RESUMEN
PURPOSE: Infantile nystagmus syndrome (INS) is characterized by involuntary eye oscillations that can assume different waveforms. Previous attempts to uncover reasons for the presence of several nystagmus waveforms have not led to a general consensus in the community. Recently, we characterized the belladonna (bel) zebrafish mutant strain, in which INS-like ocular motor abnormalities are caused by misprojection of a variable fraction of optic nerve fibers. Here we studied intrinsic and extrinsic factors influencing the occurrence of different waveforms in bel larvae. METHODS: Eye movements of bel larvae were recorded in the presence of a stationary grating pattern. Waveforms of spontaneous oscillations were grouped in three categories: "pendular," "unidirectional jerk," and "bidirectional jerk," and the occurrences of each category were compared within and between individual larvae. Moreover, the effects of the characteristics of a preceding optokinetic response (OKR), of the field of view, and of the eye orbital position were analyzed. RESULTS: The different waveform categories co-occurred in most individuals. We found waveforms being influenced by the preceding OKR and by the field of view. Moreover, we found different kinds of relationships between orbital position and initiation of a specific waveform, including pendular nystagmus in a more eccentric orbital position, and differences among jerk oscillations regarding the beating direction of the first saccade or waveform amplitude. CONCLUSIONS: Our data suggest that waveform categories in bel larvae do not reflect the severity of the morphological phenotype but rather are influenced by viewing conditions.
Asunto(s)
Modelos Animales de Enfermedad , Proteínas con Homeodominio LIM/genética , Proteínas del Tejido Nervioso/genética , Nistagmo Congénito/fisiopatología , Nistagmo Optoquinético/fisiología , Factores de Transcripción/genética , Proteínas de Pez Cebra/genética , Pez Cebra/fisiología , Animales , Larva , Mutación , Fibras Nerviosas/fisiología , Nistagmo Congénito/genética , Nervio Óptico/fisiopatología , Visión Binocular/fisiología , Campos Visuales/fisiologíaRESUMEN
Many animals change their body pigmentation according to illumination of their environment. In aquatic vertebrates, this reaction is mediated through aggregation or dispersion of melanin-filled vesicles (melanosomes) in dermal pigment cells (melanophores). The adaptive value of this behavior is usually seen in camouflage by allowing the animal to visually blend into the background. When exposed to visible light from below, however, dark-adapted zebrafish embryos at the age of 2 days post fertilization (dpf) surprisingly display dispersal instead of aggregation of melanosomes, i.e. their body coloration becomes dark on a bright background. Melanosomes of older embryos and early larvae (3-5 dpf) on the other hand aggregate as expected under these conditions. Here we provide an explanation to this puzzling finding: Melanosome dispersion in larvae 3 dpf and older is efficiently triggered by ultraviolet (UV) light, irrespective of the visual background, suggesting that the extent of pigmentation is a trade-off between threats from predation and UV irradiation. The UV light-induced dispersion of melanosomes thereby is dependent on input from retinal short wavelength-sensitive (SWS) cone photoreceptors. In young embryos still lacking a functional retina, protection from UV light predominates, and light triggers a dispersal of melanosomes via photoreceptors intrinsic to the melanophores, regardless of the actual UV content. In older embryos and early larvae with functional retinal photoreceptors in contrast, this light-induced dispersion is counteracted by a delayed aggregation in the absence of UV light. These data suggest that the primary function of melanosome dispersal has evolved as a protective adaption to prevent UV damage, which was only later co-opted for camouflage.
Asunto(s)
Melaninas/metabolismo , Melanosomas/metabolismo , Pigmentación de la Piel , Rayos Ultravioleta , Pez Cebra/metabolismo , Animales , Larva/metabolismo , Larva/efectos de la radiación , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismoRESUMEN
Large-field movements in the visual surround trigger spontaneous, compensatory eye movements known as optokinetic response (OKR) in all vertebrates. In zebrafish (Danio rerio) the OKR is well developed at 5 days post fertilization and can be used in the laboratory for screening of visual performance following genetic manipulations or pharmaceutical treatments. Several setups for measurement of the zebrafish OKR have been described. All of them are based on the presentation of moving gratings to the larva or to the adult fish. However, they differ in the way of presenting gratings and in the method of analysis. Here, we describe a detailed protocol for our newest software that enables computer-generation of the moving stripes and automatic tracking of eye movement. This protocol makes it possible to quantitatively measure OKR in both larvae and adult fishes in a fast and reliable way.
Asunto(s)
Computadores , Microscopía/métodos , Nistagmo Optoquinético , Pez Cebra/fisiología , Animales , Ojo/anatomía & histología , Ojo/crecimiento & desarrollo , Larva/anatomía & histología , Larva/fisiología , Grabación en Video/métodos , Pez Cebra/anatomía & histología , Pez Cebra/crecimiento & desarrolloRESUMEN
We present here the positional cloning of the Danio rerio albino mutant and show that the affected gene encodes Slc45a2. The human orthologous gene has previously been shown to be involved in human skin color variation, and mutations therein have been implicated in the disease OCA4. Through ultrastructural analysis of the melanosomes in albino alleles as well as the tyrosinase-deficient mutant sandy, we add new insights into the role of Slc45a2 in the production of melanin. To gain further understanding of the role of Slc45a2 and its possible interactions with other proteins involved in melanization, we further analyzed the role of the V-ATPase as a melanosomal acidifier. We show that it is possible to rescue the melanization potential of the albino melanosomes through genetic and chemical inhibition of V-ATPase, thereby increasing internal melanosome pH.
Asunto(s)
Evolución Biológica , Homeostasis , Melanosomas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Pigmentación , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Clonación Molecular , Homeostasis/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Melanocitos/metabolismo , Melanocitos/patología , Melanóforos/efectos de los fármacos , Melanóforos/metabolismo , Melanosomas/efectos de los fármacos , Melanosomas/ultraestructura , Proteínas de Transporte de Membrana/química , Modelos Biológicos , Datos de Secuencia Molecular , Monofenol Monooxigenasa/metabolismo , Morfolinos/farmacología , Mutación/genética , Cresta Neural/efectos de los fármacos , Cresta Neural/metabolismo , Cresta Neural/patología , Especificidad de Órganos/efectos de los fármacos , Pigmentación/efectos de los fármacos , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Retina/patología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Agudeza Visual/efectos de los fármacos , Proteínas de Pez Cebra/químicaRESUMEN
Larval zebrafish lacking eyes and pineal organ show elevated activity levels and undirected light-seeking behaviour upon loss of illumination. This behaviour, termed dark photokinesis, is mediated by hypothalamic deep brain photoreceptors expressing melanopsin.
Asunto(s)
Encéfalo/fisiología , Estimulación Luminosa , Células Fotorreceptoras de Vertebrados/fisiología , Percepción Visual , Pez Cebra/fisiología , AnimalesRESUMEN
PURPOSE: To characterize the expression pattern of cadherin 23 (cdh23) in the zebrafish visual system, and to determine whether zebrafish cdh23 mutants have retinal defects similar to those present in the human disease Usher syndrome 1D. METHODS: In situ hybridization and immunohistochemistry were used to characterize cdh23 expression in the zebrafish, and to evaluate cdh23 mutants for retinal degeneration. Visual function was assessed by measurement of the optokinetic response in cdh23 siblings and mutants. RESULTS: We detected cdh23 mRNA expression in multiple nuclei of both the developing and adult central nervous system. In the retina, cdh23 mRNA was expressed in a small subset of amacrine cells, beginning at 70 h postfertilization and continuing through adulthood. No expression was detected in photoreceptors. The cdh23-positive population of amacrine cells was GABAergic. Examination of homozygous larvae expressing two different mutant alleles of cdh23-cdh23(tc317e) or cdh23(tj264a)-revealed no detectable morphological retinal defects or degeneration. In addition, the optokinetic response to moving gratings of varied contrast or spatial frequency was normal in both mutants. CONCLUSIONS: Unlike in other vertebrates, cdh23 is not detectable in zebrafish photoreceptors. Instead, cdh23 is expressed by a small subset of GABAergic amacrine cells. Moreover, larvae with mutations in cdh23 do not exhibit any signs of gross retinal degeneration or dysfunction. The role played by cdh23 in human retinal function is likely performed by either a different gene or an unidentified cdh23 splice variant in the retina that is not affected by the above mutations.
Asunto(s)
Células Amacrinas/metabolismo , Cadherinas/genética , Regulación del Desarrollo de la Expresión Génica , Larva/genética , Mutación , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Alelos , Empalme Alternativo , Células Amacrinas/citología , Animales , Proteínas Relacionadas con las Cadherinas , Movimientos Oculares , Homocigoto , Humanos , Inmunohistoquímica , Hibridación in Situ , Células Fotorreceptoras/citología , Células Fotorreceptoras/metabolismo , ARN Mensajero/biosíntesis , Receptores de GABA/genética , Degeneración Retiniana/genética , Síndromes de Usher/genéticaRESUMEN
Training experimental animals to discriminate between different visual stimuli has been an important tool in cognitive neuroscience as well as in vision research for many decades. Current methods used for visual choice discrimination training of zebrafish require human observers for response tracking, stimulus presentation and reward delivery and, consequently, are very labor intensive and possibly experimenter biased. By combining video tracking of fish positions, stimulus presentation on computer monitors and food delivery by computer-controlled electromagnetic valves, we developed a method that allows for a fully automated training of multiple adult zebrafish to arbitrary visual stimuli in parallel. The standardized training procedure facilitates the comparison of results across different experiments and laboratories and contributes to the usability of zebrafish as vertebrate model organisms in behavioral brain research and vision research.
Asunto(s)
Control de la Conducta/métodos , Investigación Conductal/métodos , Conducta de Elección/fisiología , Aprendizaje Discriminativo/fisiología , Percepción Visual/fisiología , Pez Cebra/fisiología , Animales , Conducta AnimalRESUMEN
Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications. Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time. Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity. Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.
Asunto(s)
Medidas del Movimiento Ocular/instrumentación , Movimientos Oculares/fisiología , Visión Ocular/fisiología , Animales , Automatización , Larva , Fenómenos Fisiológicos Oculares , Pez CebraRESUMEN
Mutations in ubiquitously expressed metabolic genes often lead to CNS-specific effects, presumably because of the high metabolic demands of neurons. However, mutations in omnipresent metabolic pathways can conceivably also result in cell type-specific effects because of cell-specific requirements for intermediate products. One such example is the zebrafish noir mutant, which we found to be mutated in the pdhb gene, coding for the E1 beta subunit of the pyruvate dehydrogenase complex. This vision mutant is described as blind and was isolated because of its vision defect-related darker appearance. A detailed morphological, behavioral, and physiological analysis of the phenotype revealed an unexpected specific effect on the retina. Surprisingly, the cholinergic amacrine cells of the inner retina are affected earlier than the photoreceptors. This might be attributable to the inability of these cells to maintain production of their neurotransmitter acetylcholine. This is reflected in an earlier loss of motion vision, followed only later by a general loss of light perception. Since both characteristics of the phenotype are attributable to a loss of acetyl-CoA production by pyruvate dehydrogenase, we used a ketogenic diet to bypass this metabolic block and could indeed partially rescue vision and prolong survival of the larvae. The noir mutant provides a case for a systemic disease with ocular manifestation with a surprising specific effect on the retina given the ubiquitous requirement for the mutated gene.
Asunto(s)
Mutación/genética , Piruvato Descarboxilasa/deficiencia , Enfermedades de la Retina/diagnóstico , Enfermedades de la Retina/genética , Aminobutiratos/farmacología , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Ácido Aspártico/farmacología , Colina O-Acetiltransferasa/metabolismo , Análisis Mutacional de ADN , Dieta Cetogénica/métodos , Modelos Animales de Enfermedad , Electrorretinografía/métodos , Embrión no Mamífero , Agonistas de Aminoácidos Excitadores/farmacología , Larva , Movimiento/fisiología , Nistagmo Optoquinético/genética , Nistagmo Optoquinético/fisiología , Estimulación Luminosa/métodos , Retina/citología , Retina/embriología , Retina/crecimiento & desarrollo , Retina/patología , Enfermedades de la Retina/dietoterapia , Tirosina 3-Monooxigenasa/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Many arthropods including insects and spiders exploit skylight polarization for navigation. One of the four eye pairs of the spider Drassodes cupreus is dedicated to detect skylight polarization. These eyes are equipped with a tapetum that strongly plane-polarizes reflected light. This effectively enhances the polarization-sensitivity of the photoreceptors, improving orientation performance. With a multidisciplinary approach, we demonstrate that D. cupreus exploits reflective elements also present in non-polarizing tapetal eyes of other species such as Agelena labyrinthica. By approximately orthogonal arrangement of two multilayer reflectors consisting of reflecting guanine platelets, the tapetum uses the mechanism of polarization by reflection for polarizing reflected light.
Asunto(s)
Luz , Arañas/anatomía & histología , Arañas/fisiología , Animales , Conducta Animal/fisiología , Ojo/anatomía & histología , Modelos Biológicos , Modelos Teóricos , Fenómenos Fisiológicos Oculares , Visión Ocular/fisiologíaRESUMEN
BACKGROUND: Visual acuity, the ability of the visual system to distinguish two separate objects at a given angular distance, is influenced by the optical and neuronal properties of the visual system. Although many factors may contribute, the ultimate limit is photoreceptor spacing. In general, at least one unstimulated photoreceptor flanked by two stimulated ones is needed to perceive two objects as separate. This critical interval is also referred to as the Nyquist frequency and is according to the Shannon sampling theorem the highest spatial frequency where a pattern can be faithfully transmitted. We measured visual acuity in a behavioral experiment and compared the data to the physical limit given by photoreceptor spacing in zebrafish larvae. RESULTS: We determined visual acuity by using the optokinetic response (OKR), reflexive eye movements in response to whole field movements of the visual scene. By altering the spatial frequency we determined the visual acuity at approximately 0.16 cycles/degree (cpd) (minimum separable angle = 3.1 degrees ). On histological sections we measured the retinal magnification factor and the distance between double cones, that are thought to mediate motion perception. These measurements set the physical limit at 0.24 cpd (2.1 degrees ). CONCLUSION: The maximal spatial information as limited by photoreceptor spacing can not be fully utilized in a motion dependent visual behavior, arguing that the larval zebrafish visual system has not matured enough to optimally translate visual information into behavior. Nevertheless behavioral acuity is remarkable close to its maximal value, given the immature state of young zebrafish larvae.
RESUMEN
Orientation of animals towards or away from light is a simple behavior commonly found in the animal kingdom. A recent study using zebrafish larvae has revealed the underlying neural logic of this primal choice behavior, by differential use of the retinal ON- and OFF-pathways.
Asunto(s)
Luz , Actividad Motora/fisiología , Orientación/fisiología , Retina/fisiología , Transducción de Señal/fisiología , Percepción Visual/fisiología , Pez Cebra/fisiología , Animales , Larva/fisiologíaRESUMEN
Small teleost fish are increasingly used for studying the genetic basis of vision. In particular, zebrafish (Danio rerio) and medaka (Oryzias latipes) are commonly used vertebrate model organisms in developmental research, including research on the development of visual function. A multitude of behavior-based visual tests are established for larvae that have been successfully used to identify and characterize visual defects in genetically manipulated strains of these species. Testing the visual system of adult fish has proven to be more difficult for a number of reasons, including complications in restraining fish, or shoaling and dominance behavior interfering with visual behavior in population screening assays. In this paper, we present a simple and cost-effective method to quantitatively measure the optokinetic response (OKR) of individual adult zebrafish and medaka, which can be used to characterize visual capabilities of adult fish. This method can be applied to any fish species of similar size.