had no clear effect on morphology of the prosencephalic artery or trunk vessels. Ascorbic acid, an antioxidant, protected against the TCDD-induced decrease in blood flow through the prosencephalic artery, but not the abnormal morphological changes in the shape of this artery. These results indicate that activation of Ahr2/Arnt1 pathway by TCDD and BNF affects the shape of certain blood vessels in the brain of developing zebrafish. (c) 2010 Elsevier B.V. All rights reserved.

Glutathione S-Transferase Protein Expression in Different Life Stages of Zebrafish (Danio rerio)

PubMed Central

Tierbach, Alena; Groh, Ksenia J; Schönenberger, René; Schirmer, Kristin

2018-01-01

Abstract Zebrafish is a widely used animal model in biomedical sciences and toxicology. Although evidence for the presence of phases I and II xenobiotic defense mechanisms in zebrafish exists on the transcriptional and enzyme activity level, little is known about the protein expression of xenobiotic metabolizing enzymes. Given the important role of glutathione S-transferases (GSTs) in phase II biotransformation, we analyzed cytosolic GST proteins in zebrafish early life stages and different organs of adult male and female fish, using a targeted proteomics approach. The established multiple reaction monitoring-based assays enable the measurement of the relative abundance of specific GST isoenzymes and GST classes in zebrafish through a combination of proteotypic peptides and peptides shared within the same class. GSTs of the classes alpha, mu, pi and rho are expressed in zebrafish embryo as early as 4 h postfertilization (hpf). The majority of GST enzymes are present at 72 hpf followed by a continuous increase in expression thereafter. In adult zebrafish, GST expression is organ dependent, with most of the GST classes showing the highest expression in the liver. The expression of a wide range of cytosolic GST isoenzymes and classes in zebrafish early life stages and adulthood supports the use of zebrafish as a model organism in chemical-related investigations. PMID:29361160

Brief Embryonic Strychnine Exposure in Zebrafish Causes Long-Term Adult Behavioral Impairment with Indications of Embryonic Synaptic Changes

PubMed Central

Roy, Nicole M.; Arpie, Brianna; Lugo, Joseph; Linney, Elwood; Levin, Edward D.; Cerutti, Daniel

2015-01-01

Zebrafish provide a powerful model of the impacts of embryonic toxicant exposure on neural development that may result in long-term behavioral dysfunction. In this study, zebrafish embryos were treated with 1.5 mM strychnine for short embryonic time windows to induce transient changes in inhibitory neural signaling, and were subsequently raised in untreated water until adulthood. PCR analysis showed indications that strychnine exposure altered expression of some genes related to glycinergic, GABAergic and glutamatergic neuronal synapses during embryonic development. In adulthood, treated fish showed significant changes in swimming speed and tank diving behavior compared to controls. Taken together, these data show that a short embryonic exposure to a neurotoxicant can alter development of neural synapses and lead to changes in adult behavior. PMID:23022260

Behavioral and Molecular Analysis of Nicotine-Conditioned Place Preference in Zebrafish

PubMed Central

Kedikian, Ximena; Faillace, Maria Paula; Bernabeu, Ramón

2013-01-01

Studies using mice and rats have demonstrated that nicotine induces a conditioned place preference (CPP), with more effective results obtained by using biased procedures. Zebrafish have also been used as a model system to identify factors influencing nicotine-associated reward by using an unbiased design. Here, we report that zebrafish exhibited putative nicotine biased CPP to an initially aversive compartment (nicotine-paired group). A counterbalanced nicotine-exposed control group did not show a significant preference shift, providing evidence that the preference shift in the nicotine-paired group was not due to a reduction of aversion for this compartment. Zebrafish preference was corroborated by behavioral analysis of several indicators of drug preference, such as time spent in the drug-paired side, number of entries to the drug-paired side, and distance traveled. These results provided strong evidence that zebrafish may actually develop a preference for nicotine, although the drug was administrated in an aversive place for the fish, which was further supported by molecular studies. Reverse transcription-quantitative real-time PCR analysis depicted a significant increase in the expression of α7 and α6 but not α4 and β2 subunits of the nicotinic receptor in nicotine-paired zebrafish brains. In contrast, zebrafish brains from the counterbalanced nicotine group showed no significant changes. Moreover, CREB phosphorylation, an indicator of neural activity, accompanied the acquisition of nicotine-CPP. Our studies offered an incremental value to the drug addiction field, because they further describe behavioral features of CPP to nicotine in zebrafish. The results suggested that zebrafish exposed to nicotine in an unfriendly environment can develop a preference for that initially aversive place, which is likely due to the rewarding effect of nicotine. Therefore, this model can be used to screen exogenous and endogenous molecules involved in nicotine

Aspartame-fed zebrafish exhibit acute deaths with swimming defects and saccharin-fed zebrafish have elevation of cholesteryl ester transfer protein activity in hypercholesterolemia.

PubMed

Kim, Jae-Yong; Seo, Juyi; Cho, Kyung-Hyun

2011-11-01

Although many artificial sweeteners (AS) have safety issues, the AS have been widely used in industry. To determine the physiologic effect of AS in the presence of hyperlipidemia, zebrafish were fed aspartame or saccharin with a high-cholesterol diet (HCD). After 12 days, 30% of zebrafish, which consumed aspartame and HCD, died with exhibiting swimming defects. The aspartame group had 65% survivability, while the control and saccharin groups had 100% survivability. Under HCD, the saccharin-fed groups had the highest increase in the serum cholesterol level (599 mg/dL). Aspartame-fed group showed a remarkable increase in serum glucose (up to 125 mg/dL), which was 58% greater than the increase in the HCD alone group. The saccharin and HCD groups had the highest cholesteryl ester transfer protein (CETP) activity (52% CE-transfer), while the HCD alone group had 42% CE-transfer. Histologic analysis revealed that the aspartame and HCD groups showed more infiltration of inflammatory cells in the brain and liver sections. Conclusively, under presence of hyperlipidemia, aspartame-fed zebrafish exhibited acute swimming defects with an increase in brain inflammation. Saccharin-fed zebrafish had an increased atherogenic serum lipid profile with elevation of CETP activity. Copyright © 2011 Elsevier Ltd. All rights reserved.

The smell of "anxiety": Behavioral modulation by experimental anosmia in zebrafish.

PubMed

Abreu, Murilo S; Giacomini, Ana C V V; Kalueff, Allan V; Barcellos, Leonardo J G

2016-04-01

Olfaction is strongly involved in the regulation of fish behavior, including reproductive, defensive, social and migration behaviors. In fish, anosmia (the lack of olfaction) can be induced experimentally, impairing their ability to respond to various olfactory stimuli. Here, we examine the effects of experimental lidocaine-induced anosmia on anxiety-like behavior and whole-body cortisol levels in adult zebrafish (Danio rerio). We show that experimentally-induced anosmia reduces anxiolytic-like behavioral effects of fluoxetine and seems to interact with anxiogenic effect of stress also paralleling cortisol responses in zebrafish. These findings provide first experimental evidence that temporary anosmia modulates anxiety-like behaviors and physiology in adult zebrafish. Copyright © 2016 Elsevier Inc. All rights reserved.

Zebrafish Health Conditions in the China Zebrafish Resource Center and 20 Major Chinese Zebrafish Laboratories.

PubMed

Liu, Liyue; Pan, Luyuan; Li, Kuoyu; Zhang, Yun; Zhu, Zuoyan; Sun, Yonghua

2016-07-01

In China, the use of zebrafish as an experimental animal in the past 15 years has widely expanded. The China Zebrafish Resource Center (CZRC), which was established in 2012, is becoming one of the major resource centers in the global zebrafish community. Large-scale use and regular exchange of zebrafish resources have put forward higher requirements on zebrafish health issues in China. This article reports the current aquatic infrastructure design, animal husbandry, and health-monitoring programs in the CZRC. Meanwhile, through a survey of 20 Chinese zebrafish laboratories, we also describe the current health status of major zebrafish facilities in China. We conclude that it is of great importance to establish a widely accepted health standard and health-monitoring strategy in the Chinese zebrafish research community.

Zebrafish heart regeneration: 15 years of discoveries

PubMed Central

González‐Rosa, Juan Manuel; Burns, Caroline E.

2017-01-01

Abstract Cardiovascular disease is the leading cause of death worldwide. Compared to other organs such as the liver, the adult human heart lacks the capacity to regenerate on a macroscopic scale after injury. As a result, myocardial infarctions are responsible for approximately half of all cardiovascular related deaths. In contrast, the zebrafish heart regenerates efficiently upon injury through robust myocardial proliferation. Therefore, deciphering the mechanisms that underlie the zebrafish heart's endogenous regenerative capacity represents an exciting avenue to identify novel therapeutic strategies for inducing regeneration of the human heart. This review provides a historical overview of adult zebrafish heart regeneration. We summarize 15 years of research, with a special focus on recent developments from this fascinating field. We discuss experimental findings that address fundamental questions of regeneration research. What is the origin of regenerated muscle? How is regeneration controlled from a genetic and molecular perspective? How do different cell types interact to achieve organ regeneration? Understanding natural models of heart regeneration will bring us closer to answering the ultimate question: how can we stimulate myocardial regeneration in humans? PMID:28979788

Evolution of complexity in the zebrafish synapse proteome

PubMed Central

Bayés, Àlex; Collins, Mark O.; Reig-Viader, Rita; Gou, Gemma; Goulding, David; Izquierdo, Abril; Choudhary, Jyoti S.; Emes, Richard D.; Grant, Seth G. N.

2017-01-01

The proteome of human brain synapses is highly complex and is mutated in over 130 diseases. This complexity arose from two whole-genome duplications early in the vertebrate lineage. Zebrafish are used in modelling human diseases; however, its synapse proteome is uncharacterized, and whether the teleost-specific genome duplication (TSGD) influenced complexity is unknown. We report the characterization of the proteomes and ultrastructure of central synapses in zebrafish and analyse the importance of the TSGD. While the TSGD increases overall synapse proteome complexity, the postsynaptic density (PSD) proteome of zebrafish has lower complexity than mammals. A highly conserved set of ∼1,000 proteins is shared across vertebrates. PSD ultrastructural features are also conserved. Lineage-specific proteome differences indicate that vertebrate species evolved distinct synapse types and functions. The data sets are a resource for a wide range of studies and have important implications for the use of zebrafish in modelling human synaptic diseases. PMID:28252024

Contextual fear conditioning in zebrafish.

PubMed

Kenney, Justin W; Scott, Ian C; Josselyn, Sheena A; Frankland, Paul W

2017-10-01

Zebrafish are a genetically tractable vertebrate that hold considerable promise for elucidating the molecular basis of behavior. Although numerous recent advances have been made in the ability to precisely manipulate the zebrafish genome, much less is known about many aspects of learning and memory in adult fish. Here, we describe the development of a contextual fear conditioning paradigm using an electric shock as the aversive stimulus. We find that contextual fear conditioning is modulated by shock intensity, prevented by an established amnestic agent (MK-801), lasts at least 14 d, and exhibits extinction. Furthermore, fish of various background strains (AB, Tu, and TL) are able to acquire fear conditioning, but differ in fear extinction rates. Taken together, we find that contextual fear conditioning in zebrafish shares many similarities with the widely used contextual fear conditioning paradigm in rodents. Combined with the amenability of genetic manipulation in zebrafish, we anticipate that our paradigm will prove to be a useful complementary system in which to examine the molecular basis of vertebrate learning and memory. © 2017 Kenney et al.; Published by Cold Spring Harbor Laboratory Press.

Brain-wide mapping of neural activity controlling zebrafish exploratory locomotion

PubMed Central

Dunn, Timothy W; Mu, Yu; Narayan, Sujatha; Randlett, Owen; Naumann, Eva A; Yang, Chao-Tsung; Schier, Alexander F

2016-01-01

In the absence of salient sensory cues to guide behavior, animals must still execute sequences of motor actions in order to forage and explore. How such successive motor actions are coordinated to form global locomotion trajectories is unknown. We mapped the structure of larval zebrafish swim trajectories in homogeneous environments and found that trajectories were characterized by alternating sequences of repeated turns to the left and to the right. Using whole-brain light-sheet imaging, we identified activity relating to the behavior in specific neural populations that we termed the anterior rhombencephalic turning region (ARTR). ARTR perturbations biased swim direction and reduced the dependence of turn direction on turn history, indicating that the ARTR is part of a network generating the temporal correlations in turn direction. We also find suggestive evidence for ARTR mutual inhibition and ARTR projections to premotor neurons. Finally, simulations suggest the observed turn sequences may underlie efficient exploration of local environments. DOI: http://dx.doi.org/10.7554/eLife.12741.001 PMID:27003593

Adult zebrafish intestine resection: a novel model of short bowel syndrome, adaptation, and intestinal stem cell regeneration.

PubMed

Schall, K A; Holoyda, K A; Grant, C N; Levin, D E; Torres, E R; Maxwell, A; Pollack, H A; Moats, R A; Frey, M R; Darehzereshki, A; Al Alam, D; Lien, C; Grikscheit, T C

2015-08-01

Loss of significant intestinal length from congenital anomaly or disease may lead to short bowel syndrome (SBS); intestinal failure may be partially offset by a gain in epithelial surface area, termed adaptation. Current in vivo models of SBS are costly and technically challenging. Operative times and survival rates have slowed extension to transgenic models. We created a new reproducible in vivo model of SBS in zebrafish, a tractable vertebrate model, to facilitate investigation of the mechanisms of intestinal adaptation. Proximal intestinal diversion at segment 1 (S1, equivalent to jejunum) was performed in adult male zebrafish. SBS fish emptied distal intestinal contents via stoma as in the human disease. After 2 wk, S1 was dilated compared with controls and villus ridges had increased complexity, contributing to greater villus epithelial perimeter. The number of intervillus pockets, the intestinal stem cell zone of the zebrafish increased and contained a higher number of bromodeoxyuridine (BrdU)-labeled cells after 2 wk of SBS. Egf receptor and a subset of its ligands, also drivers of adaptation, were upregulated in SBS fish. Igf has been reported as a driver of intestinal adaptation in other animal models, and SBS fish exposed to a pharmacological inhibitor of the Igf receptor failed to demonstrate signs of intestinal adaptation, such as increased inner epithelial perimeter and BrdU incorporation. We describe a technically feasible model of human SBS in the zebrafish, a faster and less expensive tool to investigate intestinal stem cell plasticity as well as the mechanisms that drive intestinal adaptation. Copyright © 2015 the American Physiological Society.

Adult zebrafish intestine resection: a novel model of short bowel syndrome, adaptation, and intestinal stem cell regeneration

PubMed Central

Schall, K. A.; Holoyda, K. A.; Grant, C. N.; Levin, D. E.; Torres, E. R.; Maxwell, A.; Pollack, H. A.; Moats, R. A.; Frey, M. R.; Darehzereshki, A.; Al Alam, D.; Lien, C.

2015-01-01

Loss of significant intestinal length from congenital anomaly or disease may lead to short bowel syndrome (SBS); intestinal failure may be partially offset by a gain in epithelial surface area, termed adaptation. Current in vivo models of SBS are costly and technically challenging. Operative times and survival rates have slowed extension to transgenic models. We created a new reproducible in vivo model of SBS in zebrafish, a tractable vertebrate model, to facilitate investigation of the mechanisms of intestinal adaptation. Proximal intestinal diversion at segment 1 (S1, equivalent to jejunum) was performed in adult male zebrafish. SBS fish emptied distal intestinal contents via stoma as in the human disease. After 2 wk, S1 was dilated compared with controls and villus ridges had increased complexity, contributing to greater villus epithelial perimeter. The number of intervillus pockets, the intestinal stem cell zone of the zebrafish increased and contained a higher number of bromodeoxyuridine (BrdU)-labeled cells after 2 wk of SBS. Egf receptor and a subset of its ligands, also drivers of adaptation, were upregulated in SBS fish. Igf has been reported as a driver of intestinal adaptation in other animal models, and SBS fish exposed to a pharmacological inhibitor of the Igf receptor failed to demonstrate signs of intestinal adaptation, such as increased inner epithelial perimeter and BrdU incorporation. We describe a technically feasible model of human SBS in the zebrafish, a faster and less expensive tool to investigate intestinal stem cell plasticity as well as the mechanisms that drive intestinal adaptation. PMID:26089336

Brief embryonic strychnine exposure in zebrafish causes long-term adult behavioral impairment with indications of embryonic synaptic changes.

PubMed

Roy, Nicole M; Arpie, Brianna; Lugo, Joseph; Linney, Elwood; Levin, Edward D; Cerutti, Daniel

2012-01-01

Zebrafish provide a powerful model of the impacts of embryonic toxicant exposure on neural development that may result in long-term behavioral dysfunction. In this study, zebrafish embryos were treated with 1.5mM strychnine for short embryonic time windows to induce transient changes in inhibitory neural signaling, and were subsequently raised in untreated water until adulthood. PCR analysis showed indications that strychnine exposure altered expression of some genes related to glycinergic, GABAergic and glutamatergic neuronal synapses during embryonic development. In adulthood, treated fish showed significant changes in swimming speed and tank diving behavior compared to controls. Taken together, these data show that a short embryonic exposure to a neurotoxicant can alter development of neural synapses and lead to changes in adult behavior. Copyright © 2012 Elsevier Inc. All rights reserved.

In vivo physiological recording from the lateral line of juvenile zebrafish

PubMed Central

Olt, Jennifer; Allen, Claire E.

2016-01-01

Key points Zebrafish provide a unique opportunity to investigate in vivo sensory transduction in mature hair cells.We have developed a method for studying the biophysical properties of mature hair cells from the lateral line of juvenile zebrafish.The method involves application of the anaesthetic benzocaine and intubation to maintain ventilation and oxygenation through the gills.The same approach could be used for in vivo functional studies in other sensory and non‐sensory systems from juvenile and adult zebrafish. Abstract Hair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post‐fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l−1 was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l−1 did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS‐222, which reduces the size of basolateral membrane K+ currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live

Genome-wide Gene Expression Profiling of Acute Metal Exposures in Male Zebrafish

DTIC Science & Technology

2014-10-23

Data in Brief Genome-wide gene expression profiling of acute metal exposures in male zebrafish Christine E. Baer a,⁎, Danielle L. Ippolito b, Naissan... Zebrafish Whole organism Nickel Chromium Cobalt Toxicogenomics To capture global responses to metal poisoning and mechanistic insights into metal...toxicity, gene expression changes were evaluated in whole adult male zebrafish following acute 24 h high dose exposure to three metals with known human

Movement maintains forebrain neurogenesis via peripheral neural feedback in larval zebrafish

PubMed Central

Hall, Zachary Jonas

2018-01-01

The postembryonic brain exhibits experience-dependent development, in which sensory experience guides normal brain growth. This neuroplasticity is thought to occur primarily through structural and functional changes in pre-existing neurons. Whether neurogenesis also mediates the effects of experience on brain growth is unclear. Here, we characterized the importance of motor experience on postembryonic neurogenesis in larval zebrafish. We found that movement maintains an expanded pool of forebrain neural precursors by promoting progenitor self-renewal over the production of neurons. Physical cues associated with swimming (bodily movement) increase neurogenesis and these cues appear to be conveyed by dorsal root ganglia (DRG) in the zebrafish body: DRG-deficient larvae exhibit attenuated neurogenic responses to movement and targeted photoactivation of DRG in immobilized larvae expands the pallial pool of proliferative cells. Our results demonstrate the importance of movement in neurogenic brain growth and reveal a fundamental sensorimotor association that may couple early motor and brain development. PMID:29528285

Conserved gene regulation during acute inflammation between zebrafish and mammals

PubMed Central

Forn-Cuní, G.; Varela, M.; Pereiro, P.; Novoa, B.; Figueras, A.

2017-01-01

Zebrafish (Danio rerio), largely used as a model for studying developmental processes, has also emerged as a valuable system for modelling human inflammatory diseases. However, in a context where even mice have been questioned as a valid model for these analysis, a systematic study evaluating the reproducibility of human and mammalian inflammatory diseases in zebrafish is still lacking. In this report, we characterize the transcriptomic regulation to lipopolysaccharide in adult zebrafish kidney, liver, and muscle tissues using microarrays and demonstrate how the zebrafish genomic responses can effectively reproduce the mammalian inflammatory process induced by acute endotoxin stress. We provide evidence that immune signaling pathways and single gene expression is well conserved throughout evolution and that the zebrafish and mammal acute genomic responses after lipopolysaccharide stimulation are highly correlated despite the differential susceptibility between species to that compound. Therefore, we formally confirm that zebrafish inflammatory models are suited to study the basic mechanisms of inflammation in human inflammatory diseases, with great translational impact potential. PMID:28157230

Cytochrome P450 20A1 in zebrafish: Cloning, regulation and potential involvement in hyperactivity disorders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lemaire, Benjamin; Kubota, Akira; O'Meara, Conor M.

Cytochrome P450 (CYP) enzymes for which there is no functional information are considered “orphan” CYPs. Previous studies showed that CYP20A1, an orphan, is expressed in human hippocampus and substantia nigra, and in zebrafish (Danio rerio) CYP20A1 maternal transcript occurs in eggs, suggesting involvement in brain and in early development. Moreover, hyperactivity is reported in humans with chromosome 2 microdeletions including CYP20A1. We examined CYP20A1 in zebrafish, including impacts of chemical exposure on expression. Zebrafish CYP20A1 cDNA was cloned, sequenced, and aligned with cloned human CYP20A1 and predicted vertebrate orthologs. CYP20A1s share a highly conserved N-terminal region and unusual sequences inmore » the I-helix and the heme-binding CYP signature motifs. CYP20A1 mRNA expression was observed in adult zebrafish organs including the liver, heart, gonads, spleen and brain, as well as the eye and optic nerve. Putative binding sites in proximal promoter regions of CYP20A1s, and response of zebrafish CYP20A1 to selected nuclear and xenobiotic receptor agonists, point to up-regulation by agents involved in steroid hormone response, cholesterol and lipid metabolism. There also was a dose-dependent reduction of CYP20A1 expression in embryos exposed to environmentally relevant levels of methylmercury. Morpholino knockdown of CYP20A1 in developing zebrafish resulted in behavioral effects, including hyperactivity and a slowing of the optomotor response in larvae. The results suggest that altered expression of CYP20A1 might be part of a mechanism linking methylmercury exposure to neurobehavioral deficits. The expanded information on CYP20A1 brings us closer to “deorphanization”, that is, identifying CYP20A1 functions and its roles in health and disease. - Highlights: • The “orphan” CYP20A1 was cloned from zebrafish and its sequence analyzed. • Knockdown of CYP20A1 reduced an optomotor response and elicited bursts of activity. • Effects

Kcnh1 Voltage-gated Potassium Channels Are Essential for Early Zebrafish Development*

PubMed Central

Stengel, Rayk; Rivera-Milla, Eric; Sahoo, Nirakar; Ebert, Christina; Bollig, Frank; Heinemann, Stefan H.; Schönherr, Roland; Englert, Christoph

2012-01-01

The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning. PMID:22927438

Acute toxicity of dichloroacetonitrile (DCAN), a typical nitrogenous disinfection by-product (N-DBP), on zebrafish (Danio rerio).

PubMed

Lin, Tao; Zhou, Dongju; Dong, Jian; Jiang, Fuchun; Chen, Wei

2016-11-01

Dichloroacetonitrile (DCAN) is a typical nitrogenous disinfection by-product (N-DBP) and its toxicity on aquatic animals is investigated for the first time. The present study was designed to investigate the potential adverse effects of DCAN on zebrafish. DCAN could induce developmental toxicity to zebrafish embryos. A significant decrease in hatchability and an increase in malformation and mortality occurred when DCAN concentration was above 100µg/L. Heart function alteration and neuronal function disturbance occurred at concentration higher than 500 and 100µg/L, respectively. Further, DCAN was easily accumulated in adult zebrafish. The rank order of declining bioconcentration factor (BCF) was liver (1240-1670)> gill (1210-1430)> muscle (644-877). DCAN caused acute metabolism damage to adult zebrafish especially at 8 days exposure, at which time the "Integrated Biomarker Response" (IBR) index value reached 798 at 1mg/L DCAN dose. Acute DNA damage was induced to adult zebrafish by DCAN even at 10µg/L dose. Copyright © 2016 Elsevier Inc. All rights reserved.

Chronic exposure of μg/L range Bisphenol A to adult zebrafish (Danio rerio) leading to adipogenesis

NASA Astrophysics Data System (ADS)

Ngo, Mai Thi; Doan, Thao Thi-Phuong; Nguyen, Cong Thanh; Vo, Diem Thi-Ngoc; Do, Chi Hong-Lan; Le, Nga Phi

2017-09-01

Bisphenol A (BPA) is known as an endocrine disruptor compound and commonly found in food-packaging plastics and in aquatic environment. It is scientifically concerned that BPA may causes significant health risks to human and aquatic animals. In fact, most of scientific studies have been conducted with BPA-acute toxicity in early stages of animal development, while far lesser researches have been focused on BPA-chronic toxicity in adults. This study was to investigate the chronic effects of environmental 1, 10 and 100 µg/L BPA to four-week-old zebrafishes (Danio rerio) after 60 days of exposure in terms of changing of body morphology, hepatocyte morphology and the transcriptional expression of biomarker gene for lipid metabolism. As a result, significant effects were found in: 1/ the increase of body weight, but not body length; 2/ the increase in the number of vacuolated hepatocytes corresponding to relatively higher glycogen and lipid content; 3/ shifting hepatocyte morphology to basophilic cytoplasm and cytoplasmic and/or nuclear enlargement, but not inflammation signs (macrophages, granuloma, fibrosis/cirrhosis); 4/ the decrease of mRNA level of PPARγ and C/EBPα genes in liver. Our study here indicates that the exposure to μg/L range concentration of BPA leads to adipogensis in adult zebrafishes.

Enhanced uptake of BPA in the presence of nanoplastics can lead to neurotoxic effects in adult zebrafish.

PubMed

Chen, Qiqing; Yin, Daqiang; Jia, Yunlu; Schiwy, Sabrina; Legradi, Jessica; Yang, Shouye; Hollert, Henner

2017-12-31

Plastic particles have been proven to be abundant in the aquatic environment, raising concerns about their potential toxic effects. In the present study, we determined the bioaccumulation potential of bisphenol A (BPA) in adult zebrafish (Danio rerio) in the absence and presence of nano-sized plastic particles (nanoplastics, NPPs). Results show that BPA can accumulate in the viscera, gill, head and muscle of zebrafish with 85, 43, 20, and 3μg/g ww after 1d exposure. NPPs were also found to accumulate in different tissues of the fish. Relative equilibrium was reached after 1d exposure in different tissues with 39 to 636mg/kg ww. Co-exposure of NPPs and BPA led to a 2.2 and 2.6-fold significant increment of BPA uptake in the head and viscera, if compared with BPA alone treatment after 3d exposure. As such, we further investigated several neurotoxic biomarker alterations in the fish head. It was found that either BPA or NPPs can cause myelin basic protein (MBP)/gene up-regulation in the central nervous system (CNS); meanwhile, both contaminants exhibited significant inhibition of acetylcholinesterase (AChE) activity, which is a well-known representative biomarker for neurotoxicity. Moreover, for the co-exposure treatment, biomarkers of myeline and tubulin protein/gene expressions, dopamine content, and the mRNA expression of mesencephalic astrocyte derived neurotrophic factor (MANF) were all significantly up-regulated, suggesting that an enhanced neurotoxic effects in both CNS and dopaminergic system occurred. However, AChE activity was no more inhibited in the co-exposure treatment, which implies that solely AChE measurement may not be sufficient to identify neurotoxic effects in the cholinergic system. Overall, the present study demonstrates that the presence of NPPs can increase BPA bioavailability and cause neurotoxicity in adult zebrafish. Copyright © 2017 Elsevier B.V. All rights reserved.

Mind the fish: zebrafish as a model in cognitive social neuroscience

PubMed Central

Oliveira, Rui F.

2013-01-01

Understanding how the brain implements social behavior on one hand, and how social processes feedback on the brain to promote fine-tuning of behavioral output according to changes in the social environment is a major challenge in contemporary neuroscience. A critical step to take this challenge successfully is finding the appropriate level of analysis when relating social to biological phenomena. Given the enormous complexity of both the neural networks of the brain and social systems, the use of a cognitive level of analysis (in an information processing perspective) is proposed here as an explanatory interface between brain and behavior. A conceptual framework for a cognitive approach to comparative social neuroscience is proposed, consisting of the following steps to be taken across different species with varying social systems: (1) identification of the functional building blocks of social skills; (2) identification of the cognitive mechanisms underlying the previously identified social skills; and (3) mapping these information processing mechanisms onto the brain. Teleost fish are presented here as a group of choice to develop this approach, given the diversity of social systems present in closely related species that allows for planned phylogenetic comparisons, and the availability of neurogenetic tools that allows the visualization and manipulation of selected neural circuits in model species such as the zebrafish. Finally, the state-of-the art of zebrafish social cognition and of the tools available to map social cognitive abilities to neural circuits in zebrafish are reviewed. PMID:23964204

Mind the fish: zebrafish as a model in cognitive social neuroscience.

PubMed

Oliveira, Rui F

2013-01-01

Understanding how the brain implements social behavior on one hand, and how social processes feedback on the brain to promote fine-tuning of behavioral output according to changes in the social environment is a major challenge in contemporary neuroscience. A critical step to take this challenge successfully is finding the appropriate level of analysis when relating social to biological phenomena. Given the enormous complexity of both the neural networks of the brain and social systems, the use of a cognitive level of analysis (in an information processing perspective) is proposed here as an explanatory interface between brain and behavior. A conceptual framework for a cognitive approach to comparative social neuroscience is proposed, consisting of the following steps to be taken across different species with varying social systems: (1) identification of the functional building blocks of social skills; (2) identification of the cognitive mechanisms underlying the previously identified social skills; and (3) mapping these information processing mechanisms onto the brain. Teleost fish are presented here as a group of choice to develop this approach, given the diversity of social systems present in closely related species that allows for planned phylogenetic comparisons, and the availability of neurogenetic tools that allows the visualization and manipulation of selected neural circuits in model species such as the zebrafish. Finally, the state-of-the art of zebrafish social cognition and of the tools available to map social cognitive abilities to neural circuits in zebrafish are reviewed.

Induction of Social Behavior in Zebrafish: Live Versus Computer Animated Fish as Stimuli

PubMed Central

Qin, Meiying; Wong, Albert; Seguin, Diane

2014-01-01

Abstract The zebrafish offers an excellent compromise between system complexity and practical simplicity and has been suggested as a translational research tool for the analysis of human brain disorders associated with abnormalities of social behavior. Unlike laboratory rodents zebrafish are diurnal, thus visual cues may be easily utilized in the analysis of their behavior and brain function. Visual cues, including the sight of conspecifics, have been employed to induce social behavior in zebrafish. However, the method of presentation of these cues and the question of whether computer animated images versus live stimulus fish have differential effects have not been systematically analyzed. Here, we compare the effects of five stimulus presentation types: live conspecifics in the experimental tank or outside the tank, playback of video-recorded live conspecifics, computer animated images of conspecifics presented by two software applications, the previously employed General Fish Animator, and a new application Zebrafish Presenter. We report that all stimuli were equally effective and induced a robust social response (shoaling) manifesting as reduced distance between stimulus and experimental fish. We conclude that presentation of live stimulus fish, or 3D images, is not required and 2D computer animated images are sufficient to induce robust and consistent social behavioral responses in zebrafish. PMID:24575942

Induction of social behavior in zebrafish: live versus computer animated fish as stimuli.

PubMed

Qin, Meiying; Wong, Albert; Seguin, Diane; Gerlai, Robert

2014-06-01

The zebrafish offers an excellent compromise between system complexity and practical simplicity and has been suggested as a translational research tool for the analysis of human brain disorders associated with abnormalities of social behavior. Unlike laboratory rodents zebrafish are diurnal, thus visual cues may be easily utilized in the analysis of their behavior and brain function. Visual cues, including the sight of conspecifics, have been employed to induce social behavior in zebrafish. However, the method of presentation of these cues and the question of whether computer animated images versus live stimulus fish have differential effects have not been systematically analyzed. Here, we compare the effects of five stimulus presentation types: live conspecifics in the experimental tank or outside the tank, playback of video-recorded live conspecifics, computer animated images of conspecifics presented by two software applications, the previously employed General Fish Animator, and a new application Zebrafish Presenter. We report that all stimuli were equally effective and induced a robust social response (shoaling) manifesting as reduced distance between stimulus and experimental fish. We conclude that presentation of live stimulus fish, or 3D images, is not required and 2D computer animated images are sufficient to induce robust and consistent social behavioral responses in zebrafish.

The Morphogenesis of Cranial Sutures in Zebrafish

PubMed Central

Topczewska, Jolanta M.; Shoela, Ramy A.; Tomaszewski, Joanna P.; Mirmira, Rupa B.; Gosain, Arun K.

2016-01-01

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births. PMID:27829009

Brain Transcriptomic Response to Social Eavesdropping in Zebrafish (Danio rerio)

PubMed Central

Oliveira, Rui F.

2015-01-01

Public information is widely available at low cost to animals living in social groups. For instance, bystanders may eavesdrop on signaling interactions between conspecifics and use it to adapt their subsequent behavior towards the observed individuals. This social eavesdropping ability is expected to require specialized mechanisms such as social attention, which selects social information available for learning. To begin exploring the genetic basis of social eavesdropping, we used a previously established attention paradigm in the lab to study the brain gene expression profile of male zebrafish (Danio rerio) in relation to the attention they paid towards conspecifics involved or not involved in agonistic interactions. Microarray gene chips were used to characterize their brain transcriptomes based on differential expression of single genes and gene sets. These analyses were complemented by promoter region-based techniques. Using data from both approaches, we further drafted protein interaction networks. Our results suggest that attentiveness towards conspecifics, whether interacting or not, activates pathways linked to neuronal plasticity and memory formation. The network analyses suggested that fos and jun are key players on this response, and that npas4a, nr4a1 and egr4 may also play an important role. Furthermore, specifically observing fighting interactions further triggered pathways associated to a change in the alertness status (dnajb5) and to other genes related to memory formation (btg2, npas4b), which suggests that the acquisition of eavesdropped information about social relationships activates specific processes on top of those already activated just by observing conspecifics. PMID:26713440

Zebrafish models for the functional genomics of neurogenetic disorders.

PubMed

Kabashi, Edor; Brustein, Edna; Champagne, Nathalie; Drapeau, Pierre

2011-03-01

In this review, we consider recent work using zebrafish to validate and study the functional consequences of mutations of human genes implicated in a broad range of degenerative and developmental disorders of the brain and spinal cord. Also we present technical considerations for those wishing to study their own genes of interest by taking advantage of this easily manipulated and clinically relevant model organism. Zebrafish permit mutational analyses of genetic function (gain or loss of function) and the rapid validation of human variants as pathological mutations. In particular, neural degeneration can be characterized at genetic, cellular, functional, and behavioral levels. Zebrafish have been used to knock down or express mutations in zebrafish homologs of human genes and to directly express human genes bearing mutations related to neurodegenerative disorders such as spinal muscular atrophy, ataxia, hereditary spastic paraplegia, amyotrophic lateral sclerosis (ALS), epilepsy, Huntington's disease, Parkinson's disease, fronto-temporal dementia, and Alzheimer's disease. More recently, we have been using zebrafish to validate mutations of synaptic genes discovered by large-scale genomic approaches in developmental disorders such as autism, schizophrenia, and non-syndromic mental retardation. Advances in zebrafish genetics such as multigenic analyses and chemical genetics now offer a unique potential for disease research. Thus, zebrafish hold much promise for advancing the functional genomics of human diseases, the understanding of the genetics and cell biology of degenerative and developmental disorders, and the discovery of therapeutics. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

Defining Optimal Brain Health in Adults

PubMed Central

Gorelick, Philip B.; Furie, Karen L.; Iadecola, Costantino; Smith, Eric E.; Waddy, Salina P.; Lloyd-Jones, Donald M.; Bae, Hee-Joon; Bauman, Mary Ann; Dichgans, Martin; Duncan, Pamela W.; Girgus, Meighan; Howard, Virginia J.; Lazar, Ronald M.; Seshadri, Sudha; Testai, Fernando D.; van Gaal, Stephen; Yaffe, Kristine; Wasiak, Hank; Zerna, Charlotte

2017-01-01

Cognitive function is an important component of aging and predicts quality of life, functional independence, and risk of institutionalization. Advances in our understanding of the role of cardiovascular risks have shown them to be closely associated with cognitive impairment and dementia. Because many cardiovascular risks are modifiable, it may be possible to maintain brain health and to prevent dementia in later life. The purpose of this American Heart Association (AHA)/American Stroke Association presidential advisory is to provide an initial definition of optimal brain health in adults and guidance on how to maintain brain health. We identify metrics to define optimal brain health in adults based on inclusion of factors that could be measured, monitored, and modified. From these practical considerations, we identified 7 metrics to define optimal brain health in adults that originated from AHA’s Life’s Simple 7: 4 ideal health behaviors (nonsmoking, physical activity at goal levels, healthy diet consistent with current guideline levels, and body mass index <25 kg/m2) and 3 ideal health factors (untreated blood pressure <120/<80 mm Hg, untreated total cholesterol <200 mg/dL, and fasting blood glucose <100 mg/dL). In addition, in relation to maintenance of cognitive health, we recommend following previously published guidance from the AHA/American Stroke Association, Institute of Medicine, and Alzheimer’s Association that incorporates control of cardiovascular risks and suggest social engagement and other related strategies. We define optimal brain health but recognize that the truly ideal circumstance may be uncommon because there is a continuum of brain health as demonstrated by AHA’s Life’s Simple 7. Therefore, there is opportunity to improve brain health through primordial prevention and other interventions. Furthermore, although cardiovascular risks align well with brain health, we acknowledge that other factors differing from those related to

Pharmacological analyses of learning and memory in zebrafish (Danio rerio).

PubMed

Bailey, Jordan M; Oliveri, Anthony N; Levin, Edward D

2015-12-01

Over the last decade, zebrafish (Danio rerio) have become valuable as a complementary model in behavioral pharmacology, opening a new avenue for understanding the relationships between drug action and behavior. This species offers a useful intermediate approach bridging the gap between in vitro studies and traditional mammalian models. Zebrafish offer great advantages of economy compared to their rodent counterparts, their complex brains and behavioral repertoire offer great translational potential relative to in vitro models. The development and validation of a variety of tests to measure behavior, including cognition, in zebrafish have set the stage for the use of this animal for behavioral pharmacology studies. This has led to research into the basic mechanisms of cognitive function as well as screening for potential cognition-improving drug therapies, among other lines of research. As with all models, zebrafish have limitations, which span pharmacokinetic challenges to difficulties quantifying behavior. The use, efficacy and limitations associated with a zebrafish model of cognitive function are discussed in this review, within the context of behavioral pharmacology. Copyright © 2015 Elsevier Inc. All rights reserved.

In vivo physiological recording from the lateral line of juvenile zebrafish.

PubMed

Olt, Jennifer; Allen, Claire E; Marcotti, Walter

2016-10-01

Zebrafish provide a unique opportunity to investigate in vivo sensory transduction in mature hair cells. We have developed a method for studying the biophysical properties of mature hair cells from the lateral line of juvenile zebrafish. The method involves application of the anaesthetic benzocaine and intubation to maintain ventilation and oxygenation through the gills. The same approach could be used for in vivo functional studies in other sensory and non-sensory systems from juvenile and adult zebrafish. Hair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post-fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l(-1) was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l(-1) did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS-222, which reduces the size of basolateral membrane K(+) currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live zebrafish. More

Intrinsic Properties of Larval Zebrafish Neurons in Ethanol

PubMed Central

Ikeda, Hiromi; Delargy, Alison H.; Yokogawa, Tohei; Urban, Jason M.; Burgess, Harold A.; Ono, Fumihito

2013-01-01

The behavioral effects of ethanol have been studied in multiple animal models including zebrafish. Locomotion of zebrafish larvae is resistant to high concentrations of ethanol in bath solution. This resistance has been attributed to a lower systemic concentration of ethanol in zebrafish when compared with bath solution, although the mechanism to maintain such a steep gradient is unclear. Here we examined whether the intrinsic properties of neurons play roles in this resistance. In order to minimize the contribution of metabolism and diffusional barriers, larvae were hemisected and the anterior half immersed in a range of ethanol concentrations thereby ensuring the free access of bath ethanol to the brain. The response to vibrational stimuli of three types of reticulospinal neurons: Mauthner neurons, vestibulospinal neurons, and MiD3 neurons were examined using an intracellular calcium indicator. The intracellular [Ca2+] response in MiD3 neurons decreased in 100 mM ethanol, while Mauthner neurons and vestibulospinal neurons required >300 mM ethanol to elicit similar effects. The ethanol effect in Mauthner neurons was reversible following removal of ethanol. Interestingly, activities of MiD3 neurons displayed spontaneous recovery in 300 mM ethanol, suggestive of acute tolerance. Finally, we examined with mechanical vibration the startle response of free-swimming larvae in 300 mM ethanol. Ethanol treatment abolished long latency startle responses, suggesting a functional change in neural processing. These data support the hypothesis that individual neurons in larval zebrafish brains have distinct patterns of response to ethanol dictated by specific molecular targets. PMID:23658822

Intradiencephalon injection of histamine inhibited the recovery of locomotor function of spinal cord injured zebrafish.

PubMed

Huang, Shu-Bing; Zhao, Hou-De; Wang, Lin-Fang; Sun, Meng-Fei; Zhu, Ying-Li; Wu, Yi-Bo; Xu, Yi-Da; Peng, Shi-Xiao; Cui, Chun; Shen, Yan-Qin

2017-07-29

Human spinal cord injury (SCI) usually causes irreversible disability beneath the injured site due to poor neural regeneration. On the contrary, zebrafish show significant regenerative ability after SCI, thus is usually worked as an animal model for studying neuroregeneration. Most of the previous SCI studies focused on the local site of SCI, the supraspinal-derived signals were rarely mentioned. Here we showed that intradiencephalon injection of histamine (HA) inhibited the locomotor recovery in adult zebrafish post-SCI. Immunofluorescence results showed that intradiencephalon HA administration increased the activated microglia 3 days post injury (dpi), promoted the proliferation of radial glial cells at 7 dpi and affected the morphology of radial glial cells at 11 dpi. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) results showed that intradiencephalon HA administration also reduced the expression of neurotrophic factors including brain-derived neurotrophic factor (BDNF) and insulin-like growth factor1 (IGF-1) at the lesion site, however, had no effect on the expression of pro-inflammatory factors such as TNF-alpha and IL-1 beta. Hence, our data suggested that exogenous intradiencephalon HA retarded locomotor recovery in spinal cord injured zebrafish via modulating the repair microenvironment. Copyright © 2017 Elsevier Inc. All rights reserved.

Using visual lateralization to model learning and memory in zebrafish larvae

PubMed Central

Andersson, Madelene Åberg; Ek, Fredrik; Olsson, Roger

2015-01-01

Impaired learning and memory are common symptoms of neurodegenerative and neuropsychiatric diseases. Present, there are several behavioural test employed to assess cognitive functions in animal models, including the frequently used novel object recognition (NOR) test. However, although atypical functional brain lateralization has been associated with neuropsychiatric conditions, spanning from schizophrenia to autism, few animal models are available to study this phenomenon in learning and memory deficits. Here we present a visual lateralization NOR model (VLNOR) in zebrafish larvae as an assay that combines brain lateralization and NOR. In zebrafish larvae, learning and memory are generally assessed by habituation, sensitization, or conditioning paradigms, which are all representatives of nondeclarative memory. The VLNOR is the first model for zebrafish larvae that studies a memory similar to the declarative memory described for mammals. We demonstrate that VLNOR can be used to study memory formation, storage, and recall of novel objects, both short and long term, in 10-day-old zebrafish. Furthermore we show that the VLNOR model can be used to study chemical modulation of memory formation and maintenance using dizocilpine (MK-801), a frequently used non-competitive antagonist of the NMDA receptor, used to test putative antipsychotics in animal models. PMID:25727677

Developmental and Persistent Toxicities of Maternally Deposited Selenomethionine in Zebrafish (Danio rerio).

PubMed

Thomas, Jith K; Janz, David M

2015-08-18

The objectives of this study were (1) to establish egg selenium (Se) toxicity thresholds for mortality and deformities in early life stages of zebrafish (Danio rerio) after exposure to excess selenomethionine (SeMet, the dominant chemical species of Se in diets) via in ovo maternal transfer and (2) to investigate the persistent effects of developmental exposure to excess SeMet on swim performance and metabolic capacities in F1-generation adult zebrafish. Adult zebrafish were fed either control food (1.3 μg Se/g, dry mass or d.m.) or food spiked with increasing measured concentrations of Se (3.4, 9.8, or 27.5 μg Se/g, d.m.) in the form of SeMet for 90 d. In ovo exposure to SeMet increased mortality and deformities in larval zebrafish in a concentration-dependent fashion with threshold egg Se concentrations (EC10s) of 7.5 and 7.0 μg Se/g d.m., respectively. Impaired swim performance and greater respiration and metabolic rates were observed in F1-generation zebrafish exposed in ovo to 6.8 and 12.7 μg Se/g d.m and raised to adulthood in clean water. A species sensitivity distribution (SSD) based on egg Se developmental toxicity thresholds suggests that zebrafish are the most sensitive fish species studied to date.

Seeing the whole picture: A comprehensive imaging approach to functional mapping of circuits in behaving zebrafish.

PubMed

Feierstein, C E; Portugues, R; Orger, M B

2015-06-18

In recent years, the zebrafish has emerged as an appealing model system to tackle questions relating to the neural circuit basis of behavior. This can be attributed not just to the growing use of genetically tractable model organisms, but also in large part to the rapid advances in optical techniques for neuroscience, which are ideally suited for application to the small, transparent brain of the larval fish. Many characteristic features of vertebrate brains, from gross anatomy down to particular circuit motifs and cell-types, as well as conserved behaviors, can be found in zebrafish even just a few days post fertilization, and, at this early stage, the physical size of the brain makes it possible to analyze neural activity in a comprehensive fashion. In a recent study, we used a systematic and unbiased imaging method to record the pattern of activity dynamics throughout the whole brain of larval zebrafish during a simple visual behavior, the optokinetic response (OKR). This approach revealed the broadly distributed network of neurons that were active during the behavior and provided insights into the fine-scale functional architecture in the brain, inter-individual variability, and the spatial distribution of behaviorally relevant signals. Combined with mapping anatomical and functional connectivity, targeted electrophysiological recordings, and genetic labeling of specific populations, this comprehensive approach in zebrafish provides an unparalleled opportunity to study complete circuits in a behaving vertebrate animal. Copyright © 2014. Published by Elsevier Ltd.

Effects of the UV filter benzophenone-3 (oxybenzone) at low concentrations in zebrafish (Danio rerio)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blüthgen, Nancy; University of Basel, Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, Klingelbergstrasse 50, CH-4056 Basel; Zucchi, Sara

Organic UV filters including benzophenone-3 (BP-3) are widely used to protect humans and materials from damage by UV irradiation. Despite the environmental occurrence of BP-3 in the aquatic environment, little is known about its effects and modes of action. In the present study we assess molecular and physiological effects of BP-3 in adult male zebrafish (Danio rerio) and in eleuthero-embryos by a targeted gene expression approach focusing on the sex hormone system. Fish and embryos are exposed for 14 days and 120 hours post fertilization, respectively, to 2.4–312 μg/L and 8.2–438 μg/L BP-3. Chemical analysis of water and fish demonstratesmore » that BP-3 is partly transformed to benzophenone-1 (BP-1) and both compounds are accumulated in adult fish. Biotransformation to BP-1 is absent in eleuthero-embryos. BP-3 exposure leads to similar alterations of gene expression in both adult fish and eleuthero-embryos. In the brain of adult males esr1, ar and cyp19b are down-regulated at 84 μg/L BP-3. There is no induction of vitellogenin expression by BP-3, both at the transcriptional and protein level. An overall down-regulation of the hsd3b, hsd17b3, hsd11b2 and cyp11b2 transcripts is observed in the testes, suggesting an antiandrogenic activity. No histological changes were observed in the testes after BP-3 treatment. The study leads to the conclusion that low concentrations of BP-3 exhibit similar multiple hormonal activities at the transcription level in two different life stages of zebrafish. Forthcoming studies should show whether this translates to additional physiological effects. Highlights: ► Activity of UV filter benzophenone-3 (BP-3) is assessed in zebrafish. ► BP-3 is partly metabolized to benzophenone-1 by adult fish but not embryos. ► Alterations of gene expression are similar in adult males and embryos. ► Gene expression alterations point to multiple hormonal activity of BP-3.« less

Effects of embryonic ethanol exposure at low doses on neuronal development, voluntary ethanol consumption and related behaviors in larval and adult zebrafish: Role of hypothalamic orexigenic peptides

PubMed Central

Sterling, M.E.; Chang, G.-Q.; Karatayev, O.; Chang, S.Y.; Leibowitz, S.F.

2016-01-01

Embryonic exposure to ethanol is known to affect neurochemical systems in rodents and increase alcohol drinking and related behaviors in humans and rodents. With zebrafish emerging as a powerful tool for uncovering neural mechanisms of numerous diseases and exhibiting similarities to rodents, the present report building on our rat studies examined in zebrafish the effects of embryonic ethanol exposure on hypothalamic neurogenesis, expression of orexigenic neuropeptides, and voluntary ethanol consumption and locomotor behaviors in larval and adult zebrafish, and also effects of central neuropeptide injections on these behaviors affected by ethanol. At 24 h post-fertilization, zebrafish embryos were exposed for 2 h to ethanol, at low concentrations of 0.25% and 0.5%, in the tank water. Embryonic ethanol compared to control dose-dependently increased hypothalamic neurogenesis and the proliferation and expression of the orexigenic peptides, galanin (GAL) and orexin (OX), in the anterior hypothalamus. These changes in hypothalamic peptide neurons were accompanied by an increase in voluntary consumption of 10% ethanol-gelatin and in novelty-induced locomotor and exploratory behavior in adult zebrafish and locomotor activity in larvae. After intracerebroventricular injection, these peptides compared to vehicle had specific effects on these behaviors altered by ethanol, with GAL stimulating consumption of 10% ethanol-gelatin more than plain gelatin food and OX stimulating novelty-induced locomotor behavior while increasing intake of food and ethanol equally. These results, similar to those obtained in rats, suggest that the ethanol-induced increase in genesis and expression of these hypothalamic peptide neurons contribute to the behavioral changes induced by embryonic exposure to ethanol. PMID:26778786

Optical manipulation for optogenetics: otoliths manipulation in zebrafish (Conference Presentation)

NASA Astrophysics Data System (ADS)

Favre-Bulle, Itia A.; Scott, Ethan; Rubinsztein-Dunlop, Halina

2016-03-01

Otoliths play an important role in Zebrafish in terms of hearing and sense of balance. Many studies have been conducted to understand its structure and function, however the encoding of its movement in the brain remains unknown. Here we developed a noninvasive system capable of manipulating the otolith using optical trapping while we image its behavioral response and brain activity. We'll also present our tools for behavioral response detection and brain activity mapping. Acceleration is sensed through movements of the otoliths in the inner ear. Because experimental manipulations involve movements, electrophysiology and fluorescence microscopy are difficult. As a result, the neural codes underlying acceleration sensation are poorly understood. We have developed a technique for optically trapping otoliths, allowing us to simulate acceleration in stationary larval zebrafish. By applying forces to the otoliths, we can elicit behavioral responses consistent with compensation for perceived acceleration. Since the animal is stationary, we can use calcium imaging in these animals' brains to identify the functional circuits responsible for mediating responses to acceleration in natural settings.

Zebrafish Craniofacial Development: A Window into Early Patterning

PubMed Central

Mork, Lindsey; Crump, Gage

2016-01-01

The formation of the face and skull involves a complex series of developmental events mediated by cells derived from the neural crest, endoderm, mesoderm, and ectoderm. Although vertebrates boast an enormous diversity of adult facial morphologies, the fundamental signaling pathways and cellular events that sculpt the nascent craniofacial skeleton in the embryo have proven to be highly conserved from fish to man. The zebrafish Danio rerio, a small freshwater cyprinid fish from eastern India, has served as a popular model of craniofacial development since the 1990s. Unique strengths of the zebrafish model include a simplified skeleton during larval stages, access to rapidly developing embryos for live imaging, and amenability to transgenesis and complex genetics. In this chapter, we describe the anatomy of the zebrafish craniofacial skeleton; its applications as models for the mammalian jaw, middle ear, palate, and cranial sutures; the superior imaging technology available in fish that has provided unprecedented insights into the dynamics of facial morphogenesis; the use of the zebrafish to decipher the genetic underpinnings of craniofacial biology; and finally a glimpse into the most promising future applications of zebrafish craniofacial research. PMID:26589928

Neuronal expression of fibroblast growth factor receptors in zebrafish.

PubMed

Rohs, Patricia; Ebert, Alicia M; Zuba, Ania; McFarlane, Sarah

2013-12-01

Fibroblast growth factor (FGF) signaling is important for a host of developmental processes such as proliferation, differentiation, tissue patterning, and morphogenesis. In vertebrates, FGFs signal through a family of four fibroblast growth factor receptors (FGFR 1-4), one of which is duplicated in zebrafish (FGFR1). Here we report the mRNA expression of the five known zebrafish fibroblast growth factor receptors at five developmental time points (24, 36, 48, 60, and 72h postfertilization), focusing on expression within the central nervous system. We show that the receptors have distinct and dynamic expression in the developing zebrafish brain, eye, inner ear, lateral line, and pharynx. In many cases, the expression patterns are similar to those of homologous FGFRs in mouse, chicken, amphibians, and other teleosts. Copyright © 2013 Elsevier B.V. All rights reserved.

Time-lapse imaging of neural development: zebrafish lead the way into the fourth dimension.

PubMed

Rieger, Sandra; Wang, Fang; Sagasti, Alvaro

2011-07-01

Time-lapse imaging is often the only way to appreciate fully the many dynamic cell movements critical to neural development. Zebrafish possess many advantages that make them the best vertebrate model organism for live imaging of dynamic development events. This review will discuss technical considerations of time-lapse imaging experiments in zebrafish, describe selected examples of imaging studies in zebrafish that revealed new features or principles of neural development, and consider the promise and challenges of future time-lapse studies of neural development in zebrafish embryos and adults. Copyright © 2011 Wiley-Liss, Inc.

Fibroblast growth factor (Fgf) signaling pathway regulates liver homeostasis in zebrafish.

PubMed

Tsai, Su-Mei; Liu, Da-Wei; Wang, Wen-Pin

2013-04-01

In mammals, fibroblast growth factor (FGF) signaling controls liver specification and regulates the metabolism of lipids, cholesterol, and bile acids. FGF signaling also promotes hepatocyte proliferation, and helps detoxify hepatotoxin during liver regeneration after partial hepatectomy. However, the function of Fgf in zebrafish liver is not yet well understood, specifically for postnatal homeostasis. The current study analyzed the expression of fgf receptors (fgfrs) in the liver of zebrafish. We then investigated the function of Fgf signaling in the zebrafish liver by expressing a dominant-negative Fgf receptor in hepatocytes (lfabp:dnfgfr1-egfp, lf:dnfr). Histological analysis showed that our genetic intervention resulted in a small liver size with defected medial expansion of developing livers in transgenic (Tg) larvae. Morphologically, the liver lobe of lf:dnfr adult fish was shorter than that of control. Ballooning degeneration of hepatocytes was observed in fish as young as 3 months. Further examination revealed the development of hepatic steatosis and cholestasis. In adult Tg fish, we unexpectedly observed increased liver-to-body-weight ratios, with higher percentages of proliferating hepatocytes. Considering all these findings, we concluded that as in mammals, in adult zebrafish the metabolism of lipid and bile acids in the liver are regulated by Fgf signaling. Disruption of the Fgf signal-mediated metabolism might indirectly affect hepatocyte proliferation.

Characterization of behavioral and endocrine effects of LSD on zebrafish.

PubMed

Grossman, Leah; Utterback, Eli; Stewart, Adam; Gaikwad, Siddharth; Chung, Kyung Min; Suciu, Christopher; Wong, Keith; Elegante, Marco; Elkhayat, Salem; Tan, Julia; Gilder, Thomas; Wu, Nadine; Dileo, John; Cachat, Jonathan; Kalueff, Allan V

2010-12-25

Lysergic acid diethylamide (LSD) is a potent hallucinogenic drug that strongly affects animal and human behavior. Although adult zebrafish (Danio rerio) are emerging as a promising neurobehavioral model, the effects of LSD on zebrafish have not been investigated previously. Several behavioral paradigms (the novel tank, observation cylinder, light-dark box, open field, T-maze, social preference and shoaling tests), as well as modern video-tracking tools and whole-body cortisol assay were used to characterize the effects of acute LSD in zebrafish. While lower doses (5-100 microg/L) did not affect zebrafish behavior, 250 microg/L LSD increased top dwelling and reduced freezing in the novel tank and observation cylinder tests, also affecting spatiotemporal patterns of activity (as assessed by 3D reconstruction of zebrafish traces and ethograms). LSD evoked mild thigmotaxis in the open field test, increased light behavior in the light-dark test, reduced the number of arm entries and freezing in the T-maze and social preference test, without affecting social preference. In contrast, LSD affected zebrafish shoaling (increasing the inter-fish distance in a group), and elevated whole-body cortisol levels. Overall, our findings show sensitivity of zebrafish to LSD action, and support the use of zebrafish models to study hallucinogenic drugs of abuse. Copyright (c) 2010 Elsevier B.V. All rights reserved.

The effects of vitamin D on brain development and adult brain function.

PubMed

Kesby, James P; Eyles, Darryl W; Burne, Thomas H J; McGrath, John J

2011-12-05

A role for vitamin D in brain development and function has been gaining support over the last decade. Multiple lines of evidence suggest that this vitamin is actually a neuroactive steroid that acts on brain development, leading to alterations in brain neurochemistry and adult brain function. Early deficiencies have been linked with neuropsychiatric disorders, such as schizophrenia, and adult deficiencies have been associated with a host of adverse brain outcomes, including Parkinson's disease, Alzheimer's disease, depression and cognitive decline. This review summarises the current state of research on the actions of vitamin D in the brain and the consequences of deficiencies in this vitamin. Furthermore, we discuss specific implications of vitamin D status on the neurotransmitter, dopamine. Copyright © 2011 Elsevier Ltd. All rights reserved.

Kidney organogenesis in the zebrafish: insights into vertebrate nephrogenesis and regeneration

PubMed Central

Gerlach, Gary F.; Wingert, Rebecca A.

2012-01-01

Vertebrates form a progressive series of up to three kidney organs during development—the pronephros, mesonephros, and metanephros. Each kidney derives from the intermediate mesoderm and is comprised of conserved excretory units called nephrons. The zebrafish is a powerful model for vertebrate developmental genetics, and recent studies have illustrated that zebrafish and mammals share numerous similarities in nephron composition and physiology. The zebrafish embryo forms an architecturally simple pronephros that has two nephrons, and these eventually become a scaffold onto which a mesonephros of several hundred nephrons is constructed during larval stages. In adult zebrafish, the mesonephros exhibits ongoing nephrogenesis, generating new nephrons from a local pool of renal progenitors during periods of growth or following kidney injury. The characteristics of the zebrafish pronephros and mesonephros make them genetically tractable kidney systems in which to study the functions of renal genes and address outstanding questions about the mechanisms of nephrogenesis. Here, we provide an overview of the formation and composition of these zebrafish kidney organs, and discuss how various zebrafish mutants, gene knockdowns, and transgenic models have created frameworks in which to further delineate nephrogenesis pathways. PMID:24014448

Anxiogenic-like effects of chronic nicotine exposure in zebrafish.

PubMed

Stewart, Adam Michael; Grossman, Leah; Collier, Adam D; Echevarria, David J; Kalueff, Allan V

2015-12-01

Nicotine is one of the most widely used and abused legal drugs. Although its pharmacological profile has been extensively investigated in humans and rodents, nicotine CNS action remains poorly understood. The importance of finding evolutionarily conserved signaling pathways, and the need to apply high-throughput in vivo screens for CNS drug discovery, necessitate novel efficient experimental models for nicotine research. Zebrafish (Danio rerio) are rapidly emerging as an excellent organism for studying drug abuse, neuropharmacology and toxicology and have recently been applied to testing nicotine. Anxiolytic, rewarding and memory-modulating effects of acute nicotine treatment in zebrafish are consistently reported in the literature. However, while nicotine abuse is more relevant to long-term exposure models, little is known about chronic effects of nicotine on zebrafish behavior. In the present study, chronic 4-day exposure to 1-2mg/L nicotine mildly increased adult zebrafish shoaling but did not alter baseline cortisol levels. We also found that chronic exposure to nicotine evokes robust anxiogenic behavioral responses in zebrafish tested in the novel tank test paradigm. Generally paralleling clinical and rodent data on anxiogenic effects of chronic nicotine, our study supports the developing utility of zebrafish for nicotine research. Copyright © 2015 Elsevier Inc. All rights reserved.

Actin-Cytoskeleton- and Rock-Mediated INM Are Required for Photoreceptor Regeneration in the Adult Zebrafish Retina

PubMed Central

Lahne, Manuela; Li, Jingling; Marton, Rebecca M.

2015-01-01

Loss of retinal neurons in adult zebrafish (Danio rerio) induces a robust regenerative response mediated by the reentry of the resident Müller glia into the cell cycle. Upon initiating Müller glia proliferation, their nuclei migrate along the apicobasal axis of the retina in phase with the cell cycle in a process termed interkinetic nuclear migration (INM). We examined the mechanisms governing this cellular process and explored its function in regenerating the adult zebrafish retina. Live-cell imaging revealed that the majority of Müller glia nuclei migrated to the outer nuclear layer (ONL) to divide. These Müller glia formed prominent actin filaments at the rear of nuclei that had migrated to the ONL. Inhibiting actin filament formation or Rho-associated coiled-coil kinase (Rock) activity, which is necessary for phosphorylation of myosin light chain and actin myosin-mediated contraction, disrupted INM with increased numbers of mitotic nuclei remaining in the basal inner nuclear layer, the region where Müller glia typically reside. Double knockdown of Rho-associated coiled-coil kinase 2a (Rock2a) and Rho-associated coiled-coil kinase 2b (Rock2b) similarly disrupted INM and reduced Müller glial cell cycle reentry. In contrast, Rock inhibition immediately before the onset of INM did not affect Müller glia proliferation, but subsequently reduced neuronal progenitor cell proliferation due to early cell cycle exit. Long-term, Rock inhibition increased the generation of mislocalized ganglion/amacrine cells at the expense of rod and cone photoreceptors. In summary, INM is driven by an actin-myosin-mediated process controlled by Rock2a and Rock2b activity, which is required for sufficient proliferation and regeneration of photoreceptors after light damage. SIGNIFICANCE STATEMENT The human retina does not replace lost or damaged neurons, ultimately causing vision impairment. In contrast, zebrafish are capable of regenerating lost neurons. Understanding the mechanisms

Actin-Cytoskeleton- and Rock-Mediated INM Are Required for Photoreceptor Regeneration in the Adult Zebrafish Retina.

PubMed

Lahne, Manuela; Li, Jingling; Marton, Rebecca M; Hyde, David R

2015-11-25

Loss of retinal neurons in adult zebrafish (Danio rerio) induces a robust regenerative response mediated by the reentry of the resident Müller glia into the cell cycle. Upon initiating Müller glia proliferation, their nuclei migrate along the apicobasal axis of the retina in phase with the cell cycle in a process termed interkinetic nuclear migration (INM). We examined the mechanisms governing this cellular process and explored its function in regenerating the adult zebrafish retina. Live-cell imaging revealed that the majority of Müller glia nuclei migrated to the outer nuclear layer (ONL) to divide. These Müller glia formed prominent actin filaments at the rear of nuclei that had migrated to the ONL. Inhibiting actin filament formation or Rho-associated coiled-coil kinase (Rock) activity, which is necessary for phosphorylation of myosin light chain and actin myosin-mediated contraction, disrupted INM with increased numbers of mitotic nuclei remaining in the basal inner nuclear layer, the region where Müller glia typically reside. Double knockdown of Rho-associated coiled-coil kinase 2a (Rock2a) and Rho-associated coiled-coil kinase 2b (Rock2b) similarly disrupted INM and reduced Müller glial cell cycle reentry. In contrast, Rock inhibition immediately before the onset of INM did not affect Müller glia proliferation, but subsequently reduced neuronal progenitor cell proliferation due to early cell cycle exit. Long-term, Rock inhibition increased the generation of mislocalized ganglion/amacrine cells at the expense of rod and cone photoreceptors. In summary, INM is driven by an actin-myosin-mediated process controlled by Rock2a and Rock2b activity, which is required for sufficient proliferation and regeneration of photoreceptors after light damage. The human retina does not replace lost or damaged neurons, ultimately causing vision impairment. In contrast, zebrafish are capable of regenerating lost neurons. Understanding the mechanisms that regulate retinal

Neurobehavioral impairments produced by developmental lead exposure persisted for generations in zebrafish (Danio rerio).

PubMed

Xu, Xiaojuan; Weber, Daniel; Burge, Rebekah; VanAmberg, Kelsey

2016-01-01

The zebrafish has become a useful animal model for studying the effects of environmental contaminants on neurobehavioral development due to its ease of breeding, high number of eggs per female, short generation times, and a well-established avoidance conditioning paradigm. Using avoidance conditioning as the behavioral paradigm, the present study investigated the effects of embryonic exposure to lead (Pb) on learning in adult zebrafish and the third (F3) generation of those fish. In Experiment 1, adult zebrafish that were developmentally exposed to 0.0, 0.1, 1.0 or 10.0μM Pb (2-24h post fertilization) as embryos were trained and tested for avoidance responses. The results showed that adult zebrafish hatched from embryos exposed to 0.0 or 0.1μM Pb learned avoidance responses during training and displayed significantly increased avoidance responses during testing, while those hatched from embryos exposed to 1.0 or 10.0μM Pb displayed no significant increases in avoidance responses from training to testing. In Experiment 2, the F3 generation of zebrafish that were developmentally exposed to an identical exposure regimen as in Experiment 1 were trained and tested for avoidance responses. The results showed that the F3 generation of zebrafish developmentally exposed as embryos to 0.0 or 0.1μM Pb learned avoidance responses during training and displayed significantly increased avoidance responses during testing, while the F3 generation of zebrafish developmentally exposed as embryos to 1.0 or 10.0μM Pb displayed no significant changes in avoidance responses from training to testing. Thus, developmental Pb exposure produced learning impairments that persisted for at least three generations, demonstrating trans-generational effects of embryonic exposure to Pb. Copyright © 2015. Published by Elsevier B.V.

Effects of embryonic ethanol exposure at low doses on neuronal development, voluntary ethanol consumption and related behaviors in larval and adult zebrafish: Role of hypothalamic orexigenic peptides.

PubMed

Sterling, M E; Chang, G-Q; Karatayev, O; Chang, S Y; Leibowitz, S F

2016-05-01

Embryonic exposure to ethanol is known to affect neurochemical systems in rodents and increase alcohol drinking and related behaviors in humans and rodents. With zebrafish emerging as a powerful tool for uncovering neural mechanisms of numerous diseases and exhibiting similarities to rodents, the present report building on our rat studies examined in zebrafish the effects of embryonic ethanol exposure on hypothalamic neurogenesis, expression of orexigenic neuropeptides, and voluntary ethanol consumption and locomotor behaviors in larval and adult zebrafish, and also effects of central neuropeptide injections on these behaviors affected by ethanol. At 24h post-fertilization, zebrafish embryos were exposed for 2h to ethanol, at low concentrations of 0.25% and 0.5%, in the tank water. Embryonic ethanol compared to control dose-dependently increased hypothalamic neurogenesis and the proliferation and expression of the orexigenic peptides, galanin (GAL) and orexin (OX), in the anterior hypothalamus. These changes in hypothalamic peptide neurons were accompanied by an increase in voluntary consumption of 10% ethanol-gelatin and in novelty-induced locomotor and exploratory behavior in adult zebrafish and locomotor activity in larvae. After intracerebroventricular injection, these peptides compared to vehicle had specific effects on these behaviors altered by ethanol, with GAL stimulating consumption of 10% ethanol-gelatin more than plain gelatin food and OX stimulating novelty-induced locomotor behavior while increasing intake of food and ethanol equally. These results, similar to those obtained in rats, suggest that the ethanol-induced increase in genesis and expression of these hypothalamic peptide neurons contribute to the behavioral changes induced by embryonic exposure to ethanol. Copyright © 2016 Elsevier B.V. All rights reserved.

Anxiolytic-like effects of noribogaine in zebrafish.

PubMed

Kalueff, Allan V; Kaluyeva, Aleksandra; Maillet, Emeline L

2017-07-14

Noribogaine is the main psychoactive metabolite of the hallucinogenic drug ibogaine, and is a particularly interesting compound potentially useful to treat dependence and various psychiatric disorders. Here, we report the effects of noribogaine on anxiety and locomotion in zebrafish (Danio rerio), a new promising model organism in neurobehavioral and psychopharmacological research. Adult zebrafish were subjected to the 5min novel tank test (NTT) following an acute, 20-min drug immersion in 1, 5 and 10mg/L noribogaine. Overall, noribogaine produced robust anxiolytic-like behavior in zebrafish (increasing the time spent and transitions to the top half compartment and reducing freezing bouts) without overt effects on fish locomotion. Taken together, these results indicate that noribogaine modulates the components of the acute stress response related to emotionality and anxiety behaviors, implicating this drug as a potentially useful non-sedative anxiolytic agent. Copyright © 2017 Elsevier B.V. All rights reserved.

Stereoselective effects of ibuprofen in adult zebrafish (Danio rerio) using UPLC-TOF/MS-based metabolomics.

PubMed

Song, Yue; Chai, Tingting; Yin, Zhiqiang; Zhang, Xining; Zhang, Wei; Qian, Yongzhong; Qiu, Jing

2018-06-09

Ibuprofen (IBU), as a commonly used non-steroidal anti-inflammatory drug (NSAID) and pharmaceutical and personal care product (PPCP), is frequently prescribed by doctors to relieve pain. It is widely released into environmental water and soil in the form of chiral enantiomers by the urination and defecation of humans or animals and by sewage discharge from wastewater treatment plants. This study focused on the alteration of metabolism in the adult zebrafish (Danio rerio) brain after exposure to R-(-)-/S-(+)-/rac-IBU at 5 μg L -1 for 28 days. A total of 45 potential biomarkers and related pathways, including amino acids and their derivatives, purine and its derivatives, nucleotides and other metabolites, were observed with untargeted metabolomics. To validate the metabolic disorders induced by IBU, 22 amino acids and 3 antioxidant enzymes were selected to be quantitated and determined using targeted metabolomics and enzyme assay. Stereoselective changes were observed in the 45 identified biomarkers from the untargeted metabolomics analysis. The 22 amino acids quantitated in targeted metabolomics and 3 antioxidant enzymes determined in enzyme assay also showed stereoselective changes after R-(-)-/S-(+)-/rac-IBU exposure. Results showed that even at a low concentration of R-(-)-/S-(+)-/rac-IBU, disorders in metabolism and antioxidant defense systems were still induced with stereoselectivity. Our study may enable a better understanding of the risks of chiral PPCPs in aquatic organisms in the environment. Copyright © 2018 Elsevier Ltd. All rights reserved.

Zebrafish: an animal model for research in veterinary medicine.

PubMed

Nowik, N; Podlasz, P; Jakimiuk, A; Kasica, N; Sienkiewicz, W; Kaleczyc, J

2015-01-01

The zebrafish (Danio rerio) has become known as an excellent model organism for studies of vertebrate biology, vertebrate genetics, embryonal development, diseases and drug screening. Nevertheless, there is still lack of detailed reports about usage of the zebrafish as a model in veterinary medicine. Comparing to other vertebrates, they can lay hundreds of eggs at weekly intervals, externally fertilized zebrafish embryos are accessible to observation and manipulation at all stages of their development, which makes possible to simplify the research techniques such as fate mapping, fluorescent tracer time-lapse lineage analysis and single cell transplantation. Although zebrafish are only 2.5 cm long, they are easy to maintain. Intraperitoneal and intracerebroventricular injections, blood sampling and measurement of food intake are possible to be carry out in adult zebrafish. Danio rerio is a useful animal model for neurobiology, developmental biology, drug research, virology, microbiology and genetics. A lot of diseases, for which the zebrafish is a perfect model organism, affect aquatic animals. For a part of them, like those caused by Mycobacterium marinum or Pseudoloma neutrophila, Danio rerio is a natural host, but the zebrafish is also susceptible to the most of fish diseases including Itch, Spring viraemia of carp and Infectious spleen and kidney necrosis. The zebrafish is commonly used in research of bacterial virulence. The zebrafish embryo allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host. Plenty of common pathogens can be examined using zebrafish model: Streptococcus iniae, Vibrio anguillarum or Listeria monocytogenes. The steps are taken to use the zebrafish also in fungal research, especially that dealing with Candida albicans and Cryptococcus neoformans. Although, the zebrafish is used commonly as an animal model to study diseases caused by external agents, it is also useful in studies of metabolic

Novel Insights into the Genetic Controls of Primitive and Definitive Hematopoiesis from Zebrafish Models

PubMed Central

Sood, Raman; Liu, Paul

2012-01-01

Hematopoiesis is a dynamic process where initiation and maintenance of hematopoietic stem cells, as well as their differentiation into erythroid, myeloid and lymphoid lineages, are tightly regulated by a network of transcription factors. Understanding the genetic controls of hematopoiesis is crucial as perturbations in hematopoiesis lead to diseases such as anemia, thrombocytopenia, or cancers, including leukemias and lymphomas. Animal models, particularly conventional and conditional knockout mice, have played major roles in our understanding of the genetic controls of hematopoiesis. However, knockout mice for most of the hematopoietic transcription factors are embryonic lethal, thus precluding the analysis of their roles during the transition from embryonic to adult hematopoiesis. Zebrafish are an ideal model organism to determine the function of a gene during embryonic-to-adult transition of hematopoiesis since bloodless zebrafish embryos can develop normally into early larval stage by obtaining oxygen through diffusion. In this review, we discuss the current status of the ontogeny and regulation of hematopoiesis in zebrafish. By providing specific examples of zebrafish morphants and mutants, we have highlighted the contributions of the zebrafish model to our overall understanding of the roles of transcription factors in regulation of primitive and definitive hematopoiesis. PMID:22888355

Pan-neuronal calcium imaging with cellular resolution in freely swimming zebrafish.

PubMed

Kim, Dal Hyung; Kim, Jungsoo; Marques, João C; Grama, Abhinav; Hildebrand, David G C; Gu, Wenchao; Li, Jennifer M; Robson, Drew N

2017-11-01

Calcium imaging with cellular resolution typically requires an animal to be tethered under a microscope, which substantially restricts the range of behaviors that can be studied. To expand the behavioral repertoire amenable to imaging, we have developed a tracking microscope that enables whole-brain calcium imaging with cellular resolution in freely swimming larval zebrafish. This microscope uses infrared imaging to track a target animal in a behavior arena. On the basis of the predicted trajectory of the animal, we applied optimal control theory to a motorized stage system to cancel brain motion in three dimensions. We combined this motion-cancellation system with differential illumination focal filtering, a variant of HiLo microscopy, which enabled us to image the brain of a freely swimming larval zebrafish for more than an hour. This work expands the repertoire of natural behaviors that can be studied with cellular-resolution calcium imaging to potentially include spatial navigation, social behavior, feeding and reward.

Distribution, pharmacokinetics and primary metabolism model of tramadol in zebrafish.

PubMed

Zhuo, Huiqin; Jin, Hongwei; Peng, Huifang; Huang, Heqing

2016-12-01

The current study aimed to develop a rapid, robust and adequately sensitive method for simultaneous determination of the concentration of tramadol and its active metabolites in zebrafish. The pharmacokinetic and elimination pattern of tramadol and its major phase I metabolites following oral or intramuscular administration in zebrafish tissues was achieved using electrospray ionization‑quadrupole‑time of flight/mass spectrometry (ESI‑Q‑TOF/MS) and gas chromatography/mass spectrometry (GC‑MS). Following administration, the metabolisms were detected in the brain, eyes, muscle and gill tissues within 1 h. Two tramadol metabolites, O‑ and N‑desmethyltramadol, were detected in brain tissue, with N‑desmethyltramadol detected at a higher level. Following GC‑MS detection the curve indicated an initial rapid phase, corresponding to the detection of the tramadol within 1 min, and reached peak value in the brain at 5 min. Faster drug clearance was detected in low‑dose groups, and concentration had dropped around the to initial level (1.11 µg) at 20 min, but was detectable for up to 3 h. However, it took 80 min to fall back to the initial value (1.73 µg) in the high‑dose groups, and tramadol was detectable for up to 4 h. This study developed and validated a simple and high throughput analytical procedure to determine the distribution and pharmacokinetic profiles of tramadol, and its primary metabolites in tissues of zebrafish.

BISPHENOL A EXPOSURE DURING EARLY DEVELOPMENT INDUCES SEX-SPECIFIC CHANGES IN ADULT ZEBRAFISH SOCIAL INTERACTIONS

PubMed Central

Weber, Daniel N.; Hoffmann, Raymond G.; Hoke, Elizabeth S.; Tanguay, Robert L.

2014-01-01

Developmental bisphenol A (BPA) exposure is associated with adverse behavioral effects, although underlying modes of action remain unclear. Because BPA is a suspected xenoestrogen, the objective was to identify sex-based changes in adult zebrafish social behavior developmentally exposed to BPA (0.0, 0.1 or 1 μM) or one of two control compounds (0.1μM 17β-estradiol [E2], and 0.1 μM GSK4716, a synthetic estrogen-related receptor γ ligand). A test chamber was divided lengthwise so each arena held one fish unable to detect the presence of the other fish. A mirror was inserted at one end of each arena; baseline activity levels were determined without mirror. Arenas were divided into 3, computer-generated zones to represent different distances from mirror image. Circadian rhythm patterns were evaluated at 1–3 (= AM) and 5–8 (= PM) hr postprandial. Adult zebrafish were placed into arenas and monitored by digital camera for 5 min. Total distance traveled, % time spent at mirror image, and number of attacks on mirror image were quantified. E2, GSK4716, and all BPA treatments dampened male activity and altered male circadian activity patterns; there was no marked effect on female activity. BPA induced non-monotonic effects (response curve changes direction within range of concentrations examined) on male % time at mirror only in AM. All treatments produced increased % time at the mirror during PM. Male attacks on the mirror were reduced by BPA exposure only during AM. There were sex-specific effects of developmental BPA on social interactions and time-of-day of observation affected results. PMID:25424546

A Brain-wide Circuit Model of Heat-Evoked Swimming Behavior in Larval Zebrafish.

PubMed

Haesemeyer, Martin; Robson, Drew N; Li, Jennifer M; Schier, Alexander F; Engert, Florian

2018-05-16

Thermosensation provides crucial information, but how temperature representation is transformed from sensation to behavior is poorly understood. Here, we report a preparation that allows control of heat delivery to zebrafish larvae while monitoring motor output and imaging whole-brain calcium signals, thereby uncovering algorithmic and computational rules that couple dynamics of heat modulation, neural activity and swimming behavior. This approach identifies a critical step in the transformation of temperature representation between the sensory trigeminal ganglia and the hindbrain: A simple sustained trigeminal stimulus representation is transformed into a representation of absolute temperature as well as temperature changes in the hindbrain that explains the observed motor output. An activity constrained dynamic circuit model captures the most prominent aspects of these sensori-motor transformations and predicts both behavior and neural activity in response to novel heat stimuli. These findings provide the first algorithmic description of heat processing from sensory input to behavioral output. Copyright © 2018 Elsevier Inc. All rights reserved.

An integrative analysis of ethanol tolerance and withdrawal in zebrafish (Danio rerio)

PubMed Central

Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert

2014-01-01

The zebrafish is emerging as a popular animal model for alcohol (ethanol or EtOH) addiction due to its simplicity and practical advantages. Two phenomena associated with ethanol addiction are the development of tolerance and withdrawal. Using a multi-level approach in the current study, we characterize ethanol tolerance and withdrawal in zebrafish. We first investigate the temporal trajectory of ethanol concentration in the zebrafish brain in response to an acute exposure and during withdrawal. We report that ethanol concentrations approach a steady state within 60 minutes of exposure to 0.50% and 1.00% v/v ethanol and rapidly decline and return to zero within 60 minutes following withdrawal from chronic ethanol exposure (0.50% v/v). We characterize the changes associated with ethanol tolerance and withdrawal in zebrafish by focusing on 3 domains relevant to ethanol addiction: motor patterns, physiological responses (i.e. cortisol levels), and neurochemical alterations. The use of multiple domains of investigation allowed an in-depth analysis of ethanol induced changes in zebrafish. PMID:24598276

Dopamine receptor antagonism disrupts social preference in zebrafish, a strain comparison study

PubMed Central

Scerbina, Tanya; Chatterjee, Diptendu; Gerlai, Robert

2012-01-01

Zebrafish form shoals in nature and in the laboratory. The sight of conspecifics has been found reinforcing in zebrafish learning tasks. However, the mechanisms of shoaling, and those of its reinforcing properties, are not known. The dopaminergic system has been implicated in reward among other functions and it is also engaged by drugs of abuse as shown in a variety of vertebrates including zebrafish. The ontogenetic changes in dopamine levels and, to a lesser degree, in serotonin levels, have been found to accompany the maturation of shoaling in zebrafish. Thus, we hypothesized that the dopaminergic system may contribute to shoaling in zebrafish. To test this we employed a D1-receptor antagonist and quantified behavioral responses of our subjects using a social preference (shoaling) paradigm. We found significant reduction of social preference induced by the D1-R antagonist, SCH23390, in the AB strain of zebrafish, an alteration that was not accompanied by changes in motor function or vision. We also detected D1-R antagonist induced changes in the level of dopamine, DOPAC, serotonin and 5HIAA, respectively, in the brain of AB zebrafish as quantified by HPLC with electrochemical detection. We found the antagonist induced behavioral changes to be absent and the levels of these neurochemicals to be lower in another zebrafish population, SF, demonstrating naturally occurring genetic variability in these traits. We conclude that this variability may be utilized to unravel the mechanisms of social behavior in zebrafish, a line of research that may be extended to other vertebrates including our own species. PMID:22491827

Towards child versus adult brain mechanical properties.

PubMed

Chatelin, S; Vappou, J; Roth, S; Raul, J S; Willinger, R

2012-02-01

The characterization of brain tissue mechanical properties is of crucial importance in the development of realistic numerical models of the human head. While the mechanical behavior of the adult brain has been extensively investigated in several studies, there is a considerable paucity of data concerning the influence of age on mechanical properties of the brain. Therefore, the implementation of child and infant head models often involves restrictive assumptions like properties scaling from adult or animal data. The present study presents a step towards the investigation of the effects of age on viscoelastic properties of human brain tissue from a first set of dynamic oscillatory shear experiments. Tests were also performed on three different locations of brain (corona radiata, thalamus and brainstem) in order to investigate regional differences. Despite the limited number of child brain samples a significant increase in both storage and loss moduli occurring between the age of 5 months and the age of 22 months was found, confirmed by statistical Student's t-tests (p=0.104,0.038 and 0.054 for respectively corona radiata, thalamus and brain stem samples locations respectively). The adult brain appears to be 3-4 times stiffer than the young child one. Moreover, the brainstem was found to be approximately 2-3 times stiffer than both gray and white matter from corona radiata and thalamus. As a tentative conclusion, this study provides the first rheological data on the human brain at different ages and brain regions. This data could be implemented in numerical models of the human head, especially in models concerning pediatric population. Copyright © 2011 Elsevier Ltd. All rights reserved.

The Mammalian “Obesogen” Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish

PubMed Central

Lyssimachou, Angeliki; Santos, Joana G.; André, Ana; Soares, Joana; Lima, Daniela; Guimarães, Laura; Almeida, C. Marisa R.; Teixeira, Catarina; Castro, L. Filipe C.; Santos, Miguel M.

2015-01-01

Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an “obesogenic” phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound. PMID:26633012

Transient Overexpression of adh8a Increases Allyl Alcohol Toxicity in Zebrafish Embryos

PubMed Central

Klüver, Nils; Ortmann, Julia; Paschke, Heidrun; Renner, Patrick; Ritter, Axel P.; Scholz, Stefan

2014-01-01

Fish embryos are widely used as an alternative model to study toxicity in vertebrates. Due to their complexity, embryos are believed to more resemble an adult organism than in vitro cellular models. However, concerns have been raised with respect to the embryo's metabolic capacity. We recently identified allyl alcohol, an industrial chemical, to be several orders of magnitude less toxic to zebrafish embryo than to adult zebrafish (embryo LC50 = 478 mg/L vs. fish LC50 = 0.28 mg/L). Reports on mammals have indicated that allyl alcohol requires activation by alcohol dehydrogenases (Adh) to form the highly reactive and toxic metabolite acrolein, which shows similar toxicity in zebrafish embryos and adults. To identify if a limited metabolic capacity of embryos indeed can explain the low allyl alcohol sensitivity of zebrafish embryos, we compared the mRNA expression levels of Adh isoenzymes (adh5, adh8a, adh8b and adhfe1) during embryo development to that in adult fish. The greatest difference between embryo and adult fish was found for adh8a and adh8b expression. Therefore, we hypothesized that these genes might be required for allyl alcohol activation. Microinjection of adh8a, but not adh8b mRNA led to a significant increase of allyl alcohol toxicity in embryos similar to levels reported for adults (LC50 = 0.42 mg/L in adh8a mRNA-injected embryos). Furthermore, GC/MS analysis of adh8a-injected embryos indicated a significant decline of internal allyl alcohol concentrations from 0.23-58 ng/embryo to levels below the limit of detection (< 4.6 µg/L). Injection of neither adh8b nor gfp mRNA had an impact on internal allyl alcohol levels supporting that the increased allyl alcohol toxicity was mediated by an increase in its metabolization. These results underline the necessity to critically consider metabolic activation in the zebrafish embryo. As demonstrated here, mRNA injection is one useful approach to study the role of candidate enzymes involved in

Reduced synaptic density and deficient locomotor response in neuronal activity-regulated pentraxin 2a mutant zebrafish.

PubMed

Elbaz, Idan; Lerer-Goldshtein, Tali; Okamoto, Hitoshi; Appelbaum, Lior

2015-04-01

Neuronal-activity-regulated pentraxin (NARP/NPTX2/NP2) is a secreted synaptic protein that regulates the trafficking of glutamate receptors and mediates learning, memory, and drug addiction. The role of NPTX2 in regulating structural synaptic plasticity and behavior in a developing vertebrate is indefinite. We characterized the expression of nptx2a in larvae and adult zebrafish and established a transcription activator-like effector nuclease (TALEN)-mediated nptx2a mutant (nptx2a(-/-)) to study the role of Nptx2a in regulating structural synaptic plasticity and behavior. Similar to mammals, the zebrafish nptx2a was expressed in excitatory neurons in the brain and spinal cord. Its expression was induced in response to a mechanosensory stimulus but did not change during day and night. Behavioral assays showed that loss of Nptx2a results in reduced locomotor response to light-to-dark transition states and to a sound stimulus. Live imaging of synapses using the transgenic nptx2a:GAL4VP16 zebrafish and a fluorescent presynaptic synaptophysin (SYP) marker revealed reduced synaptic density in the axons of the spinal motor neurons and the anterodorsal lateral-line ganglion (gAD), which regulate locomotor activity and locomotor response to mechanosensory stimuli, respectively. These results suggest that Nptx2a affects locomotor response to external stimuli by mediating structural synaptic plasticity in excitatory neuronal circuits. © FASEB.

Cadherin-17 is required to maintain pronephric duct integrity during zebrafish development.

PubMed

Horsfield, Julia; Ramachandran, Anassuya; Reuter, Katja; LaVallie, Edward; Collins-Racie, Lisa; Crosier, Kathryn; Crosier, Philip

2002-07-01

We have isolated a zebrafish cadherin that is orthologous to human LI-cadherin (CDH17). Zebrafish cdh17 is expressed exclusively in the pronephric ducts during embryogenesis, and in the mesonephros during larval development and adulthood. Like its mammalian ortholog, cdh17 is also expressed in liver and intestine in adult zebrafish. We show that cdh17-positive mesodermal cells do not contribute to the hematopoietic system. Consistent with a cell adhesion role for Cdh17, depletion of Cdh17 function using antisense morpholino oligonucleotides compromised cell cohesion during pronephric duct formation. Our results indicate that Cdh17 is necessary for maintaining the integrity of the pronephric ducts during zebrafish embryogenesis. This finding contrasts with the role of mammalian CDH17, which does not appear to be involved in nephric development.

Effect of acute ethanol administration on zebrafish tail-beat motion.

PubMed

Bartolini, Tiziana; Mwaffo, Violet; Butail, Sachit; Porfiri, Maurizio

2015-11-01

Zebrafish is becoming a species of choice in neurobiological and behavioral studies of alcohol-related disorders. In these efforts, the activity of adult zebrafish is typically quantified using indirect activity measures that are either scored manually or identified automatically from the fish trajectory. The analysis of such activity measures has produced important insight into the effect of acute ethanol exposure on individual and social behavior of this vertebrate species. Here, we leverage a recently developed tracking algorithm that reconstructs fish body shape to investigate the effect of acute ethanol administration on zebrafish tail-beat motion in terms of amplitude and frequency. Our results demonstrate a significant effect of ethanol on the tail-beat amplitude as well as the tail-beat frequency, both of which were found to robustly decrease for high ethanol concentrations. Such a direct measurement of zebrafish motor functions is in agreement with evidence based on indirect activity measures, offering a complementary perspective in behavioral screening. Copyright © 2015 Elsevier Inc. All rights reserved.

Developmental exposure to low concentrations of two brominated flame retardants, BDE-47 and BDE-99, causes life-long behavioral alterations in zebrafish.

PubMed

Glazer, Lilah; Wells, Corinne N; Drastal, Meghan; Odamah, Kathryn-Ann; Galat, Richard E; Behl, Mamta; Levin, Edward D

2018-05-01

Polybrominated diphenyl ethers (PBDEs) were widely used as flame retardants until the early 2000s, mainly in home furnishings and electronics. The persistence of PBDEs in the environment leads to continued ubiquitous exposure to low levels, with infants and children experiencing higher exposures than adults. Accumulating evidence suggest that low-level exposures during early life stages can affect brain development and lead to long-term behavioral impairments. We investigated the effects of zebrafish exposure to low doses of the two prominent PBDEs; 2,2',4,4',5,-Pentabromodiphenyl ether (BDE-99) and 2,2',4,4',-Tetrabromodiphenyl ether (BDE-47), during embryo-development on short- and long-term behavioral endpoints. We included the organophosphate pesticide chlorpyrifos (CPF) due to its well documented neurotoxicity across species from zebrafish to humans. Zebrafish embryos were exposed to the following individual treatments; 0.1% DMSO (vehicle control); 0.3μM CPF; 0.01, 0.03, 0.1, 0.3μM BDE-47; 0.003, 0.03, 0.3, 1, 3, 10, 20μM BDE-99 from 5 until 120h post fertilization (hpf). Low exposure levels were determined as those not causing immediate overt toxicity, and behavior assays were conducted in the low-level range. At 144 hpf the larvae were tested for locomotor activity. At approximately 6 months of age adult zebrafish were tested in a behavioral battery including assays for anxiety-related behavior, sensorimotor response and habituation, social interaction, and predator avoidance. In the short-term, larval locomotor activity was reduced in larvae treated with 0.3μM CPF and 0.1μM BDE-47. BDE-99 treatment caused non-monotonic dose effects, with 0.3μM causing hyperactivity and 1μM or higher causing hypoactivity. In the long-term, adult anxiety-related behavior was reduced in all treatments as measured in both the novel tank dive test and tap test. We show that exposure of zebrafish embryos to low concentrations of the brominated flame retardants BDE-47 and

Unique and potent effects of acute ibogaine on zebrafish: the developing utility of novel aquatic models for hallucinogenic drug research.

PubMed

Cachat, Jonathan; Kyzar, Evan J; Collins, Christopher; Gaikwad, Siddharth; Green, Jeremy; Roth, Andrew; El-Ounsi, Mohamed; Davis, Ari; Pham, Mimi; Landsman, Samuel; Stewart, Adam Michael; Kalueff, Allan V

2013-01-01

An indole alkaloid, ibogaine is the principal psychoactive component of the iboga plant, used by indigenous peoples in West Africa for centuries. Modulating multiple neurotransmitter systems, the drug is a potent hallucinogen in humans, although its psychotropic effects remain poorly understood. Expanding the range of model species is an important strategy for translational neuroscience research. Here we exposed adult zebrafish (Danio rerio) to 10 and 20mg/L of ibogaine, testing them in the novel tank, light-dark box, open field, mirror stimulation, social preference and shoaling tests. In the novel tank test, the zebrafish natural diving response (geotaxis) was reversed by ibogaine, inducing initial top swimming followed by bottom dwelling. Ibogaine also attenuated the innate preference for dark environments (scototaxis) in the light-dark box test. While it did not exert overt locomotor or thigmotaxic responses in the open field test, the drug altered spatiotemporal exploration of novel environment, inducing clear preference of some areas over others. Ibogaine also promoted 'mirror' exploration in the mirror stimulation test, disrupted group cohesion in the shoaling test, and evoked strong coloration responses due to melanophore aggregation, but did not alter brain c-fos expression or whole-body cortisol levels. Overall, our results support the complex pharmacological profile of ibogaine and its high sensitivity in zebrafish models, dose-dependently affecting multiple behavioral domains. While future investigations in zebrafish may help elucidate the mechanisms underlying these unique behavioral effects, our study strongly supports the developing utility of aquatic models in hallucinogenic drug research. High sensitivity of three-dimensional phenotyping approaches applied here to behavioral effects of ibogaine in zebrafish provides further evidence of how 3D reconstructions of zebrafish swimming paths may be useful for high-throughput pharmacological screening

Chemokine guided angiogenesis directs coronary vasculature formation in zebrafish

PubMed Central

Harrison, Michael R.M.; Bussmann, Jeroen; Huang, Ying; Zhao, Long; Osorio, Arthela; Burns, C. Geoffrey; Burns, Caroline E.; Sucov, Henry M.; Siekmann, Arndt F.; Lien, Ching-Ling

2015-01-01

SUMMARY Interruption of coronary blood supply severely impairs heart function with often-fatal consequences for heart disease patients. However the formation and maturation of these coronary vessels is not fully understood. Here we provide a detailed analysis of coronary vessel development in zebrafish. We observe that coronary vessels form in zebrafish by angiogenic sprouting of arterial cells derived from the endocardium at the atrioventricular canal. Endothelial cells express the CXC-motif chemokine receptor Cxcr4a and migrate to vascularize the ventricle under the guidance of the myocardium-expressed ligand Cxcl12b. cxcr4a mutant zebrafish fail to form a vascular network, whereas ectopic expression of Cxcl12b ligand induces coronary vessel formation. Importantly, cxcr4a mutant zebrafish fail to undergo heart regeneration following injury. Our results suggest that chemokine-signaling has an essential role in coronary vessel formation by directing migration of endocardium-derived endothelial cells. Poorly developed vasculature in cxcr4a mutants likely underlies decreased regenerative potential in adults. PMID:26017769

Advances in the Study of Heart Development and Disease Using Zebrafish

PubMed Central

Brown, Daniel R.; Samsa, Leigh Ann; Qian, Li; Liu, Jiandong

2016-01-01

Animal models of cardiovascular disease are key players in the translational medicine pipeline used to define the conserved genetic and molecular basis of disease. Congenital heart diseases (CHDs) are the most common type of human birth defect and feature structural abnormalities that arise during cardiac development and maturation. The zebrafish, Danio rerio, is a valuable vertebrate model organism, offering advantages over traditional mammalian models. These advantages include the rapid, stereotyped and external development of transparent embryos produced in large numbers from inexpensively housed adults, vast capacity for genetic manipulation, and amenability to high-throughput screening. With the help of modern genetics and a sequenced genome, zebrafish have led to insights in cardiovascular diseases ranging from CHDs to arrhythmia and cardiomyopathy. Here, we discuss the utility of zebrafish as a model system and summarize zebrafish cardiac morphogenesis with emphasis on parallels to human heart diseases. Additionally, we discuss the specific tools and experimental platforms utilized in the zebrafish model including forward screens, functional characterization of candidate genes, and high throughput applications. PMID:27335817

A zebrafish (Danio rerio) model of infectious spleen and kidney necrosis virus (ISKNV) infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu Xiaopeng; Zhang Lichun; Weng Shaoping

2008-06-20

Zebrafish is a model animal for studies of genetics, development, toxicology, oncology, and immunology. In this study, infectious spleen and kidney necrosis virus (ISKNV) was used to establish an infection in zebrafish, and the experimental conditions were established and characterized. Mortality of adult zebrafish infected with ISKNV by intraperitoneal (i.p.) injection exceeded 60%. ISKNV can be passed stably in zebrafish for over ten passages. The ailing zebrafish displayed petechial hemorrhaging and scale protrusion. Histological analysis of moribund fish revealed necrosis of tissue and enlarged cells in kidney and spleen. The real-time RT-PCR analysis of mRNA level confirmed that ISKNV wasmore » replicated in zebrafish. Immunohistochemistry and immunofluorescence analyses further confirmed the presence of ISKNV-infected cells in almost all organs of the infected fish. Electron microscope analyses showed that the ISKNV particle was present in the infected tissues. The establishment of zebrafish infection model of ISKNV can offer a valuable tool for studying the interactions between ISKNV and its host.« less

Antibiotic toxicity and absorption in zebrafish using liquid chromatography-tandem mass spectrometry.

PubMed

Zhang, Fan; Qin, Wei; Zhang, Jing-Pu; Hu, Chang-Qin

2015-01-01

Evaluation of drug toxicity is necessary for drug safety, but in vivo drug absorption is varied; therefore, a rapid, sensitive and reliable method for measuring drugs is needed. Zebrafish are acceptable drug toxicity screening models; we used these animals with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a multiple reaction monitoring mode to quantify drug uptake in zebrafish to better estimate drug toxicity. Analytes were recovered from zebrafish homogenate by collecting supernatant. Measurements were confirmed for drugs in the range of 10-1,000 ng/mL. Four antibiotics with different polarities were tested to explore any correlation of drug polarity, absorption, and toxicity. Zebrafish at 3 days post-fertilization (dpf) absorbed more drug than those at 6 h post-fertilization (hpf), and different developmental periods appeared to be differentially sensitive to the same compound. By observing abnormal embryos and LD50 values, zebrafish embryos at 6 hpf were considered to be suitable for evaluating embryotoxicity. Also, larvae at 3 dpf were adapted to measure acute drug toxicity in adult mammals. Thus, we can exploit zebrafish to study drug toxicity and can reliably quantify drug uptake with LC-MS/MS. This approach will be helpful for future studies of toxicology in zebrafish.

Altered gene expression in the brain and ovaries of zebrafish (Danio rerio) exposed to the aromatase inhibitor fadrozole: microarray analysis and hypothesis generation.

PubMed

Villeneuve, L; Wang, Rong-Lin; Bencic, David C; Biales, Adam D; Martinović, Dalma; Lazorchak, James M; Toth, Gregory; Ankley, Gerald T

2009-08-01

As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine-active chemicals (EACs) with different modes of action, zebrafish (Danio rerio) were exposed to 25 or 100 microg/L of the aromatase inhibitor fadrozole for 24, 48, or 96 h. Global transcriptional response in brain and ovarian tissue of fish exposed to 25 microg/L of fadrozole was compared to that in control fish using a commercially available, 22,000-gene oligonucleotide microarray. Transcripts altered in brain were functionally linked to differentiation, development, DNA replication, and cell cycle. Additionally, multiple genes associated with the one-carbon pool by folate pathway (KEGG 00670) were significantly up-regulated. Transcripts altered in ovary were functionally linked to cell-cell adhesion, extracellular matrix, vasculogenesis, and development. Promoter motif analysis identified GATA-binding factor 2, Ikaros 2, alcohol dehydrogenase gene regulator 1, myoblast-determining factor, and several heat shock factors as being associated with coexpressed gene clusters that were differentially expressed following exposure to fadrozole. Based on the transcriptional changes observed, it was hypothesized that fadrozole elicits neurodegenerative stress in brain tissue and that fish cope with this stress through proliferation of radial glial cells. Additionally, it was hypothesized that changes of gene expression in the ovary of fadrozole-exposed zebrafish reflect disruption of oocyte maturation and ovulation because of impaired vitellogenesis. These hypotheses and others derived from the microarray results provide a foundation for future studies aimed at understanding responses of the HPG axis to EACs and other chemical stressors.

Ionic channels underlying the ventricular action potential in zebrafish embryo.

PubMed

Alday, Aintzane; Alonso, Hiart; Gallego, Monica; Urrutia, Janire; Letamendia, Ainhoa; Callol, Carles; Casis, Oscar

2014-06-01

Over the last years zebrafish has become a popular model in the study of cardiac physiology, pathology and pharmacology. Recently, the application of the 3Rs regulation and the characteristics of the embryo have reduced the use of adult zebrafish use in many studies. However, the zebrafish embryo cardiac physiology is poorly characterized since most works have used indirect techniques and direct recordings of cardiac action potential and ionic currents are scarce. In order to optimize the zebrafish embryo model, we used electrophysiological, pharmacological and immunofluorescence tools to identify the characteristics and the ionic channels involved in the ventricular action potentials of zebrafish embryos. The application of Na(+) or T-type Ca(+2) channel blockers eliminated the cardiac electrical activity, indicating that the action potential upstroke depends on Na(+) and T-type Ca(+2) currents. The plateau phase depends on L-type Ca(+2) channels since it is abolished by specific blockade. The direct channel blockade indicates that the action potential repolarization and diastolic potential depends on ERG K(+) channels. The presence in the embryonic heart of the Nav1.5, Cav1.2, Cav3.2 and ERG channels was also confirmed by immunofluorescence, while the absence of effect of specific blockers and immunostaining indicate that two K(+) repolarizing currents present in human heart, Ito and IKs, are absent in the embryonic zebrafish heart. Our results describe the ionic channels present and its role in the zebrafish embryo heart and support the use of zebrafish embryos to study human diseases and their use for drug testing. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Generation and phenotype analysis of zebrafish mutations of obesity-related genes lepr and mc4r].

PubMed

Fei, Fei; Sun, Shao-Yang; Yao, Yu-Xiao; Wang, Xu

2017-02-25

Obesity has become a severe public health problem across the world, and seriously affects the health and life quality of human beings. Here we generated lepr and mc4r mutant zebrafish via the CRISPR/Cas9 technique, and performed morphological and functional characterizations of those mutants. We observed that there was no significant phenotypic difference between homozygous mutants and wild-type controls before 2.5 months post-fertilization (mpf). However, the adult lepr -/- and mc4r -/- individuals displayed increased food intake, heavier weight, and higher body fat percentage, the characteristics of obesity phenotypes. Blood glucose test showed that overfeeding induced significantly impaired glucose tolerance in adult lepr -/- and mc4r -/- zebrafish. Furthermore, we analyzed 76 energy metabolism-related transcripts in lepr -/- and mc4r -/- zebrafish livers by using real-time RT-PCR, and compared the results with the published microarray data of Lep ob/ob mouse livers, and found that the changes in the expression of insulin/IGF signaling (IIS) pathway genes in lepr -/- zebrafish and Lep ob/ob mouse were positively correlated, suggesting that the IIS pathway maintains functional conservation between zebrafish and mammals during the evolution of the obesity-regulating molecule network.

Two-photon-based photoactivation in live zebrafish embryos.

PubMed

Russek-Blum, Niva; Nabel-Rosen, Helit; Levkowitz, Gil

2010-12-24

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Graph theoretical model of a sensorimotor connectome in zebrafish.

PubMed

Stobb, Michael; Peterson, Joshua M; Mazzag, Borbala; Gahtan, Ethan

2012-01-01

Mapping the detailed connectivity patterns (connectomes) of neural circuits is a central goal of neuroscience. The best quantitative approach to analyzing connectome data is still unclear but graph theory has been used with success. We present a graph theoretical model of the posterior lateral line sensorimotor pathway in zebrafish. The model includes 2,616 neurons and 167,114 synaptic connections. Model neurons represent known cell types in zebrafish larvae, and connections were set stochastically following rules based on biological literature. Thus, our model is a uniquely detailed computational representation of a vertebrate connectome. The connectome has low overall connection density, with 2.45% of all possible connections, a value within the physiological range. We used graph theoretical tools to compare the zebrafish connectome graph to small-world, random and structured random graphs of the same size. For each type of graph, 100 randomly generated instantiations were considered. Degree distribution (the number of connections per neuron) varied more in the zebrafish graph than in same size graphs with less biological detail. There was high local clustering and a short average path length between nodes, implying a small-world structure similar to other neural connectomes and complex networks. The graph was found not to be scale-free, in agreement with some other neural connectomes. An experimental lesion was performed that targeted three model brain neurons, including the Mauthner neuron, known to control fast escape turns. The lesion decreased the number of short paths between sensory and motor neurons analogous to the behavioral effects of the same lesion in zebrafish. This model is expandable and can be used to organize and interpret a growing database of information on the zebrafish connectome.

Acid-sensing ion channels (ASICs) in the taste buds of adult zebrafish.

PubMed

Viña, E; Parisi, V; Cabo, R; Laurà, R; López-Velasco, S; López-Muñiz, A; García-Suárez, O; Germanà, A; Vega, J A

2013-03-01

In detecting chemical properties of food, different molecules and ion channels are involved including members of the acid-sensing ion channels (ASICs) family. Consistently ASICs are present in sensory cells of taste buds of mammals. In the present study the presence of ASICs (ASIC1, ASIC2, ASIC3 and ASIC4) was investigated in the taste buds of adult zebrafish (zASICs) using Western blot and immunohistochemistry. zASIC1 and zASIC3 were regularly absent from taste buds, whereas faint zASIC2 and robust zASIC4 immunoreactivities were detected in sensory cells. Moreover, zASIC2 also immunolabelled nerves supplying taste buds. The present results demonstrate for the first time the presence of zASICs in taste buds of teleosts, with different patterns to that occurring in mammals, probably due to the function of taste buds in aquatic environment and feeding. Nevertheless, the role of zASICs in taste remains to be demonstrated. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior.

PubMed

Ben-Moshe Livne, Zohar; Alon, Shahar; Vallone, Daniela; Bayleyen, Yared; Tovin, Adi; Shainer, Inbal; Nisembaum, Laura G; Aviram, Idit; Smadja-Storz, Sima; Fuentes, Michael; Falcón, Jack; Eisenberg, Eli; Klein, David C; Burgess, Harold A; Foulkes, Nicholas S; Gothilf, Yoav

2016-11-01

The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior.

Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior

PubMed Central

Alon, Shahar; Vallone, Daniela; Tovin, Adi; Shainer, Inbal; Nisembaum, Laura G.; Aviram, Idit; Smadja-Storz, Sima; Fuentes, Michael; Falcón, Jack; Eisenberg, Eli; Klein, David C.; Burgess, Harold A.; Foulkes, Nicholas S.; Gothilf, Yoav

2016-01-01

The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior. PMID:27870848

Oceans of Opportunity: Exploring Vertebrate Hematopoiesis in Zebrafish

PubMed Central

Carroll, Kelli J.; North, Trista E.

2015-01-01

Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell (HSPC) biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor and effector cell emergence, expansion and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell-types can be identified and characterized. Further, a myriad of transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of HSPC biology during development, in response to infection or injury, or in the setting of hematological malignancy, continues to deepen, zebrafish will remain essential for exploring the spatio-temporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease. PMID:24816275

Molecular and functional characterization of the scavenger receptor CD36 in zebrafish and common carp.

PubMed

Fink, Inge R; Benard, Erica L; Hermsen, Trudi; Meijer, Annemarie H; Forlenza, Maria; Wiegertjes, Geert F

2015-02-01

CD36 is a scavenger receptor which has been studied closely in mammals where it is expressed by many different cell types and plays a role in highly diverse processes, both homeostatic and pathologic. It is among other things important in the innate immune system, in angiogenesis, and in clearance of apoptotic cells, and it is also involved in lipid metabolism and atherosclerosis. Recently, in the cephalochordate amphioxus a primitive CD36 family member was described, which was present before the divergence of CD36 from other scavenger receptor B family members, SCARB1 and SCARB2. Not much is known on the Cd36 molecule in teleost fish. We therefore studied Cd36 in both zebrafish and common carp, two closely related cyprinid fish species. Whereas a single cd36 gene is present in zebrafish, carp has two cd36 genes, and all show conserved synteny compared to mammalian CD36. The gene expression of carp cd36 is high in brain, ovary and testis but absent in immune organs. Although in mammals CD36 expression in erythrocytes, monocytes and macrophages is high, gene expression studies in leukocyte subtypes of adult carp and zebrafish larvae, including thrombocytes and macrophages provided no indication for any substantial expression of cd36 in immune cell types. Surprisingly, analysis of the cd36 promoter region does show the presence of several binding sites for transcription factors known to regulate immune responses. Overexpression of carp cd36 locates the receptor on the cell surface of mammalian cell lines consistent with the predicted topology of cyprinid Cd36 with a large extracellular domain, two transmembrane domains, and short cytoplasmic tails at both ends. Gene expression of cd36 is down-regulated during infection of zebrafish with Mycobacterium marinum, whereas knockdown of cd36 in zebrafish larvae led to higher bacterial burden upon such infection. We discuss the putative role for Cd36 in immune responses of fish in the context of other members of the

Progenitor potential of nkx6.1-expressing cells throughout zebrafish life and during beta cell regeneration.

PubMed

Ghaye, Aurélie P; Bergemann, David; Tarifeño-Saldivia, Estefania; Flasse, Lydie C; Von Berg, Virginie; Peers, Bernard; Voz, Marianne L; Manfroid, Isabelle

2015-09-02

In contrast to mammals, the zebrafish has the remarkable capacity to regenerate its pancreatic beta cells very efficiently. Understanding the mechanisms of regeneration in the zebrafish and the differences with mammals will be fundamental to discovering molecules able to stimulate the regeneration process in mammals. To identify the pancreatic cells able to give rise to new beta cells in the zebrafish, we generated new transgenic lines allowing the tracing of multipotent pancreatic progenitors and endocrine precursors. Using novel bacterial artificial chromosome transgenic nkx6.1 and ascl1b reporter lines, we established that nkx6.1-positive cells give rise to all the pancreatic cell types and ascl1b-positive cells give rise to all the endocrine cell types in the zebrafish embryo. These two genes are initially co-expressed in the pancreatic primordium and their domains segregate, not as a result of mutual repression, but through the opposite effects of Notch signaling, maintaining nkx6.1 expression while repressing ascl1b in progenitors. In the adult zebrafish, nkx6.1 expression persists exclusively in the ductal tree at the tip of which its expression coincides with Notch active signaling in centroacinar/terminal end duct cells. Tracing these cells reveals that they are able to differentiate into other ductal cells and into insulin-expressing cells in normal (non-diabetic) animals. This capacity of ductal cells to generate endocrine cells is supported by the detection of ascl1b in the nkx6.1:GFP ductal cell transcriptome. This transcriptome also reveals, besides actors of the Notch and Wnt pathways, several novel markers such as id2a. Finally, we show that beta cell ablation in the adult zebrafish triggers proliferation of ductal cells and their differentiation into insulin-expressing cells. We have shown that, in the zebrafish embryo, nkx6.1+ cells are bona fide multipotent pancreatic progenitors, while ascl1b+ cells represent committed endocrine precursors. In

Chronic PFOS exposures induce life stage-specific behavioral deficits in adult zebrafish and produce malformation and behavioral deficits in F1 offspring.

PubMed

Chen, Jiangfei; Das, Siba R; La Du, Jane; Corvi, Margaret M; Bai, Chenglian; Chen, Yuanhong; Liu, Xiaojuan; Zhu, Guonian; Tanguay, Robert L; Dong, Qiaoxiang; Huang, Changjiang

2013-01-01

Perfluorooctane sulfonic acid (PFOS) is an organic contaminant that is ubiquitous in the environment. Few studies have assessed the behavioral effects of chronic PFOS exposure in aquatic organisms. The present study defined the behavioral effects of varying life span chronic exposures to PFOS in zebrafish. Specifically, zebrafish were exposed to control or 0.5 µM PFOS during 1 to 20, 21 to 120, or 1 to 120 d postfertilization (dpf). Exposure to PFOS impaired the adult zebrafish behavior mode under the tapping stimulus. The movement speed of male and female fish exposed for 1 to 120 dpf was significantly increased compared with control before and after tapping, whereas in the groups exposed for 1 to 20 and 21 to 120 dpf, only the males exhibited elevated swim speed before tapping. Residues of PFOS in F1 embryos derived from parental exposure for 1 to 120 and 21 to 120 dpf were significantly higher than control, and F1 embryos in these two groups also showed high malformation and mortality. The F1 larvae of parental fish exposed to PFOS for 1 to 20 or 21 to 120 dpf exhibited a higher swimming speed than control larvae in a light-to-dark behavior assessment test. The F1 larvae derived from parental fish exposed to PFOS for 1 to 120 dpf showed a significantly lower speed in the light period and a higher speed in the dark period compared with controls. Although there was little PFOS residue in embryos derived from the 1- to 20-dpf parental PFOS-exposed group, the adverse behavioral effects on both adult and F1 larvae indicate that exposure during the first 21 dpf induces long-term neurobehaviorial toxicity. The authors' findings demonstrate that chronic PFOS exposure during different life stages adversely affects adult behavior and F1 offspring morphology, behavior, and survival. Copyright © 2012 SETAC.

Automated analysis of brain activity for seizure detection in zebrafish models of epilepsy.

PubMed

Hunyadi, Borbála; Siekierska, Aleksandra; Sourbron, Jo; Copmans, Daniëlle; de Witte, Peter A M

2017-08-01

Epilepsy is a chronic neurological condition, with over 30% of cases unresponsive to treatment. Zebrafish larvae show great potential to serve as an animal model of epilepsy in drug discovery. Thanks to their high fecundity and relatively low cost, they are amenable to high-throughput screening. However, the assessment of seizure occurrences in zebrafish larvae remains a bottleneck, as visual analysis is subjective and time-consuming. For the first time, we present an automated algorithm to detect epileptic discharges in single-channel local field potential (LFP) recordings in zebrafish. First, candidate seizure segments are selected based on their energy and length. Afterwards, discriminative features are extracted from each segment. Using a labeled dataset, a support vector machine (SVM) classifier is trained to learn an optimal feature mapping. Finally, this SVM classifier is used to detect seizure segments in new signals. We tested the proposed algorithm both in a chemically-induced seizure model and a genetic epilepsy model. In both cases, the algorithm delivered similar results to visual analysis and found a significant difference in number of seizures between the epileptic and control group. Direct comparison with multichannel techniques or methods developed for different animal models is not feasible. Nevertheless, a literature review shows that our algorithm outperforms state-of-the-art techniques in terms of accuracy, precision and specificity, while maintaining a reasonable sensitivity. Our seizure detection system is a generic, time-saving and objective method to analyze zebrafish LPF, which can replace visual analysis and facilitate true high-throughput studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Abnormal cerebellar development and ataxia in CARP VIII morphant zebrafish.

PubMed

Aspatwar, Ashok; Tolvanen, Martti E E; Jokitalo, Eija; Parikka, Mataleena; Ortutay, Csaba; Harjula, Sanna-Kaisa E; Rämet, Mika; Vihinen, Mauno; Parkkila, Seppo

2013-02-01

Congenital ataxia and mental retardation are mainly caused by variations in the genes that affect brain development. Recent reports have shown that mutations in the CA8 gene are associated with mental retardation and ataxia in humans and ataxia in mice. The gene product, carbonic anhydrase-related protein VIII (CARP VIII), is predominantly present in cerebellar Purkinje cells, where it interacts with the inositol 1,4,5-trisphosphate receptor type 1, a calcium channel. In this study, we investigated the effects of the loss of function of CARP VIII during embryonic development in zebrafish using antisense morpholino oligonucleotides against the CA8 gene. Knockdown of CA8 in zebrafish larvae resulted in a curved body axis, pericardial edema and abnormal movement patterns. Histologic examination revealed gross morphologic defects in the cerebellar region and in the muscle. Electron microscopy studies showed increased neuronal cell death in developing larvae injected with CA8 antisense morpholinos. These data suggest a pivotal role for CARP VIII during embryonic development. Furthermore, suppression of CA8 expression leads to defects in motor and coordination functions, mimicking the ataxic human phenotype. This work reveals an evolutionarily conserved function of CARP VIII in brain development and introduces a novel zebrafish model in which to investigate the mechanisms of CARP VIII-related ataxia and mental retardation in humans.

Comparative Analyses of Zebrafish Anxiety-Like Behavior Using Conflict-Based Novelty Tests.

PubMed

Kysil, Elana V; Meshalkina, Darya A; Frick, Erin E; Echevarria, David J; Rosemberg, Denis B; Maximino, Caio; Lima, Monica Gomes; Abreu, Murilo S; Giacomini, Ana C; Barcellos, Leonardo J G; Song, Cai; Kalueff, Allan V

2017-06-01

Modeling of stress and anxiety in adult zebrafish (Danio rerio) is increasingly utilized in neuroscience research and central nervous system (CNS) drug discovery. Representing the most commonly used zebrafish anxiety models, the novel tank test (NTT) focuses on zebrafish diving in response to potentially threatening stimuli, whereas the light-dark test (LDT) is based on fish scototaxis (innate preference for dark vs. bright areas). Here, we systematically evaluate the utility of these two tests, combining meta-analyses of published literature with comparative in vivo behavioral and whole-body endocrine (cortisol) testing. Overall, the NTT and LDT behaviors demonstrate a generally good cross-test correlation in vivo, whereas meta-analyses of published literature show that both tests have similar sensitivity to zebrafish anxiety-like states. Finally, NTT evokes higher levels of cortisol, likely representing a more stressful procedure than LDT. Collectively, our study reappraises NTT and LDT for studying anxiety-like states in zebrafish, and emphasizes their developing utility for neurobehavioral research. These findings can help optimize drug screening procedures by choosing more appropriate models for testing anxiolytic or anxiogenic drugs.

Cannabinoid modulation of zebrafish fear learning and its functional analysis investigated by c-Fos expression.

PubMed

Ruhl, Tim; Zeymer, Malou; von der Emde, Gerhard

2017-02-01

It has been shown that zebrafish fear learning proceeds in the same way as reported for rodents. However, in zebrafish fear learning it is possible to substitute the use of electric shocks as unconditioned stimulus and utilize the inborn fear responses to the alarm substance Schreckstoff, instead. The skin extract Schreckstoff elicits typical fear reactions such as preferred bottom dwelling, swimming in a tighter shoal, erratic movements and freezing. This natural fear behavior can be transferred from Schreckstoff to any other sensory stimulus by associative conditioning (fear learning). We presented Schreckstoff simultaneously with a red light stimulus and tested the effectiveness of fear learning during memory retrieval. The two brain regions known to be relevant for learning in zebrafish are the medial and the lateral pallium of the dorsal telencephalon, both containing rich expressions of the endocannabinoid receptor CB1. To test the influence of the zebrafish endocannabinoid system on fear acquisition learning, an experimental group of ten fish was pretreated with the CB1 receptor agonist THC (Δ 9 -tetrahydrocannabinol; 100nM for 1h). We found that CB1 activation significantly inhibited acquisition of fear learning, possibly by impairing stimulus encoding processes in pallial areas. This was supported by analyzes of c-Fos expression in the brains of experimental animals. Schreckstoff exposure during fear acquisition learning and memory retrieval during red light presentation increased the number of labelled cells in pallial structures, but in no other brain region investigated (e.g. striatum, thalamus, and habenula). THC administration before fear conditioning significantly decreased c-Fos expression in these structures to a level similar to the control group without Schreckstoff experience, suggesting that Schreckstoff induced fear learning requires brain circuits restricted mainly to pallial regions of the dorsal telencephalon. Copyright © 2016 Elsevier

Behavioral and neurogenomic transcriptome changes in wild-derived zebrafish with fluoxetine treatment

PubMed Central

2013-01-01

Background Stress and anxiety-related behaviors are seen in many organisms. Studies have shown that in humans and other animals, treatment with selective serotonin reuptake inhibitors (e.g. fluoxetine) can reduce anxiety and anxiety-related behaviors. The efficacies and side effects, however, can vary between individuals. Fluoxetine can modulate anxiety in a stereospecific manner or with equal efficacy regardless of stereoisomer depending on the mechanism of action (e.g. serotonergic or GABAergic effects). Zebrafish are an emerging and valuable translational model for understanding human health related issues such as anxiety. In this study we present data showing the behavioral and whole brain transcriptome changes with fluoxetine treatment in wild-derived zebrafish and suggest additional molecular mechanisms of this widely-prescribed drug. Results We used automated behavioral analyses to assess the effects of racemic and stereoisomeric fluoxetine on male wild-derived zebrafish. Both racemic and the individual isomers of fluoxetine reduced anxiety-related behaviors relative to controls and we did not observe stereospecific fluoxetine effects. Using RNA-sequencing of the whole brain, we identified 411 genes showing differential expression with racemic fluoxetine treatment. Several neuropeptides (neuropeptide Y, isotocin, urocortin 3, prolactin) showed consistent expression patterns with the alleviation of stress and anxiety when anxiety-related behavior was reduced with fluoxetine treatment. With gene ontology and KEGG pathway analyses, we identified lipid and amino acid metabolic processes, and steroid biosynthesis among other terms to be over-enriched. Conclusion Our results demonstrate that fluoxetine reduces anxiety-related behaviors in wild-derived zebrafish and alters their neurogenomic state. We identify two biological processes, lipid and amino acid metabolic synthesis that characterize differences in the fluoxetine treated fish. Fluoxetine may be acting on

Effects of the UV filter benzophenone-3 (oxybenzone) at low concentrations in zebrafish (Danio rerio).

PubMed

Blüthgen, Nancy; Zucchi, Sara; Fent, Karl

2012-09-01

Organic UV filters including benzophenone-3 (BP-3) are widely used to protect humans and materials from damage by UV irradiation. Despite the environmental occurrence of BP-3 in the aquatic environment, little is known about its effects and modes of action. In the present study we assess molecular and physiological effects of BP-3 in adult male zebrafish (Danio rerio) and in eleuthero-embryos by a targeted gene expression approach focusing on the sex hormone system. Fish and embryos are exposed for 14 days and 120 hours post fertilization, respectively, to 2.4-312 μg/L and 8.2-438 μg/L BP-3. Chemical analysis of water and fish demonstrates that BP-3 is partly transformed to benzophenone-1 (BP-1) and both compounds are accumulated in adult fish. Biotransformation to BP-1 is absent in eleuthero-embryos. BP-3 exposure leads to similar alterations of gene expression in both adult fish and eleuthero-embryos. In the brain of adult males esr1, ar and cyp19b are down-regulated at 84 μg/L BP-3. There is no induction of vitellogenin expression by BP-3, both at the transcriptional and protein level. An overall down-regulation of the hsd3b, hsd17b3, hsd11b2 and cyp11b2 transcripts is observed in the testes, suggesting an antiandrogenic activity. No histological changes were observed in the testes after BP-3 treatment. The study leads to the conclusion that low concentrations of BP-3 exhibit similar multiple hormonal activities at the transcription level in two different life stages of zebrafish. Forthcoming studies should show whether this translates to additional physiological effects. Copyright © 2012 Elsevier Inc. All rights reserved.

Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia.

PubMed

Eachus, Helen; Zaucker, Andreas; Oakes, James A; Griffin, Aliesha; Weger, Meltem; Güran, Tülay; Taylor, Angela; Harris, Abigail; Greenfield, Andy; Quanson, Jonathan L; Storbeck, Karl-Heinz; Cunliffe, Vincent T; Müller, Ferenc; Krone, Nils

2017-12-01

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

Editor's Highlight: Transgenic Zebrafish Reporter Lines as Alternative In Vivo Organ Toxicity Models.

PubMed

Poon, Kar Lai; Wang, Xingang; Lee, Serene G P; Ng, Ashley S; Goh, Wei Huang; Zhao, Zhonghua; Al-Haddawi, Muthafar; Wang, Haishan; Mathavan, Sinnakaruppan; Ingham, Philip W; McGinnis, Claudia; Carney, Tom J

2017-03-01

Organ toxicity, particularly liver toxicity, remains one of the major reasons for the termination of drug candidates in the development pipeline as well as withdrawal or restrictions of marketed drugs. A screening-amenable alternative in vivo model such as zebrafish would, therefore, find immediate application in the early prediction of unacceptable organ toxicity. To identify highly upregulated genes as biomarkers of toxic responses in the zebrafish model, a set of well-characterized reference drugs that cause drug-induced liver injury (DILI) in the clinic were applied to zebrafish larvae and adults. Transcriptome microarray analysis was performed on whole larvae or dissected adult livers. Integration of data sets from different drug treatments at different stages identified common upregulated detoxification pathways. Within these were candidate biomarkers which recurred in multiple treatments. We prioritized 4 highly upregulated genes encoding enzymes acting in distinct phases of the drug metabolism pathway. Through promoter isolation and fosmid recombineering, eGFP reporter transgenic zebrafish lines were generated and evaluated for their response to DILI drugs. Three of the 4 generated reporter lines showed a dose and time-dependent induction in endodermal organs to reference drugs and an expanded drug set. In conclusion, through integrated transcriptomics and transgenic approaches, we have developed parallel independent zebrafish in vivo screening platforms able to predict organ toxicities of preclinical drugs. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Sprouting Buds of Zebrafish Research in Malaysia: First Malaysia Zebrafish Disease Model Workshop.

PubMed

Okuda, Kazuhide Shaun; Tan, Pei Jean; Patel, Vyomesh

2016-04-01

Zebrafish is gaining prominence as an important vertebrate model for investigating various human diseases. Zebrafish provides unique advantages such as optical clarity of embryos, high fecundity rate, and low cost of maintenance, making it a perfect complement to the murine model equivalent in biomedical research. Due to these advantages, researchers in Malaysia are starting to take notice and incorporate the zebrafish model into their research activities. However, zebrafish research in Malaysia is still in its infancy stage and many researchers still remain unaware of the full potential of the zebrafish model or have limited access to related tools and techniques that are widely utilized in many zebrafish laboratories worldwide. To overcome this, we organized the First Malaysia Zebrafish Disease Model Workshop in Malaysia that took place on 11th and 12th of November 2015. In this workshop, we showcased how the zebrafish model is being utilized in the biomedical field in international settings as well as in Malaysia. For this, notable international speakers and those from local universities known to be carrying out impactful research using zebrafish were invited to share some of the cutting edge techniques that are used in their laboratories that may one day be incorporated in the Malaysian scientific community.

Proteomics Identification of Potential Candidates Involved in Cell Proliferation for Early Stage of Brain Regeneration in the Adult Zebrafish.

PubMed

Lim, Fei Tieng; Ogawa, Satoshi; Smith, A Ian; Parhar, Ishwar S

2017-02-01

The central nervous system (CNS) of the non-mammalian vertebrates has better neuroregenerative capability as compared with the mammalian CNS. Regeneration of habenula was observed 40 days after damage in zebrafish. During the early stage of regeneration, we found a significant increase of apoptotic cells on day-1 post-damage and of proliferative cells on day-3 post-damage. To identify the molecular factor(s) involved in the early stages of neuroregeneration, differentially expressed proteins during sham, 20- and 40-h post-habenula damage were investigated by proteomic approach by using two-dimensional differential gel electrophoresis (2D-DIGE) coupled with Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-ToF) and tandem mass spectrometry. Protein profiles revealed 17 differentially (>1.5-fold) expressed proteins: 10 upregulated, 4 downregulated, 2 proteins were found to be downregulated at the early stage but upregulated at a later stage, and 1 protein was found to be upregulated at 2 different time points. All proteins identified can be summarized under few molecular processes involved in the early stages of neuroregeneration in zebrafish CNS: apoptosis regulation (Wnt inhibitory factor 1 [WIF1]), neuroprotection (metallothionein), cell proliferation (Spred2, ependymin, Lhx1, and Wnts), differentiation (Spred2, Lhx9, and Wnts), and morphogenesis (cytoplasmic actins and draculin). These protein profiling results suggest that drastic molecular changes occur in the neuroregenerative process during this period, which includes cell proliferation, differentiation, and protection.

Biological Uptake, Distribution, and Depuration of Radio-Labeled Graphene in Adult Zebrafish: Effects of Graphene Size and Natural Organic Matter.

PubMed

Lu, Kun; Dong, Shipeng; Petersen, Elijah J; Niu, Junfeng; Chang, Xiaofeng; Wang, Peng; Lin, Sijie; Gao, Shixiang; Mao, Liang

2017-03-28

The exciting commercial application potential of graphene materials may inevitably lead to their increasing release into the environment where they may pose ecological risks. This study focused on using carbon-14-labeled few-layer graphene (FLG) to determine whether the size of graphene plays a role in its uptake, depuration, and biodistribution in adult zebrafish. After 48 h exposure to larger FLG (L-FLG) at 250 μg/L, the amount of graphene in the organism was close to 48 mg/kg fish dry mass, which was more than 170-fold greater than the body burden of those exposed to the same concentration of smaller FLG (S-FLG). The amount of uptake for both L-FLG and S-FLG increased by a factor of 2.5 and 16, respectively, when natural organic matter (NOM) was added in the exposure suspension. While the L-FLG mainly accumulated in the gut of adult zebrafish, the S-FLG was found in both the gut and liver after exposure with or without NOM. Strikingly, the S-FLG was able to pass through the intestinal wall and enter the intestinal epithelial cells and blood. The presence of NOM increased the quantity of S-FLG in these cells. Exposure to L-FLG or S-FLG also had a significantly different impact on the intestinal microbial community structure.

Glucocorticoid activity detected by in vivo zebrafish assay and in vitro glucocorticoid receptor bioassay at environmental relevant concentrations.

PubMed

Chen, Qiyu; Jia, Ai; Snyder, Shane A; Gong, Zhiyuan; Lam, Siew Hong

2016-02-01

Glucocorticoids are pharmaceutical contaminants of emerging concern due to their incomplete removal during wastewater treatment, increased presence in aquatic environment and their biological potency. The zebrafish is a popular model for aquatic toxicology and environmental risk assessment. This study aimed to determine if glucocorticoids at environmental concentrations would perturb expression of selected glucocorticoid-responsive genes in zebrafish and to investigate their potentials as an in vivo zebrafish assay in complementing in vitro glucocorticoid receptor bioassay. The relative expression of eleven glucocorticoid-responsive genes in zebrafish larvae and liver of adult male zebrafish exposed to three representative glucocorticoids (dexamethasone, prednisolone and triamcinolone) was determined. The expression of pepck, baiap2 and pxr was up-regulated in zebrafish larvae and the expression of baiap2, pxr and mmp-2 was up-regulated in adult zebrafish exposed to glucocorticoids at concentrations equivalent to total glucocorticoids reported in environmental samples. The responsiveness of the specific genes were sufficiently robust in zebrafish larvae exposed to a complex environmental sample detected with in vitro glucocorticoid activity equivalent to 478 pM dexamethasone (DEX-EQ) and confirmed to contain low concentration (0.2 ng/L or less) of the targeted glucocorticoids, and possibly other glucocorticoid-active compounds. The findings provided in vivo relevance to the in vitro glucocorticoid activity and suggested that the environmental sample can perturb glucocorticoid-responsive genes in its original, or half the diluted, concentration as may be found in the environment. The study demonstrated the important complementary roles of in vivo zebrafish and in vitro bioassays coupled with analytical chemistry in monitoring environmental glucocorticoid contaminants. Copyright © 2015 Elsevier Ltd. All rights reserved.

The contribution of the swimbladder to buoyancy in the adult zebrafish (Danio rerio): a morphometric analysis.

PubMed

Robertson, George N; Lindsey, Benjamin W; Dumbarton, Tristan C; Croll, Roger P; Smith, Frank M

2008-06-01

Many teleost fishes use a swimbladder, a gas-filled organ in the coelomic cavity, to reduce body density toward neutral buoyancy, thus minimizing the locomotory cost of maintaining a constant depth in the water column. However, for most swimbladder-bearing teleosts, the contribution of this organ to the attainment of neutral buoyancy has not been quantified. Here, we examined the quantitative contribution of the swimbladder to buoyancy and three-dimensional stability in a small cyprinid, the zebrafish (Danio rerio). In aquaria during daylight hours, adult animals were observed at mean depths from 10.1 +/- 6.0 to 14.2 +/- 5.6 cm below the surface. Fish mass and whole-body volume were linearly correlated (r(2) = 0.96) over a wide range of body size (0.16-0.73 g); mean whole-body density was 1.01 +/- 0.09 g cm(-3). Stereological estimations of swimbladder volume from linear dimensions of lateral X-ray images and direct measurements of gas volumes recovered by puncture from the same swimbladders showed that results from these two methods were highly correlated (r(2) = 0.85). The geometric regularity of the swimbladder thus permitted its volume to be accurately estimated from a single lateral image. Mean body density in the absence of the swimbladder was 1.05 +/- 0.04 g cm(-3). The swimbladder occupied 5.1 +/- 1.4% of total body volume, thus reducing whole-body density significantly. The location of the centers of mass and buoyancy along rostro-caudal and dorso-ventral axes overlapped near the ductus communicans, a constriction between the anterior and posterior swimbladder chambers. Our work demonstrates that the swimbladder of the adult zebrafish contributes significantly to buoyancy and attitude stability. Furthermore, we describe and verify a stereological method for estimating swimbladder volume that will aid future studies of the functions of this organ. 2008 Wiley-Liss, Inc

Guidelines for Better Communication with Brain Impaired Adults

MedlinePlus

... are here Home Guidelines for Better Communication with Brain-Impaired Adults Printer-friendly version Communicating with a loved one with a brain disorder can indeed be challenging. Finding the right ...

The behavior of larval zebrafish reveals stressor-mediated anorexia during early vertebrate development.

PubMed

De Marco, Rodrigo J; Groneberg, Antonia H; Yeh, Chen-Min; Treviño, Mario; Ryu, Soojin

2014-01-01

The relationship between stress and food consumption has been well documented in adults but less so in developing vertebrates. Here we demonstrate that an encounter with a stressor can suppress food consumption in larval zebrafish. Furthermore, we provide indication that food intake suppression cannot be accounted for by changes in locomotion, oxygen consumption and visual responses, as they remain unaffected after exposure to a potent stressor. We also show that feeding reoccurs when basal levels of cortisol (stress hormone in humans and teleosts) are re-established. The results present evidence that the onset of stress can switch off the drive for feeding very early in vertebrate development, and add a novel endpoint for analyses of metabolic and behavioral disorders in an organism suitable for high-throughput genetics and non-invasive brain imaging.

The behavior of larval zebrafish reveals stressor-mediated anorexia during early vertebrate development

PubMed Central

De Marco, Rodrigo J.; Groneberg, Antonia H.; Yeh, Chen-Min; Treviño, Mario; Ryu, Soojin

2014-01-01

The relationship between stress and food consumption has been well documented in adults but less so in developing vertebrates. Here we demonstrate that an encounter with a stressor can suppress food consumption in larval zebrafish. Furthermore, we provide indication that food intake suppression cannot be accounted for by changes in locomotion, oxygen consumption and visual responses, as they remain unaffected after exposure to a potent stressor. We also show that feeding reoccurs when basal levels of cortisol (stress hormone in humans and teleosts) are re-established. The results present evidence that the onset of stress can switch off the drive for feeding very early in vertebrate development, and add a novel endpoint for analyses of metabolic and behavioral disorders in an organism suitable for high-throughput genetics and non-invasive brain imaging. PMID:25368561

The power of projectomes: genetic mosaic labeling in the larval zebrafish brain reveals organizing principles of sensory circuits.

PubMed

Robles, Estuardo

2017-09-01

In no vertebrate species do we possess an accurate, comprehensive tally of neuron types in the brain. This is in no small part due to the vast diversity of neuronal types that comprise complex vertebrate nervous systems. A fundamental goal of neuroscience is to construct comprehensive catalogs of cell types defined by structure, connectivity, and physiological response properties. This type of information will be invaluable for generating models of how assemblies of neurons encode and distribute sensory information and correspondingly alter behavior. This review summarizes recent efforts in the larval zebrafish to construct sensory projectomes, comprehensive analyses of axonal morphologies in sensory axon tracts. Focusing on the olfactory and optic tract, these studies revealed principles of sensory information processing in the olfactory and visual systems that could not have been directly quantified by other methods. In essence, these studies reconstructed the optic and olfactory tract in a virtual manner, providing insights into patterns of neuronal growth that underlie the formation of sensory axon tracts. Quantitative analysis of neuronal diversity revealed organizing principles that determine information flow through sensory systems in the zebrafish that are likely to be conserved across vertebrate species. The generation of comprehensive cell type classifications based on structural, physiological, and molecular features will lead to testable hypotheses on the functional role of individual sensory neuron subtypes in controlling specific sensory-evoked behaviors.

Acute exposure to waterborne cadmium induced oxidative stress and immunotoxicity in the brain, ovary and liver of zebrafish (Danio rerio).

PubMed

Zheng, Jia-Lang; Yuan, Shuang-Shuang; Wu, Chang-Wen; Lv, Zhen-Ming

2016-11-01

Cadmium (Cd) is an environmental contaminant that poses serious risks to aquatic organisms and their associated ecosystem. The mechanisms underlying Cd-induced oxidative stress and immunotoxicity in fish remain largely unknown. In this study, adult female zebrafish were exposed to 0 (control), 1mgL -1 Cd for 24h and 96h, and the oxidative stress and inflammatory responses induced by Cd were evaluated in the brain, liver and ovary. Reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA) increased in a time-dependent manner after treatment with Cd in the brain and liver. The increase may result from the disturbance of genes including copper and zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), inducible nitric oxide synthase (iNOS), and ciclooxigenase-2 (COX-2) at mRNA, protein and activity levels. Although ROS, NO and MDA were not significantly affected by Cd in the ovary, the up-regulation of Cu/Zn-SOD, CAT, iNOS, and COX-2 was observed. Exposure to Cd induced a sharp increase in the protein levels of tumor necrosis factor alpha (TNF-α) in the brain, liver and ovary, possibly contributing to activate inflammatory responses. Furthermore, we also found a dramatic increase in mRNA levels of NF-E2-related factor 2 (Nrf2) and nuclear transcription factor κB (NF-κB) at 24h in the liver and ovary. The corresponding changes in the mRNA levels of Kelch-like-ECH-associated protein 1 (Keap1a and Keap1b) and the inhibitor of κBα (IκBαa and IκBαb) may contribute to regulate the transcriptional activity of Nrf2 and NF-κB, respectively. Contrarily, mRNA levels of Nrf2, NF-κB, Keap1, Keap1b, IκBαa and IκBαb remained stable at 24 and 96h in the brain. Taken together, we demonstrated Cd-induced oxidative stress and immunotoxicity in fish, possibly through transcriptional regulation of Nrf2 and NF-κB and gene modifications at transcriptional, translational, post-translational levels, which would greatly extend our understanding on the Cd

Oceans of opportunity: exploring vertebrate hematopoiesis in zebrafish.

PubMed

Carroll, Kelli J; North, Trista E

2014-08-01

Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor, and effector cell emergence, expansion, and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell types can be identified and characterized. Further, myriad transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of blood stem and progenitor cell biology during development, in response to infection or injury, or in the setting of hematologic malignancy continues to deepen, zebrafish will remain essential for exploring the spatiotemporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease. Copyright © 2014 ISEH - International

High-Resolution Tissue Doppler Imaging of the Zebrafish Heart During Its Regeneration

PubMed Central

Su, Ta-Han; Shih, Cho-Chiang

2015-01-01

Abstract The human heart cannot regenerate after injury, whereas the adult zebrafish can fully regenerate its heart even after 20% of the ventricle is amputated. Many studies have begun to reveal the cellular and molecular mechanisms underlying this regenerative process, which have exciting implications for human cardiac diseases. However, the dynamic functions of the zebrafish heart during regeneration are not yet understood. This study established a high-resolution echocardiography for tissue Doppler imaging (TDI) of the zebrafish heart to explore the cardiac functions during different regeneration phases. Experiments were performed on AB-line adult zebrafish (n=40) in which 15% of the ventricle was surgically removed. An 80-MHz ultrasound TDI based on color M-mode imaging technology was employed. The cardiac flow velocities and patterns from both the ventricular chamber and myocardium were measured at different regeneration phases relative to the day of amputation. The peak velocities of early diastolic inflow, early diastolic myocardial motion, late diastolic myocardial motion, early diastolic deceleration slope, and heart rate were increased at 3 days after the myocardium amputation, but these parameters gradually returned to close to their baseline values for the normal heart at 7 days after amputation. The peak velocities of late diastolic inflow, ventricular systolic outflow, and systolic myocardial motion did not significantly differ during the heart regeneration. PMID:25517185

Temperature differentially regulates the two kisspeptin systems in the brain of zebrafish.

PubMed

Shahjahan, Md; Kitahashi, Takashi; Ogawa, Satoshi; Parhar, Ishwar S

2013-11-01

Kisspeptins encoded by the kiss1 and kiss2 genes play an important role in reproduction through the stimulation of gonadotropin-releasing hormone (GnRH) secretion by activating their receptors (KissR1 EU047918 and KissR2 EU047917). To understand the mechanism through which temperature affects reproduction, we examined kiss1 and kiss2 and their respective receptor (kissr1 and kissr2) gene expression in the brain of male zebrafish exposed to a low temperature (15°C), normal temperature (27°C), and high temperature (35°C) for 7-days. kiss1 mRNA levels in the brain were significantly increased (2.9-fold) in the low temperature compared to the control (27°C), while no noticeable change was observed in the high temperature conditions. Similarly, kissr1 mRNA levels were significantly increased (1.5-2.2-folds) in the low temperature conditions in the habenula, the nucleus of the medial longitudinal fascicle, oculomotor nucleus, and the interpeduncular nucleus. kiss2 mRNA levels were significantly decreased (0.5-fold) in the low and high temperature conditions, concomitant with kissr2 mRNA levels (0.5-fold) in the caudal zone of the periventricular hypothalamus and the posterior tuberal nucleus. gnrh3 but not gnrh2 mRNA levels were also decreased (0.5-fold) in the low and high temperature conditions. These findings suggest that while the kiss1/kissr1 system is sensitive to low temperature, the kiss2/kissr2 system is sensitive to both extremes of temperature, which leads to failure in reproduction. Copyright © 2013. Published by Elsevier Inc.

In vivo optoacoustic monitoring of calcium activity in the brain (Conference Presentation)

NASA Astrophysics Data System (ADS)

Deán-Ben, Xose Luís.; Gottschalk, Sven; Sela, Gali; Lauri, Antonella; Kneipp, Moritz; Ntziachristos, Vasilis; Westmeyer, Gil G.; Shoham, Shy; Razansky, Daniel

2017-03-01

Non-invasive observation of spatio-temporal neural activity of large neural populations distributed over the entire brain of complex organisms is a longstanding goal of neuroscience [1,2]. Recently, genetically encoded calcium indicators (GECIs) have revolutionized neuroimaging by enabling mapping the activity of entire neuronal populations in vivo [3]. Visualization of these powerful sensors with fluorescence microscopy has however been limited to superficial regions while deep brain areas have so far remained unreachable [4]. We have developed a volumetric multispectral optoacoustic tomography platform for imaging neural activation deep in scattering brains [5]. The developed methodology can render 100 volumetric frames per second across scalable fields of view ranging between 50-1000 mm3 with respective spatial resolution of 35-150µm. Experiments performed in immobilized and freely swimming larvae and in adult zebrafish brains expressing the genetically-encoded calcium indicator GCaMP5G demonstrated, for the first time, the fundamental ability to directly track neural dynamics using optoacoustics while overcoming the depth barrier of optical imaging in scattering brains [6]. It was further possible to monitor calcium transients in a scattering brain of a living adult transgenic zebrafish expressing GCaMP5G calcium indicator [7]. Fast changes in optoacoustic traces associated to GCaMP5G activity were detectable in the presence of other strongly absorbing endogenous chromophores, such as hemoglobin. The results indicate that the optoacoustic signal traces generally follow the GCaMP5G fluorescence dynamics and further enable overcoming the longstanding optical-diffusion penetration barrier associated to scattering in biological tissues [6]. The new functional optoacoustic neuroimaging method can visualize neural activity at penetration depths and spatio-temporal resolution scales not covered with the existing neuroimaging techniques. Thus, in addition to the well

Brain size and limits to adult neurogenesis.

PubMed

Paredes, Mercedes F; Sorrells, Shawn F; Garcia-Verdugo, Jose M; Alvarez-Buylla, Arturo

2016-02-15

The walls of the cerebral ventricles in the developing embryo harbor the primary neural stem cells from which most neurons and glia derive. In many vertebrates, neurogenesis continues postnatally and into adulthood in this region. Adult neurogenesis at the ventricle has been most extensively studied in organisms with small brains, such as reptiles, birds, and rodents. In reptiles and birds, these progenitor cells give rise to young neurons that migrate into many regions of the forebrain. Neurogenesis in adult rodents is also relatively widespread along the lateral ventricles, but migration is largely restricted to the rostral migratory stream into the olfactory bulb. Recent work indicates that the wall of the lateral ventricle is highly regionalized, with progenitor cells giving rise to different types of neurons depending on their location. In species with larger brains, young neurons born in these spatially specified domains become dramatically separated from potential final destinations. Here we hypothesize that the increase in size and topographical complexity (e.g., intervening white matter tracts) in larger brains may severely limit the long-term contribution of new neurons born close to, or in, the ventricular wall. We compare the process of adult neuronal birth, migration, and integration across species with different brain sizes, and discuss how early regional specification of progenitor cells may interact with brain size and affect where and when new neurons are added. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Lidocaine Hydrochloride Compared with MS222 for the Euthanasia of Zebrafish (Danio rerio).

PubMed

Collymore, Chereen; Banks, E Kate; Turner, Patricia V

2016-11-01

Despite several shortcomings, MS222 is the most commonly used chemical agent for euthanasia of zebrafish. Although lidocaine hydrochloride has some advantages over MS222, its effectiveness as a euthanasia agent for zebrafish is unknown. Larvae at 9 to 16 d postfertilization were exposed to 250 mg/L MS222 or 400, 500, 600, 700, 800, 900, or 1000 mg/L lidocaine and observed for cessation of heartbeat. Adult zebrafish were exposed to 250 mg/L MS222 or 400, 500, or 600 mg/L lidocaine; times to loss of righting reflex, cessation of opercular movement, and complete recovery; body length; aversive behavior; and gross and microscopic evidence of acute toxicity were evaluated. The heartbeat was not lost from any larvae in any group, regardless of drug or dosage. For adults, time to loss of righting reflex was greatest in the 500-mg/L lidocaine group. Opercular movement ceased earlier in all lidocaine groups compared with the MS222 group. Fish in the 500-mg/L lidocaine group were smaller than those in other groups. Fewer fish in the lidocaine groups displayed aversive behavior (erratic swimming and piping) compared with the MS222 group. No fish in the lidocaine hydrochloride groups (n = 30) recovered from euthanasia, whereas one fish in the MS222 group did (n = 10). Neither the MS222 nor lidocaine groups showed any gross or histologic changes suggestive of acute toxicity. Our results suggest that lidocaine hydrochloride may be an effective alternative chemical euthanasia agent for adult zebrafish but should not be used in larval fish.

Lidocaine Hydrochloride Compared with MS222 for the Euthanasia of Zebrafish (Danio rerio)

PubMed Central

Collymore, Chereen; Banks, E Kate; Turner, Patricia V

2016-01-01

Despite several shortcomings, MS222 is the most commonly used chemical agent for euthanasia of zebrafish. Although lidocaine hydrochloride has some advantages over MS222, its effectiveness as a euthanasia agent for zebrafish is unknown. Larvae at 9 to 16 d postfertilization were exposed to 250 mg/L MS222 or 400, 500, 600, 700, 800, 900, or 1000 mg/L lidocaine and observed for cessation of heartbeat. Adult zebrafish were exposed to 250 mg/L MS222 or 400, 500, or 600 mg/L lidocaine; times to loss of righting reflex, cessation of opercular movement, and complete recovery; body length; aversive behavior; and gross and microscopic evidence of acute toxicity were evaluated. The heartbeat was not lost from any larvae in any group, regardless of drug or dosage. For adults, time to loss of righting reflex was greatest in the 500-mg/L lidocaine group. Opercular movement ceased earlier in all lidocaine groups compared with the MS222 group. Fish in the 500-mg/L lidocaine group were smaller than those in other groups. Fewer fish in the lidocaine groups displayed aversive behavior (erratic swimming and piping) compared with the MS222 group. No fish in the lidocaine hydrochloride groups (n = 30) recovered from euthanasia, whereas one fish in the MS222 group did (n = 10). Neither the MS222 nor lidocaine groups showed any gross or histologic changes suggestive of acute toxicity. Our results suggest that lidocaine hydrochloride may be an effective alternative chemical euthanasia agent for adult zebrafish but should not be used in larval fish. PMID:27931323

Brain activation during mental rotation in school children and adults.

PubMed

Kucian, K; von Aster, M; Loenneker, T; Dietrich, T; Mast, F W; Martin, E

2007-01-01

Mental rotation is a complex cognitive skill depending on the manipulation of mental representations. We aimed to investigate the maturing neuronal network for mental rotation by measuring brain activation in 20 children and 20 adults using functional magnetic resonance imaging. Our results indicate that brain activation patterns are very similar between children and adults. However, adults exhibit stronger activation in the left intraparietal sulcus compared to children. This finding suggests a shift of activation from a predominantly right parietal activation in children to a bilateral activation pattern in adults. Furthermore, adults show a deactivation of the posterior cingulate gyrus and precuneus, which is not observed in children. In conclusion, developmental changes of brain activation during mental rotation are leading to a bilateral parietal activation pattern and faster performance.

Automatic zebrafish heartbeat detection and analysis for zebrafish embryos.

PubMed

Pylatiuk, Christian; Sanchez, Daniela; Mikut, Ralf; Alshut, Rüdiger; Reischl, Markus; Hirth, Sofia; Rottbauer, Wolfgang; Just, Steffen

2014-08-01

A fully automatic detection and analysis method of heartbeats in videos of nonfixed and nonanesthetized zebrafish embryos is presented. This method reduces the manual workload and time needed for preparation and imaging of the zebrafish embryos, as well as for evaluating heartbeat parameters such as frequency, beat-to-beat intervals, and arrhythmicity. The method is validated by a comparison of the results from automatic and manual detection of the heart rates of wild-type zebrafish embryos 36-120 h postfertilization and of embryonic hearts with bradycardia and pauses in the cardiac contraction.

Osteoblast Production by Reserved Progenitor Cells in Zebrafish Bone Regeneration and Maintenance.

PubMed

Ando, Kazunori; Shibata, Eri; Hans, Stefan; Brand, Michael; Kawakami, Atsushi

2017-12-04

Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Adolescent binge drinking alters adult brain neurotransmitter gene expression, behavior, brain regional volumes, and neurochemistry in mice

PubMed Central

Coleman, Leon G.; He, Jun; Lee, Joohwi; Styner, Martin; Crews, Fulton T.

2013-01-01

Background Binge-drinking is common in human adolescents. The adolescent brain is undergoing structural maturation and has a unique sensitivity to alcohol neurotoxicity. Therefore, adolescent binge ethanol may have long-term effects on the adult brain that alter brain structure and behaviors that are relevant to alcohol use disorders. Methods In order to determine if adolescent ethanol binge drinking alters the adult brain, male C57BL/6 mice were treated with either water or ethanol during adolescence (5g/kg/day i.g., post-natal days P28-37) and assessed during adulthood (P60-P88). An array of neurotransmitter-specific genes, behavioral tests (i.e. reversal learning, prepulse inhibition, and open field), and post-mortem brain structure using MRI and immunohistochemistry, were employed to assess persistent alterations in adult brain. Results At P38, 24 hours after adolescent ethanol (AE) binge, many neurotransmitter genes, particularly cholinergic and dopaminergic, were reduced by ethanol treatment. Interestingly, dopamine receptor type 4 mRNA was reduced and confirmed using immunohistochemistry. Normal control maturation (P38-P88) resulted in decreased neurotransmitter mRNA, e.g. an average decrease of 56%. Following adolescent ethanol treatment, adults showed greater gene expression reductions than controls, averaging 73%. Adult spatial learning assessed in the Morris water maze was not changed by adolescent ethanol treatment, but reversal learning experiments revealed deficits. Assessment of adult brain region volumes using MRI indicated that the olfactory bulb and basal forebrain were smaller in adults following adolescent ethanol. Immunohistochemical analyses found reduced basal forebrain area and fewer basal forebrain cholinergic neurons. Conclusions Adolescent binge ethanol treatment reduces adult neurotransmitter gene expression, particularly cholinergic genes, reduces basal forebrain and olfactory bulb volumes, and causes a reduction in the density of basal

Behavioural responses to novelty or to a predator stimulus are not altered in adult zebrafish by early embryonic alcohol exposure

PubMed Central

Seguin, Diane; Shams, Soaleha; Gerlai, Robert

2016-01-01

Background Fetal Alcohol Spectrum Disorders (FASD) may vary in symptoms and severity. In the milder and more prevalent forms of the disease, behavioural abnormalities may include impaired social behaviour, e.g. difficulty interpreting social cues. FASD patients remain often undiagnosed due to lack of biomarkers, and treatment is unavailable because the mechanisms of the disease are not yet understood. Animal models have been proposed to facilitate addressing these problems. More recently, short exposure of the zebrafish embryo to low concentrations of alcohol was shown to lead to significant and lasting impairment of behaviour in response to social stimuli. The impairment may be the result of abnormal social behaviour or altered fear/anxiety. The goal of the current study was to investigate the latter. Methods Here, we employed the alcohol exposure regimen used previously (exposure of 24th hour post-fertilization embryos to 0.00, 0.25, 0.50, 0.75 or 1.00 vol/vol % alcohol for 2 hours), allowed the fish to reach adulthood, and measured the behavioural responses of these adults to a novel tank (anxiety related behaviours) as well as to an animated image of a sympatric predator of zebrafish (fear related behaviours). Results We found behavioural responses of embryonic alcohol exposed adult fish to remain statistically indistinguishable from those of controls, suggesting unaltered anxiety and fear in the embryonic alcohol treated fish. Conclusions Given that motor and perceptual function was previously shown to be also unaltered in the adults after embryonic alcohol exposure, our current results suggest that the impaired response of these fish to social stimuli may be the result of abnormal social behaviour. PMID:27790739

Infection of zebrafish embryos with live fluorescent Streptococcus pneumoniae as a real-time pneumococcal meningitis model.

PubMed

Jim, Kin Ki; Engelen-Lee, JooYeon; van der Sar, Astrid M; Bitter, Wilbert; Brouwer, Matthijs C; van der Ende, Arie; Veening, Jan-Willem; van de Beek, Diederik; Vandenbroucke-Grauls, Christina M J E

2016-08-19

Streptococcus pneumoniae is one of the most important causes of bacterial meningitis, an infection where unfavourable outcome is driven by bacterial and host-derived toxins. In this study, we developed and characterized a pneumococcal meningitis model in zebrafish embryos that allows for real-time investigation of early host-microbe interaction. Zebrafish embryos were infected in the caudal vein or hindbrain ventricle with green fluorescent wild-type S. pneumoniae D39 or a pneumolysin-deficient mutant. The kdrl:mCherry transgenic zebrafish line was used to visualize the blood vessels, whereas phagocytic cells were visualized by staining with far red anti-L-plastin or in mpx:GFP/mpeg1:mCherry zebrafish, that have green fluorescent neutrophils and red fluorescent macrophages. Imaging was performed by fluorescence confocal and time-lapse microscopy. After infection by caudal vein, we saw focal clogging of the pneumococci in the blood vessels and migration of bacteria through the blood-brain barrier into the subarachnoid space and brain tissue. Infection with pneumolysin-deficient S. pneumoniae in the hindbrain ventricle showed attenuated growth and migration through the brain as compared to the wild-type strain. Time-lapse and confocal imaging revealed that the initial innate immune response to S. pneumoniae in the subarachnoid space mainly consisted of neutrophils and that pneumolysin-mediated cytolytic activity caused a marked reduction of phagocytes. This new meningitis model permits detailed analysis and visualization of host-microbe interaction in pneumococcal meningitis in real time and is a very promising tool to further our insights in the pathogenesis of pneumococcal meningitis.

Expansion of Multipotent Stem Cells from the Adult Human Brain

PubMed Central

Murrell, Wayne; Palmero, Emily; Bianco, John; Stangeland, Biljana; Joel, Mrinal; Paulson, Linda; Thiede, Bernd; Grieg, Zanina; Ramsnes, Ingunn; Skjellegrind, Håvard K.; Nygård, Ståle; Brandal, Petter; Sandberg, Cecilie; Vik-Mo, Einar; Palmero, Sheryl; Langmoen, Iver A.

2013-01-01

The discovery of stem cells in the adult human brain has revealed new possible scenarios for treatment of the sick or injured brain. Both clinical use of and preclinical research on human adult neural stem cells have, however, been seriously hampered by the fact that it has been impossible to passage these cells more than a very few times and with little expansion of cell numbers. Having explored a number of alternative culturing conditions we here present an efficient method for the establishment and propagation of human brain stem cells from whatever brain tissue samples we have tried. We describe virtually unlimited expansion of an authentic stem cell phenotype. Pluripotency proteins Sox2 and Oct4 are expressed without artificial induction. For the first time multipotency of adult human brain-derived stem cells is demonstrated beyond tissue boundaries. We characterize these cells in detail in vitro including microarray and proteomic approaches. Whilst clarification of these cells’ behavior is ongoing, results so far portend well for the future repair of tissues by transplantation of an adult patient’s own-derived stem cells. PMID:23967194

Expression of sall4 in taste buds of zebrafish.

PubMed

Jackson, Robyn; Braubach, Oliver R; Bilkey, Jessica; Zhang, Jing; Akimenko, Marie-Andrée; Fine, Alan; Croll, Roger P; Jonz, Michael G

2013-07-01

We characterized the expression of sall4, a gene encoding a zinc finger transcription factor involved in the maintenance of embryonic stem cells, in taste buds of zebrafish (Danio rerio). Using an enhancer trap line (ET5), we detected enhanced green fluorescent protein (EGFP) in developing and adult transgenic zebrafish in regions containing taste buds: the lips, branchial arches, and the nasal and maxillary barbels. Localization of EGFP to taste cells of the branchial arches and lips was confirmed by co-immunolabeling with antibodies against calretinin and serotonin, and a zebrafish-derived neuronal marker (zn-12). Transgenic insertion of the ET construct into the zebrafish genome was evaluated and mapped to chromosome 23 in proximity (i.e. 23 kb) to the sall4 gene. In situ hybridization and expression analysis between 24 and 96 h post-fertilization (hpf) demonstrated that transgenic egfp expression in ET5 zebrafish was correlated with the spatial and temporal pattern of expression of sall4 in the wild-type. Expression was first observed in the central nervous system and branchial arches at 24 hpf. At 48 hpf, sall4 and egfp expression was observed in taste bud primordia surrounding the mouth and branchial arches. At 72 and 96 hpf, expression was detected in the upper and lower lips and branchial arches. Double fluorescence in situ hybridization at 3 and 10 dpf confirmed colocalization of sall4 and egfp in the lips and branchial arches. These studies reveal sall4 expression in chemosensory cells and implicate this transcription factor in the development and renewal of taste epithelia in zebrafish. Copyright © 2013 Wiley Periodicals, Inc.

Using the zebrafish to understand tendon development and repair

PubMed Central

Chen, Jessica W.; Galloway, Jenna L.

2017-01-01

Tendons are important components of our musculoskeletal system. Injuries to these tissues are very common, resulting from occupational-related injuries, sports-related trauma, and age-related degeneration. Unfortunately, there are few treatment options, and current therapies rarely restore injured tendons to their original function. An improved understanding of the pathways regulating their development and repair would have significant impact in stimulating the formulation of regenerative-based approaches for tendon injury. The zebrafish provides an ideal system in which to perform genetic and chemical screens to identify new pathways involved in tendon biology. Until recently, there had been few descriptions of tendons and ligaments in the zebrafish and their similarity to mammalian tendon tissues. In this chapter, we describe the development of the zebrafish tendon and ligament tissues in the context of their gene expression, structure, and interactions with neighboring musculoskeletal tissues. We highlight the similarities with tendon development in higher vertebrates, showing that the craniofacial tendons and ligaments in zebrafish morphologically, molecularly, and structurally resemble mammalian tendons and ligaments from embryonic to adult stages. We detail methods for fluorescent in situ hybridization and immunohistochemistry as an assay to examine morphological changes in the zebrafish musculoskeleton. Staining assays such as these could provide the foundation for screen-based approaches to identify new regulators of tendon development, morphogenesis, and repair. These discoveries would provide new targets and pathways to study in the context of regenerative medicine-based approaches to improve tendon healing. PMID:28129848

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera

NASA Astrophysics Data System (ADS)

Cruz Perez, Carlos; Lauri, Antonella; Symvoulidis, Panagiotis; Cappetta, Michele; Erdmann, Arne; Westmeyer, Gil Gregor

2015-09-01

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera.

PubMed

Perez, Carlos Cruz; Lauri, Antonella; Symvoulidis, Panagiotis; Cappetta, Michele; Erdmann, Arne; Westmeyer, Gil Gregor

2015-09-01

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

Dose-Specific Effects of Di-Isononyl Phthalate on the Endocannabinoid System and on Liver of Female Zebrafish.

PubMed

Forner-Piquer, Isabel; Maradonna, Francesca; Gioacchini, Giorgia; Santangeli, Stefania; Allarà, Marco; Piscitelli, Fabiana; Habibi, Hamid R; Di Marzo, Vincenzo; Carnevali, Oliana

2017-10-01

Phthalates, used as plasticizers, have become a ubiquitous contaminant and have been reported for their potential to induce toxicity in living organisms. Among them, di-isononyl phthalate (DiNP) has been recently used to replace di(2-ethylhexyl) phthalate (DEHP). Nowadays, there is evidence that DiNP is an endocrine-disrupting chemical; however, little is known about its effects on the endocannabinoid system (ECS) and lipid metabolism. Hence, the aim of our study was to investigate the effects of DiNP on the ECS in zebrafish liver and brain and on hepatic lipid storage. To do so, adult female zebrafish were exposed to three concentrations (0.42 µg/L, 4.2 µg/L, and 42 µg/L) of DiNP via water for 3 weeks. Afterwards, we investigated transcript levels for genes involved in the ECS of the brain and liver as well as liver histology and image analysis, Fourier-transform infrared spectroscopy imaging, and measurement of endocannabinoid levels. Our results demonstrate that DiNP upregulates orexigenic signals and causes hepatosteatosis together with deregulation of the peripheral ECS and lipid metabolism. A decrease in the levels of ECS components at the central level was observed after exposure to the highest DiNP concentration tested. These findings suggest that replacement of DEHP with DiNP should be considered with caution because of observed adverse DiNP effects on aquatic organisms. Copyright © 2017 Endocrine Society.

Changes in Brain Monoamines Underlie Behavioural Disruptions after Zebrafish Diet Exposure to Polycyclic Aromatic Hydrocarbons Environmental Mixtures

PubMed Central

Vignet, Caroline; Trenkel, Verena M.; Vouillarmet, Annick; Bricca, Giampiero; Bégout, Marie-Laure; Cousin, Xavier

2017-01-01

Zebrafish were exposed through diet to two environmentally relevant polycyclic aromatic hydrocarbons (PAHs) mixtures of contrasted compositions, one of pyrolytic (PY) origin and one from light crude oil (LO). Monoamine concentrations were quantified in the brains of the fish after six month of exposure. A significant decrease in noradrenaline (NA) was observed in fish exposed to both mixtures, while a decrease in serotonin (5HT) and dopamine (DA) was observed only in LO-exposed fish. A decrease in metabolites of 5HT and DA was observed in fish exposed to both mixtures. Several behavioural disruptions were observed that depended on mixtures, and parallels were made with changes in monoamine concentrations. Indeed, we observed an increase in anxiety in fish exposed to both mixtures, which could be related to the decrease in 5HT and/or NA, while disruptions of daily activity rhythms were observed in LO fish, which could be related to the decrease in DA. Taken together, these results showed that (i) chronic exposures to PAHs mixtures disrupted brain monoamine contents, which could underlie behavioural disruptions, and that (ii) the biological responses depended on mixture compositions. PMID:28273853

Characterization of Sleep in Zebrafish and Insomnia in Hypocretin Receptor Mutants

PubMed Central

Yokogawa, Tohei; Marin, Wilfredo; Faraco, Juliette; Pézeron, Guillaume; Appelbaum, Lior; Zhang, Jian; Rosa, Frédéric; Mourrain, Philippe; Mignot, Emmanuel

2007-01-01

Sleep is a fundamental biological process conserved across the animal kingdom. The study of how sleep regulatory networks are conserved is needed to better understand sleep across evolution. We present a detailed description of a sleep state in adult zebrafish characterized by reversible periods of immobility, increased arousal threshold, and place preference. Rest deprivation using gentle electrical stimulation is followed by a sleep rebound, indicating homeostatic regulation. In contrast to mammals and similarly to birds, light suppresses sleep in zebrafish, with no evidence for a sleep rebound. We also identify a null mutation in the sole receptor for the wake-promoting neuropeptide hypocretin (orexin) in zebrafish. Fish lacking this receptor demonstrate short and fragmented sleep in the dark, in striking contrast to the excessive sleepiness and cataplexy of narcolepsy in mammals. Consistent with this observation, we find that the hypocretin receptor does not colocalize with known major wake-promoting monoaminergic and cholinergic cell groups in the zebrafish. Instead, it colocalizes with large populations of GABAergic neurons, including a subpopulation of Adra2a-positive GABAergic cells in the anterior hypothalamic area, neurons that could assume a sleep modulatory role. Our study validates the use of zebrafish for the study of sleep and indicates molecular diversity in sleep regulatory networks across vertebrates. PMID:17941721

Diving into the world of alcohol teratogenesis: a review of zebrafish models of fetal alcohol spectrum disorder.

PubMed

Fernandes, Yohaan; Buckley, Desire M; Eberhart, Johann K

2018-04-01

The term fetal alcohol spectrum disorder (FASD) refers to the entire suite of deleterious outcomes resulting from embryonic exposure to alcohol. Along with other reviews in this special issue, we provide insight into how animal models, specifically the zebrafish, have informed our understanding of FASD. We first provide a brief introduction to FASD. We discuss the zebrafish as a model organism and its strengths for alcohol research. We detail how zebrafish has been used to model some of the major defects present in FASD. These include behavioral defects, such as social behavior as well as learning and memory, and structural defects, disrupting organs such as the brain, sensory organs, heart, and craniofacial skeleton. We provide insights into how zebrafish research has aided in our understanding of the mechanisms of ethanol teratogenesis. We end by providing some relatively recent advances that zebrafish has provided in characterizing gene-ethanol interactions that may underlie FASD.

The hydrodynamics of predator-prey interactions in zebrafish

NASA Astrophysics Data System (ADS)

McHenry, Matthew; Soto, Alberto; Carrillo, Andres; Byron, Margaret

2017-11-01

Hydrodynamics govern the behavior of fishes when they operate as predators or prey. In addition to the role of fluid forces in propulsion, fishes relay on flow stimuli to sense a predatory threat and to localize palatable prey. We have performed a series of experiments on zebrafish (Danio rerio) that aim to resolve the major factors that determine whether prey survive an encounter with a predator. Zebrafish serve as a model system in this pursuit because the adults prey on larvae of the same species and the larvae are often successful in evading the attacks of the adults. We use a combination of theoretical and experimental approaches to resolve the behavioral algorithms and kinematics that determined the outcome of these interactions. In this context, the hydrodynamics of intermediate Reynolds numbers largely determines the range of flow stimuli and the limits to locomotor performance at dictate prey survival. These principles have the potential to apply to a broad diversity of fishes and other aquatic animals. ONR: N00014-15-1-2249.

GABA regulates synaptic integration of newly generated neurons in the adult brain

NASA Astrophysics Data System (ADS)

Ge, Shaoyu; Goh, Eyleen L. K.; Sailor, Kurt A.; Kitabatake, Yasuji; Ming, Guo-Li; Song, Hongjun

2006-02-01

Adult neurogenesis, the birth and integration of new neurons from adult neural stem cells, is a striking form of structural plasticity and highlights the regenerative capacity of the adult mammalian brain. Accumulating evidence suggests that neuronal activity regulates adult neurogenesis and that new neurons contribute to specific brain functions. The mechanism that regulates the integration of newly generated neurons into the pre-existing functional circuitry in the adult brain is unknown. Here we show that newborn granule cells in the dentate gyrus of the adult hippocampus are tonically activated by ambient GABA (γ-aminobutyric acid) before being sequentially innervated by GABA- and glutamate-mediated synaptic inputs. GABA, the major inhibitory neurotransmitter in the adult brain, initially exerts an excitatory action on newborn neurons owing to their high cytoplasmic chloride ion content. Conversion of GABA-induced depolarization (excitation) into hyperpolarization (inhibition) in newborn neurons leads to marked defects in their synapse formation and dendritic development in vivo. Our study identifies an essential role for GABA in the synaptic integration of newly generated neurons in the adult brain, and suggests an unexpected mechanism for activity-dependent regulation of adult neurogenesis, in which newborn neurons may sense neuronal network activity through tonic and phasic GABA activation.

Acute and chronic ethanol exposure differentially alters alcohol dehydrogenase and aldehyde dehydrogenase activity in the zebrafish liver.

PubMed

Tran, Steven; Nowicki, Magda; Chatterjee, Diptendu; Gerlai, Robert

2015-01-02

Chronic ethanol exposure paradigms have been successfully used in the past to induce behavioral and central nervous system related changes in zebrafish. However, it is currently unknown whether chronic ethanol exposure alters ethanol metabolism in adult zebrafish. In the current study we examine the effect of acute ethanol exposure on adult zebrafish behavioral responses, as well as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity in the liver. We then examine how two different chronic ethanol exposure paradigms (continuous and repeated ethanol exposure) alter behavioral responses and liver enzyme activity during a subsequent acute ethanol challenge. Acute ethanol exposure increased locomotor activity in a dose-dependent manner. ADH activity was shown to exhibit an inverted U-shaped curve and ALDH activity was decreased by ethanol exposure at all doses. During the acute ethanol challenge, animals that were continuously housed in ethanol exhibited a significantly reduced locomotor response and increased ADH activity, however, ALDH activity did not change. Zebrafish that were repeatedly exposed to ethanol demonstrated a small but significant attenuation of the locomotor response during the acute ethanol challenge but ADH and ALDH activity was similar to controls. Overall, we identified two different chronic ethanol exposure paradigms that differentially alter behavioral and physiological responses in zebrafish. We speculate that these two paradigms may allow dissociation of central nervous system-related and liver enzyme-dependent ethanol induced changes in zebrafish. Copyright © 2014 Elsevier Inc. All rights reserved.

Impact of stress, fear and anxiety on the nociceptive responses of larval zebrafish.

PubMed

Lopez-Luna, Javier; Al-Jubouri, Qussay; Al-Nuaimy, Waleed; Sneddon, Lynne U

2017-01-01

Both adult and larval zebrafish have been demonstrated to show behavioural responses to noxious stimulation but also to potentially stress- and fear or anxiety- eliciting situations. The pain or nociceptive response can be altered and modulated by these situations in adult fish through a mechanism called stress-induced analgesia. However, this phenomenon has not been described in larval fish yet. Therefore, this study explores the behavioural changes in larval zebrafish after noxious stimulation and exposure to challenges that can trigger a stress, fear or anxiety reaction. Five-day post fertilization zebrafish were exposed to either a stressor (air emersion), a predatory fear cue (alarm substance) or an anxiogenic (caffeine) alone or prior to immersion in acetic acid 0.1%. Pre- and post-stimulation behaviour (swimming velocity and time spent active) was recorded using a novel tracking software in 25 fish at once. Results show that larvae reduced both velocity and activity after exposure to the air emersion and alarm substance challenges and that these changes were attenuated using etomidate and diazepam, respectively. Exposure to acetic acid decreased velocity and activity as well, whereas air emersion and alarm substance inhibited these responses, showing no differences between pre- and post-stimulation. Therefore, we hypothesize that an antinociceptive mechanism, activated by stress and/or fear, occur in 5dpf zebrafish, which could have prevented the larvae to display the characteristic responses to pain.

Melanophore migration and survival during zebrafish adult pigment stripe development require the immunoglobulin superfamily adhesion molecule Igsf11.

PubMed

Eom, Dae Seok; Inoue, Shinya; Patterson, Larissa B; Gordon, Tiffany N; Slingwine, Rebecca; Kondo, Shigeru; Watanabe, Masakatsu; Parichy, David M

2012-01-01

The zebrafish adult pigment pattern has emerged as a useful model for understanding the development and evolution of adult form as well as pattern-forming mechanisms more generally. In this species, a series of horizontal melanophore stripes arises during the larval-to-adult transformation, but the genetic and cellular bases for stripe formation remain largely unknown. Here, we show that the seurat mutant phenotype, consisting of an irregular spotted pattern, arises from lesions in the gene encoding Immunoglobulin superfamily member 11 (Igsf11). We find that Igsf11 is expressed by melanophores and their precursors, and we demonstrate by cell transplantation and genetic rescue that igsf11 functions autonomously to this lineage in promoting adult stripe development. Further analyses of cell behaviors in vitro, in vivo, and in explant cultures ex vivo demonstrate that Igsf11 mediates adhesive interactions and that mutants for igsf11 exhibit defects in both the migration and survival of melanophores and their precursors. These findings identify the first in vivo requirements for igsf11 as well as the first instance of an immunoglobulin superfamily member functioning in pigment cell development and patterning. Our results provide new insights into adult pigment pattern morphogenesis and how cellular interactions mediate pattern formation.

Melanophore Migration and Survival during Zebrafish Adult Pigment Stripe Development Require the Immunoglobulin Superfamily Adhesion Molecule Igsf11

PubMed Central

Patterson, Larissa B.; Gordon, Tiffany N.; Slingwine, Rebecca; Kondo, Shigeru; Watanabe, Masakatsu; Parichy, David M.

2012-01-01

The zebrafish adult pigment pattern has emerged as a useful model for understanding the development and evolution of adult form as well as pattern-forming mechanisms more generally. In this species, a series of horizontal melanophore stripes arises during the larval-to-adult transformation, but the genetic and cellular bases for stripe formation remain largely unknown. Here, we show that the seurat mutant phenotype, consisting of an irregular spotted pattern, arises from lesions in the gene encoding Immunoglobulin superfamily member 11 (Igsf11). We find that Igsf11 is expressed by melanophores and their precursors, and we demonstrate by cell transplantation and genetic rescue that igsf11 functions autonomously to this lineage in promoting adult stripe development. Further analyses of cell behaviors in vitro, in vivo, and in explant cultures ex vivo demonstrate that Igsf11 mediates adhesive interactions and that mutants for igsf11 exhibit defects in both the migration and survival of melanophores and their precursors. These findings identify the first in vivo requirements for igsf11 as well as the first instance of an immunoglobulin superfamily member functioning in pigment cell development and patterning. Our results provide new insights into adult pigment pattern morphogenesis and how cellular interactions mediate pattern formation. PMID:22916035

Acute administration of THC impairs spatial but not associative memory function in zebrafish.

PubMed

Ruhl, Tim; Prinz, Nicole; Oellers, Nadine; Seidel, Nathan Ian; Jonas, Annika; Albayram, Onder; Bilkei-Gorzo, Andras; von der Emde, Gerhard

2014-10-01

The present study examined the effect of acute administration of endocannabinoid receptor CB1 ligand ∆-9-tetrahydrocannabinol (THC) on intracellular signalling in the brain and retrieval from two different memory systems in the zebrafish (Danio rerio). First, fish were treated with THC and changes in the phosphorylation level of mitogen-activated protein (MAP) kinases Akt and Erk in the brain were determined 1 h after drug treatment. Next, animals of a second group learned in a two-alternative choice paradigm to discriminate between two colours, whereas a third group solved a spatial cognition task in an open-field maze by use of an ego-allocentric strategy. After memory acquisition and consolidation, animals were pharmacologically treated using the treatment regime as in the first group and then tested again for memory retrieval. We found an enhanced Erk but not Akt phosphorylation suggesting that THC treatment specifically activated Erk signalling in the zebrafish telencephalon. While CB1 agonist THC did not affect behavioural performance of animals in the colour discrimination paradigm, spatial memory was significantly impaired. The effect of THC on spatial learning is probably specific, since neither motor activity nor anxiety-related behaviour was influenced by the drug treatment. That indicates a striking influence of the endocannabinoid system (ECS) on spatial cognition in zebrafish. The results are very coincident with reports on mammals, demonstrating that the ECS is functional highly conserved during vertebrate evolution. We further conclude that the zebrafish provides a promising model organism for ongoing research on the ECS.

Orthosiphon stamineus Leaf Extract Affects TNF-α and Seizures in a Zebrafish Model

PubMed Central

Choo, Brandon Kar Meng; Kundap, Uday P.; Kumari, Yatinesh; Hue, Seow-Mun; Othman, Iekhsan; Shaikh, Mohd Farooq

2018-01-01

Epileptic seizures result from abnormal brain activity and can affect motor, autonomic and sensory function; as well as, memory, cognition, behavior, or emotional state. Effective anti-epileptic drugs (AEDs) are available but have tolerability issues due to their side effects. The Malaysian herb Orthosiphon stamineus, is a traditional epilepsy remedy and possesses anti-inflammatory, anti-oxidant and free-radical scavenging abilities, all of which are known to protect against seizures. This experiment thus aimed to explore if an ethanolic leaf extract of O. stamineus has the potential to be a novel symptomatic treatment for epileptic seizures in a zebrafish model; and the effects of the extract on the expression levels of several genes in the zebrafish brain which are associated with seizures. The results of this study indicate that O. stamineus has the potential to be a novel symptomatic treatment for epileptic seizures as it is pharmacologically active against seizures in a zebrafish model. The anti-convulsive effect of this extract is also comparable to that of diazepam at higher doses and can surpass diazepam in certain cases. Treatment with the extract also counteracts the upregulation of NF-κB, NPY and TNF-α as a result of a Pentylenetetrazol (PTZ) treated seizure. The anti-convulsive action for this extract could be at least partially due to its downregulation of TNF-α. Future work could include the discovery of the active anti-convulsive compound, as well as determine if the extract does not cause cognitive impairment in zebrafish. PMID:29527169

Adult mouse brain gene expression patterns bear an embryologic imprint

PubMed Central

Zapala, Matthew A.; Hovatta, Iiris; Ellison, Julie A.; Wodicka, Lisa; Del Rio, Jo A.; Tennant, Richard; Tynan, Wendy; Broide, Ron S.; Helton, Rob; Stoveken, Barbara S.; Winrow, Christopher; Lockhart, Daniel J.; Reilly, John F.; Young, Warren G.; Bloom, Floyd E.; Lockhart, David J.; Barlow, Carrolee

2005-01-01

The current model to explain the organization of the mammalian nervous system is based on studies of anatomy, embryology, and evolution. To further investigate the molecular organization of the adult mammalian brain, we have built a gene expression-based brain map. We measured gene expression patterns for 24 neural tissues covering the mouse central nervous system and found, surprisingly, that the adult brain bears a transcriptional “imprint” consistent with both embryological origins and classic evolutionary relationships. Embryonic cellular position along the anterior–posterior axis of the neural tube was shown to be closely associated with, and possibly a determinant of, the gene expression patterns in adult structures. We also observed a significant number of embryonic patterning and homeobox genes with region-specific expression in the adult nervous system. The relationships between global expression patterns for different anatomical regions and the nature of the observed region-specific genes suggest that the adult brain retains a degree of overall gene expression established during embryogenesis that is important for regional specificity and the functional relationships between regions in the adult. The complete collection of extensively annotated gene expression data along with data mining and visualization tools have been made available on a publicly accessible web site (www.barlow-lockhart-brainmapnimhgrant.org). PMID:16002470

Elucidating the mechanism of action of tributyltin (TBT) in zebrafish.

PubMed

McGinnis, Courtney L; Crivello, Joseph F

2011-05-01

Tributyltin (TBT), an antifouling agent, has been implicated in the masculinization of fish species worldwide, but the masculinizing mechanism is not fully understood. We have examined the actions of TBT as an endocrine disruptor in zebrafish (Danio rerio). In HeLa cells transiently co-transfected with plasmid constructs containing the zebrafish estrogen receptors (zfERα, zfERβ(1) and zfERβ(2)) and the zebrafish estrogen response element (zfERE-tk-luc), ethinyl estradiol (EE2) induced luciferase activity 4 to 6-fold and was inhibited by TBT. In HeLa cells transiently co-transfected with the zebrafish androgen receptor (zfAR) and the murine androgen receptor response element (ARE-slp-luc), testosterone induced luciferase activity was not inhibited by TBT. In HeLa cells co-transfected with zfERα, zfERβ(1) and zfERβ(2) and a plasmid containing zebrafish aromatase (zfCyp19b-luc), TBT inhibited luciferase activity. In zebrafish exposed to 1mg/kg and 5mg/kg TBT in vivo, there was a increase in liver sulfotransferase and a decrease acyl-CoA testosterone acyltransferase activity. Real-time PCR analysis of sexual differentiation markers in fish exposed to TBT in vivo revealed a tissue-specific response. In brain there was increased production of Sox9, Dax1, and SF1 mRNA, an androgenizing effect, while in the liver there was increased production of Dax1, Cyp19a and zfERβ(1) mRNA but decreased production of Sox9 mRNA, a feminizing effect. In the gonads there was increased production of zfERα and zfCyp19a mRNA, again a feminizing effect. TBT has an overall masculinizing effect but the masculinizing effect is tempered by a feminizing effect on gene transcription in certain tissues. These results are discussed in the context of TBT as an endocrine disruptor in zebrafish. Copyright © 2011 Elsevier B.V. All rights reserved.

Maternal thyroid hormones are essential for neural development in zebrafish.

PubMed

Campinho, Marco A; Saraiva, João; Florindo, Claudia; Power, Deborah M

2014-07-01

Teleost eggs contain an abundant store of maternal thyroid hormones (THs), and early in zebrafish embryonic development, all the genes necessary for TH signaling are expressed. Nonetheless the function of THs in embryonic development remains elusive. To test the hypothesis that THs are fundamental for zebrafish embryonic development, an monocarboxilic transporter 8 (Mct8) knockdown strategy was deployed to prevent maternal TH uptake. Absence of maternal THs did not affect early specification of the neural epithelia but profoundly modified later dorsal specification of the brain and spinal cord as well as specific neuron differentiation. Maternal THs acted upstream of pax2a, pax7, and pax8 genes but downstream of shha and fgf8a signaling. The lack of inhibitory spinal cord interneurons and increased motoneurons in the mct8 morphants is consistent with their stiff axial body and impaired mobility. The mct8 mutations are associated with X-linked mental retardation in humans, and the cellular and molecular consequences of MCT8 knockdown during embryonic development in zebrafish provides new insight into the potential role of THs in this condition.

Zebrafish as a systems toxicology model for developmental neurotoxicity testing.

PubMed

Nishimura, Yuhei; Murakami, Soichiro; Ashikawa, Yoshifumi; Sasagawa, Shota; Umemoto, Noriko; Shimada, Yasuhito; Tanaka, Toshio

2015-02-01

The developing brain is extremely sensitive to many chemicals. Exposure to neurotoxicants during development has been implicated in various neuropsychiatric and neurological disorders, including autism spectrum disorder, attention deficit hyperactive disorder, schizophrenia, Parkinson's disease, and Alzheimer's disease. Although rodents have been widely used for developmental neurotoxicity testing, experiments using large numbers of rodents are time-consuming, expensive, and raise ethical concerns. Using alternative non-mammalian animal models may relieve some of these pressures by allowing testing of large numbers of subjects while reducing expenses and minimizing the use of mammalian subjects. In this review, we discuss some of the advantages of using zebrafish in developmental neurotoxicity testing, focusing on central nervous system development, neurobehavior, toxicokinetics, and toxicodynamics in this species. We also describe some important examples of developmental neurotoxicity testing using zebrafish combined with gene expression profiling, neuroimaging, or neurobehavioral assessment. Zebrafish may be a systems toxicology model that has the potential to reveal the pathways of developmental neurotoxicity and to provide a sound basis for human risk assessments. © 2014 Japanese Teratology Society.

Botulinum Toxin Induces Muscle Paralysis and Inhibits Bone Regeneration in Zebrafish

PubMed Central

Recidoro, Anthony M.; Roof, Amanda C.; Schmitt, Michael; Worton, Leah E.; Petrie, Timothy; Strand, Nicholas; Ausk, Brandon J.; Srinivasan, Sundar; Moon, Randall T.; Gardiner, Edith M.; Kaminsky, Werner; Bain, Steven D.; Allan, Christopher H.; Gross, Ted S.; Kwon, Ronald Y.

2016-01-01

Intramuscular administration of Botulinum toxin (BTx) has been associated with impaired osteogenesis in diverse conditions of bone formation (e.g., development, growth, and healing), yet the mechanisms of neuromuscular-bone crosstalk underlying these deficits have yet to be identified. Motivated by the emerging utility of zebrafish (Danio rerio) as a rapid, genetically tractable, and optically transparent model for human pathologies (as well as the potential to interrogate neuromuscular-mediated bone disorders in a simple model that bridges in vitro and more complex in vivo model systems), in this study we developed a model of BTx-induced muscle paralysis in adult zebrafish, and examined its effects on intramembranous ossification during tail fin regeneration. BTx administration induced rapid muscle paralysis in adult zebrafish in a manner that was dose-dependent, transient, and focal, mirroring the paralytic phenotype observed in animal and human studies. During fin regeneration, BTx impaired continued bone ray outgrowth, morphology, and patterning, indicating defects in early osteogenesis. Further, BTx significantly decreased mineralizing activity and crystalline mineral accumulation, suggesting delayed late-stage osteoblast differentiation and/or altered secondary bone apposition. Bone ray transection proximal to the amputation site focally inhibited bone outgrowth in the affected ray, implicating intra- and/or inter-ray nerves in this process. Taken together, these studies demonstrate the potential to interrogate pathological features of BTx-induced osteoanabolic dysfunction in the regenerating zebrafish fin, define the technological toolbox for detecting bone growth and mineralization deficits in this process, and suggest that pathways mediating neuromuscular regulation of osteogenesis may be conserved beyond established mammalian models of bone anabolic disorders. PMID:24806738

Removal of the precursors of N-nitrosodiethylamine (NDEA), an emerging disinfection byproduct, in drinking water treatment process and its toxicity to adult zebrafish (Danio rerio).

PubMed

Zheng, Jian; Lin, Tao; Chen, Wei

2018-01-01

N-nitrosodiethylamine (NDEA) is one of the emerging nitrogenous disinfection byproducts with probable cytotoxicity, genotoxicity, and carcinogenesis. Its potential toxicological effects have received extensive attention but remain to be poorly understood. In this study, changes in NDEA precursors in drinking water treatment process were studied using the trial of its formation potential (FP), and the toxicity induced by NDEA to adult zebrafish was investigated. NDEA FP in the raw water of Taihu Lake ranged from 46.9 to 68.3 ng/L. The NDEA precursors were removed effectively by O 3 /BAC process. Hydrophilic fraction and low-molecular-weight fraction (<1 kDa) had the highest NDEA FP. The toxicity results demonstrated that the acute lethal concentration of NDEA causing 50% mortality in 96 h (96-h LC50) was 210.4 mg/L, and NDEA was more likely to be accumulated in kidney, followed by liver and gill. NDEA induced oxidative stress and antioxidant defense to zebrafish metabolism system at concentrations over 5 μg/L. After a 42-day exposure, a significant DNA damage was observed in zebrafish liver cells at NDEA concentrations beyond 500 μg/L. This study investigated NDEA properties in both engineering prospective and toxicity evaluation, thus providing comprehensive information on its control in drinking water treatment process and its toxicity effect on zebrafish as a model animal. Copyright © 2017 Elsevier Ltd. All rights reserved.

Adult Neurogenesis in the Mammalian Brain: Significant Answers and Significant Questions

PubMed Central

Ming, Guo-li; Song, Hongjun

2011-01-01

Summary Adult neurogenesis, a process of generating functional neurons from adult neural precursors, occurs throughout life in restricted brain regions in mammals. The past decade has witnessed tremendous progress in addressing questions related to almost every aspect of adult neurogenesis in the mammalian brain. Here we review major advances in our understanding of adult mammalian neurogenesis in the dentate gyrus of the hippocampus and from the subventricular zone of the lateral ventricle, the rostral migratory stream to the olfactory bulb. We highlight emerging principles that have significant implications for stem cell biology, developmental neurobiology, neural plasticity, and disease mechanisms. We also discuss remaining questions related to adult neural stem cells and their niches, underlying regulatory mechanisms and potential functions of newborn neurons in the adult brain. Building upon the recent progress and aided by new technologies, the adult neurogenesis field is poised to leap forward in the next decade. PMID:21609825

The small molecule probe PT-Yellow labels the renal proximal tubules in zebrafish.

PubMed

Sander, Veronika; Patke, Shantanu; Sahu, Srikanta; Teoh, Chai Lean; Peng, Zhenzhen; Chang, Young-Tae; Davidson, Alan J

2015-01-01

We report the development of a small fluorescent molecule, BDNCA3-D2, herein referred to as PT-Yellow. Soaking zebrafish embryos in PT-Yellow or intraperitoneal injection into adults results in non-toxic in vivo fluorescent labeling of the renal proximal tubules, the major site of blood filtrate reabsorption and a common target of injury in acute kidney injury. We demonstrate the applicability of this new compound as a rapid and simple readout for zebrafish kidney filtration and proximal tubule reabsorption function.

Myotonia congenita-associated mutations in chloride channel-1 affect zebrafish body wave swimming kinematics.

PubMed

Cheng, Wei; Tian, Jing; Burgunder, Jean-Marc; Hunziker, Walter; Eng, How-Lung

2014-01-01

Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1). Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita.

Myotonia Congenita-Associated Mutations in Chloride Channel-1 Affect Zebrafish Body Wave Swimming Kinematics

PubMed Central

Cheng, Wei; Tian, Jing; Burgunder, Jean-Marc; Hunziker, Walter; Eng, How-Lung

2014-01-01

Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1). Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita. PMID:25083883

Embryonic senescence and laminopathies in a progeroid zebrafish model.

PubMed

Koshimizu, Eriko; Imamura, Shintaro; Qi, Jie; Toure, Jamal; Valdez, Delgado M; Carr, Christopher E; Hanai, Jun-ichi; Kishi, Shuji

2011-03-30

Mutations that disrupt the conversion of prelamin A to mature lamin A cause the rare genetic disorder Hutchinson-Gilford progeria syndrome and a group of laminopathies. Our understanding of how A-type lamins function in vivo during early vertebrate development through aging remains limited, and would benefit from a suitable experimental model. The zebrafish has proven to be a tractable model organism for studying both development and aging at the molecular genetic level. Zebrafish show an array of senescence symptoms resembling those in humans, which can be targeted to specific aging pathways conserved in vertebrates. However, no zebrafish models bearing human premature senescence currently exist. We describe the induction of embryonic senescence and laminopathies in zebrafish harboring disturbed expressions of the lamin A gene (LMNA). Impairments in these fish arise in the skin, muscle and adipose tissue, and sometimes in the cartilage. Reduced function of lamin A/C by translational blocking of the LMNA gene induced apoptosis, cell-cycle arrest, and craniofacial abnormalities/cartilage defects. By contrast, induced cryptic splicing of LMNA, which generates the deletion of 8 amino acid residues lamin A (zlamin A-Δ8), showed embryonic senescence and S-phase accumulation/arrest. Interestingly, the abnormal muscle and lipodystrophic phenotypes were common in both cases. Hence, both decrease-of-function of lamin A/C and gain-of-function of aberrant lamin A protein induced laminopathies that are associated with mesenchymal cell lineages during zebrafish early development. Visualization of individual cells expressing zebrafish progerin (zProgerin/zlamin A-Δ37) fused to green fluorescent protein further revealed misshapen nuclear membrane. A farnesyltransferase inhibitor reduced these nuclear abnormalities and significantly prevented embryonic senescence and muscle fiber damage induced by zProgerin. Importantly, the adult Progerin fish survived and remained fertile with

In vivo spectroscopic photoacoustic tomography imaging of a far red fluorescent protein expressed in the exocrine pancreas of adult zebrafish

NASA Astrophysics Data System (ADS)

Liu, Mengyang; Schmitner, Nicole; Sandrian, Michelle G.; Zabihian, Behrooz; Hermann, Boris; Salvenmoser, Willi; Meyer, Dirk; Drexler, Wolfgang

2014-03-01

Fluorescent proteins brought a revolution in life sciences and biological research in that they make a powerful tool for researchers to study not only the structural and morphological information, but also dynamic and functional information in living cells and organisms. While green fluorescent proteins (GFP) have become a common labeling tool, red-shifted or even near infrared fluorescent proteins are becoming the research focus due to the fact that longer excitation wavelengths are more suitable for deep tissue imaging. In this study, E2-Crimson, a far red fluorescent protein whose excitation wavelength is 611 nm, was genetically expressed in the exocrine pancreas of adult zebrafish. Using spectroscopic all optical detection photoacoustic tomography, we mapped the distribution of E2-Crimson in 3D after imaging the transgenic zebrafish in vivo using two different wavelengths. With complementary morphological information provided by imaging the same fish using a spectral domain optical coherence tomography system, the E2-Crimson distribution acquired from spectroscopic photoacoustic tomography was confirmed in 2D by epifluorescence microscopy and in 3D by histology. To the authors' knowledge, this is the first time a far red fluorescent protein is imaged in vivo by spectroscopic photoacoustic tomography. Due to the regeneration feature of zebrafish pancreas, this work preludes the longitudinal studies of animal models of diseases such as pancreatitis by spectroscopic photoacoustic tomography. Since the effective penetration depth of photoacoustic tomography is beyond the transport mean free path length, other E2-Crimson labeled inner organs will also be able to be studied dynamically using spectroscopic photoacoustic tomography.

PFOS induces behavioral alterations, including spontaneous hyperactivity that is corrected by dexamfetamine in zebrafish larvae.

PubMed

Spulber, Stefan; Kilian, Pascal; Wan Ibrahim, Wan Norhamidah; Onishchenko, Natalia; Ulhaq, Mazhar; Norrgren, Leif; Negri, Sara; Di Tuccio, Marcello; Ceccatelli, Sandra

2014-01-01

Perfluorooctane sulfonate (PFOS) is a widely spread environmental contaminant. It accumulates in the brain and has potential neurotoxic effects. The exposure to PFOS has been associated with higher impulsivity and increased ADHD prevalence. We investigated the effects of developmental exposure to PFOS in zebrafish larvae, focusing on the modulation of activity by the dopaminergic system. We exposed zebrafish embryos to 0.1 or 1 mg/L PFOS (0.186 or 1.858 µM, respectively) and assessed swimming activity at 6 dpf. We analyzed the structure of spontaneous activity, the hyperactivity and the habituation during a brief dark period (visual motor response), and the vibrational startle response. The findings in zebrafish larvae were compared with historical data from 3 months old male mice exposed to 0.3 or 3 mg/kg/day PFOS throughout gestation. Finally, we investigated the effects of dexamfetamine on the alterations in spontaneous activity and startle response in zebrafish larvae. We found that zebrafish larvae exposed to 0.1 mg/L PFOS habituate faster than controls during a dark pulse, while the larvae exposed to 1 mg/L PFOS display a disorganized pattern of spontaneous activity and persistent hyperactivity. Similarly, mice exposed to 0.3 mg/kg/day PFOS habituated faster than controls to a new environment, while mice exposed to 3 mg/kg/day PFOS displayed more intense and disorganized spontaneous activity. Dexamfetamine partly corrected the hyperactive phenotype in zebrafish larvae. In conclusion, developmental exposure to PFOS in zebrafish induces spontaneous hyperactivity mediated by a dopaminergic deficit, which can be partially reversed by dexamfetamine in zebrafish larvae.

Inhibiting effects of rhynchophylline on methamphetamine-dependent zebrafish are related with the expression of tyrosine hydroxylase (TH).

PubMed

Zhu, Chen; Liu, Wei; Luo, Chaohua; Liu, Yi; Li, Chan; Fang, Miao; Lin, Yingbo; Ou, Jinying; Chen, Minting; Zhu, Daoqi; Yung, Ken Kin-Lam; Mo, Zhixian

2017-03-01

In this study, to study the effect of rhynchophylline on TH in midbrain of methamphetamine-induced conditioned place preference (CPP) adult zebrafish, place preference adult zebrafish models were established by methamphetamine (40μg/g) and the expression of TH was observed by immunohistochemistry technique and Western blot. Ketamine (150μg/g), high dose of rhynchophylline (100μg/g) group can significantly reduce the place preference; immunohistochemistry results showed that the number of TH-positive neurons in midbrain was increased in the methamphetamine model group, whereas less TH-positive neurons were found in the ketamine group and high dosage rhynchophylline group. Western blot results showed that the expression of TH protein was significantly increased in the model group, whereas less expression was found in the ketamine group, high dosage rhynchophylline group. Our data pointed out that TH plays an important role in the formation of methamphetamine-induced place preference in adult zebrafish. Rhynchophylline reversed the expression of TH in the midbrain demonstrates the potential effect of mediates methamphetamine induced rewarding effect. Copyright © 2017 Elsevier B.V. All rights reserved.

Neurogenesis in the embryonic and adult brain: same regulators, different roles

PubMed Central

Urbán, Noelia; Guillemot, François

2014-01-01

Neurogenesis persists in adult mammals in specific brain areas, known as neurogenic niches. Adult neurogenesis is highly dynamic and is modulated by multiple physiological stimuli and pathological states. There is a strong interest in understanding how this process is regulated, particularly since active neuronal production has been demonstrated in both the hippocampus and the subventricular zone (SVZ) of adult humans. The molecular mechanisms that control neurogenesis have been extensively studied during embryonic development. Therefore, we have a broad knowledge of the intrinsic factors and extracellular signaling pathways driving proliferation and differentiation of embryonic neural precursors. Many of these factors also play important roles during adult neurogenesis, but essential differences exist in the biological responses of neural precursors in the embryonic and adult contexts. Because adult neural stem cells (NSCs) are normally found in a quiescent state, regulatory pathways can affect adult neurogenesis in ways that have no clear counterpart during embryogenesis. BMP signaling, for instance, regulates NSC behavior both during embryonic and adult neurogenesis. However, this pathway maintains stem cell proliferation in the embryo, while it promotes quiescence to prevent stem cell exhaustion in the adult brain. In this review, we will compare and contrast the functions of transcription factors (TFs) and other regulatory molecules in the embryonic brain and in adult neurogenic regions of the adult brain in the mouse, with a special focus on the hippocampal niche and on the regulation of the balance between quiescence and activation of adult NSCs in this region. PMID:25505873

Optogenetics: a new enlightenment age for zebrafish neurobiology.

PubMed

Del Bene, Filippo; Wyart, Claire

2012-03-01

Zebrafish became a model of choice for neurobiology because of the transparency of its brain and because of its amenability to genetic manipulation. In particular, at early stages of development the intact larva is an ideal system to apply optical techniques for deep imaging in the nervous system, as well as genetically encoded tools for targeting subsets of neurons and monitoring and manipulating their activity. For these applications,new genetically encoded optical tools, fluorescent sensors, and light-gated channels have been generated,creating the field of "optogenetics." It is now possible to monitor and control neuronal activity with minimal perturbation and unprecedented spatio-temporal resolution.We describe here the main achievements that have occurred in the last decade in imaging and manipulating neuronal activity in intact zebrafish larvae. We provide also examples of functional dissection of neuronal circuits achieved with the applications of these techniques in the visual and locomotor systems.

Monitoring Toxic Ionic Liquids in Zebrafish ( Danio rerio) with Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI-MSI)

NASA Astrophysics Data System (ADS)

Perez, Consuelo J.; Tata, Alessandra; de Campos, Michel L.; Peng, Chun; Ifa, Demian R.

2017-06-01

Ambient mass spectrometry imaging has become an increasingly powerful technique for the direct analysis of biological tissues in the open environment with minimal sample preparation and fast analysis times. In this study, we introduce desorption electrospray ionization mass spectrometry imaging (DESI-MSI) as a novel, rapid, and sensitive approach to localize the accumulation of a mildly toxic ionic liquid (IL), AMMOENG 130 in zebrafish ( Danio rerio). The work demonstrates that DESI-MSI has the potential to rapidly monitor the accumulation of IL pollutants in aquatic organisms. AMMOENG 130 is a quaternary ammonium-based IL reported to be broadly used as a surfactant in commercialized detergents. It is known to exhibit acute toxicity to zebrafish causing extensive damage to gill secondary lamellae and increasing membrane permeability. Zebrafish were exposed to the IL in a static 96-h exposure study in concentrations near the LC50 of 1.25, 2.5, and 5.0 mg/L. DESI-MS analysis of zebrafish gills demonstrated the appearance of a dealkylated AMMOENG 130 metabolite in the lowest concentration of exposure identified by a high resolution hybrid LTQ-Orbitrap mass spectrometer as the trimethylstearylammonium ion, [C21H46N]+. With DESI-MSI, the accumulation of AMMOENG 130 and its dealkylated metabolite in zebrafish tissue was found in the nervous and respiratory systems. AMMOENG 130 and the metabolite were capable of penetrating the blood brain barrier of the fish with significant accumulation in the brain. Hence, we report for the first time the simultaneous characterization, distribution, and metabolism of a toxic IL in whole body zebrafish analyzed by DESI-MSI. This ambient mass spectrometry imaging technique shows great promise for the direct analysis of biological tissues to qualitatively monitor foreign, toxic, and persistent compounds in aquatic organisms from the environment. [Figure not available: see fulltext.

Brain Volume Differences Associated With Hearing Impairment in Adults

PubMed Central

Vriend, Chris; Heslenfeld, Dirk J.; Versfeld, Niek J.; Kramer, Sophia E.

2018-01-01

Speech comprehension depends on the successful operation of a network of brain regions. Processing of degraded speech is associated with different patterns of brain activity in comparison with that of high-quality speech. In this exploratory study, we studied whether processing degraded auditory input in daily life because of hearing impairment is associated with differences in brain volume. We compared T1-weighted structural magnetic resonance images of 17 hearing-impaired (HI) adults with those of 17 normal-hearing (NH) controls using a voxel-based morphometry analysis. HI adults were individually matched with NH adults based on age and educational level. Gray and white matter brain volumes were compared between the groups by region-of-interest analyses in structures associated with speech processing, and by whole-brain analyses. The results suggest increased gray matter volume in the right angular gyrus and decreased white matter volume in the left fusiform gyrus in HI listeners as compared with NH ones. In the HI group, there was a significant correlation between hearing acuity and cluster volume of the gray matter cluster in the right angular gyrus. This correlation supports the link between partial hearing loss and altered brain volume. The alterations in volume may reflect the operation of compensatory mechanisms that are related to decoding meaning from degraded auditory input. PMID:29557274

Cell Migration During Heart Regeneration in Zebrafish

PubMed Central

Tahara, Naoyuki; Brush, Michael; Kawakami, Yasuhiko

2018-01-01

Zebrafish possess the remarkable ability to regenerate injured hearts as adults, which contrasts the very limited ability in mammals. Although very limited, mammalian hearts do in fact have measurable levels of cardiomyocyte regeneration. Therefore, elucidating mechanisms of zebrafish heart regeneration would provide information of naturally occurring regeneration to potentially apply to mammalian studies, in addition to addressing this biologically interesting phenomenon in itself. Studies over the past 13 years have identified processes and mechanisms of heart regeneration in zebrafish. After heart injury, preexisting cardiomyocytes dedifferentiate, enter the cell cycle, and repair the injured myocardium. This process requires interaction with epicardial cells, endocardial cells, and vascular endothelial cells. Epicardial cells envelope the heart, while endocardial cells make up the inner lining of the heart. They provide paracrine signals to cardiomyocytes to regenerate the injured myocardium, which is vascularized during heart regeneration. In addition, accumulating results suggest that local migration of these major cardiac cell types have roles in heart regeneration. In this review, we summarize the characteristics of various heart injury methods used in the research community and regeneration of the major cardiac cell types. Then, we discuss local migration of these cardiac cell types and immune cells during heart regeneration. PMID:27085002

Magnetite-Based Magnetoreceptor Cells in the Olfactory Organ of Rainbow Trout and Zebrafish

NASA Astrophysics Data System (ADS)

Kirschvink, J. L.; Cadiou, H.; Dixson, A. D.; Eder, S.; Kobayashi, A.; McNaughton, P. A.; Muhamad, A. N.; Raub, T. D.; Walker, M. M.; Winklhofer, M.; Yuen, B. B.

2011-12-01

Many vertebrate and invertebrate animals have a geomagnetic sensory system, but the biophysics and anatomy of how magnetic stimuli are transduced to the nervous system is a challenging problem. Previous work in our laboratories identified single-domain magnetite chains in olfactory epithelium in cells proximal to the ros V nerve, which, in rainbow trout, responds to magnetic fields. Our objectives are to characterize these magnetite-containing cells and determine whether they form part of the mechanism of magnetic field transduction in teleost fishes, as a model for other Vertebrates. Using a combination of reflection mode confocal microscopy and a Prussian Blue technique modified to stain specifically for magnetite, our Auckland group estimated that both juvenile rainbow trout (ca. 7 cm total length) olfactory rosettes have ~200 magnetite-containing cells. The magnetite present in two types of cells within the olfactory epithelium appears to be arranged in intracellular chains. All of our groups (Munich, Auckland, Cambridge and Caltech) have obtained different types of structural evidence that magnetite chains closely associate with the plasma membrane in the cells, even in disaggregated tissues. In addition, our Cambridge group used Ca2+ imaging to demonstrate a clear response by individual magnetite-containing cells to a step change in the intensity of the external magnetic field and a slow change in Ca2+ activity when the external magnetic field was cancelled. In the teleost, zebrafish (Danio rerio), a small (~4 cm adult length in captivity) genetic and developmental biology model organism, our Caltech group detected ferromagnetic material throughout the body, but concentrated in the rostral trunk, using NRM and IRM scans of whole adults. Our analysis suggests greater than one million, 80-100 nm crystals, with Lowrie-Fuller curves strongly consistent with single-domain magnetite in 100-100,000 magnetocytes. Ferromagentic resonance (FMR) spectra show crystals

Extraction Protocols for Individual Zebrafish's Ventricle Myosin and Skeletal Muscle Actin for In vitro Motility Assays

PubMed Central

Scheid, Lisa-Mareike; Weber, Cornelia; Bopp, Nasrin; Mosqueira, Matias; Fink, Rainer H. A.

2017-01-01

The in vitro motility assay (IVMA) is a technique that enables the measurement of the interaction between actin and myosin providing a relatively simple model to understand the mechanical muscle function. For actin-myosin IVMA, myosin is immobilized in a measurement chamber, where it converts chemical energy provided by ATP hydrolysis into mechanical energy. The result is the movement of fluorescently labeled actin filaments that can be recorded microscopically and analyzed quantitatively. Resulting sliding speeds and patterns help to characterize the underlying actin-myosin interaction that can be affected by different factors such as mutations or active compounds. Additionally, modulatory actions of the regulatory proteins tropomyosin and troponin in the presence of calcium on actin-myosin interaction can be studied with the IVMA. Zebrafish is considered a suitable model organism for cardiovascular and skeletal muscle research. In this context, straightforward protocols for the isolation and use of zebrafish muscle proteins in the IVMA would provide a useful tool in molecular studies. Currently, there are no protocols available for the mentioned purpose. Therefore, we developed fast and easy protocols for characterization of zebrafish proteins in the IVMA. Our protocols enable the interested researcher to (i) isolate actin from zebrafish skeletal muscle and (ii) extract functionally intact myosin from cardiac and skeletal muscle of individual adult zebrafish. Zebrafish tail muscle actin is isolated after acetone powder preparation, polymerized, and labeled with Rhodamine-Phalloidin. Myosin from ventricles of adult zebrafish is extracted directly into IVMA flow-cells. The same extraction protocol is applicable for comparably small tissue pieces as from zebrafish tail, mouse and frog muscle. After addition of the fluorescently labeled F-actin from zebrafish—or other origin—and ATP, sliding movement can be visualized using a fluorescence microscope and an

Model of voluntary ethanol intake in zebrafish: Effect on behavior and hypothalamic orexigenic peptides

PubMed Central

Sterling, M.E.; Karatayev, O.; Chang, G.-Q.; Algava, D.B.; Leibowitz, S.F

2014-01-01

Recent studies in zebrafish have shown that exposure to ethanol in tank water affects various behaviors, including locomotion, anxiety and aggression, and produces changes in brain neurotransmitters, such as serotonin and dopamine. Building on these investigations, the present study had two goals: first, to develop a method for inducing voluntary ethanol intake in individual zebrafish, which can be used as a model in future studies to examine how this behavior is affected by various manipulations, and second, to characterize the effects of this ethanol intake on different behaviors and the expression of hypothalamic orexigenic peptides, galanin (GAL) and orexin (OX), which are known in rodents to stimulate consumption of ethanol and alter behaviors associated with alcohol abuse. Thus, we first developed a new model of voluntary intake of ethanol in fish by presenting this ethanol mixed with gelatin, which they readily consume. Using this model, we found that individual zebrafish can be trained in a short period of time to consume stable levels of 10% or 20% ethanol (v/v) mixed with gelatin and that their intake of this ethanol-gelatin mixture leads to pharmacologically-relevant blood ethanol concentrations which are strongly, positively correlated with the amount ingested. Intake of this ethanol-gelatin mixture increased locomotion, reduced anxiety, and stimulated aggressive behavior, while increasing expression of GAL and OX in specific hypothalamic areas. These findings, confirming results in rats, provide a method in zebrafish for investigating with forward genetics and pharmacological techniques the role of different brain mechanisms in controlling ethanol intake. PMID:25257106

Development of mandibular, hyoid and hypobranchial muscles in the zebrafish: homologies and evolution of these muscles within bony fishes and tetrapods

PubMed Central

Diogo, Rui; Hinits, Yaniv; Hughes, Simon M

2008-01-01

Background During vertebrate head evolution, muscle changes accompanied radical modification of the skeleton. Recent studies have suggested that muscles and their innervation evolve less rapidly than cartilage. The freshwater teleostean zebrafish (Danio rerio) is the most studied actinopterygian model organism, and is sometimes taken to represent osteichthyans as a whole, which include bony fishes and tetrapods. Most work concerning zebrafish cranial muscles has focused on larval stages. We set out to describe the later development of zebrafish head muscles and compare muscle homologies across the Osteichthyes. Results We describe one new muscle and show that the number of mandibular, hyoid and hypobranchial muscles found in four day-old zebrafish larvae is similar to that found in the adult. However, the overall configuration and/or the number of divisions of these muscles change during development. For example, the undivided adductor mandibulae of early larvae gives rise to the adductor mandibulae sections A0, A1-OST, A2 and Aω, and the protractor hyoideus becomes divided into dorsal and ventral portions in adults. There is not always a correspondence between the ontogeny of these muscles in the zebrafish and their evolution within the Osteichthyes. All of the 13 mandibular, hyoid and hypobranchial muscles present in the adult zebrafish are found in at least some other living teleosts, and all except the protractor hyoideus are found in at least some extant non-teleost actinopterygians. Of these muscles, about a quarter (intermandibularis anterior, adductor mandibulae, sternohyoideus) are found in at least some living tetrapods, and a further quarter (levator arcus palatini, adductor arcus palatini, adductor operculi) in at least some extant sarcopterygian fish. Conclusion Although the zebrafish occupies a rather derived phylogenetic position within actinopterygians and even within teleosts, with respect to the mandibular, hyoid and hypobranchial muscles it

Adult Brain and Spine Tumor Research and Development

Cancer.gov

Chief, Dr. Mark Gilbert and Senior Investigator, Dr. Terri Armstrong, of the NCI Center for Cancer Research, Neuro-Oncology Branch, will be joined by moderator and Chief Executive Officer, David Arons of the National Brain Tumor Society led a discussion on adult brain and spine tumor research and treatment.

Predicting adult fish acute lethality with the zebrafish embryo: relevance of test duration, endpoints, compound properties, and exposure concentration analysis.

PubMed

Knöbel, Melanie; Busser, Frans J M; Rico-Rico, Angeles; Kramer, Nynke I; Hermens, Joop L M; Hafner, Christoph; Tanneberger, Katrin; Schirmer, Kristin; Scholz, Stefan

2012-09-04

The zebrafish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, which is required by various regulations for environmental risk assessment of chemicals. We investigated the reliability of the embryo test by probing organic industrial chemicals with a wide range of physicochemical properties, toxicities, and modes of toxic action. Moreover, the relevance of using measured versus nominal (intended) exposure concentrations, inclusion of sublethal endpoints, and different exposure durations for the comparability with reported fish acute toxicity was explored. Our results confirm a very strong correlation of zebrafish embryo to fish acute toxicity. When toxicity values were calculated based on measured exposure concentrations, the slope of the type II regression line was 1 and nearly passed through the origin (1 to 1 correlation). Measured concentrations also explained several apparent outliers. Neither prolonged exposure (up to 120 h) nor consideration of sublethal effects led to a reduced number of outliers. Yet, two types of compounds were less lethal to embryos than to adult fish: a neurotoxic compound acting via sodium channels (permethrin) and a compound requiring metabolic activation (allyl alcohol).

Characterization of a cis-acting element involved in cell-specific expression of the zebrafish brain aromatase gene.

PubMed

Le Page, Yann; Menuet, Arnaud; Kah, Olivier; Pakdel, Farzad

2008-10-01

The cytochrome P450 Aromatase is the key enzyme catalyzing the conversion of androgens into estrogens. In zebrafish, the brain aromatase is encoded by cyp19b. Expression of cyp19b is restricted to radial glial cells bordering forebrain ventricles and is strongly stimulated by estrogens during development. At the promoter level, we have previously shown that an estrogen responsive element (ERE) is required for induction by estrogens. Here, we investigated the role of ERE flanking regions in the control of cell-specific expression. First, we show that a 20 bp length motif, named G x RE (glial x responsive element), acts in synergy with the ERE to mediate the estrogenic induction specifically in glial cells. Second, we demonstrate that, in vitro, this sequence binds factors exclusively present in glial or neuro-glial cells and is able to confer a glial specificity to an artificial estrogen-dependent gene. Taken together, these results contribute to the understanding of the molecular mechanisms allowing cyp19b regulation by estrogens and allowed to identify a promoter sequence involved in the strong estrogen inducibility of cyp19b which is specific for glial cells. The exceptional aromatase activity measured in the brain of teleost fish could rely on such mechanisms.

Matrix metalloproteinase-9 plays a role in protecting zebrafish from lethal infection with Listeria monocytogenes by enhancing macrophage migration.

PubMed

Shan, Ying; Zhang, Yikai; Zhuo, Xunhui; Li, Xiaoliang; Peng, Jinrong; Fang, Weihuan

2016-07-01

Zebrafish could serve as an alternative animal model for pathogenic bacteria in multiple infectious routes. Our previous study showed that immersion infection in zebrafish with Listeria monocytogenes did not cause lethality but induced transient expression of several immune response genes. We used an Affymetrix gene chip to examine the expression profiles of genes of zebrafish immersion-infected with L. monocytogenes. A total of 239 genes were up-regulated and 56 genes down-regulated compared with uninfected fish. Highest expression (>20-fold) was seen with the mmp-9 gene encoding the matrix metalloproteinase-9 (Mmp-9) known to degrade the extracellular matrix proteins. By morpholino knockdown of mmp-9, we found that the morphants showed rapid death with much higher bacterial load after intravenous or intraventricular (brain ventricle) infection with L. monocytogenes. Macrophages in mmp-9-knockdown morphants had significant defect in migrating to the brain cavity upon intraventricular infection. Decreased migration of murine macrophages with knockdown of mmp-9 and cd44 was also seen in transwell inserts with 8-μm pore polycarbonate membrane, as compared with the scrambled RNA. These findings suggest that Mmp-9 is a protective molecule against infection by L. monocytogenes by engaging in migration of zebrafish macrophages to the site of infection via a non-proteolytic role. Further work is required on the molecular mechanisms governing Mmp-9-driven macrophage migration in zebrafish. Copyright © 2016 Elsevier Ltd. All rights reserved.

Small interfering RNA mediated knockdown of irisin suppresses food intake and modulates appetite regulatory peptides in zebrafish.

PubMed

Sundarrajan, Lakshminarasimhan; Unniappan, Suraj

2017-10-01

Irisin is a myokine encoded in fibronectin type III domain containing 5 (FNDC5). FNDC5 forms an integral part of the muscle post-exercise, and causes an increase in energy expenditure in mammals. Irisin is abundantly expressed in cardiac and skeletal muscles and is secreted upon activation of peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1 alpha). Irisin regulates feeding behaviour and cardiovascular function in mammals. More recently, irisin has gained importance as a potential biomarker for myocardial infarction due to its abundance in cardiac muscle. The goal of this research was to determine whether irisin influences feeding, and regulates appetite regulatory peptides in zebrafish. Intraperitoneal injection of irisin [0.1, 1, 10 and 100ng/g body weight (BW)] did not affect feeding, but its knockdown using siRNA (10ng/g BW) caused a significant reduction in food intake. Knockdown of irisin reduced ghrelin and orexin-A mRNA expression, and increased cocaine and amphetamine regulated transcript mRNA expression in zebrafish brain and gut. siRNA mediated knockdown of irisin also downregulated brain derived neurotrophic factor mRNA in zebrafish. The role of endogenous irisin on food intake is likely mediated by its actions on other metabolic peptides. Collectively, these results indicate that unaltered endogenous irisin is required to maintain food intake in zebrafish. Copyright © 2017 Elsevier Inc. All rights reserved.

Zebrafish as tools for drug discovery.

PubMed

MacRae, Calum A; Peterson, Randall T

2015-10-01

The zebrafish has become a prominent vertebrate model for disease and has already contributed to several examples of successful phenotype-based drug discovery. For the zebrafish to become useful in drug development more broadly, key hurdles must be overcome, including a more comprehensive elucidation of the similarities and differences between human and zebrafish biology. Recent studies have begun to establish the capabilities and limitations of zebrafish for disease modelling, drug screening, target identification, pharmacology, and toxicology. As our understanding increases and as the technologies for manipulating zebrafish improve, it is hoped that the zebrafish will have a key role in accelerating the emergence of precision medicine.

From the Cover: Zebrafish Larvae Are Insensitive to Stimulation by Cocaine: Importance of Exposure Route and Toxicokinetics.

PubMed

Kirla, Krishna Tulasi; Groh, Ksenia J; Steuer, Andrea E; Poetzsch, Michael; Banote, Rakesh Kumar; Stadnicka-Michalak, Julita; Eggen, Rik I L; Schirmer, Kristin; Kraemer, Thomas

2016-11-01

Zebrafish (Danio rerio) larvae have been suggested as vertebrate model to complement or even replace mammals for rapidly assessing behavioral effects of psychoactive drugs. Yet, divergent responses have been reported in mammals and fish despite the conservation of many drug targets. Cocaine, eg, acts as stimulant in mammals but no such response has been documented for zebrafish larvae. We hypothesized that differences in exposure routes (inhalation or injection in mammals vs waterborne in fish) may be a reason for differences in behavioral responses. We characterized cocaine toxicokinetics by liquid chromatography-mass spectrometry and found its rapid uptake into larvae. We used Matrix-assisted laser desorption ionization-mass spectrometry imaging for the first time to characterize internal distribution of cocaine in zebrafish larvae. Surprisingly, eyes accumulated the highest amount of cocaine and retained most of it even after 48 h depuration. We attribute this to trapping by pigment melanin, a thus far little explored mechanism that may also be relevant for other basic drugs. Cocaine also reached the brain but with levels similar to those in trunk indicating simple passive diffusion as means of distribution which was supported by toxicokinetic models. Although brain levels covered those known to cause hyperactivity in mammals, only hypoactivity (decreased locomotion) was recorded in zebrafish larvae. Our results therefore point to cocaine's anesthetic properties as the dominant mechanism of interaction in the fish: upon entry through the fish skin and gills, it first acts on peripheral nerves rapidly overriding any potential stimulatory response in the brain. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Analyzing notochord segmentation and intervertebral disc formation using the twhh:gfp transgenic zebrafish model.

PubMed

Haga, Yutaka; Dominique, Vincent J; Du, Shao Jun

2009-10-01

To characterize the process of vertebral segmentation and disc formation in living animals, we analyzed tiggy-winkle hedgehog (twhh):green fluorescent protein (gfp) and sonic hedgehog (shh):gfp transgenic zebrafish models that display notochord-specific GFP expression. We found that they showed distinct patterns of expression in the intervertebral discs of late stage fish larvae and adult zebrafish. A segmented pattern of GFP expression was detected in the intervertebral disc of twhh:gfp transgenic fish. In contrast, little GFP expression was found in the intervertebral disc of shh:gfp transgenic fish. Treating twhh:gfp transgenic zebrafish larvae with exogenous retinoic acid (RA), a teratogenic factor on normal development, resulted in disruption of notochord segmentation and formation of oversized vertebrae. Histological analysis revealed that the oversized vertebrae are likely due to vertebral fusion. These studies demonstrate that the twhh:gfp transgenic zebrafish is a useful model for studying vertebral segmentation and disc formation, and moreover, that RA signaling may play a role in this process.

Characterization of Light Lesion Paradigms and Optical Coherence Tomography as Tools to Study Adult Retina Regeneration in Zebrafish

PubMed Central

Weber, Anke; Hochmann, Sarah; Cimalla, Peter; Gärtner, Maria; Kuscha, Veronika; Hans, Stefan; Geffarth, Michaela; Kaslin, Jan; Koch, Edmund; Brand, Michael

2013-01-01

Light-induced lesions are a powerful tool to study the amazing ability of photoreceptors to regenerate in the adult zebrafish retina. However, the specificity of the lesion towards photoreceptors or regional differences within the retina are still incompletely understood. We therefore characterized the process of degeneration and regeneration in an established paradigm, using intense white light from a fluorescence lamp on swimming fish (diffuse light lesion). We also designed a new light lesion paradigm where light is focused through a microscope onto the retina of an immobilized fish (focused light lesion). Focused light lesion has the advantage of creating a locally restricted area of damage, with the additional benefit of an untreated control eye in the same animal. In both paradigms, cell death is observed as an immediate early response, and proliferation is initiated around 2 days post lesion (dpl), peaking at 3 dpl. We furthermore find that two photoreceptor subtypes (UV and blue sensitive cones) are more susceptible towards intense white light than red/green double cones and rods. We also observed specific differences within light lesioned areas with respect to the process of photoreceptor degeneration: UV cone debris is removed later than any other type of photoreceptor in light lesions. Unspecific damage to retinal neurons occurs at the center of a focused light lesion territory, but not in the diffuse light lesion areas. We simulated the fish eye optical properties using software simulation, and show that the optical properties may explain the light lesion patterns that we observe. Furthermore, as a new tool to study retinal degeneration and regeneration in individual fish in vivo, we use spectral domain optical coherence tomography. Collectively, the light lesion and imaging assays described here represent powerful tools for studying degeneration and regeneration processes in the adult zebrafish retina. PMID:24303018

Regulation of zebrafish sleep and arousal states: current and prospective approaches

PubMed Central

Chiu, Cindy N.; Prober, David A.

2013-01-01

Every day, we shift among various states of sleep and arousal to meet the many demands of our bodies and environment. A central puzzle in neurobiology is how the brain controls these behavioral states, which are essential to an animal's well-being and survival. Mammalian models have predominated sleep and arousal research, although in the past decade, invertebrate models have made significant contributions to our understanding of the genetic underpinnings of behavioral states. More recently, the zebrafish has emerged as a promising model system for sleep and arousal research. Here we review experimental evidence that the zebrafish, a diurnal vertebrate, exhibits fundamental behavioral and neurochemical characteristics of mammalian sleep and arousal. We also propose how specific advantages of the zebrafish can be harnessed to advance the field. These include tractable genetics to identify and manipulate molecular and cellular regulators of behavioral states, optical transparency to facilitate in vivo observation of neural structure and function, and amenability to high-throughput drug screens to discover novel therapies for neurological disorders. PMID:23576957

Experience-Dependent Neural Plasticity in the Adult Damaged Brain

ERIC Educational Resources Information Center

Kerr, Abigail L.; Cheng, Shao-Ying; Jones, Theresa A.

2011-01-01

Behavioral experience is at work modifying the structure and function of the brain throughout the lifespan, but it has a particularly dramatic influence after brain injury. This review summarizes recent findings on the role of experience in reorganizing the adult damaged brain, with a focus on findings from rodent stroke models of chronic upper…

The synthetic substance hypoxanthine 3-N-oxide elicits alarm reactions in zebrafish (Danio rerio)

PubMed Central

Parra, Kevin V.; Adrian, James C.; Gerlai, Robert

2009-01-01

Zebrafish, one of the preferred study species of geneticists, is gaining increasing popularity in behavioral neuroscience. This small and prolific species may be an excellent tool with which the biological mechanisms of vertebrate brain function and behavior are investigated. Zebrafish has been proposed as a model organism in the analysis of fear responses and human anxiety disorders. Species-specific cues signaling the presence of predators have been successfully utilized in such research. Zebrafish has been shown to respond to its natural alarm substance with species-typical fear reactions. However, the extraction of this alarm substance and ascertaining its consistent dosing has been problematic. A synthetic substance with a known chemical identity and molecular weight would allow precise dosing and experimental control. Previously, the chemical component, hypoxanthine 3-N-oxide, common to several fish alarm substances has been identified and has been shown to elicit alarm reactions in fish species belonging to the Osteriophysan superorder. In the current study we investigate the effect of hypoxanthine 3-N-oxide by exposing zebrafish to three different concentrations of this synthetic substance. Our results show that the substance efficaciously induces species-typical fear reactions increasing the number of erratic movement episodes and jumps in zebrafish. We discuss the translational relevance of our findings and conclude that hypoxanthine 3-N-oxide will have utility to elicit fear responses in the laboratory in a precisely controlled manner in zebrafish. PMID:19583985

Chlorella diet alters mitochondrial cardiolipin contents differentially in organs of Danio rerio analyzed by a lipidomics approach

PubMed Central

Chao, Yu-Jen; Wu, Wen-Hsin; Balazova, Maria; Wu, Ting-Yuan; Lin, Jamie; Liu, Yi-Wen

2018-01-01

The zebrafish (Danio rerio) is an important and widely used vertebrate model organism for the study of human diseases which include disorders caused by dysfunctional mitochondria. Mitochondria play an essential role in both energy metabolism and apoptosis, which are mediated through a mitochondrial phospholipid cardiolipin (CL). In order to examine the cardiolipin profile in the zebrafish model, we developed a CL analysis platform by using liquid chromatography-mass spectrometry (LC-MS). Meanwhile, we tested whether chlorella diet would alter the CL profile in the larval fish, and in various organs of the adult fish. The results showed that chlorella diet increased the chain length of CL in larval fish. In the adult zebrafish, the distribution patterns of CL species were similar between the adult brain and eye tissues, and between the heart and muscles. Interestingly, monolyso-cardiolipin (MLCL) was not detected in brain and eyes but found in other examined tissues, indicating a different remodeling mechanism to maintain the CL integrity. While the adult zebrafish were fed with chlorella for four weeks, the CL distribution showed an increase of the species of saturated acyl chains in the brain and eyes, but a decrease in the other organs. Moreover, chlorella diet led to a decrease of MLCL percentage in organs except the non-MLCL-containing brain and eyes. The CL analysis in the zebrafish provides an important tool for studying the mechanism of mitochondria diseases, and may also be useful for testing medical regimens targeting against the Barth Syndrome. PMID:29494608