RESUMEN
Cell adhesion molecules are membrane-bound proteins predominantly expressed in the central nervous system along principal axonal pathways with key roles in nervous system development, neural cell differentiation and migration, axonal growth and guidance, myelination, and synapse formation. Here, we describe ten affected individuals with bi-allelic variants in the neuronal cell adhesion molecule NRCAM that lead to a neurodevelopmental syndrome of varying severity; the individuals are from eight families. This syndrome is characterized by developmental delay/intellectual disability, hypotonia, peripheral neuropathy, and/or spasticity. Computational analyses of NRCAM variants, many of which cluster in the third fibronectin type III (Fn-III) domain, strongly suggest a deleterious effect on NRCAM structure and function, including possible disruption of its interactions with other proteins. These findings are corroborated by previous in vitro studies of murine Nrcam-deficient cells, revealing abnormal neurite outgrowth, synaptogenesis, and formation of nodes of Ranvier on myelinated axons. Our studies on zebrafish nrcamaΔ mutants lacking the third Fn-III domain revealed that mutant larvae displayed significantly altered swimming behavior compared to wild-type larvae (p < 0.03). Moreover, nrcamaΔ mutants displayed a trend toward increased amounts of α-tubulin fibers in the dorsal telencephalon, demonstrating an alteration in white matter tracts and projections. Taken together, our study provides evidence that NRCAM disruption causes a variable form of a neurodevelopmental disorder and broadens the knowledge on the growing role of the cell adhesion molecule family in the nervous system.
Asunto(s)
Trastornos del Neurodesarrollo , Enfermedades del Sistema Nervioso Periférico , Animales , Axones/metabolismo , Adhesión Celular/genética , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular Neuronal , Humanos , Ratones , Hipotonía Muscular/genética , Hipotonía Muscular/metabolismo , Espasticidad Muscular/metabolismo , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
LRRK2 gain-of-function is considered a major cause of Parkinson's disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.
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Monoaminas Biogénicas/metabolismo , Encéfalo/metabolismo , Eliminación de Gen , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Ansiedad/genética , Encéfalo/embriología , Encéfalo/enzimología , Sistemas CRISPR-Cas , Larva/metabolismo , Monoaminooxidasa/metabolismo , Olfato/genética , Natación , Pez Cebra/embriologíaRESUMEN
The vertebrate inner ear employs sensory hair cells and neurons to mediate hearing and balance. In mammals, damaged hair cells and neurons are not regenerated. In contrast, hair cells in the inner ear of zebrafish are produced throughout life and regenerate after trauma. However, it is unknown whether new sensory neurons are also formed in the adult zebrafish statoacoustic ganglion (SAG), the sensory ganglion connecting the inner ear to the brain. Using transgenic lines and marker analysis, we identify distinct cell populations and anatomical landmarks in the juvenile and adult SAG. In particular, we analyze a Neurod/Nestin-positive progenitor pool that produces large amounts of new neurons at juvenile stages, which transitions to a quiescent state in the adult SAG. Moreover, BrdU pulse chase experiments reveal the existence of a proliferative but otherwise marker-negative cell population that replenishes the Neurod/Nestin-positive progenitor pool at adult stages. Taken together, our study represents the first comprehensive characterization of the adult zebrafish SAG showing that zebrafish, in sharp contrast to mammals, display continued neurogenesis in the SAG well beyond embryonic and larval stages.
Asunto(s)
Células Madre Adultas/fisiología , Oído Interno/fisiología , Ganglios Sensoriales/citología , Células Ciliadas Auditivas/fisiología , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Pez Cebra , Células Madre Adultas/citología , Envejecimiento/fisiología , Animales , Animales Modificados Genéticamente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/genética , Oído Interno/citología , Embrión no Mamífero , Ganglios Sensoriales/fisiología , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/metabolismo , Larva , Proteínas del Tejido Nervioso/metabolismo , Nestina/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/fisiología , Nicho de Células Madre/fisiología , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismoRESUMEN
Aquatic eco-neurotoxicology is an emerging field that requires new analytical systems to study the effects of pollutants on animal behaviors. This is especially true if we are to gain insights into one of the least studied aspects: the potential perturbations that neurotoxicants can have on cognitive behaviors. The paucity of experimental data is partly caused by a lack of low-cost technologies for the analysis of higher-level neurological functions (e.g., associative learning) in small aquatic organisms. Here, we present a proof-of-concept prototype that utilizes a new real-time animal tracking software for on-the-fly video analysis and closed-loop, external hardware communications to deliver stimuli based on specific behaviors in aquatic organisms, spanning three animal phyla: chordates (fish, frog), platyhelminthes (flatworm), and arthropods (crustacean). The system's open-source software features an intuitive graphical user interface and advanced adaptive threshold-based image segmentation for precise animal detection. We demonstrate the precision of animal tracking across multiple aquatic species with varying modes of locomotion. The presented technology interfaces easily with low-cost and open-source hardware such as the Arduino microcontroller family for closed-loop stimuli control. The new system has potential future applications in eco-neurotoxicology, where it could enable new opportunities for cognitive research in diverse small aquatic model organisms.
Asunto(s)
Artrópodos , Programas Informáticos , Animales , Conducta AnimalRESUMEN
Many targeted natural product isolation approaches rely on the use of pre-existing bioactivity information to inform the strategy used for the isolation of new bioactive compounds. Bioactivity information can be available either in the form of prior assay data or via Structure Activity Relationship (SAR) information which can indicate a potential chemotype that exhibits a desired bioactivity. The work described herein utilizes a unique method of targeted isolation using structure-based virtual screening to identify potential antibacterial compounds active against MRSA within the marine sponge order Verongiida. This is coupled with molecular networking-guided, targeted isolation to provide a novel drug discovery procedure. A total of 12 previously reported bromotyrosine-derived alkaloids were isolated from the marine sponge species Pseudoceratina durissima, and the compound, (+)-aeroplysinin-1 (1) displayed activity against the MRSA pathogen (MIC: <32 µg/mL). The compounds (1−3, 6 and 9) were assessed for their central nervous system (CNS) interaction and behavioral toxicity to zebrafish (Danio rerio) larvae, whereby several of the compounds were shown to induce significant hyperactivity. Anthelmintic activity against the parasitic nematode Haemonchus contorutus was also evaluated (2−4, 6−8).
Asunto(s)
Alcaloides , Antihelmínticos , Productos Biológicos , Poríferos , Alcaloides/química , Animales , Antibacterianos/química , Antibacterianos/farmacología , Estructura Molecular , Poríferos/química , Pez CebraRESUMEN
Due to increasing numbers of anthropogenic chemicals with unknown neurotoxic properties, there is an increasing need for a paradigm shift toward rapid and higher throughput behavioral bioassays. In this work, we demonstrate application of a purpose-built high throughput multidimensional behavioral test battery on larval stages of Danio rerio (zebrafish) at 5 days post fertilization (dpf). The automated battery comprised of the established spontaneous swimming (SS), simulated predator response (SPR), larval photomotor response (LPR) assays as well as a new thermotaxis (TX) assay. We applied the novel system to characterize environmentally relevant concentrations of emerging pharmaceutical micropollutants including anticonvulsants (gabapentin: 400 ng/L; carbamazepine: 3000 ng/L), inflammatory drugs (ibuprofen: 9800 ng/L), and antidepressants (fluoxetine: 300 ng/L; venlafaxine: 2200 ng/L). The successful integration of the thermal preference assay into a multidimensional behavioral test battery provided means to reveal ibuprofen-induced perturbations of thermal preference behaviors upon exposure during embryogenesis. Moreover, we discovered that photomotor responses in larval stages of fish are also altered by the as yet understudied anticonvulsant gabapentin. Collectively our results demonstrate the utility of high-throughput multidimensional behavioral ecotoxicity test batteries in prioritizing emerging risks associated with neuroactive drugs that can perturb neurodevelopment. Moreover, we showcase the added value of thermotaxis bioassays for preliminary screening of emerging contaminants.
Asunto(s)
Contaminantes Químicos del Agua , Pez Cebra , Animales , Gabapentina/farmacología , Ibuprofeno/farmacología , Larva , Natación , Contaminantes Químicos del Agua/análisis , Contaminantes Químicos del Agua/toxicidad , Pez Cebra/fisiologíaRESUMEN
Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgshΔex5-6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgshΔex5-6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgshΔex5-6 zebrafish is largely dependent on interleukin-1ß and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgshΔex5-6 mutant larvae in a context-dependent manner. We expect the sgshΔex5-6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.
Asunto(s)
Modelos Animales de Enfermedad , Hidrolasas/genética , Mucopolisacaridosis III , Animales , Humanos , Mucopolisacaridosis III/metabolismo , Mucopolisacaridosis III/patología , Mutación , Fenotipo , Pez CebraRESUMEN
Zebrafish can regenerate after brain injury, and the regenerative process is driven by resident stem cells. Stem cells are heterogeneous in the vertebrate brain, but the significance of having heterogeneous stem cells in regeneration is not understood. Limited availability of specific stem cells might impair the regeneration of particular cell lineages. We studied regeneration of the adult zebrafish cerebellum, which contains two major stem and progenitor cell types: ventricular zone and neuroepithelial cells. Using conditional lineage tracing we demonstrate that cerebellar regeneration depends on the availability of specific stem cells. Radial glia-like cells are thought to be the predominant stem cell type in homeostasis and after injury. However, we find that radial glia-like cells play a minor role in adult cerebellar neurogenesis and in recovery after injury. Instead, we find that neuroepithelial cells are the predominant stem cell type supporting cerebellar regeneration after injury. Zebrafish are able to regenerate many, but not all, cell types in the cerebellum, which emphasizes the need to understand the contribution of different adult neural stem and progenitor cell subtypes in the vertebrate central nervous system.
Asunto(s)
Cerebelo/fisiología , Células-Madre Neurales/citología , Células Neuroepiteliales/citología , Neuroglía/citología , Regeneración , Pez Cebra/fisiología , Animales , Conducta Animal , Linaje de la Célula , Cerebelo/patología , Homeostasis , Modelos Biológicos , Células-Madre Neurales/metabolismo , Células Neuroepiteliales/metabolismo , Neurogénesis , Neuroglía/metabolismoRESUMEN
Human amyloids and plaques uncovered post mortem are highly heterogeneous in structure and composition, yet literature concerning the heteroaggregation of amyloid proteins is extremely scarce. This knowledge deficiency is further exacerbated by the fact that peptide delivery is a major therapeutic strategy for targeting their full-length counterparts associated with the pathologies of a range of human diseases, including dementia and type 2 diabetes (T2D). Accordingly, here we examined the coaggregation of full-length human islet amyloid polypeptide (IAPP), a peptide associated with type 2 diabetes, with its primary and secondary amyloidogenic fragments 19-29 S20G and 8-20. Single-molecular aggregation dynamics was obtained by high-speed atomic force microscopy, augmented by transmission electron microscopy, X-ray diffraction, and super-resolution stimulated emission depletion microscopy. The coaggregation significantly prolonged the pause phase of fibril elongation, increasing its dwell time by 3-fold. Surprisingly, unidirectional elongation of mature fibrils, instead of protofilaments, was observed for the coaggregation, indicating a new form of tertiary protein aggregation unknown to existing theoretical models. Further in vivo zebrafish embryonic assay indicated improved survival and hatching, as well as decreased frequency and severity of developmental abnormalities for embryos treated with the heteroaggregates of IAPP with 19-29 S20G, but not with 8-20, compared to the control, indicating the therapeutic potential of 19-29 S20G against T2D.
Asunto(s)
Amiloidosis/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Agregación Patológica de Proteínas/tratamiento farmacológico , Amiloidosis/metabolismo , Animales , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/farmacología , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Agregación Patológica de Proteínas/metabolismo , Pez Cebra/metabolismoRESUMEN
The fish embryo toxicity (FET) biotest has gained popularity as one of the alternative approaches to acute fish toxicity tests in chemical hazard and risk assessment. Despite the importance and common acceptance of FET, it is still performed in multiwell plates and requires laborious and time-consuming manual manipulation of specimens and solutions. This work describes the design and validation of a microfluidic Lab-on-a-Chip technology for automation of the zebrafish embryo toxicity test common in aquatic ecotoxicology. The innovative device supports rapid loading and immobilization of large numbers of zebrafish embryos suspended in a continuous microfluidic perfusion as a means of toxicant delivery. Furthermore, we also present development of a customized mechatronic automation interface that includes a high-resolution USB microscope, LED cold light illumination, and miniaturized 3D printed pumping manifolds that were integrated to enable time-resolved in situ analysis of developing fish embryos. To investigate the applicability of the microfluidic FET (µFET) in toxicity testing, copper sulfate, phenol, ethanol, caffeine, nicotine, and dimethyl sulfoxide were tested as model chemical stressors. Results obtained on a chip-based system were compared with static protocols performed in microtiter plates. This work provides evidence that FET analysis performed under microperfusion opens a brand new alternative for inexpensive automation in aquatic ecotoxicology.
Asunto(s)
Dispositivos Laboratorio en un Chip , Microfluídica/métodos , Pruebas de Toxicidad/instrumentación , Pez Cebra/embriología , Animales , Cafeína/toxicidad , Sulfato de Cobre/toxicidad , Dimetilsulfóxido/toxicidad , Embrión no Mamífero/efectos de los fármacos , Etanol/toxicidad , Microfluídica/instrumentación , Nicotina/toxicidad , Impresión Tridimensional , Pruebas de Toxicidad/métodosRESUMEN
Teleost fishes retain populations of adult stem/progenitor cells within multiple primary sensory processing structures of the mature brain. Though it has commonly been thought that their ability to give rise to adult-born neurons is mainly associated with continuous growth throughout life, whether a relationship exists between the processing function of these structures and the addition of new neurons remains unexplored. We investigated the ultrastructural organisation and modality-specific neurogenic plasticity of niches located in chemosensory (olfactory bulb, vagal lobe) and visual processing (periventricular grey zone, torus longitudinalis) structures of the adult zebrafish (Danio rerio) brain. Transmission electron microscopy showed that the cytoarchitecture of sensory niches includes many of the same cellular morphologies described in forebrain niches. We demonstrate that cells with a radial-glial phenotype are present in chemosensory niches, while the niche of the caudal tectum contains putative neuroepithelial-like cells instead. This was supported by immunohistochemical evidence showing an absence of glial markers, including glial fibrillary acidic protein, glutamine synthetase, and S100ß in the tectum. By exposing animals to sensory assays we further illustrate that stem/progenitor cells and their neuronal progeny within sensory structures respond to modality-specific stimulation at distinct stages in the process of adult neurogenesis - chemosensory niches at the level of neuronal survival and visual niches in the size of the stem/progenitor population. Our data suggest that the adult brain has the capacity for sensory-specific modulation of adult neurogenesis and that this property may be associated with the type of stem cell present in the niche.
Asunto(s)
Células Madre Adultas/fisiología , Neurogénesis/fisiología , Prosencéfalo/fisiología , Nicho de Células Madre/fisiología , Pez Cebra/fisiología , Células Madre Adultas/ultraestructura , Animales , Bromodesoxiuridina , Recuento de Células , Supervivencia Celular/fisiología , Femenino , Inmunohistoquímica , Masculino , Microscopía Electrónica de Transmisión , Células-Madre Neurales/fisiología , Células-Madre Neurales/ultraestructura , Neuroglía/fisiología , Neuroglía/ultraestructura , Neuronas/fisiología , Neuronas/ultraestructura , Percepción Olfatoria/fisiología , Prosencéfalo/ultraestructura , Percepción del Gusto/fisiología , Percepción Visual/fisiologíaRESUMEN
Severe traumatic injury to the adult mammalian CNS leads to life-long loss of function. By contrast, several non-mammalian vertebrate species, including adult zebrafish, have a remarkable ability to regenerate injured organs, including the CNS. However, the cellular and molecular mechanisms that enable or prevent CNS regeneration are largely unknown. To study brain regeneration mechanisms in adult zebrafish, we developed a traumatic lesion assay, analyzed cellular reactions to injury and show that adult zebrafish can efficiently regenerate brain lesions and lack permanent glial scarring. Using Cre-loxP-based genetic lineage-tracing, we demonstrate that her4.1-positive ventricular radial glia progenitor cells react to injury, proliferate and generate neuroblasts that migrate to the lesion site. The newly generated neurons survive for more than 3 months, are decorated with synaptic contacts and express mature neuronal markers. Thus, regeneration after traumatic lesion of the adult zebrafish brain occurs efficiently from radial glia-type stem/progenitor cells.
Asunto(s)
Encéfalo/fisiología , Regeneración Nerviosa/fisiología , Neuroglía/fisiología , Nervio Radial/citología , Células Madre/fisiología , Pez Cebra/fisiología , Factores de Edad , Animales , Animales Modificados Genéticamente , Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Diferenciación Celular/fisiología , Rastreo Celular/métodos , Transdiferenciación Celular/fisiología , Embrión no Mamífero , Modelos Biológicos , Células-Madre Neurales/fisiología , Neuroglía/citología , Heridas Punzantes/fisiopatología , Pez Cebra/embriologíaRESUMEN
Chemobehavioural phenotyping presents unique opportunities for analyzing neurotoxicants and discovering behavior-modifying neuroceuticals in small aquatic model organisms such as zebrafish (Danio rerio). A recently popularized approach in this field involves the utilization of zebrafish embryos for a photo-motor response (PMR) bioassay. The PMR bioassay entails stimulating zebrafish embryos between 24 and 36 h post fertilization (hpf) with a high-intensity light stimulus, inducing a transient increase in the frequency of photo-induced embryo body flexions. These flexions can be computationally analyzed to derive behavioral signatures, enabling the categorization of neuromodulating chemicals. Despite the significant advantages of the PMR bioassay, its widespread implementation is hindered by lack of well described and straightforward high-throughput bioinformatic analysis of behavioral data. In this methods article, we present an easily implementable bioinformatics protocol specifically designed for rapid behavioral analysis of large cohorts of zebrafish specimens in PMR bioassays. We also address common pitfalls encountered during PMR analysis, discuss its limitations, and propose future directions for developing next-generation biometric analysis techniques in chemobehavioural assays utilizing zebrafish embryos.
Asunto(s)
Síndromes de Neurotoxicidad , Pez Cebra , Animales , Pez Cebra/fisiología , Embrión no MamíferoRESUMEN
Assessment of animals' sensory-motor functions requires precise and electronically controlled stimuli to induce and quantify specific behavioral phenotypes. However, accessible and inexpensive tools for conducting diverse sensory-motor biotests with fish are lacking. In this work, we present an open-source software and hardware interface that enables automated delivery of three independent and fully programmable stimuli for behavioral bioassays. We demonstrate the proof-of-concept application of this low-cost technology in establishing reproducible fear responses using a mechanical tap-startle stimulus in larval zebrafish. This response is characterized by a sudden burst of motion in response to a nondirectional mechanical stimulus delivered to the fish chamber. We propose that the simplicity and flexibility of this interface offer innovative opportunities for studying sensory-motor functions in various fields, including neurobiology, neuropharmacology, neurotoxicology, and aquatic ecotoxicology.
Asunto(s)
Perciformes , Pez Cebra , Animales , Pez Cebra/fisiología , Conducta Animal/fisiología , Larva/fisiología , Programas InformáticosRESUMEN
Unlike mammals, adult and larval zebrafish exhibit robust regeneration following traumatic spinal cord injury. This remarkable regenerative capacity, combined with exquisite imaging capabilities and an abundance of powerful genetic techniques, has established the zebrafish as an important vertebrate model for the study of neural regeneration. Here, we describe a protocol for the complete mechanical ablation of the larval zebrafish spinal cord. With practice, this protocol can be used to reproducibly injure upward of 100 samples per hour, facilitating the high-throughput screening of factors involved in spinal cord regeneration and repair.
Asunto(s)
Traumatismos de la Médula Espinal , Regeneración de la Medula Espinal , Animales , Pez Cebra , Larva , Médula Espinal , Regeneración Nerviosa , MamíferosRESUMEN
Cre-mediated site-specific recombination has emerged as an indispensable tool for the precise manipulation of the mammalian genome. Recently, we showed that Cre is also highly efficient in zebrafish and temporal control of recombination can be achieved by using the ligand-inducible CreER(T2). Previous attempts have been made to control recombination by using the temperature inducible hsp70l promoter to conditionally drive the expression of Cre or EGFP-Cre, respectively. However, in this study we demonstrate that the hsp70l promoter possesses a basal leakiness resulting in Cre-mediated recombination even at permissive temperatures. In order to prevent non-conditional recombination, we combined the hsp70l promoter with a mCherry-tagged ligand-inducible CreER(T2). At permissive temperatures and in the absence of the ligand tamoxifen (TAM), no non-conditional recombination is observed indicating tight regulation of CreER(T2). Instead, comprehensive site-specific recombination is mediated following heat induction and administration of TAM.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Calor , Integrasas/genética , Activación Transcripcional/fisiología , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Embrión no Mamífero , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Masculino , Modelos Biológicos , Regiones Promotoras Genéticas/genética , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transgenes/genética , Transgenes/fisiología , Pez Cebra/embriologíaRESUMEN
Due to technical limitations, there have been minimal studies performed on thermal preferences and thermotactic behaviors of aquatic ectotherm species commonly used in ecotoxicity testing. In this work, we demonstrate an innovative, purpose-built and miniaturized electrothermal array for rapid thermal preference behavioral tests. We applied the novel platform to define thermal preferences in multiple invertebrate and vertebrate species. Specifically, Dugesia notogaea (freshwater planarians), Chironomus tepperi (nonbiting midge larvae), Ostracoda (seed shrimp), Artemia franciscana (brine shrimp), Daphnia carinata (water flea), Austrochiltonia subtenuis (freshwater amphipod), Physa acuta (freshwater snail), Potamopyrgus antipodarum (New Zealand mud snail) and larval stage of Danio rerio (zebrafish) were tested. The Australian freshwater water fleas, amphipods, snail Physa acuta as well as zebrafish exhibited the most consistent preference to cool zones and clear avoidance of zones >27 °C out of nine species tested. Our results indicate the larval stage of zebrafish as the most responsive species highly suitable for prospective development of multidimensional behavioral test batteries. We also showcase preliminary data that environmentally relevant concentrations of pharmaceutical pollutants such as non-steroidal anti-inflammatory drug (NSAID) ibuprofen (9800 ng/L) and insecticide imidacloprid (4600 ng/L) but not anti-depressant venlafaxine (2200 ng/L) and (iv) anticonvulsant medications gabapentin (400 ng/L) can perturb thermal preference behavior of larval zebrafish. Collectively our results demonstrate the utility of simple and inexpensive thermoelectric technology in rapid exploration of thermal preference in diverse species of aquatic animals. We postulate that more broadly such technologies can also have added value in ecotoxicity testing of emerging contaminants.
Asunto(s)
Anfípodos , Chironomidae , Insecticidas , Contaminantes Químicos del Agua , Animales , Ecotoxicología , Pez Cebra , Insecticidas/farmacología , Temperatura , Ibuprofeno , Clorhidrato de Venlafaxina/farmacología , Gabapentina , Anticonvulsivantes/farmacología , Estudios Prospectivos , Contaminantes Químicos del Agua/toxicidad , Australia , Larva , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios/farmacología , Preparaciones FarmacéuticasRESUMEN
Large-scale chemobehavioral phenotyping with zebrafish embryos is a promising avenue for accelerated neurotoxicity testing and discovery of behavior-modifying neuroceuticals. These strategies are hampered by lack of effective embryo in-test positioning, wide-field imaging, and high-throughput bioinformatic analytics. In this study, we demonstrate advantages of using custom large-density embryo arrays in conjunction with an open-source ultra-high-definition video imaging system. Moreover, we present a high-throughput bioinformatics workflow for rapid behavioral analysis of large cohorts of specimens in photomotor response bioassays. The system validation was showcased in a proof-of-concept neurotoxicity analysis.