成年斑马鱼OKR行为学分析

作为视功能检测和与视觉有关突变体筛选的方法, 眼动(Optokinetic response, OKR)和视动(Optomoter response, OMR)行为学是简单有效的视功能检测手段, 广泛用于幼年斑马鱼研究中, 而成年斑马鱼OKR的分析方法却很少有报道。文章介绍了成年斑马鱼眼动反应诱导方式, 以及使用模板匹配(Pattern match)的方法程序跟踪眼部运动, 实现了成年斑马鱼OKR的定量分析。使用该方法, 检测到斑马鱼双眼视觉区对OKR行为的产生具有一定的贡献作用, 并且成年斑马鱼单眼对运动光栅表现出一定的方向敏感性。同样的方法也可适用于幼年斑马鱼的OKR行为学分析。利用此方法初步检测到了钟基因period1b突变体幼鱼的OKR异常。     

The Effect of Zeaxanthin on the Visual Acuity of Zebrafish

Oral supplementation of carotenoids such as zeaxanthin or lutein which naturally occur in human retina have been shown to improve vision and prevent progression of damage to advanced AMD in some studies. The zebrafish eye shares many physiological similarities with the human eye and is increasingly being used as model for vision research. We hypothesized that injection of zeaxanthin into the zebrafish eye would improve the visual acuity of the zebrafish over time. Visual acuity, calculated in cycles per degree, was measured in adult zebrafish to establish a consistent baseline using the optokinetic response. Zeaxanthin dissolved into phosphate buffered saline (PBS) or PBS only was injected into the anterior chamber of the right and left eyes of the Zebrafish. Visual acuities were measured at 1 week and 3, 8 and 12 weeks post-injection to compare to baseline values. Repeated measures ANOVA was used to compare visual acuities between fish injected with PBS and zeaxanthin. A significant improvement in visual acuity, 14% better than before the injection (baseline levels), was observed one week after injection with zeaxanthin (p = 0.04). This improvement peaked at more than 30% for some fish a few weeks after the injection and improvement in vision persisted at 3 weeks after injection (p = 0.006). The enhanced visual function was not significantly better than baseline at 8 weeks (p = 0.19) and returned to baseline levels 12 weeks after the initial injection (p = 0.50). Zeaxanthin can improve visual acuity in zebrafish eyes. Further studies are required to develop a better understanding of the role zeaxanthin and other carotenoids play during normal visual function.     

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish^1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.Su-Qi Zou and Wu Yin contributed equally to this paper.Open in a separate windowClick here to view.^{(35M, flv)}     

OMR-Arena: Automated Measurement and Stimulation System to Determine Mouse Visual Thresholds Based on Optomotor Responses

Measurement of the optomotor response is a common way to determine thresholds of the visual system in animals. Particularly in mice, it is frequently used to characterize the visual performance of different genetically modified strains or to test the effect of various drugs on visual performance. Several methods have been developed to facilitate the presentation of stimuli using computer screens or projectors. Common methods are either based on the measurement of eye movement during optokinetic reflex behavior or rely on the measurement of head and/or body-movements during optomotor responses. Eye-movements can easily and objectively be quantified, but their measurement requires invasive fixation of the animals. Head movements can be observed in freely moving animals, but until now depended on the judgment of a human observer who reported the counted tracking movements of the animal during an experiment. In this study we present a novel measurement and stimulation system based on open source building plans and software. This system presents appropriate 360 stimuli while simultaneously video-tracking the animal''s head-movements without fixation. The on-line determined head gaze is used to adjust the stimulus to the head position, as well as to automatically calculate visual acuity. Exemplary, we show that automatically measured visual response curves of mice match the results obtained by a human observer very well. The spatial acuity thresholds yielded by the automatic analysis are also consistent with the human observer approach and with published results. Hence, OMR-arena provides an affordable, convenient and objective way to measure mouse visual performance.     

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial world. Animal models are of pivotal importance to study such diseases. In this regard the photoreceptor-specific toxin N-methyl-N-nitrosourea (MNU) has been widely used in rodents to pharmacologically induce retinal degeneration. Previously, we have established a MNU-induced retinal degeneration model in the zebrafish, another popular model system in visual research.A fascinating difference to mammals is the persistent neurogenesis in the adult zebrafish retina and its regeneration after damage. To quantify this observation we have employed visual acuity measurements in the adult zebrafish. Thereby, the optokinetic reflex was used to follow functional changes in non-anesthetized fish. This was supplemented with histology as well as immunohistochemical staining for apoptosis (TUNEL) and proliferation (PCNA) to correlate the developing morphological changes.In summary, apoptosis of photoreceptors occurs three days after MNU treatment, which is followed by a marked reduction of cells in the outer nuclear layer (ONL). Thereafter, proliferation of cells in the inner nuclear layer (INL) and ONL is observed. Herein, we reveal that not only a complete histological but also a functional regeneration occurs over a time course of 30 days. Now we illustrate the methods to quantify and follow up zebrafish retinal de- and regeneration using MNU in a video-format.     

Visual acuity and contrast sensitivity of adult zebrafish

Background

The aim of this study was to evaluate the visual acuity of adult zebrafish by assessing the optokinetic reflex. Using a modified commercially available optomotor device (OptoMotry?), virtual three-dimensional gratings of variable spatial frequency or contrast were presented to adult zebrafish. In a first experiment, visual acuity was evaluated by changing the spatial frequency at different angular velocities. Thereafter, contrast sensitivity was evaluated by changing the contrast level at different spatial frequencies.

Results

At the different tested angular velocities (10, 15, 20, 25, and 30 d/s) and a contrast of 100%, visual acuity values ranged from 0.56 to 0.58 c/d. Contrast sensitivity measured at different spatial frequencies (0.011, 0.025, 0.5, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.55 c/d) with an angular velocity of 10 d/s and 25 d/s revealed an inverted U-shaped contrast sensitivity curve. The highest mean contrast sensitivity (±SD) values of 20.49?±?4.13 and 25.24?±?8.89 were found for a spatial frequency of 0.05 c/d (angular velocity 10 d/s) and 0.1 c/d (angular velocity 25 d/s), respectively.

Conclusions

Visual acuity and contrast sensitivity measurements in adult zebrafish with the OptoMotry? device are feasible and reveal a remarkably higher VA compared to larval zebrafish and mice.     

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

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.     

Aerial and aquatic visual acuity of the grey bichir Polypterus senegalus,as estimated by optokinetic response

The present study assessed the aerial and aquatic visual abilities of juvenile grey bichir Polypterus senegalus, fish capable of terrestrial locomotion, by measuring the optokinetic response to stimuli of varying speed and spatial frequency. In water, fish tracked slow-moving (2° s⁻¹) stimuli moderately well and fast-moving stimuli very poorly. Spatial acuity was very low compared with many other species, with maximum response observed at 0.05–0.075 stimulus cycles per degree of visual arc; however, it should be noted that adult fish, with their larger eyes, are likely to have somewhat improved spatial acuity. Low spatial acuity and limited stimulus tracking ability might be expected in a nocturnal ambush predator such as P. senegalus, where gaze stabilization may be less crucial and other sensory inputs may have greater importance in perception of the environment. In air, spatial and temporal acuity were both poorer by every measure, but some visual ability persisted. As the eye shows no anatomical specialization for aerial vision, poor vision was expected; however, the large decrease in saccade velocity observed in air trials was unexpected. Stimulus parameters typically have little effect on the characteristics of the saccade, so this finding may suggest that the function of the reflex system itself could be compromised in the aerial vision of some fishes capable of terrestrial locomotion.     

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.     

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 μl of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.     

Transparent adult zebrafish as a tool for in vivo transplantation analysis

The zebrafish is a useful model for understanding normal and cancer stem cells, but analysis has been limited to embryogenesis due to the opacity of the adult fish. To address this, we have created a transparent adult zebrafish in which we transplanted either hematopoietic stem/progenitor cells or tumor cells. In a hematopoiesis radiation recovery assay, transplantation of GFP-labeled marrow cells allowed for striking in vivo visual assessment of engraftment from 2 hr-5 weeks posttransplant. Using FACS analysis, both transparent and wild-type fish had equal engraftment, but this could only be visualized in the transparent recipient. In a tumor engraftment model, transplantation of RAS-melanoma cells allowed for visualization of tumor engraftment, proliferation, and distant metastases in as little as 5 days, which is not seen in wild-type recipients until 3 to 4 weeks. This transparent adult zebrafish serves as the ideal combination of both sensitivity and resolution for in vivo stem cell analyses.     

VisioTracker,an Innovative Automated Approach to Oculomotor Analysis

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. Download video file.^{(36M, mov)}     

Measuring the optokinetic response of zebrafish larvae

Our laboratory screens for visual mutants by examining larval eye movements in response to rotating illuminated stripes. This behavior, which is termed an optokinetic response (OKR), is a reflex that appears in zebrafish at the same time as the development of the visual system. The OKR can be accurately measured by 4 d post-fertilization, which is the age when larvae begin foraging for food. The OKR requires approximately 1 min per larva analyzed. After identifying fish with defective eye movements, we conduct secondary screens (such as histological analysis and electroretinography) to identify the subset of fish with disruptions in the function of the outer retina. This paper describes our protocol for the OKR. Our setup is simple to construct and the materials needed are inexpensive. This makes our system especially useful for new undergraduate and graduate students, as well as introductory science lecturers.     

Measuring behavioral and endocrine responses to novelty stress in adult zebrafish

Several behavioral assays are currently used for high-throughput neurophenotyping and screening of genetic mutations and psychotropic drugs in zebrafish (Danio rerio). In this protocol, we describe a battery of two assays to characterize anxiety-related behavioral and endocrine phenotypes in adult zebrafish. Here, we detail how to use the 'novel tank' test to assess behavioral indices of anxiety (including reduced exploration, increased freezing behavior and erratic movement), which are quantifiable using manual registration and computer-aided video-tracking analyses. In addition, we describe how to analyze whole-body zebrafish cortisol concentrations that correspond to their behavior in the novel tank test. This protocol is an easy, inexpensive and effective alternative to other methods of measuring stress responses in zebrafish, thus enabling the rapid acquisition and analysis of large amounts of data. As will be shown here, fish anxiety-like behavior can be either attenuated or exaggerated depending on stress or drug exposure, with cortisol levels generally expected to parallel anxiety behaviors. This protocol can be completed over the course of 2 d, with a variable testing duration depending on the number of fish used.     

Heart Dissection in Larval,Juvenile and Adult Zebrafish,Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews^1-6), however, work examining post-embryonic through adult cardiac development has been limited^7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish''s lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis¹¹. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100μm ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)^{twu34 (12)}, which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.Download video file.^{(42M, mov)}     

An Optimized Whole-Body Cortisol Quantification Method for Assessing Stress Levels in Larval Zebrafish

Glucocorticoids serve important regulatory functions for many physiological processes and are critical mediators of the stress response. The stress response is a set of bodily processes aimed at counteracting a state of threatened homeostasis. Proper stress response is critical for the survival of an animal, however prolonged or abnormal stress response can be detrimental and is implicated in a number of human diseases such as depression and metabolic diseases. To dissect the underlying mechanism of this complex and important response, the zebrafish, Danio rerio offer important advantages such as ease of genetic manipulations and high-throughput behavioral analyses. However, there is a paucity of suitable methods to measure stress level in larval zebrafish. Therefore, an efficient low-cost method to monitor stress hormone levels will greatly facilitate stress research in zebrafish larvae. In this study, we optimized sample collection as well as cortisol extraction methods and developed a home-made ELISA protocol for measuring whole-body cortisol level in zebrafish larvae. Further, using our customized protocols, we characterized the response of larval zebrafish to a variety of stressors. This assay, developed for efficient cortisol quantification, will be useful for systematic and large-scale stress analyses in larval zebrafish.     

THE VISUAL ACUITY OF THE FIDDLER-CRAB,UCA PUGNAX

The visual acuity of the fiddler-crab can be measured at various illuminations by means of its response to a moving visual pattern. The method, although similar to that used by Hecht and Wolf for the bee and Hecht and Wald for Drosophila, must be modified to give consistent results. An explanation of the response to a visual pattern is given in terms of the structure of the eye. Visual acuity of the crab varies with log I as in man, the bee, and Drosophila. Hecht and Wolf''s explanation of the varying visual acuity with illumination in terms of the distribution of functional ommatidia in the eye is supported to that extent. In the fiddler-crab as in man, monocular and binocular visual acuity is similar with a maximum of 0.0042 for the fiddler-crab. This agrees fairly well with visual acuities of 0.0041, 0.0038, and 0.0032 as found in the field. In man and the bee, the minimum visual angle corresponds to the minimum angle of two adjacent receptors; in Drosophila and the fiddler-crab the minimum visual angle corresponds to approximately twice the minimum angle between two adjacent receptors.     

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases¹. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease². Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells³. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.     

Death-associated odors induce stress in zebrafish

Living animals exploit information released from dead animals to conduct adaptive biological responses. For instance, a recently published study has shown that avoidance behavior is triggered by death-associated odors in zebrafish. Stress can clearly act as an adaptive response that allows an organism to deal with an imminent threat. However, it has not been demonstrated whether these chemical cues are stressful for fish. Here, we confirmed that dead zebrafish scents induce defensive behavior in live conspecifics. Additionally, we show for the first time in fish that these scents increase cortisol in conspecifics. To reach this conclusion, firstly, we exposed zebrafish to multi-sensorial cues (e.g., visual, tactile, chemical cues) from dead conspecifics that displayed defensive behaviors and increased cortisol. Also, when we limited zebrafish to chemical cues from dead conspecifics, similar responses arose. These responses coincide with the decaying destruction of epidermal cells, indicating that defensive and stress responses could take place as an effect of substances emanating from decaying flesh, as well as alarm substance released due to rupture of epidermal cells. Taken together, these results illustrate that living zebrafish utilize cues from dead conspecific to avoid or to cope with danger and ensure survival.     

Brain-Stimulation Induced Blindsight: Unconscious Vision or Response Bias?

A dissociation between visual awareness and visual discrimination is referred to as “blindsight”. Blindsight results from loss of function of the primary visual cortex (V1) which can occur due to cerebrovascular accidents (i.e. stroke-related lesions). There are also numerous reports of similar, though reversible, effects on vision induced by transcranial Magnetic Stimulation (TMS) to early visual cortex. These effects point to V1 as the “gate” of visual awareness and have strong implications for understanding the neurological underpinnings of consciousness. It has been argued that evidence for the dissociation between awareness of, and responses to, visual stimuli can be a measurement artifact of the use of a high response criterion under yes-no measures of visual awareness when compared with the criterion free forced-choice responses. This difference between yes-no and forced-choice measures suggests that evidence for a dissociation may actually be normal near-threshold conscious vision. Here we describe three experiments that tested visual performance in normal subjects when their visual awareness was suppressed by applying TMS to the occipital pole. The nature of subjects’ performance whilst undergoing occipital TMS was then verified by use of a psychophysical measure (d'') that is independent of response criteria. This showed that there was no genuine dissociation in visual sensitivity measured by yes-no and forced-choice responses. These results highlight that evidence for visual sensitivity in the absence of awareness must be analysed using a bias-free psychophysical measure, such as d'', In order to confirm whether or not visual performance is truly unconscious.