No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
Zebrafish mutants: Behavioral genetic studies of visual system defects
Developmental Dynamics
Abstract
Zebrafish are a promising model for behavioral and genetic studies of vertebrate visual system development and retinal degeneration. In the past few years, numerous studies on zebrafish vision have been published. While most of the studies focus on the molecular and cellular characterization of mutations that disrupt zebrafish visual system structure in early development, others examine the mechanisms that underlie inherited visual system disorders in adults. Behavioral assays, along with morphologic and electrophysiological methods, are powerful tools for functional analyses of zebrafish visual development and performance. © 2001 Wiley-Liss, Inc.
... Escape response is the ability of zebrafish to escape from a threatening stimulus (Li 2001). To test this response, a round container is used. ...
... Threatening stimulus in the form of a vertical black paper is utilised to cover a part of the apparatus. Zebrafish usually avoids that area and when forced with it usually turns and swims in the opposite direction (Fleisch and Neuhauss 2006;Li 2001). This study can be elicited in adult zebrafish of 2 to 3 months. ...
... This response can be utilised to study retinal degeneration (nba, nbb, nbc mutant) (Li 2001;Dowling 1998, 2000). Among these three mutants, nbc mutant does not possess a mutation in a retina-specific gene. ...
Tumor angiogenesis is the most crucial step in the progression of all types of cancers. Preexisted blood vessel vascularizes into new one through sprouting or intussusceptive (splitting) mechanism. This process would facilitate the growth in the size of tumors regulated by VEGF (vascular endothelial growth factor), leading to metastasis, which ultimately increases the severity of cancer. So, it is very important to suppress tumor angiogenesis before the situation gets worse in a cancer patient. A wide variety of in vitro and in vivo models have been used to study the process of tumor angiogenesis and metastasis of cancer. It has helped us to discover new drugs and to find novel therapies for cancer, including anti-angiogenic therapy. Mainly angiogenesis is traditionally modeled in rodents and chick embryo, but of late zebrafish is emerging as the preferred model due its several advantages over the other animals. Zebrafish (Danio rerio) serves as the ideal model to study the various cancers, since it is possible to induce tumor growth or suppression easily, when compared to the other animal models. Also, tumor xenograft model has been studied in zebrafish extensively using many human cancer cell lines. So, in this chapter, we have reviewed some literatures that appreciate zebrafish model to study tumor angiogenesis.KeywordsZebrafishAngiogenesisVEGFTumorXenograftAnti-angiogenic therapy
... The following question emerged from the above findings: what are the functional changes following il7r knockout? The behavioural analysis of zebrafish larvae is a very intuitive and quick approach 33,34 . Zebrafish vision begins to function at 4 dpf 35 , and zebrafish larvae have the ability to swim at 5 dpf 36 . ...
... The swimming speed of WT and il7r −/− mutants was similar in the light environment. Physiologically, when there is a light stimulus, zebrafish prefer to swim from the dark to the light; this phenomenon is called phototaxis 34 . We concluded that the weak response to the light stimulus indicated that the vision of il7r −/− mutants was impaired. ...
Interleukin 7 receptor (il7r), a transmembrane receptor, belongs to the type I cytokine receptor family. Il7r is involved in the pathogenesis of neurodegenerative disorders, such as multiple sclerosis. Targeted knockdown of il7r leads to delayed myelination, highlighting the potential role of il7r in the development of the nervous system. Zebrafish is an ideal model for the study of neurogenesis; moreover, the il7r gene is highly conserved between zebrafish and human. The aim of the present study was to investigate the novel function of il7r in neurogenesis. First, an il7r-/-homozygous mutant line was generated by clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (CRISPR/Cas9) technology. Second, the gross development of il7r-/-mutants revealed remarkably smaller eyes and delayed retinal neurodifferentiation. Third, microarray analysis revealed that genes associated with the phototransduction signalling pathway were strongly down-regulated in il7r-/-mutants. Finally, the results from behavioural tests indicated that visual function was impaired in il7r-/-mutant larvae. Overall, our data demonstrate that a lack of il7r retards the development of the retina. Thus, il7r is an essential molecule for maintaining normal retinal development in zebrafish.
... A genetic analysis of the development of the monoamine system The developmental ontogeny of DA, NA and 5HT neurons in zebrafish has been characterized (Bellipanni et al. 2002; Guo et al. 1999b; Ma 2003; Rink & Wullimann 2001). Similar to all teleosts examined to date, the majority of DA neurons are found in the basal forebrain while absent from the midbrain. ...
... Similar to all teleosts examined to date, the majority of DA neurons are found in the basal forebrain while absent from the midbrain. Anatomical as well as tracing experiments have shown that the teleost basal forebrain DA neurons send ascending projections to telencephalon, particularly to the proposed area of striatum (Parent et al. 1984; Rink & Wullimann 2001). Thus, the telesot basal forebrain DA neurons may carry out functions similar to the mammalian midbrain DA neurons. ...
How our brain is wired and subsequently generates functional output, ranging from sensing and locomotion to emotion, decision-making and learning and memory, remains poorly understood. Dys-regulation of these processes can lead to neurodegenerative, as well as neuro-psychiatric, disorders. Molecular genetic together with behavioral analyses in model organisms identify genes involved in the formation of neuronal circuits, the execution of behavior and mechanisms involved in neuro-pathogenesis. In this review I will discuss the current progress and future potential for study in a newly established vertebrate model organism for genetics, the zebrafish Danio rerio. Where available, schemes and results of genetic screens will be reviewed concerning the sensory, motor and neuromodulatory monoamine systems. Genetic analyses in zebrafish have the potential to provide important insights into the relationship between genes, neuronal circuits and behavior in normal as well as diseased states.
... In Drosophila, for example, adult flies bearing the optomotor blind (omb) mutant allele, omb H31 , show an impaired optomotor behavioral response; yet, more severe mutant alleles are lethal (Pflugfelder and Heisenberg, 1995). Likewise, in zebrafish, a behavioral screen based on the fish's escape response was used to isolate the night blind a (nba) mutation (Li and Dowling, 1997;Li, 2001). Interestingly, nba heterozygotes show degeneration that is restricted to the retina, whereas homozygotes die as embryos, show degeneration throughout the CNS, as well as abnormalities in other organs such as the heart (Li, 2001;Maaswinkel et al., 2003), indicating that nba has functions outside of the retina. ...
... Likewise, in zebrafish, a behavioral screen based on the fish's escape response was used to isolate the night blind a (nba) mutation (Li and Dowling, 1997;Li, 2001). Interestingly, nba heterozygotes show degeneration that is restricted to the retina, whereas homozygotes die as embryos, show degeneration throughout the CNS, as well as abnormalities in other organs such as the heart (Li, 2001;Maaswinkel et al., 2003), indicating that nba has functions outside of the retina. Future isolation of additional lre alleles, and mapping and molecular identification of the lre gene will aid in resolving these important questions. ...
To initiate a genetic analysis of olfactory development and function in the zebrafish, Danio rerio, we developed a behavioral genetic screen for mutations affecting the olfactory sensory system. First, we characterized olfactory responses of wild-type zebrafish to various odors. We found that 3-day-old juvenile zebrafish reacted to the amino acid L-cysteine with an aversive behavioral response. We isolated one mutant, laure (lre), which showed no aversive behavioral response to L-cysteine at 3 days of development, and carried out a preliminary characterization of this mutant's defects. We found that lre mutant fish were also defective in their response to L-serine and L-alanine, but not to taurocholic acid, as young adults. In addition, lre mutant fish had significantly fewer primary olfactory sensory neurons than normal, and the axons of these neurons did not form the characteristic axon termination pattern in the developing olfactory bulb. Nevertheless, the olfactory epithelium of lre mutant fish showed normal or near normal electrophysiological responses to several odorants. Our data suggest that the behavioral defects observed in the lre mutant result from the disruption of the developing olfactory sensory neurons and their axonal connections within the olfactory bulb. The isolation of the lre mutant shows that our behavior-based screen represents a viable approach for carrying out a genetic dissection of olfactory behaviors in this vertebrate model system.
... The drugs can be effectively administered to the fish by immersing them into the drug solution at controlled concentrations and for the desired duration of time [21,30]. Additionally, the effects of mutations and drugs can be efficiently detected by the growing number of behavioral test paradigms developed for this species [19,[31][32][33][34][35]. ...
Anxiety continues to represent a major unmet medical need. Despite the availability of numerous anxiolytic drugs, a large proportion of patients do not respond well to current pharmacotherapy, or their response diminishes with chronic drug application. To discover novel compounds and to investigate the mode of action of anxiolytic drugs, animal models have been proposed. The zebrafish is a novel animal model in this research. It is particularly appropriate, as it has evolutionarily conserved features, and drug administration can be employed in a non-invasive manner by immersing the fish into the drug solution. The first step in the analysis of anxiolytic drugs with zebrafish is to test reference compounds. Here, we investigate the effects of buspirone hydrochloride, an anxiolytic drug often employed in the human clinic. We utilize two genetically distinct populations of zebrafish, ABSK, derived from the quasi-inbred AB strain, and WT, a genetically heterogeneous wild-type population. We placed juvenile (10–13-day, post-fertilization, old) zebrafish singly in petri dishes containing one of four buspirone concentrations (0 mg/L control, 5 mg/L, 20 mg/L or 80 mg/L) for 1 h, with each fish receiving a single exposure to one concentration, a between subject experimental design. Subsequently, we recorded the behavior of the zebrafish for 30 min using video-tracking. Buspirone decreased distance moved, number of immobility episodes and thigmotaxis, and it increased immobility duration and turn angle in a quasi-linear dose dependent but genotype independent manner. Although it is unclear whether these changes represent anxiolysis in zebrafish, the results demonstrate that behavioral analysis of juvenile zebrafish may be a sensitive and simple way to quantify the effects of human anxiolytic drugs.
... The zebrafish is a standard model system for vision studies because its retina is strikingly similar to that of mammals 36,37 . Sensory integration in zebrafish is mediated by the olfacto-retinal centrifugal pathway. ...
The analysis of fish behavior in response to odor stimulation is a crucial component of the general study of cross-modal sensory integration in vertebrates. In zebrafish, the centrifugal pathway runs between the olfactory bulb and the neural retina, originating at the terminalis neuron in the olfactory bulb. Any changes in the ambient odor of a fish’s environment warrant a change in visual sensitivity and can trigger mating-like behavior in males due to increased GnRH signaling in the terminalis neuron. Behavioral experiments to study this phenomenon are commonly conducted in a controlled environment where a video of the fish is recorded over time before and after the application of chemicals to the water. Given the subtleties of behavioral change, trained biologists are currently required to annotate such videos as part of a study. This process of manually analyzing the videos is time-consuming, requires multiple experts to avoid human error/bias and cannot be easily crowdsourced on the Internet. Machine learning algorithms from computer vision, on the other hand, have proven to be effective for video annotation tasks because they are fast, accurate, and, if designed properly, can be less biased than humans. In this work, we propose to automate the entire process of analyzing videos of behavior changes in zebrafish by using tools from computer vision, relying on minimal expert supervision. The overall objective of this work is to create a generalized tool to predict animal behaviors from videos using state-of-the-art deep learning models, with the dual goal of advancing understanding in biology and engineering a more robust and powerful artificial information processing system for biologists.
... Zebrafish offer a wide range of genetic models, including various skin to visual mutants, to probe the visual system functioning Gestri et al., 2012;Li, 2001;Ren et al., 2002). For example, zebrafish that lack iridophores (e.g., roy mutants) display a low contrast sensitivity to light, whereas genetic ablation of melanophores in albino mutants or both melanophores and iridophores in ruby mutants impairs escape response under dim, bright or low-contrast illumination (Ren et al., 2002). ...
Color is an important environmental factor that in multiple ways affects human and animal behavior and physiology. Widely used in neuroscience research, various experimental (animal) models may also improve our understanding of how different colors impact brain and behavioral processes. The zebrafish (Danio rerio) is rapidly emerging as an important novel model species to explore complex neurobehavioral processes. The growing utility of zebrafish in biomedicine makes it timely to consider the role of colors in their behavioral and physiological responses. Here, we summarize mounting evidence implicating colors as a critical variable in zebrafish models and neurobehavioral traits, with a particular relevance to CNS disease modeling, genetic and pharmacological modulation, as well as environmental enrichment and animal welfare. We also discuss the growing value of zebrafish models to study color neurobiology and neurobehavioral phenomics, and outline future directions of research in this field.
... Motor behaviors occur sequentially during the development of zebrafish , which includes early spontaneous tail curling, escape responses to touch and free swimming. Common visual behaviors, such as the visual startle response, phototaxis, and the optomotor and optokinetic responses have been extensively studied in zebrafish (Li, 2001;Mueller and Neuhauss, 2010). The visual system extracts optical information from the environment to guide common behaviors in fish, including predatory responses, socializing and breeding (Haug et al., 2010;Chen and Fernald, 2011). ...
Boscalid is a persistent fungicide that is frequently detected in surface waters and may be neurotoxic to aquatic organisms. Herein, we evaluated the effects of environmentally relevant boscalid concentrations to zebrafish to explore its potentially neurotoxic mechanisms of effect. Behavioral responses (swimming, phototaxis, and predation), histopathology, transcriptomics, biochemical parameter analysis and gene expression of larval and adult zebrafish following boscalid treatment were assessed. We found that boscalid significantly inhibited the locomotor ability and phototactic response of larvae after an 8-d exposure, and altered the locomotor activity, predation trajectories and ability in adults after a 21-d exposure. It was noted that predation rates of zebrafish were significantly decreased by 30% and 100% after exposure to 0.1 and 1.0 mg/L boscalid, respectively. Adverse alterations in the cell differentiation of eyes and brain injury were also observed in both larvae and adults following boscalid exposure. The expression of genes related to neurodevelopment, neurotransmission, eye development, and visual function, in conjunction with RNA-Seq results, indicated that boscalid may impair visual phototransduction and nervous system processes in larval zebrafish. Conclusively, boscalid exposure may affect the neurobehavioral response of zebrafish by impairing proper visual and nervous system function.
... Motor behaviors occur sequentially during the development of zebrafish , which includes early spontaneous tail curling, escape responses to touch and free swimming. Common visual behaviors, such as the visual startle response, phototaxis, and the optomotor and optokinetic responses have been extensively studied in zebrafish (Li, 2001;Mueller and Neuhauss, 2010). The visual system extracts optical information from the environment to guide common behaviors in fish, including predatory responses, socializing and breeding (Haug et al., 2010;Chen and Fernald, 2011). ...
... The zebrafish is a standard model system for vision studies because its retina is strikingly similar to that of mammals [27,45]. Sensory integration in zebrafish is 6/18 . ...
The analysis of fish behavior in response to odor stimulation is a crucial component of the general study of cross-modal sensory integration in vertebrates. In zebrafish, the centrifugal pathway runs between the olfactory bulb and the neural retina, originating at the terminalis neuron in the olfactory bulb. Any changes in the ambient odor of a fish's environment warrants a change in visual sensitivity and can trigger mating-like behavior in males due to increased GnRH signaling in the terminalis neuron. Behavioral experiments to study this phenomenon are commonly conducted in a controlled environment where a video of the fish is recorded over time before and after the application of chemicals to the water. Given the subtleties of behavioral change, trained biologists are currently required to annotate such videos as part of a study. This process of manually analyzing the videos is time-consuming, requires multiple experts to avoid human error/bias and cannot be easily crowdsourced on the Internet. Machine learning algorithms from computer vision, on the other hand, have proven to be effective for video annotation tasks because they are fast, accurate, and, if designed properly, can be less biased than humans. In this work, we propose to automate the entire process of analyzing videos of behavior changes in zebrafish by using tools from computer vision, relying on minimal expert supervision. The overall objective of this work is to create a generalized tool to predict animal behaviors from videos using state-of-the-art deep learning models, with the dual goal of advancing understanding in biology and engineering a more robust and powerful artificial information processing system for biologists.
... Based on these findings, it is conceivable that in response to olfactory stimulation, some of the dopamine-mediated inhibition of neural activity will be lifted and, as such, retinal functions such as visual sensitivity will be increased. To test this hypothesis, Li and coworkers (Maaswinkel and Li 2003;Huang et al. 2005;Li et al. 2017) measured the visual sensitivity of zebrafish in response to olfactory stimulation using a behavioral assay based on visually mediated escape responses (Li 2001). In those studies, amino acids (e.g., alanine, arginine, aspartate, and methionine) were chosen to stimulate olfactory neurons, because they are among the most effective odors for zebrafish (Edwards and Michel 2002). ...
Cross-modal sensory communication is an innate biological process that refers to the combination and/or interpretation of different types of sensory input in the brain. Often, this process conjugates with neural modulation, by which the neural signals that convey sensory information are adjusted, such as intensity, frequency, complexity, and/or novelty. Although the anatomic pathways involved in cross-modal sensory integration have been previously described, the course of development and the physiological roles of multisensory signaling integration in brain functions remain to be elucidated. In this article, I review some of the recent findings in sensory integration from research using the zebrafish models. In zebrafish, cross-modal sensory integration occurs between the olfactory and visual systems. It is mediated by the olfacto-retinal centrifugal (ORC) pathway, which originates from the terminalis nerve (TN) in the olfactory bulb and terminates in the neural retina. In the retina, the TNs synapse with the inner nuclear layer dopaminergic interplexiform cells (DA-IPCs). Through the ORC pathway, stimulation of the olfactory neurons alters the cellular activity of TNs and DA-IPCs, which in turn modulates retinal neural function and increases behavioral visual sensitivity.
... Zebrafish maintain high evolutionary proximity to mammals, and their retinas share great similarities to humans (e.g., structure, cellular organization, neural circuitry and signaling transmission) (Li, 2001;Vacaru et al., 2014). While much progress has been made to understand the anatomy of crossmodal circuitry in zebrafish, our knowledge of the underlying regulatory mechanism and physiological roles of centrifugal input to the retina is still in its nascent stage. ...
We propose a computational model of vision that describes the integration of cross-modal sensory information between the olfactory and visual systems in zebrafish based on the principles of the statistical extreme value theory. The integration of olfacto-retinal information is mediated by the centrifugal pathway that originates from the olfactory bulb and terminates in the neural retina. Motivation for using extreme value theory stems from physiological evidence suggesting that extremes and not the mean of the cell responses direct cellular activity in the vertebrate brain. We argue that the visual system, as measured by retinal ganglion cell responses in spikes/sec, follows an extreme value process for sensory integration and the increase in visual sensitivity from the olfactory input can be better modeled using extreme value distributions. As zebrafish maintains high evolutionary proximity to mammals, our model can be extended to other vertebrates as well.
... Zebrafish maintain high evolutionary proximity to mammals, and their retinas share great similarities to humans (e.g., structure, cellular organization, neural circuitry and signaling transmission) [26,47]. While much progress has been made to understand the anatomy of cross-modal circuitry in zebrafish, our knowledge of the underlying regulatory mechanism and physiological roles of centrifugal input to the retina is still in its nascent stage. ...
We propose a computational model of vision that describes the relationship between olfactory input and visual sensitivity of zebrafish based on the principles of the statistical extreme value theory. This relationship is mediated through centrifugal input which makes possible the physical connections between the neural retina and olfactory bulb. Motivation for using extreme value theory stems from physiological evidence suggesting that extremes and not the mean of the cell responses direct cellular activity in the vertebrate brain. We argue that the visual system, as measured by retinal ganglion cell responses in spikes/sec, follows an extreme value process for sensory integration and the increase in visual sensitivity from the olfactory input can be better modeled using extreme value distributions. As zebrafish maintains high evolutionary proximity to mammals, our model can be extended to other vertebrates as well.
... Hereto, fish are placed in a transparent round aquarium, with an opaque column in its center. A rotating drum containing a black segment in a white background positioned around the aquarium will elicit an escape response, as it triggers the fish to hide behind the central pole (Li 2001;Neuhauss 2003). Although the ER is a robust response, a major drawback of this assay is the fact that it takes large defects in visual function to be effectively picked up, making it difficult to ascribe it to an exact visual function (Cameron et al. 2013). ...
Due to the lack of axonal regeneration, age-related deterioration in the central nervous system (CNS) poses a significant burden on the wellbeing of a growing number of elderly. To overcome this regenerative failure and to improve the patient’s life quality, the search for novel regenerative treatment strategies requires valuable (animal) models and techniques. As an extension of the CNS, the retinofugal system, consisting of retinal ganglion cells that send their axons along the optic nerve to the visual brain areas, has importantly contributed to the current knowledge on mechanisms underlying the restricted regenerative capacities and to the development of novel strategies to enhance axonal regeneration. It provides an extensively used research tool, not only in amniote vertebrates including rodents, but also in anamniote vertebrates, such as zebrafish. Indeed, the latter show robust regeneration capacities, thereby providing insights into the factors that contribute to axonal regrowth and proper guidance, complementing studies in mammals. This review provides an integrative and critical overview of the classical and state-of-the-art models and methods that have been employed in the retinofugal system to advance our knowledge on the signaling pathways underlying the restricted versus robust axonal regeneration in rodents and zebrafish, respectively. In vitro, ex vivo and in vivo models and techniques to improve the visualization and analysis of regenerating axons are summarized. As such, the retinofugal system is presented as a valuable model to further facilitate research on axonal regeneration and to open novel therapeutic avenues for CNS pathologies.
... However, only ten percent of these mutations are thought to have functional consequences, i.e. the induced point mutations lead to a nonsense, missense or splice site mutation resulting in a phenotype (Beier, 2000;Kettleborough et al., 2013;Zan et al., 2003). A subsequent assessment of visual function in these mutant animals can reveal many new suitable model systems for retinal degeneration, e.g. in zebrafish and mice (Li, 2001;Maaswinkel et al., 2005;Won et al., 2011). An unavoidable coincidence of using such mutagenic agents however, is the chance of having multiple induced mutations per genome, potentially leading to a combined phenotype or the involvement of different genes contributing to one phenotype in some cases. ...
Over the last decade, huge progress has been made in the understanding of the molecular mechanisms underlying inherited retinal dystrophy (IRD), as well as in the development and implementation of novel therapies, especially in the field of gene therapy. The use of mutant animal models, either naturally occurring or generated by genetic modification, have contributed greatly to our knowledge on IRD. Yet, these mutant animal models do not always mimic the retinal phenotype that is observed in humans with mutations in the orthologous gene, often due to species-specific characteristics of the retina, and/or diverse functions of the gene products in different species. In this manuscript, we compare general and ocular characteristics of a series of widely used vertebrate animal models, i.e. zebrafish, chicken, rodents, cats, dogs, sheep, pigs and monkeys, in terms of genetic architecture and sequence homology, methods to modify genomes, anatomy of the eye, and structural details of the retina. Furthermore, we present an overview of mutant vertebrate animal models that have been used to study or develop treatments for the various genetic subtypes of IRD, and correlate the suitability of these models to the specific characteristics of each animal. Herewith, we provide tools that will help to select the most suitable animal model for specific research questions on IRDs in the future, and thereby assist in an optimal use of animals and resources to further increase our understanding of inherited retinal dystrophies, and develop novel treatments for these disorders.
Copyright © 2015. Published by Elsevier Ltd.
... The difference in recovery time between OKR (14 days) and OMR (25 days) after optic nerve injury may be due to the different neuronal circuits responsible for the OKR and OMR. In ontogenic studies of developing zebrafish, the OKR appears more quickly than OMR (Li, 2001). ...
The fish optic nerve regeneration process takes more than 100 days after axotomy and comprises four stages: neurite sprouting (1-4 days), axonal elongation (5-30 days), synaptic refinement (35-80 days) and functional recovery (100-120 days). We screened genes specifically upregulated in each stage from axotomized fish retina. The mRNAs for heat shock protein 70 and insulin-like growth factor-1 rapidly increased in the retinal ganglion cells soon after axotomy and function as cell-survival factors. Purpurin mRNA rapidly and transiently increased in the photoreceptors and purpurin protein diffusely increased in all nuclear layers at 1-4 days after injury. The purpurin gene has an active retinol-binding site and a signal peptide. Purpurin with retinol functions as a sprouting factor for thin neurites. This neurite-sprouting effect was closely mimicked by retinoic acid and blocked by its inhibitor. We propose that purpurin works as a retinol transporter to supply retinoic acid to damaged RGCs which in turn activates target genes. We also searched for genes involved in the second stage of regeneration. The mRNA of retinoid-signaling molecules increased in retinal ganglion cells at 7-14 days after injury and tissue transglutaminase and neuronal nitric oxide synthase mRNAs, RA-target genes, increased in retinal ganglion cells at 10-30 days after injury. They function as factors for the outgrowth of thick, long neurites. Here we present a retinoid-signaling hypothesis to explain molecular events during the early stages of optic nerve regeneration in fish.
... There are voluminous data of these sorts (see, e.g. Herdegen & Leah, 1998;Li, 2001;Gong et al., 2003;Hochheiser & Yanowitz, 2007), but it is often difficult to relate data obtained in one experiment (e.g. in an electrophysiology experiment) to data obtained in other experiments (e.g. morphological or gene expression experiments) (Yen & Jones, 1983;Okazaki et al., 2001;De Filipe, 2004;Gal et al., 2006;Jinno et al., 2007;Marmigere & Ernfors, 2007;Schulz et al., 2007). ...
Many kinds of neuroscience data are being acquired regarding the dynamic behaviour and phenotypic diversity of nerve cells. But as the size, complexity and numbers of 3D neuroanatomical datasets grow ever larger, the need for automated detection and analysis of individual neurons takes on greater importance. We describe here a method that detects and identifies neurons within confocal image stacks acquired from the zebrafish brainstem. The first step is to create a template that incorporates the location of all known neurons within a population – in this case the population of reticulospinal cells. Once created, the template is used in conjunction with a sequence of algorithms to determine the 3D location and identity of all fluorescent neurons in each confocal dataset. After an image registration step, neurons are segmented within the confocal image stack and subsequently localized to specific locations within the brainstem template – in many instances identifying neurons as specific, individual reticulospinal cells. This image-processing sequence is fully automated except for the initial selection of three registration points on a maximum projection image. In analysing confocal image stacks that ranged considerably in image quality, we found that this method correctly identified on average ∼80% of the neurons (if we assume that manual detection by experts constitutes ‘ground truth’). Because this identification can be generated approximately 100 times faster than manual identification, it offers a considerable time savings for the investigation of zebrafish reticulospinal neurons. In addition to its cell identification function, this protocol might also be integrated with stereological techniques to enhance quantification of neurons in larger databases. Our focus has been on zebrafish brainstem systems, but the methods described should be applicable to diverse neural architectures including retina, hippocampus and cerebral cortex.
... Similarly, sphingosylphosphorylcholine (SPC, a lipid mediator) KO zebrafish perform spontaneous erratic movements and escape behaviors (e.g., rapid turning) without provocation from stressful stimuli (Patton and Zon 2001). with morphological, electrophysiological, cellular, and molecular methods have proven to be powerful tools for examining this complex system (Li 2001). Advances have been made in regards to the isolation and classification of zebrafish with visual system deficits (Brockerhoff et al 1995). ...
Zebrafish have traditionally been used as effective genetic and developmental models in biomedical research. Recently, the
scope and utility of zebrafish in biomedical research has been further expanded with the implementation of new genetic techniques
aimed at developing translational models of human pathogenesis. Additionally, screens measuring specific neurobehavioral and
developmental phenotypes have proven to be very robust. This chapter further discusses the utility of zebrafish in biomedical
research and highlights some of the genetic techniques used in the creation of transgenic and mutant strains. Behavioral phenotypes
of genetically altered zebrafish are also discussed with respect to both their robust stress responses and similarity to human
disorders. Specific emphasis is placed on human brain pathogenesis and neurodevelopmental abnormalities, especially as they
relate to stress and anxiety spectrum disorders.
Key wordsZebrafish-Genetics-Translational models
... At 52 hpf, newly differentiating rod and cone photoreceptors begin expressing opsin genes ( Nawrocki et al. 1985;Raymond et al. 1995;Schmitt and Dowling 1996;Schmitt and Dowling 1999;Vihtelic et al. 1999;Soules and Link 2005;Dahm et al. 2007). By 72 hpf, the lens is spherical and the visual system is functional based on behavioral tests (Soules and Link 2005;Easter and Nicola 1996;Bilotta 2000;Li 2001;Neuhauss 2003;Greiling and Clark 2009). ...
The zebrafish lens opaque (lop) mutant was previously isolated in a genetic screen and shown to lack rod and cone photoreceptors and exhibit lens opacity, or cataract, at 7 days post-fertilization (dpf). In this manuscript, we provide four different lines of evidence demonstrating that the lop phenotype results from a defect in the cdipt (phosphatidylinositol (PI) synthase; CDP-diacylglycerol-inositol 3-phosphatidyltransferase) gene. First, DNA sequence analysis revealed that the lop mutant contained a missense mutation in the lop open reading frame, which yields a nonconservative amino acid substitution (Ser-111-Cys) within the PI synthase catalytic domain. Second, morpholino-mediated knockdown of the cdipt-encoded PI synthase protein phenocopied the cdiptlop/lop mutant, with abnormal lens epithelial and secondary fiber cell morphologies and reduced numbers of photoreceptors. Third, microinjection of in vitro transcribed, wild-type cdipt mRNA into 1–4 cell stage cdiptlop/lop embryos significantly reduced the percentage of larvae displaying lens opacity at 7 dpf. Fourth, a cdipt retroviral-insertion allele, cdipthi559, exhibited similar lens and retinal abnormalities and failed to complement the cdiptlop mutant phenotype.
... What is particularly attractive about the RGC as a model neuron is the accessibility of the entire cell-the soma, dendrites, axons, and synapses in visual centers-for both visualization and experimental manipulation. For these reasons, more so than with any other system, the complete establishment of the connectivity of this neuron has been explored: from its connections with upstream retinal targets, to all guidance choice points in the path out to brain targets (Mann et al., 2004;Erskine and Herrera, 2007;Haupt and Huber, 2008;Petros et al., 2008), to the specific formation of synapses with postsynaptic partners (McLaughlin and O'Leary, 2005;Torborg and Feller, 2005;Huberman et al., 2008), to the behavioral consequences of defects in the development of visual circuitry (Baier, 2000;Li, 2001;Neuhauss, 2003;Portugues and Engert, 2009). As such, it is becoming clear that single extrinsic factors play multiple roles in their control of the establishment of connectivity. ...
Neurons acquire a unique cell-type dependent morphology during development that is critical for their function in a neural circuit. The process involves a neuron sending out an axon that grows in a directed fashion to its target, and the elaboration of multiple, branched dendrites. The ultimate morphology of the neuron is sculpted by factors in the environment that act directly or indirectly to influence the behavior of the growing axon and dendrites. The output neuron of the retina, the retinal ganglion cell (RGC), has served as a useful model for the identification of molecular signals that control neuronal morphogenesis, because the entire development of the neuron, from the initiation of neurites to the establishment of synapses, is accessible for experimental manipulation and visualization. In this review we discuss data which argue that the visual system uses a limited number of signals to control RGC morphogenesis, with single molecules being reused multiple times to control distinct events in axon and dendrite outgrowth.
... In the following, a diverse array of endpoints using apparatus from simple, one chambered tests, to complex multi-chamber, decisionbased challenges are discussed (Table 1). The majority of the behavioural endpoints can be conducted on zebrafish 3–4 days post fertilization [17,18], excepting learning/memory-based endpoints. ...
Altered neurological function will generally be behaviourally apparent. Many of the behavioural models pioneered in mammalian models are portable to zebrafish. Tests are available to capture alterations in basic motor function, changes associated with exteroceptive and interoceptive sensory cues, and alterations in learning and memory performance. Excepting some endpoints involving learning, behavioural tests can be carried out at 4 days post fertilization. Given larvae can be reared quickly and in large numbers, and that software solutions are readily available from multiple vendors to automatically test behavioural responses in 96 larvae simultaneously, zebrafish are a potent and rapid model for screening neurological impairments. Coupling current and emerging behavioural endpoints with molecular techniques will permit and accelerate the determination of the mechanisms behind neurotoxicity and degeneration, as well as provide numerous means to test remedial drugs and other therapies. The emphasis of this review is to highlight unexplored/underutilized behavioural assays for future studies. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.
... Zebrafish (Danio rerio) have become an important model for visual neuroscience (reviewed in Taylor et al., 2000;Li, 2001;Goldsmith & Harris, 2003). Knowledge of zebrafish photoreceptor spectral absorbance properties have important implications for methodologies that employ spectral sensitivity measures or spectral stimuli to elicit innate behaviors for the purposes of screening and evaluating the visual system mutant fish (Neuhauss et al., 1999;Van Epps et al., 2001;Krauss & Neumeyer, 2003;Neuhauss, 2003). ...
... Retinal ganglion cells are restricted to the ganglion cell layer. The neural processes and synapses between different retinal cell types are mainly found in the outer-and inner-plexiform layers [1] [2]. ...
The zebrafish (Danio rerio) has recently become a mainstream model system for genetic studies of human diseases, such as neurological degenerative diseases, heart diseases, immuno-system disorders, etc. In this article, we will review some recent findings of the usefulness of zebrafish as a model vertebrate for behavioral screening of mutations in vertebrate visual system, for example, genes involved in age-related retinal degeneration.
... In larval zebrafish, there are two categories of behavior that have been studied effectively and in detail: visual behaviors and the motor control of swimming and escape. As zebrafish larvae need to blend into different backgrounds, capture prey, and avoid predators at early stages, a functional visual system is established by 5 dpf (reviewed by Baier 2000;Fleisch and Neuhauss 2006;Li 2001;Neuhauss 2003). The simplest assay is for visual background adaptation, where larvae on dark surfaces disperse melanin granules so as to appear dark while they constrict these granules to blend into a light background. ...
Over recent years, several groundbreaking techniques have been developed that allow for the anatomical description of neurons, and the observation and manipulation of their activity. Combined, these approaches should provide a great leap forward in our understanding of the structure and connectivity of the nervous system and how, as a network of individual neurons, it generates behavior. Zebrafish, given their external development and optical transparency, are an appealing system in which to employ these methods. These traits allow for direct observation of fluorescence in describing anatomy and observing neural activity, and for the manipulation of neurons using a host of light-triggered proteins. Gal4/Upstream Activating Sequence techniques, as they are based on a binary system, allow for the flexible deployment of a range of transgenes in expression patterns of interest. As such, they provide a promising approach for viewing neurons in a variety of ways, each of which can reveal something different about their structure, connectivity, or function. In this study, the author will review recent progress in the development of the Gal4/Upstream Activating Sequence system in zebrafish, feature examples of promising studies to date, and examine how various new technologies can be used in the future to untangle the complex mechanisms by which behavior is generated.
... Finally, a variety of genomic resources are available for working with zebrafish, including databases of expressed sequence tags, high-density genetic linkage and radiation hybrid maps, and an ongoing effort to sequence the zebrafish genome (Amemiya et al., 1999; Clark et al., 2001; Hukriede et al., 1999; Shimoda et al., 1999; Vogel, 2000). For these reasons, zebrafish have become a model organism for studies of such disparate subjects as immunology, gastrulation, and the function and evolution of visual systems (Kodjabachian et al., 1999; Li, 2001; Trede et al., 2001). Although recent work emphasizes the zebrafish as a model organism in the laboratory, D. rerio occurs in nature, as do 45–50 other described species in the genus Danio, as well as an untold number of undescribed species from India to southeast Asia (Fang, 1997aFang, ,b, 1998Fang, , 2000 Fang and Kottelat, 1999; McClure, 1999; Parichy and Johnson, 2001). ...
The zebrafish Danio rerio is an emerging model organism for understanding vertebrate development and genetics. One trait of both historical and recent interest is the pattern formed by neural crest-derived pigment cells, or chromatophores, which include black melanophores, yellow xanthophores, and iridescent iridophores. In zebrafish, an embryonic and early larval pigment pattern consists of several stripes of melanophores and iridophores, whereas xanthophores are scattered widely over the flank. During metamorphosis, however, this pattern is transformed into that of the adult, which comprises several dark stripes of melanophores and iridophores that alternate with light stripes of xanthophores and iridophores. In this review, we place zebrafish relative to other model and non-model species; we review what is known about the processes of chromatophore specification, differentiation, and morphogenesis during the development of embryonic and adult pigment patterns, and we address how future studies of zebrafish will likely aid our understanding of human disease and the evolution of form.
... This, however, requires a profound knowledge of the wildtype . Optomotor response and optomotor nystagmus are behaviors which can be applied in zebrafish rather easily (Bilotta & Saszik, 2001; Li, 2001). ...
The action spectrum of motion detection in zebrafish (Danio rerio) was measured using the optomotor response in the light adapted state. The function has a single maximum at 550-600 nm, and is similar to the spectral sensitivity function of the L-cone type in the mid and long wavelength range. At shorter wavelengths the values of three of the five fish tested are lower. As in goldfish [Vis. Res. 36 (1996) 4025], the result indicates a dominance of the L-cone type with an inhibitory influence of M- or S-cones. Experiments with a red/green striped cylinder showed that the optomotor response was at minimum whenever the L-cone type was not modulated by the moving pattern. This demonstrates that motion vision in zebrafish is "color blind", using mainly one of the four cone types probably involved in color vision.
... The teleost retina has various properties, including a layered structure, a regular mosaic of photoreceptors, and continuous growth throughout life (Lyall, 1957a,b), which have been particularly valuable in this regard. For example, the study of the visual system in goldfish and carp has revealed much about the mechanisms underlying color vision (Kamermans and Spekreijse, 1999), whereas the zebrafish retina has become a popular model to study CNS development (Bilotta and Saszik, 2001;Li, 2001). Furthermore, the diversity of habitats and life histories that teleosts have exploited allow an examination of the evolution of CNS function and development. ...
In order to facilitate emerging models of retinal development, we developed electroretinogram and in situ hybridization protocols to examine the ontogeny of photoreceptors in the retina of a land-locked salmonid, the rainbow trout (Oncorhynchus mykiss). We cloned cDNA fragments corresponding to the rod opsin and each of the four cone opsin gene families, which we utilized to produce riboprobes. We established the specificity of the in situ hybridization protocol by examining subcellular signal localization and through double-labeling experiments. We confirm the assumption that the accessory corner cones in the square mosaic are the ultraviolet wavelength-sensitive (UVS) cone photoreceptor (i.e., they express an SWS1 opsin) and observed UVS cones throughout the retina of small trout. Larger fish have a decrease in sensitivity to short wavelength light stimuli and the distribution of UVS cones in the mature retina is limited to the dorsal-temporal quadrant. These larger fish also possess differentiated UVS cones in the peripheral germinal zone (PGZ), including within areas peripheral to mature retina lacking UVS cones. These data are consistent with the loss of putative UVS cones from the PGZ of a migratory salmonid of another genus, and thus the disappearance of UVS cones appears to be general to the Family Salmonidae, regardless of life history strategy. The generation, differentiation, and subsequent loss of UVS cones in the smolt PGZ is a dramatic example of the supposition that the mechanisms of PGZ development recapitulate the retinal embryogenesis of that species.
... The visual system is particularly well suited for a genetic analysis, since it develops extraordinary rapidly in the zebrafish and is functional early on in larval development. Only 5 days post fertilization (dpf), the retina is well layered with all cell types present, retinal ganglion cell axons have arborized on their main target region in the brain, the optic tectum, and the maturity of the visual system is evidenced by a number of visually mediated behaviors [1][2][3][4]. Additionally, sum field potentials of the retina in response to light can readily be recorded by electroretinography [2,[5][6][7]. ...
The recessive zebrafish mutant bleached has, apart from its defects in pigmentation, a heritable defect leading to larval blindness. Here, we analyze the retina of homozygous bleached larvae, employing morphological and electrophysiological methods. Electroretinography revealed a complete lack of electrical signals in response to light. Histological analysis of mutant retinae showed a severely affected outer retina with a hypopigmented pigment epithelium and a disorganized outer nuclear layer containing few or no intact photoreceptors. Using the TUNEL assay for cell death detection, we noticed a strong increase of apoptotic cells in all retinal cell layers, starting in young larvae even before retinal support of visual function. At later stages cell death is most pronounced at the marginal zone, where new cells are constantly added to the retina. At early stages increased apoptosis is mainly confined to the retina, while at later stages elevated cell death is al so apparent in extra-retinal tissues, particularly in the brain. Hence, the lack of visual responses in homozygous bleached larvae can be attributed to a severe defect of the outer retina, preceded by increased levels of apoptotic cell death in all retinal cell layers.
... Finding too many mutants is potentially disquieting (recall ''mouse number 25''). But this rarely happens, and that that's so is instructive: One usually isolates only a handful of mutants per year that are defective in a given behavior (exception: nematode, with its very high rate of genetic throughput, which may be on the cusp of being rivaled by zebrafish, vertebrate-neurogenetics-wise, e.g., Baier, 2000; Li, 2001). This indicates further that not any sort of gene change can disrupt a given process. ...
... Methods for behavioral analysis of zebrafish visual sensitivity have been previously described (Li and Dowling, 1997; for a review, see Li, 2001). The test apparatus consisted of a circular plastic container, surrounded by a rotating drum. ...
Using a behavioral assay based on visually mediated escape responses, we measured long-wavelength-sensitive red cone (LC) sensitivities in zebrafish. In a 24 h period, the zebrafish were least sensitive to red light in the early morning and most sensitive in the late afternoon. To investigate if the fluctuation of behavioral cone sensitivity correlates with opsin gene expression, we measured LC opsin mRNA expression at different times in the day and night under different lighting conditions. Under a normal light-dark cycle, the expression of LC opsin mRNA determined by real-time RT-PCR was low in the early morning and high in the late afternoon, similar to the fluctuation of behavioral cone sensitivity. This rhythm of LC opsin mRNA expression, however, dampened out gradually in constant conditions. After 24 h of constant light (LL), the expression of LC opsin mRNA dropped to levels similar to those determined in the early morning in control animals. By contrast, when the zebrafish were kept in constant dark (DD), the expression of LC opsin mRNA increased, to levels about 30-fold higher than the expression in the early morning in control animals. This day-night fluctuation in LC opsin mRNA expression was correlated to changes in opsin density in the outer segment of cone photoreceptor cells. Microspectrophotometry (MSP) measurements found significant differences in red cone outer segment optical density with a rhythm following the behavioral sensitivity. Furthermore, dopamine modulated the circadian rhythms in expression of LC opsin mRNA. Administration of dopamine increased LC opsin mRNA expression, but only in the early morning.
... Zebrafish (Danio rerio) have become an important model for visual neuroscience (reviewed in Taylor et al., 2000;Li, 2001;Goldsmith & Harris, 2003). Knowledge of zebrafish photoreceptor spectral absorbance properties have important implications for methodologies that employ spectral sensitivity measures or spectral stimuli to elicit innate behaviors for the purposes of screening and evaluating the visual system mutant fish (Neuhauss et al., 1999;Van Epps et al., 2001;Krauss & Neumeyer, 2003;Neuhauss, 2003). ...
Numerous reports have concluded that zebrafish ( Danio rerio ) possesses A 1 -based visual pigments in their rod and cone photoreceptors. In the present study, we investigated the possibility that zebrafish have a paired visual pigment system. We measured the spectral absorption characteristics of photoreceptors from zebrafish maintained in different temperature regimes and those treated with exogenous thyroid hormone using CCD-based microspectrophotometry. Rods from fish housed at 15°C and 28°C were not significantly different, having λ max values of 503 ± 5 nm ( n = 106) and 504 ± 6 nm ( n = 88), respectively. Thyroid hormone treatment (held at 28°C), however, significantly shifted the λ max of rods from 503 ± 5 nm ( n = 194) to 527 ± 8 nm ( n = 212). Cone photoreceptors in fish housed at 28°C (without thyroid hormone treatment) had λ max values of 361 ± 3 nm ( n = 2) for ultraviolet-, 411 ± 5 nm ( n = 18) for short-, 482 ± 6 nm ( n = 9) for medium-, and 565 ± 10 nm ( n = 14) for long-wavelength sensitive cones. Thyroid hormone treatment of fish held at 28°C significantly shifted the λ max of long-wavelength sensitive cones to 613 ± 11 nm ( n = 20), substantially beyond that of the λ max of the longest possible A 1 -based visual pigment (∼580 nm). Thyroid hormone treatment produced smaller shifts of λ max in other cone types and increased the half-band width. All shifts in photoreceptor λ max values resulting from thyroid hormone treatment matched predictions for an A 1 - to A 2 -based visual pigment system. We therefore conclude that zebrafish possess a rhodopsin–porphyropsin interchange system that functions to spectrally tune rod and cone photoreceptors. We believe that these observations should be carefully considered during analysis of zebrafish spectral sensitivity.
... By understanding the molecular basis for the pathological changes that occur during hereditary diseases of the eye anterior segment, rational approaches to treatment may be formulated. Although zebrafish mutants were identified with developmental and age-related forms of retinal degeneration (Brockerhoff et al., 1995;Malicki et al., 1996;Fadool et al., 1997;Li, 2001;Maaswinkel et al., 2003), relatively few mutants were characterized with abnormalities in the other eye tissues, such as the lens (Link et al., 2001;Vihtelic and Hyde, 2002). The identification of additional lens mutants using tools to detect more subtle phenotypes will be valuable to further dissect the interactions that occur during development and maintenance of the lens and its affect on the other eye tissues. ...
The zebrafish lens opaque (lop) mutant was identified in a chemical mutagenesis screen. The lop mutant, which develops normally through 4 days postfertilization (dpf), exhibits several signs of lens and retinal degeneration at 7 dpf. Histology revealed disrupted lens fibers and increased numbers of nucleated cells within the mutant lens and anterior chamber. The mutant lens also exhibited aberrant epithelial cell morphologies and lacked a definitive transition zone, which suggests that secondary fiber differentiation was interrupted. In addition, the mutant exhibits severely reduced photoreceptors and a reduction in the number of horizontal cells at 7 dpf. Other retinal cell classes appeared unaffected in the mutant. Transmission electron microscopy and opsin immunohistochemistry showed that the different photoreceptor types were generated at the retinal margin, but the rods and cones failed to mature and disappeared. The mutant lens and retina also displayed increased cell proliferation based on proliferating cell nuclear antigen immunolabeling, suggesting that the lens opacity was due to unregulated cell proliferation and undifferentiated cell accumulation within the mutant lens. The lop mutant phenotype supports recent studies showing the lens has a role in regulating teleost retinal development.
... GnRH receptors were found to be expressed in both DA-IPCs and RGCs (Grens et al. 2005). Zebrafish (Danio rerio) have recently emerged as a model vertebrate for genetic and physiological studies of visual modulation (for reviews, see Baier, 2000; Li, 2001). By screening the F1 generation of chemically mutagenized zebrafish, Li & Dowling (2000a) isolated a dominant mutant (nbb) characterized by disruption ...
The vertebrate retina receives centrifugal input from the brain. In zebrafish, the major centrifugal input originates in the terminal nerve (TN). TN cell bodies are located in the olfactory bulb and ventral telencephalon. The TN projects axons to the retina where they branch in the inner plexiform layer (IPL) and synapse onto several inner retinal cell types, including dopaminergic interplexiform cells (DA-IPCs). This olfactoretinal centrifugal input plays a role in modulating retinal ganglion cell (RGC) activity, probably via dopamine-mediated Ca2+ signalling pathways. Normally, dopamine inhibits RGC firing by decreasing the inward Ca2+ current. Olfactory stimulation with amino acids decreases dopamine release in the retina, thereby reducing dopaminergic inhibition of RGCs. This model of olfacto-visual integration was directly tested by recording single-unit RGC activity in response to olfactory stimulation in the presence or absence of dopamine receptor blockers. Stimulation of the olfactory neurones increased RGC activity. However, this effect diminished when the dopamine D1 receptors were pharmacologically blocked. In isolated RGCs, the application of dopamine or a dopamine D1 receptor agonist decreased voltage-activated Ca2+ current and lowered Ca2+ influx. Together, the data suggest that olfactory input has a modulatory effect on RGC firing, and that this effect is mediated by dopamine D1 receptor-coupled Ca2+ signalling pathways.
... The amenability of zebrafish to forward genetic screens offers the potential to link genes to neuronal circuits and to behavior (Guo 2004). For example, assays for studying behavior related to vision and locomotion have been established, and mutations affecting these processes have been identified (Baier 2000;Drapeau et al. 2002;Goldsmith & Harris 2003;Li 2001;Neuhauss 2003;Watkins et al. 2004). In contrast, rather little has been done on reward-associated behavior in zebrafish (Darland & Dowling 2001;Gerlai et al. 2000;Lockwood et al. 2004). ...
Both natural rewards and addictive substances have the ability to reinforce behaviors. It has been unclear whether identical neural pathways mediate the actions of both. In addition, little is known about these behaviors and the underlying neural mechanisms in a genetically tractable vertebrate, the zebrafish Danio rerio. Using a conditioned place preference paradigm, we demonstrate that wildtype zebrafish exhibit a robust preference for food as well as the opiate drug morphine that can be blocked by the opioid receptor antagonist naloxone. Moreover, we show that the too few mutant, which disrupts a conserved zinc finger-containing gene and exhibits a reduction of selective groups of dopaminergic and serotonergic neurons in the basal diencephalon, displays normal food preference but shows no preference for morphine. Pretreatment with dopamine receptor antagonists abolishes morphine preference in the wildtype. These studies demonstrate that zebrafish display measurable preference behavior for reward and show that the preference for natural reward and addictive drug is dissociable by a single-gene mutation that alters subregions of brain monoamine neurotransmitter systems. Future genetic analysis in zebrafish shall uncover further molecular and cellular mechanisms underlying the formation and function of neural circuitry that regulate opiate and food preference behavior.
... The function of the olfactoretinal projection in fish is not well understood, making the nbb/ phenotype especially valuable. While a previous study suggested that olfactoretinal input alters the receptive field size of retinal horizontal cells (Umino and Dowling, 1991), studies in the nbb mutant were the first to show effects on visual behavior, dark adaptation, and circadian fluctuation of visual sensitivity (Li and Dowling, 2000; Li, 2001). A more recent study in wild-type zebrafish provided evidence that amino acid odorants increase retinal sensitivity, an effect that could very well be mediated by the terminal nerve. ...
Behavioral functions are carried out by localized circuits in the brain. Although this modular principle is clearly established, the boundaries of modules, and sometimes even their existence, are still debated. Zebrafish might offer distinct advantages in localizing behaviors to discrete brain regions because of the ability to visualize, record from, and lesion precisely identified populations of neurons in the brain. In addition, genetic screens in zebrafish enable the isolation of mutations that disrupt neural pathways and/or behaviors, as an alternative lesioning technique with complementary strengths to laser ablations. For example, the Mauthner cell, a large identified neuron in the hindbrain, has been postulated to be both necessary and sufficient for the execution of escapes. We discuss in this review how experiments, using laser ablations, calcium imaging, and mutants have eroded this notion. Even in a simple behavior, such as escape, many parallel pathways appear to be involved with no single one being absolutely necessary. Lesion studies and the analysis of behavioral mutants are now also beginning to elucidate the functional architecture of the zebrafish visual system. Although still in an embryonic stage, the neuroanatomy of behaviors in zebrafish has a bright future.
Ophthalmological disorders causing impaired vision is a global problem affecting millions of individuals. Animal models having the same type of ocular pathology as humans and allowing high- to medium-throughput screening are needed to understand the pathophysiology of ocular disorders. High prolificity, less cost, ease of mutagenesis and similar ocular morphology render zebrafish as a better animal model compared to rodents. Zebrafish can be used as an animal model in several ocular diseases. Thus zebrafish can be useful not only to understand the pathophysiology of ophthalmological diseases but also to facilitate the development of gene-mediated and regeneration therapies for the successful management of the disease. This review discusses the advantages of zebrafish as an animal model, visual system of zebrafish with its similarities and differences from the human eye, embryology of the eye in zebrafish, visual behavioural assays, and role of zebrafish as an animal model in several ocular diseases, including aniridia, retinitis pigmentosa, Leber congenital amaurosis, diabetic retinopathy, age-related macular degeneration, corneal dystrophy, cataract, glaucoma, coloboma, microphthalmia, anophthalmia, cyclopia, etc. This review also reveals the role of zebrafish in preclinical ocular toxicity test, gene-based therapeutic management, ocular drug discovery, and the study of retinal regeneration.KeywordsZebrafishOcular diseaseVisual behavioural assayGene therapyDrug developmentRegeneration
Millions of people are affected by visual impairment and blindness globally, and the prevalence of vision loss is likely to increase as we are living longer. However, many ocular diseases remain poorly controlled due to lack of proper understanding of the pathogenesis and the corresponding lack of effective therapies. Consequently, there is a major need for animal models that closely mirror the human eye pathology and at the same time allow higher-throughput drug screening approaches. In this context, zebrafish as an animal model organism not only address these needs but can in many respects reflect the human situation better than the current rodent models. Over the past decade, zebrafish have become an established model to study a variety of human diseases and are more recently becoming a valuable tool for the study of human ophthalmological disorders. Many human ocular diseases such as cataract, glaucoma, diabetic retinopathy, and age-related macular degeneration have already been modelled in zebrafish. In addition, zebrafish have become an attractive model for pre-clinical drug toxicity testing and are now increasingly used by scientists worldwide for the discovery of novel treatment approaches. This review presents the advantages and uses of zebrafish for ophthalmological research.Eye advance online publication, 7 February 2014; doi:10.1038/eye.2014.19.
Fish have adapted to a wide range of habitats, including estuaries, deep sea, lakes, and caves. Accordingly, the characteristics of the visual system vary considerably among species. This chapter presents the research carried out in a small number of species belonging to the cyprinids, such as goldfish (Carassius auratus) and zebrafish (Danio rerio). Visual sensitivity can be analyzed along three dimensions: absolute, contrast, and spectral sensitivity. The chapter focuses on absolute sensitivity. It describes the characteristics of the visual system in teleost fish and presents current research about absolute visual sensitivity. Circadian modulation of absolute sensitivity and the roles of neurotransmitters, such as dopamine and melatonin are described. The chapter also describes some of the factors that may impair absolute sensitivity. When assessing absolute, contrast, or spectral visual sensitivity, one has to distinguish the level of processing. Visual sensitivities, as determined by photon catch, electroretinogram (ERG) or retinal ganglion cells (RGC) recordings, or behavioral escape responses, optomotor response (OMR), optokinetic response (OKR), are different parameters that do not always correlate.
Zebrafish, as a new model of animal, has been gradually accepted and widely used in biology research due to its special characteristics. Besides its convenience used in development research, the application in ethology is more and more extensively. Because of the transparency before 2 days post-fertilization, the eye size nearly half of the brain and its conspicuous circadian rhythm, zebrafish has been significantly applied in vision research. The organs of olfactory and auditory are both visible on the surface, such structure makes it very easy to detect the olfactory and auditory function by means of behavior experiments. Observation of motion is very convenience as zebrafish's propensity is active. It is also used more and more in social biology research. The behavior test of zebrafish is a simple and effective method to analyze the integrative neuronal function in vivo while some experimental models have been established which reviewed as follows.
Visual sensitivity to ultraviolet (UV) light is widespread in the animal kingdom. Many studies on UV vision have utilized physiological and/or anatomical methods to determine whether animals are visually sensitive to UV wavelengths. However, ultimately behavioral methods can reveal whether retinal UV sensitivity results in perceptual detection of UV stimuli. For the widely studied zebrafish (Danio rerio), the adult retina possesses cone photoreceptors that are sensitive to UV light. Here, we used a behavioral technique, the escape response assay, to test whether adult zebrafish can visually detect and behaviorally respond to visual cues that reflect UV. We found that adult zebrafish robustly respond to UV reflecting cues under UV light while showing no responses to the same cues under no UV light. From our results, we confirm that adult zebrafish can visually detect UV reflecting cues and show that UV perceptual sensitivity is functional in adult zebrafish. Our study highlights the utility of the fish escape response assay for UV visual behavior research.
Visual impairment and blindness is widespread across the human population, and the development of therapies for ocular pathologies is of high priority. The zebrafish represents a valuable model organism for studying human ocular disease; it is utilized in eye research to understand underlying developmental processes, to identify potential causative genes for human disorders, and to develop therapies. Zebrafish eyes are similar in morphology, physiology, gene expression, and function to human eyes. Furthermore, zebrafish are highly amenable to laboratory research. This review outlines the use of zebrafish as a model for human ocular diseases such as colobomas, glaucoma, cataracts, photoreceptor degeneration, as well as dystrophies of the cornea and retinal pigmented epithelium.
In the development and use of animal models of cognitive dysfunction, it is important to develop complementary models to exploit the unique advantages of the different species. Nonmammalian vertebrates such as fish provide the opportunity to directly observe neurodevelopmental processes and determine the impact of developmental permutations on learning and memory. Zebrafish in particular are valuable because of the availability of morpholine techniques to transiently suppress specific parts of genomic expression. Invertebrate models such as C. elegans and drosophila provide other advantages, particularly the elegant genetic manipulations available. The simple nervous systems in these models are useful in determining mechanisms of cognitive function. The development of new methods for high-throughput tests of cognitive function for fish can provide a means for rapid screening of potential toxic agents as well as promising therapeutic agents. It is equally important to develop specific tests of various aspects of cognitive function, including habituation, associative learning, memory, and attention as well as to be able to differentiate changes in sensorimotor function from cognition. Key in the use of nonmammalian models is the determination of which mechanisms of cognitive function are similar to mammals and which are different. Nonmammalian models can be used in concert with classic mammalian models to determine the neural bases of cognitive function and to aid in the discovery of toxicants and potential therapeutic agents.
Vertebrate central nervous systems (CNS) contain hundreds or thousands of distinct nerve cell types with specialized morphologies and functions. As neural systems of the CNS are disrupted in diverse situations, including neurodegenerative diseases, stroke, and spinal cord injury, it is important to analyze and understand the organization and function of these systems and their components, i.e. neurons, in detail. Current imaging technology provides neurobiologists possibility of looking into the brain of an intact vertebrate to observe in vivo activities at the single cell level. However, the huge amount of data generated by these new imaging technologies makes it challenging for a neurobiologist to extract the desired information out of these large datasets. The need for automated neuron detection and analysis techniques in these datasets becomes increasingly important as the number and size of datasets grow at an increasing pace. In this work we describe a method that was developed to detect and identify neurons within 3D confocal z-stacks acquired from the zebrafish brainstem. In our method we first register z-stacks into a normalized space and design a pattern in the normalized space that determines the location of all known neurons in the brainstem. The next step is to segment neurons in the 3D z-sacks using a contour based segmentation method. The algorithm then assigns all segmented neurons to specific locations within the brainstem. The registration requires user interaction while the segmentation and neuron identification stages are fully automated.
Zebrafish possess three classes of chromatophores that include iridophores, melanophores, and xanthophores. Mutations that lack one or two classes of chromatophores have been isolated or genetically constructed. Using a behavioral assay based on visually mediated escape responses, we measured the visual response of fully and partially pigmented zebrafish. In zebrafish that lack iridophores (roy mutants), the behavioral visual responses were similar to those of wild-type animals except at low contrast stimulation. In the absence of melanophores (albino mutants) or both melanophores and iridophores (ruby mutants), the behavioral visual responses were normal under moderate illumination but reduced when tested under dim or bright conditions or under low contrast stimulation. Together, the data suggest that screening pigments in the retina play a role in the regulation of behavioral visual responses and are necessary for avoiding "scatter" under bright light conditions.
The developmental history of the vertebrate eye begins at an early embryonic stage, with the formation of the body axes and induction of neural tissue. Several recent experimental embryological and genetic studies in teleost fish have produced new insights into the morphogenetic and molecular regulation of eye formation. Molecular signaling pathways and patterned expression of transcription factors implicated in eye determination are discussed, and the importance of morphogenetic cell movements is emphasized.
The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1-2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology.
Excerpt
The next step following completion of the first draft ofthe sequence of the human genome is to overlay this information with a complementary functional genomic mapof the location of disease-causing genes and those involved in the regulation of normal function. To this end,our group has been developing two chromosomal substitution panels of 44 strains of consomic rats in which onechromosome at a time from a normal BN (Brown Norway) rat has been introgressed onto the genetic backgrounds of the SS/JrHsd/Mcw Dahl salt-sensitive (SS)and FHH/Eur Fawn Hooded Hypertensive rats. Thesestrains were chosen because they exhibit >60% of all thegenetic variation observed among 48 inbred strains ofrats. In addition, the SS rat is a well-accepted model forsalt-sensitive hypertension, insulin resistance, hyperlipidemia, endothelial dysfunction, vascular injury, cardiachypertrophy, and glomerulosclerosis, whereas the FHHrat is a model of systolic hypertension, renal disease, pulmonary hypertension, a bleeding disorder, alcoholism,and depression. The chromosomal location of genes andpathways involved in the regulation of many cardiovascular functions can be identified by simply phenotyping6–8 rats of each of the consomic strains and comparingthe response to the parental strain and the other consomicstrains. The region of the genome mediating the trait canbe narrowed by phenotyping an F2 population generatedfrom the consomic and parental strains and isolated to asingle locus model in congenic sublines in 6 months. Thedisease-causing or disease-resistance gene can then beidentified by expression profiling and/or direct sequencing. The consomic strains are currently being screenedfor 203 traits related to renal, pulmonary, vascular, andcardiac function. We have already found that transfer ofBN chromosome 13 or 18 attenuates the development ofhypertension, proteinuria, and renal disease in SS.BN-13and SS.BN-18 rats. These consomic panels will soon becommercially available and will provide investigatorswith a powerful tool to link functional data to the genome...
This chapter describes some of the behavioral screening assays devised in laboratory to isolate mutations affecting the zebrafish nervous system. Zebrafish are ideally suited for a behavioral genetic approach. The larvae show a wide range of interesting behaviors, yet are small and can be produced in large numbers. Importantly, larvae do not need to be fed to survive until 8 days postfertilization, and all the assays described in this chapter can be carried out in this period. Therefore, millions of fish can be tested in a large-scale screen, requiring relatively little maintenance. Adult behaviors can also be used for screens, on a smaller scale. Dozens of larval and adult behaviors have been described in the chapter. Well-designed behavioral screens are focused to find mutations specific to a particular neural system. Yet behavior is the endpoint of neural processing often involving tens or hundreds of cell types in a neural circuit or pathway, and therefore mutations might exert their effects at many places in the brain. Behavioral screens require responses to be reliably evoked in the laboratory. Innate behaviors are generally more robust than learned responses and therefore better suited for genetic approaches.
To characterize gene expression patterns in various tissues of the zebrafish (Danio rerio) eye and identify zebrafish orthologs of human genes by expressed sequence tag (EST) analysis for NEIBank.
mRNA was extracted from adult zebrafish eye tissues, including lenses, anterior segments (minus lens), retinas, posterior segments lacking retinas, and whole eyes. Five different cDNA libraries were constructed in the pCMVSport6 vector. Approximately 4,000 clones from each library were sequenced and analyzed using various bioinformatics programs.
The analysis yielded approximately 2,500 different gene clusters for each library. Combining data from the five libraries produced 10,392 unique gene clusters. GenBank accession numbers were identified for 37.6% (3,906) of the total gene clusters in the combined libraries and approximately 50% were linked to Unigene clusters in the current database. Several new crystallin genes, including two gammaN-crystallins, and a second major intrinsic protein (MIP) were identified in the lens library. In addition, a zebrafish homolog of cochlin (COCH), a gene that may play a role in the pathogenesis of human glaucoma, was identified in the anterior segment library. Surprisingly, no clear ortholog of the major retinal transcription factor Nrl was identified.
The zebrafish eye tissue cDNA libraries are a useful resource for comparative gene expression analysis. These libraries will complement the cDNA libraries made for the Zebrafish Gene Collection (ZGC) and provide an additional source for gene identification and characterization in the vertebrate eye.
Ethanol intake during pregnancy can produce a wide range of adverse effects on nervous system development including fetal alcohol syndrome (FAS). The most severe congenital malformation observed in newborns with FAS is cyclopia. In this study, we have exposed zebrafish embryos to different ethanol concentrations (2.4%, 1.5% or 1.0%) during eye morphogenesis in four zebrafish strains (AB, EK, GL and TL). In addition, we have studied the survival rate of the cyclopic animals to the end of larval development. The zebrafish strains GL and AB generated the higher percentage of cyclopic animals after exposure to 2.4% ethanol, while EK showed the higher percent cyclopic animals using 1.5% and 1.0% ethanol. The EK strain showed the higher percent survival during the larval period at all ethanol concentrations (2.4%, 1.5% and 1.0%). Moreover, we have investigated cytoarchitectural alterations in the main components of the visual pathway-retina and optic tectum-and ethanol treatment affects both the retina and the optic tectum. The lamination of neural retina is clearly delayed in treated larvae 3 days postfertilization and the thickness of the pigmented epithelium is considerably reduced. With regard to the optic tectum, treatment with ethanol alters the normal pattern of tectal lamination. The use of zebrafish EK strain is a suitable in vivo vertebrate model system for analyzing the teratogenic effect of ethanol during vertebrate visual system morphogenesis as it relates to both cyclopia and FAS.
We examined optokinetic and optomotor responses of 450 zebrafish mutants, which were isolated previously based on defects in organ formation, tissue patterning, pigmentation, axon guidance, or other visible phenotypes. These strains carry single point mutations in >400 essential loci. We asked which fraction of the mutants develop blindness or other types of impairments specific to the visual system. Twelve mutants failed to respond in either one or both of our assays. Subsequent histological and electroretinographic analysis revealed unique deficits at various stages of the visual pathway, including lens degeneration ( bumper ), melanin deficiency ( sandy ), lack of ganglion cells ( lakritz ), ipsilateral misrouting of axons ( belladonna ), optic-nerve disorganization ( grumpy and sleepy ), inner nuclear layer or outer plexiform layer malfunction ( noir , dropje , and possibly steifftier ), and disruption of retinotectal impulse activity ( macho and blumenkohl ). Surprisingly, mutants with abnormally large or small eyes or severe wiring defects frequently exhibit no discernible behavioral deficits. In addition, we identified 13 blind mutants that display outer-retina dystrophy, making this syndrome the single-most common cause of inherited blindness in zebrafish. Our screen showed that a significant fraction (∼5%) of the essential loci also participate in visual functions but did not reveal any systematic genetic linkage to particular morphological traits. The mutations uncovered by our behavioral assays provide distinct entry points for the study of visual pathways and set the stage for a genetic dissection of vertebrate vision.
Two genes in Drosophila, rdgA and rdgB, which when defective cause retinal degeneration, were discovered by Hotta and Benzer (Hotta, Y., and S. Benzer. 1970. Proc. Natl, Acad. Sci. U. S, A. 67:1156-1163). These mutants have photoreceptor cells that are histologically normal upon eclosion but subsequently degenerate. The defects in the rdgA and rdgB mutants were localized by the study of genetic mosaics to the photoreceptor cells. In rdgB mutants retinal degeneration is light induced. It can be prevented by rearing the flies in the dark or by blocking the receptor potential with a no-receptor-potential mutation, norpA. Vitamin A deprivation and genetic elimination of the lysosomal enzyme acid phosphatase alsoprotect the photoreceptors of rdgB flies against light-induced damage. The photopigment kinetics of dark-reared rdgB flies appear normal in vitro by spectrophotometric measurements, and in vivo by measurements of the M potential. In normal Drosophila, a 1-s exposure to intense 470-nm light produces a prolonged depolarizing afterpotential (PDA) which can last for several hours. In dark-reared rdgB mutants the PDA lasts less than 2 min;; it appears to initiate the degeneration process, since the photoreceptors become permanently unresponsive after a single such exposure. Another mutant was isolated which prevents degeneration in rdgB flies but which has a normal receptor potential. This suppressor of degeneration is an allele of norpA. It is proposed that the normal norpA gene codes for a product which, when activated, leads to the receptor potential, and which is inactivated by the product of the normal rdgB gene.
In teleost fish, dopaminergic interplexiform cells provide an intraretinal centrifugal pathway from the inner to the outer plexiform layer, where they make abundant synapses on cone-related horizontal cells. The interplexiform cells receive all their input in the inner plexiform layer from centrifugal fibers and amacrine cells. In fish, centrifugal fibers contain gonadotropin hormone-releasing hormone (GnRH)-like and FMRFamide-like peptides (Munz et al., 1982; Stell et al., 1984), whereas amacrine cells contain a variety of neuroactive substances, including a number of peptides. In this study, we examined the effects of GnRH, FMRFamide, bicuculline, and enkephalin on horizontal cell activity in the white perch retina in an attempt to understand the synaptic inputs to the interplexiform cells. When the retina was superfused with Ringer's solution containing GnRH, horizontal cells depolarized (approximately 10 mV), and their responses to small spots increased, whereas their responses to full-field lights decreased. Thus, GnRH closely mimicked the effects of dopamine on horizontal cells. The GnRH antagonist [D-Phe2, Pro3, D-Phe6]-GnRH blocked the effects of GnRH, as did haloperidol. GnRH also had no effect on horizontal cells in retinas treated with 6-hydroxydopamine. The results indicate that GnRH acts by stimulating the release of dopamine from interplexiform cells. FMRFamide alone produced no changes on either the membrane potential or light responses of horizontal cells, but it did suppress the effects of GnRH on horizontal cells in some experiments. FRMFamide also reversed the effects of prolonged darkness on horizontal cell responses. When bicuculline was applied to the retina, horizontal cells also depolarized (approximately 10 mV), responses to full-field illumination decreased, and responses to small spots increased. Most of the effects of bicuculline were suppressed by haloperidol, indicating that bicuculline also stimulates the release of dopamine from interplexiform cells. Similar results were obtained when [D-Ala2]-met-enkephalinamide was applied to the retina; horizontal cells depolarized (approximately 10 mV), responses to full-field stimuli decreased, and responses to the light spots increased. On the other hand, [D-Ala2]-leuenkephalinamide and [D-Ala2, D-Leu5]-enkephalin had no effects on horizontal cells. Both haloperidol and naloxone blocked the effects of [D-Ala2]-met-enkephalinamide on horizontal cells, indicating that [D-Ala2]-met-enkephalinamide stimulates dopamine release from interplexiform cells via specific opiate receptors.
Unusual rod electroretinogram (ERG) intensity-response functions were recorded from three female patients with retinal degeneration who had visual acuities of 20/200, retinal arteriolar narrowing, and diffuse granularity of the retinal pigment epithelium. All three patients had rod b-waves that were profoundly subnormal in amplitude and markedly delayed in implicit time to bright stimuli. Rod a-wave slopes were reduced 50% below normal, indicating photoreceptor involvement. These unusual rod ERG intensity-response functions are similar to those previously reported for the isolated cat eye with elevated retinal cyclic guanosine monophosphate (cGMP) after perfusion with isobutylmethylxanthine. This finding supports the idea that these three patients may have an elevation of retinal cGMP. Their rod ERG intensity-response functions are contrasted with those recorded from some patients with retinitis pigmentosa.
We searched for point mutations in every exon of the rhodopsin gene in 150 patients from separate families with autosomal dominant retinitis pigmentosa. Including the 4 mutations we reported previously, we found a total of 17 different mutations that correlate with the disease. Each of these mutations is a single-base substitution corresponding to a single amino acid substitution. Based on current models for the structure of rhodopsin, 3 of the 17 mutant amino acids are normally located on the cytoplasmic side of the protein, 6 in transmembrane domains, and 8 on the intradiscal side. Forty-three of the 150 patients (29%) carry 1 of these mutations, and no patient has more than 1 mutation. In every family with a mutation so far analyzed, the mutation cosegregates with the disease. We found one instance of a mutation in an affected patient that was absent in both unaffected parents (i.e., a new germ-line mutation), indicating that some "isolate" cases of retinitis pigmentosa carry a mutation of the rhodopsin gene.
The regular arrangement of retinal cone cells in a mosaic pattern is a common feature of teleosts. In the zebrafish, Brachydanio rerio, the retinal cone mosaic comprises parallel rows consisting of a repeating motif of four cone types. In order to elucidate the temporal and spatial aspects of the genesis of the cone mosaic in the developing retina, we generated a monoclonal antibody that specifically binds to the double cone photoreceptor of the adult. We first saw staining in the developing retina with this antibody, FRet 43, at 48 hours postfertilization, the time at which the first photoreceptor cells undergo their final mitotic division. We then injected embryonic fish with the thymidine analog, 5-bromo-2'-deoxyuridine (BrdU), confirming with a double-labeling experiment that the onset of FRet 43 antigenicity occurs within three hours of the cellular division that generates the double cone photoreceptors. Then we stained tangential sections of the 54-hour embryonic retina with FRet 43, further showing that cells devoid of staining alternate with stained pairs of cells in a pattern that is consistent with the arrangement of photoreceptors in the adult cone mosaic. These results indicate that a marker of the double cone phenotype is expressed at approximately the same time as cellular birthday and that the mosaic patterning is present within 6 hours of this expression.
Antisera to two putative neurotransmitters, luteinizing hormone-releasing hormone (LHRH) and molluscan cardioexcitatory tetrapeptide (H-Phe-Met-Arg-Phe-NH2; FMRF-amide), bind specifically to neurites in the inner nuclear and inner plexiform layers of the goldfish retina. Retrograde labeling showed that intraocular axon terminals originate from the nervus terminalis, whose cell bodies are located in the olfactory nerves. Double immunocytochemical and retrograde labeling showed that some terminalis neurons project to the retina; others may project only within the brain. All terminalis neurons having proven retinal projections were both LHRH- and FMRF-amide-immunoreactive. The activity of retinal ganglion cells was recorded with microelectrodes in isolated superfused goldfish retinas. In ON- and OFF-center double-color-opponent cells, micromolar FMRF-amide and salmon brain gonadotropin-releasing factor ( [Trp7, Leu8] LHRH) caused increased spontaneous activity in the dark, loss of light-induced inhibition, and increased incidence of light-entrained pulsatile response. The nervus terminalis is therefore a putatively peptidergic retinopetal projection. Sex-related olfactory stimuli may act through it, thereby modulating the output of ganglion cells responsive to color contrast.
Optokinetic and phototactic behaviors of zebrafish larvae were examined for their usefulness in screening for recessive defects in the visual system. The optokinetic response can be reliably and rapidly detected in 5-day larvae, whereas the phototactic response of larvae is variable and not robust enough to be useful for screening. We therefore measured optokinetic responses of mutagenized larvae as a genetic screen for visual system defects. Third-generation larvae, representing 266 mutagenized genomes, were examined for abnormal optokinetic responses. Eighteen optokinetic-defective mutants were identified and two mutants that did not show obvious morphological defects, no optokinetic response a (noa) and partial optokinetic response a (poa), were studied further. We recorded the electroretinogram (ERG) to determine whether these two mutations affect the retina. The b-wave of noa larvae was grossly abnormal, being delayed in onset and significantly reduced in amplitude. In contrast, the ERG waveform of poa larvae was normal, although the b-wave was reduced in amplitude in bright light. Histologically, the retinas of noa and poa larvae appeared normal. We conclude that noa larvae have a functional defect in the outer retina, whereas the outer retina of poa larvae is likely to be normal.
Cone photoreceptors in the zebrafish retina are arranged in a crystalline lattice, with each spectral subtype at a specific position in the array; rod photoreceptors are inserted around the cones. Patterning events and developmental mechanisms that lead to the formation of the cone mosaic are not known. To begin investigating this issue, we examined the initial stages of opsin expression in zebrafish embryos by in situ hybridization with goldfish opsin cRNA probes to determine how and when the cone mosaic pattern arises. We found both differences and similarities in the spatiotemporal patterns of rod and cone development, which suggest the following: 1) Expression of opsin message (including rod opsin, blue and red cone opsins) was found in a ventral patch of retina located nasal to the choroid fissure. 2) The cone mosaic pattern was generated by a crystallization-like process initiated in the precocial ventral patch and secondarily in nasal retina, which then swept like a wave into dorsotemporal retina. 3) The remainder of the retina, suggesting that these precocial rods might differ from typical rods. 4) Developmental maturation of rods in zebrafish, as reflected by expression of opsin, may be accelerated compared to cones, which are thought to become postmitotic before rods. These data are consistent with a model in which lateral inductive interactions among differentiating photoreceptors lead to patterning of the array.
In a large scale screen for genetic defects in zebrafish embryogenesis we identified 49 mutations affecting development of the retina. Based on analysis of living embryos as well as histological sections, we grouped the isolated mutations into six phenotypic categories. (1) Mutations in three loci result in a loss of wild-type laminar pattern of the neural retina. (2) Defects in four loci lead to an abnormal specification of the eye anlagen. Only one eye frequently forms in this class of mutants. (3) Seven loci predominantly affect development of the outer retinal layers. Mutants in this category display cell loss mainly in the photoreceptor cell layer. (4) Nine mutations cause retardation of eye growth without any other obvious abnormalities in the retina. (5) A group of twelve mutations is characterized by nonspecific retinal degeneration. (6) Four mutations display retinal degeneration associated with a pigmentation defect. Finally, two mutations, one with absence of the ventral retina and one with an eye-specific pigmentation defect, are not classified in any of the above groups. The identified mutations affect numerous aspects of eye development, including: specification of the eye anlage, growth rate of the optic cup, establishment of retinal stratification, specification or differentiation of retinal neurons and formation of the dorsoventral axis in the developing eye.
Complex as it is, much of the vast network of cellular functions has been successfully dissected, on a microscopic scale, by the use of mutants in which one element is altered at a time. A similar approach may be fruitful in tackling the complex structures and events underlying behavior, using behavioral mutations to indicate modifications of the nervous system. Drosophila offers the same advantages to such a study as it did to classical genetics, namely, large numbers and short generation time, to which may now be added an enormous store of accumulated knowledge concerning the organism. Containing about 105 neurons, the fly's nervous system is roughly halfway, on a logarithmic scale, between a single neuron and the human brain, and the fly is possessed of a rich repertoire of behavior. The considerable literature on Drosophila behavior since Carpenter's 1905 paper [1] has recently been reviewed by Manning.[2]
• The ocular histopathologic and electron microscopic findings were determined in eyes obtained at autopsy from twins with dominant olivopontocerebellar atrophy (OPCA) and retinal degeneration (OPCA type III). On light microscopy, a retinal degeneration that involved primarily the photoreceptor layer was present and appeared to start in the macular area and spread to involve the peripheral fundus. The retinal pigment epithelium was variably hypopigmented and hyperpigmented. On electron microscopy, osmiophilic, multimembranous, and complex lipofuscin inclusions were present in conjunctival cells, keratocytes, lens epithelium, iris and ciliary body fibrocytes, occasional outer retinal cells, and retinal pigment epithelial cells. The twins' father and an older sister were also affected and had classic neurologic and ophthalmologic abnormalities. The similarities were noted between the clinical and ultrastructural findings between OPCA type III and the neuronal ceroid lipofuscinoses.
An effect simulating broken cream in whole milk was produced when Bacillus cereus was grown in washed cream at 22° C. A similar but less marked effect was obtained when a concentrated culture filtrate of B. cereus was added to washed cream.
Microscopic examination of washed cream to which B. cereus or its concentrated filtrate had been added showed that the fat-globule membranes had been broken down. The hydrolysis of lecithin in washed cream in which B. cereus had grown was demonstrated by qualitative estimation of free choline.
Since these results could not be reproduced when a non-lecithinase-producing strain of B. cereus or its concentrated culture filtrate was added to washed cream, it appears that the hydrolysis of the lecithin of the fat-globule membrane is at least partly responsible for the formation of broken cream.
The zebrafish has recently assumed a central position
in the study of vertebrate development. Numerous studies
of other fish have shown that their central nervous systems,
and especially their visual systems, continue to add new
neurons throughout life, which is probably related to their
abilities to regenerate axons and whole nervous tissue.
Retinal neurogenesis had not been examined in adult zebrafish,
and two reports concluded that the optic tectum ceased
neurogenesis early in life, so the question arose whether
the zebrafish was anomalous in this regard. We labeled
embryonic (24- and 48-h postfertilization) and adult zebrafish
with the thymidine analog, bromo-deoxyuridine, and, after
short and long survivals, examined the retina and brain
for labeled cells. They were abundant in both the optic
tectum and the retina. Although the rate of retinal growth
slows considerably between embryonic and adult stages,
the patterns of neurogenesis in both the embryo and the
adult are similar to those described in other fish, so
these “fish-specific” features of general interest
can justifiably be studied in zebrafish.
Cobaltous-lysine was applied to one optic nerve of normal goldfish in order to determine the source of the centrifugal innervation of the retina. Cobalt-filled cells were not observed in the optic tectum, pretectum, thal-amus, or hypothalamus. However, filled cells were observed outside the central nervous system either interspersed between the olfactory nerve fibers orrostrally along the ventromedial aspect of the olfactory bulbs. These cells appear to correspond to the ganglion cells of the nervus terminalis. The cells were located bilaterally and had dendrites that branched in close proximity to the cell body and axons that coursed caudally through the medial olfactory tract. The axons traveled in the ventral forebrain and entered the optic tracts. The axons also gave off fine branches that appeared to terminate in the vicinity of the anterior commissure and in the preoptic region. Application of cobaltous-lysine to a cut olfactory tract resulted in cobalt-filled fibers in the optic tracts, retinal optic fiber layer, and retinal ganglion cell layer. However, the precise terminations of these fibers within the retina could not be readily established. The results are discussed with respect to the plethora of sources of retinopetal cells observed by others in fish and with respect to the innervation of the retina by luteinizing hormone-releasing hormone axons.
Morphological development of photoreceptors in the central retina of the zebrafish, Brachydanio rerio, was studied by using light and electron microscopic techniques. Outer segments (OS) first appeared at 2.5 days postfertilization (d2.5). On d3, synaptic elaborations were seen. By d8, two OS types were present and were identified as cones. The first indication of rod formation was also evidenced at this time, when vitreally positioned nuclei were observed and rodlike cells were infrequently detected in electron micrographs. At d12, the full complement of zebrafish photoreceptors, rods and four cone types, was identified. From this time on cells grew until adult dimensions were reached at d24. These structural observations are consistent with results of functional studies which utilized physiological and behavioral techniques.
The functional development of rod and cone photoreceptors in the zebrafish (Brachydanio rerio) was studied by electroretinographically measuring flicker fusion frequencies. Two to 3 days after fertilization, fish gave little or no response to high intensity stimuli. Increases in sensitivity and flicker resolution were observed after this time. Biphasic response curves typical of adults could be elicited from fish as young as 2 weeks postfertilization, thus indicating a functional divergence of rods and cones. The results are consistent with behavioral and anatomical analyses of zebrafish photoreceptor development.
The morphological differentiation of the zebrafish retina was analyzed by using light (LM) and transmission electron (TEM) microscopy between the time of initial ganglion cell differentiation (≈32 hours postfertilization; hpf) and shortly after the point when the retina appears functional (≈74 hpf), i.e., when all major cell types and basic synaptic connections are in place. The results show that the inner retinal neurons, like the photoreceptor and ganglion cells, differentiate first within the ventronasal region, and differentiation subsequently spreads asymmetrically into the nasal and dorsal regions before reaching the ventrotemporal retina. In addition, we show that the attenuation of the optic stalk occurs in parallel with ganglion cell differentiation between 32 and 40 hpf. The first conventional synapses appear within the inner plexiform layer simultaneously with the first photoreceptor outer segment discs at 60 hpf; functional ribbon triads arise within photoreceptor synaptic terminals at 65 hpf; and synaptic ribbons occur within bipolar cell axon terminals at the time larvae exhibit their first visual responses (≈70 hpf). Although development is initially more advanced within the ventronasal region between 50 and 60 hpf, development across the retina rapidly equilibrates such that it is relatively comparable within all quadrants of the central retina by 70 hpf. An area within the temporal retina characterized by tightly packed and highly tiered cones emerges with subsequent development. Retinal differentiation in the zebrafish corresponds with that generally described in other vertebrates and can be correlated with the development of visual and electroretinographic responses in the animal. J. Comp. Neurol. 404:515–536, 1999. © 1999 Wiley-Liss, Inc.
We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis—the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts. ©1995 Wiley-Liss, Inc.
We describe light-microscopically the development of the embryonic zebrafish eye with particular attention to cell number, cell proliferation, and cell death. The period from 16 to 36 hr post fertilization (hpf) comprises two phases; during the first (16–27 hpf) the optic vesicle becomes the eye cup, and during the second (27–36 hpf) the eye cup begins to differentiate into the neural retina and pigmented epithelium. All cells in the eye primordium are proliferative prior to 28 hpf, and the length of the cell cycle has been estimated to be 10 hr at 24–28 hpf (Nawrocki, 1985). Our cell counts are consistent with that estimate at that age, but not at earlier ages. A 10-hr cell cycle predicts that the cell number should increase by 7% per hr, but during 16–24 hpf the cell number increased by only 1.5% per hr. Despite the low rate of increase, all cells labeled with bromo-deoxyuridine, so all were proliferative. We considered three possible explanations for the nearly-constant cell number in the first phase: proliferation balanced by cell emigration from the eye, proliferation balanced by cell death, and low proliferation caused by a transient prolongation of the cell cycle. We excluded the first two, and found direct support for the third. Previous examinations of the cell cycle length in vertebrate central nervous system have concluded that it increases monotonically, in contrast to the modulation that we have shown. Modulation of the cell cycle length is well-known in flies, but it is generally effected by a prolonged arrest at one phase, in contrast to the general deceleration that we have shown. © 2000 Wiley-Liss, Inc.
We have examined the morphogenesis of the zebrafish eye, from the flat optic vesicle at 16 hours post fertilization (hpf) to the functional hemispheric eye at 72 hpf. We have produced three-dimensional reconstructions from semithin sections, measured volumes and areas, and produced a fate map by labeling clusters of cells at 14–15 hpf and finding them in the 24 hpf eye cup. Both volume and area increased sevenfold, with different schedules. Initially (16–33 hpf), area increased but volume remained constant; later (33–72 hpf) both increased. When the volume remained constant, the presumptive pigmented epithelium (PE) shrank and the presumptive neural retina (NR) enlarged. The fate map revealed that during 14–24 hpf cells changed layers, moving from the PE into the NR, probably through involution around the margin of the eye. The transformation of the flat epithelial layers of the vesicle into their cup-shaped counterparts in the eye was also accompanied by cellular rearrangements; most cells in a cluster labeled in the vesicle remained neighbors in the eye cup, but occasionally they were separated widely. This description of normal zebrafish eye development provides explanations for some mutant phenotypes and for the effects of altered retinoic acid. Dev Dyn;218:175–188. © 2000 Wiley-Liss, Inc.
We have initiated a genetic analysis of the zebrafish visual system to identify novel molecules involved in vertebrate retinal function. Zebrafish are highly visual; they have four types of cones as well as rod photoreceptors, making it possible to study both rod and cone-mediated visual responses. To identify visual mutants, optokinetic responses of mutagenized larvae are measured in a three-generation screen for recessive mutations. By measuring visual behavior our genetic screen has been targeted towards identifying mutants that do not have gross morphological abnormalities. The electroretinogram (ERG) of optokinetic-defective mutants is recorded and their retinas are examined histologically to localize defects to the retina. In this report, we summarize our screening results and ERG and histological analyses of the five morphologically normal mutants we have analyzed to date. Additionally, the more detailed characterization of a red-blind mutant that we have isolated is summarized. Our results indicate that mutants with defects in various processes such as photoreceptor synaptic transmission, photoreceptor adaptation and cell-type specific survival and/or function can be identified using this approach.
Recently gonadotropin-releasing hormone (GnRF)-like and molluscan cardioexcitatory peptide (FMRFamide)-like compounds have been colocalized immunocytochemically to the terminal nerve, a presumed olfactoretinal efferent system in goldfish. In the present study these and related neuropeptides were shown to affect ganglion cell activity, recorded extracellularly, when applied to the isolated superfused goldfish retina. GnRF was usually excitatory. Salmon GnRF (sGnRF) was 10–30× more potent than chicken or mammalian GnRF. FMRFamide and enkephalin also were often excitatory but caused more varied responses than sGnRF. M Met5-enkephalin-Arg6-Phe7-NH2 (YGGFMRFamide), which contains both enkephalin and FMRFamide sequences, tended to act like both of these peptides but with mainly enkephalin-like properties. Neuropeptide Y and the C-terminal hexapeptide of pancreatic polypeptides, whose C-terminus (-Arg-Tyr-NH2) is closely related to that of FMRFamide (-Arg-Phe-NH2), gave no consistent responses. Threshold doses were equivalent to: 0.1 μM for sGnRF; 0.5 μM for YGGFMRFamide; 1.5 μM for FMRFamide and enkephalin. Rapid, complete and irreversible desensitization was induced by single, 10–20× threshold doses of sGnRF; but desensitization was infrequent and limited with the other peptides. In general, all peptides tested affected the spatially and chromatically antagonistic receptive field components similarly, but selective actions were seen in a few cases with FMRFamide and with the opioid antagonist, naloxone. Responses, especially to sGnRF and FMRFamide, tended to be most frequently obtained and pronounced in winter and spring, suggesting a correlation with seasonally regulated sexual and reproductive activity. Our observations provide further evidence for transmitter-like roles of neuropeptides related to sGnRF and FMRFamide in the teleostean terminal nerve. The actions of agonists and antagonists, singly and in combination, imply strongly that there are distinctive postsynaptic receptors and/or neural pathways for GnRF-, FMRFamide- and enkephalin-like peptides in the goldfish retina.
Interphotoreceptor retinoid-binding protein (IRBP) was studied using immunochemical and immunocytochemical techniques in retinae of mice with allelic combinations at the rd and rds loci at different stages of development and degeneration. Until postnatal day 7 (P7), IRBP is located intracellularly in developing retinae of the different genotypes. Thereafter, IRBP is present mainly in the interphotoreceptor matrix. As previously noted, cell death is slowest in the heterozygous +/+,rds/+ mutant with loss increasing in order in +/+, , , and , +/+ animals. The IRBP content of the total retina also approximates this pattern, with lowest amounts by far in , and , +/+ mutants (after P14). Interestingly though, IRBP loss significantly precedes visual cell loss in the , retina. In all the mutants, the remaining rod cells in the outer nuclear layer exhibit synthesis of intracellularly located IRBP at late stages of degeneration. In the single homozygous , +/+ and the double homozygous , mutants, IRBP is present intracellularly during the entire degenerative process with somewhat less intracellular IRBP in the , mutant. Retinae of homozygous +/+, and heterozygous +/+, rds/+ animals exhibit a normal distribution pattern of IRBP immunoreactivity until loss of photoreceptor cells becomes pronounced at later stages of the disease. Many of the remaining cells at this time are probably cone elements although they are structurally changed. Double labeling with IRBP and S-antigen demonstrates, in many but not all, the presence of both proteins in the same cell body. Immunocytochemistry clearly demonstrated the presence of IRBP in remaining photoreceptor cells at late stages of the disease. Thus, the biochemically measured loss of IRBP appears to be a complex process neither directly dependent on the loss of photoreceptor outer segments and reduced interphotoreceptor matrix space (e.g. there is a sustained IRBP level in rodless rds mutants) nor simply due to cell death (e.g. in the , mutant, IRBP loss significantly precedes cell loss). That this IRBP is mainly intracellular, however, may indicate an abnormality in secretion which, combined with other factors, induces a degenerated and less differentiated phenotype.
Cyclic nucleotide metabolism was examined in the retina and in the retinal pigment epithelium (RPE)-choroid complex of the rds mouse (020/A), a mutant in which discrete photoreceptor outer segment disc structures fail to develop. In retinas of both rds and control (Balb/c) mice, cyclic AMP levels peak at 10–15 days (20–25 pmol mg−1 protein). The level drops to about 10 pmol mg−1 at about one month in normal retinas but remains high in affected retinas. Cyclic GMP levels increase five-fold in Balb/c retinas as ROS develop whereas, in affected retinas, the levels remain constant and low (about 5 pmol mg−1). In RPE-choroid, cyclic nucleotide levels are similar in control and affected mice. Cyclic AMP phosphodiesterase (PDE) activity is somewhat higher in affected than in control retinas; conversely, cyclic GMP-PDE is lower. Both cyclic AMP-PDE and cyclic GMP-PDE activities are different in normal and affected RPE-choroid. Thus, although the rds (020/A) mouse belongs to the early-onset photoreceptor dysplasia group of hereditary retinal degenerations on a morphological basis, it does not exhibit high retinal cyclic GMP levels and thus appears to be distinct from other animals exhibiting early postnatal photoreceptor dysfunction.
Determination of cell fate in the vertebrate retina has been shown to be largely independent of lineage. After cell fates are determined, retinal neurons become organized in a precise laminar pattern. The mechanisms for this patterning could involve morphogens distributed in gradients or, alternatively, direct cell-cell interactions. In the zebrafish mutant cyclops (Cyc(b16)), most embryos have two partial retinas joined in the ventral midline. This presents developing retinal cells near the midline with abnormal cellular environments, whereas laterally the pattern of developing cells is normal. We examined the consequences of this for patterning in the mutant's retina. We found that the retinas are joined in the midline at the apical surfaces of the photoreceptor layers. A laminar pattern emerges in the midline that preserves normal positional relationships between retinal cell types locally but is abnormal with respect to patterning over the entire retina. Lateral to the midline, retinal patterning appears normal. Metabolic labeling experiments showed that late rounds of DNA synthesis precede the emergence of the novel pattern in this midline region. We conclude that these observations in the cyclops mutant are compatible with mechanisms of pattern formation in the retina involving local cell interactions.
The notochord is a midline mesodermal structure with an essential patterning function in all vertebrate embryos. Zebrafish floating head (flh) mutants lack a notochord, but develop with prechordal plate and other mesodermal derivatives, indicating that flh functions specifically in notochord development. We show that floating head is the zebrafish homologue of Xnot, a homeobox gene expressed in the amphibian organizer and notochord. We propose that flh regulates notochord precursor cell fate.
HUMAN retinitis pigmentosa represents a class of diseases, the principal characteristic of which is the slow and progressive degeneration of photoreceptor cells. In several human neurological syndromes, photoreceptor degeneration accompanies cerebellar ataxia, deafness, mental retardation or other abnormalities. Some examples of such syndromes are BattenSpielmeyer-Vogt disease, Refsum's disease, Laurence-Moon Biedl syndrome, Bassen-Kornzweig disease, Usher's syndrome and several of the mucopolysaccharidoses1-4. As far as we know laboratory animals with neurological syndromes that include photoreceptor degeneration have not been available.
We inserted into the germline of mice either a mutant or wild-type allele from a patient with retinitis pigmentosa and a missense mutation (P23H) in the rhodopsin gene. All three lines of transgenic mice with the mutant allele developed photoreceptor degeneration; the one with the least severe retinal photoreceptor degeneration had the lowest transgene expression, which was one-sixth the level of endogenous murine rod opsin. Of two lines of mice with the wild-type allele, one expressed approximately equal amounts of transgenic and murine opsin and maintained normal retinal function and structure. The other expressed approximately 5 times more transgenic than murine opsin and developed a retinal degeneration similar to that found in mice carrying a mutant allele, presumably due to the overexpression of this protein. Our findings help to establish the pathogenicity of mutant human P23H rod opsin and suggest that overexpression of wild-type human rod opsin leads to a remarkably similar photoreceptor degeneration.
The gene for autosomal dominant retinitis pigmentosa in a large pedigree of Irish origin has recently been found to be linked to an anonymous polymorphic sequence, D3S47 (C17), from the long arm of chromosome 3. As the gene coding for rhodopsin is also assigned to the long arm of chromosome 3 and is expressed in rod photoreceptors that are affected early in this blinding disease, we searched for a mutation of the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. We found a C----A transversion in codon 23 (corresponding to a proline----histidine substitution) in 17 of 148 unrelated patients and not in any of 102 unaffected individuals. This result, coupled with the fact that the proline normally present at position 23 is highly conserved among the opsins and related G-protein receptors, indicates that this mutation could be the cause of one form of autosomal dominant retinitis pigmentosa.
Opsin phosphorylation in light was detected in three retinas from autopsy eyes with retinitis pigmentosa (RP) including one with sex-linked RP and two with autosomal recessive RP, that were studied at postmortem intervals of 1-4 hr. In these retinas from RP eyes, opsin phosphorylation in light was reduced compared with that in normal human retinas, a finding that is compatible with reduced amounts of opsin due to extensive loss of photoreceptor cells. ATP and GTP levels, although reduced below normal, also were easily detectable, supporting the idea that the reduction in opsin phosphorylation was due to loss of photoreceptor cells and not to a reduced capacity for energy metabolism. These findings in these RP retinas contrast with those in rd mice and Irish setters with rod-cone dysplasia in which a failure of opsin phosphorylation has been detected prior to onset of photoreceptor cell degeneration.
Drosophila rdgC (retinal degeneration-C) mutants show normal retinal morphology and photoreceptor physiology at young ages. Dark-reared rdgC flies retain this wild-type phenotype, but light-reared mutants undergo retinal degeneration. rdgC photoreceptors with low levels of rhodopsin as a result of vitamin A deprivation or a mutant rhodopsin (ninaE) gene fail to show rdgC-induced degeneration even after prolonged light treatment, demonstrating that degeneration occurs as a result of light stimulation of rhodopsin. Analysis of norpA; rdgC flies shows that the norpA-encoded phospholipase C, the target enzyme of the G protein activated by rhodopsin, is not required for rdgC-induced degeneration. Thus the rdgC+ gene product is required to prevent retinal degeneration that results from a previously unrecognized consequence of rhodopsin stimulation.
The determination of cell fates during the assembly of the ommatidia in the compound eye of Drosophila appears to be controlled by cell-cell interactions. In this process, the sevenless gene is essential for the development of a single type of photoreceptor cell. In the absence of proper sevenless function the cells that would normally become the R7 photoreceptors instead become nonneuronal cells. Previous morphological and genetic analysis has indicated that the product of the sevenless gene is involved in reading or interpreting the positional information that specifies this particular developmental pathway. The sevenless gene has now been isolated and characterized. The data indicate that sevenless encodes a transmembrane protein with a tyrosine kinase domain. This structural similarity between sevenless and certain hormone receptors suggests that similar mechanisms are involved in developmental decisions based on cell-cell interaction and physiological or developmental changes induced by diffusible factors.
In teleost fish, centrifugal fibres originating in the olfactory bulb and containing FMRFamide-like and luteinizing hormone releasing hormone (LHRH)-like peptides project to the retina and terminate along the border of the inner nuclear and inner plexiform layers. Using a novel simultaneous two-colour immunolabelling technique, we have found that these centrifugal fibres are often closely apposed to the dopaminergic interplexiform cells. Contacts between centrifugal fibres and dopaminergic interplexiform cells were observed by electron microscopy to be conventional type synaptic junctions. Since the dopaminergic interplexiform cells make synapses on horizontal and bipolar cells, providing an intraretinal centrifugal pathway for information flow from the inner to the outer plexiform layers, we conclude that every neuron in the teleost retina is potentially susceptible to central influences via these centrifugal fibres and dopaminergic interplexiform cells.
We have explored the structure and actions of terminal nerve (TN) fibers in the teleostean retina, the most accessible of TN projections. Using immunocytochemistry we have shown that the goldfish TN contains neuropeptides related to the molluscan cardioexcitatory peptide (FMRFamide) as well as luteinizing hormone-releasing hormone (LHRH). Retinal TN terminals were found upon major dendrites in the distal inner plexiform layer and neuronal cell bodies in the amacrine cell layer. Electron-microscopic double-labeling revealed TN terminals applied to the surface of [3H]-dopamine-, glycine-, and gamma-aminobutyric acid (GABA)-accumulating cells. Synthetic LHRH and FMRFamide at less than 1 microM modified spontaneous and light-evoked activity of ganglion cells in isolated superfused goldfish retina, especially during the active breeding season. Salmon(I)-LHRH was 10-30 times as potent as mammalian LHRH and caused rapid, prolonged desensitization. We conclude that LHRH- and FMRFamide-like peptides may be released by retinal TN endings, probably in concert with reproductive activity, and that they act independently through horizontal and/or amacrine cell pathways to modify visual information processing in the retina.
The first photoreceptor outer segments in the retina of the zebrafish Brachydanio rerio appear in the embryo 2.5 days after fertilization, as revealed by scanning and transmission electron microscopy. These outer segments arise in a small region ventral to the exit of the optic nerve. Ultrastructural features of the developing photoreceptor cells, especially those that distinguish the rods and cones, are described. By 3 days after fertilization, the time of hatching, photoreceptor outer segments are widespread in the retina. However, at this time and in the young larva the early developing ventral region remains distinctive because of its conspicuous population of rods.
The behavioral responses of developing larvae of the zebra fish (Brachydanio rerio) to vibratory stimulation are described. Larvae begin to respond after 4-5 days from the time of fertilization (the approximate time of hatching). Once this response appears, the characteristic movement, the latency of the response, and the sensitivity of the larva to sound do not change for a period of several days. A number of related behavioral changes are observed at the same time the response to sound appears.
The larval startle response is a characteristic pronounced tail flip. It is very similar to the adult startle response which is believed to be mediated via Mauthner's neuron. The data suggest that the usual response may be due to a wave of muscular contractions along one side of the larva, as would be expected to result from an action potential in a Mauthner cell. More complex responses were also observed and are described.
In previous work we described 4 types of visual response among tectal cells of the zebrafish. Cells of one class, type I, have no spontaneous activity, but respond phasically at ON and OFF. Their responses to moving edges, to stimuli that grow in size, and to stimuli equal in size and shape to the whole receptive field (RF) suggest that these cells may receive inhibitory input from near neighbor cells of the same type in the tectum, as well as excitatory input from retinal fibers. In order to further investigate this hypothesis we have studied the effects of drugs on physiological properties of type I cells recorded in the stratum periventriculare layer of the zebrafish tectum. Small (10-50 nl) injections of drugs were made in the tectum while recording 100-500 microns away with extracellular microelectrodes. Both picrotoxin and bicuculline produce the following effects: (1) onset of spontaneous bursting multiunit activity. This noise can be recorded at all depths within the tectum; (2) abolition of the second postsynaptic wave of the optic nerve shock field potential and the current source responsible for it, which occurs in the upper tectal layers at 8 ms latency. This probably represents the secondary activation of inhibitory synapses in those layers; (3) alteration of visual response properties of individual type I tectal cells. The duration of response to small flashing spots and to stimuli that grow in size both increase significantly. Responses to moving edges, which normally occur mostly as the significantly. Responses to moving edges, which normally occur mostly as the edge is crossing the RF border, become extended to encompass the entire RF. Finally, the cells show reduced negative spatial summation following drug injection. All of these effects are fully reversible with time after injection as the drugs wash out. Control injections (of teleost Ringer's solution, 100 mM HCl, 165 mM NaCl, and strychnine 2 mM or 5 mM) do not elicit any of these effects. The results reported here are consistent with the hypothesis that tectal type I cells receive a delayed inhibitory input, probably via GABA synapses, which determines major properties of the visual response.
Between 1976 and 1980, medical and social service sources were used to ascertain cases of retinitis pigmentosa in Maine (1980 population, I, 124,660). As of July 1, 1980, 241 clinically prevalent cases of retinitis pigmentosa were ascertained. Extensive pedigrees were collected for 185 of the subjects and medical records were obtained. One hundred fourteen cases were further evaluated by clinical examination including electroretinography. Adjusting for incorrect diagnosis (eight of 114, 7%) and underascertainment (23 of 185, 12.5%), we estimated that prevalence of retinitis pigmentosa in Maine is 236 cases, 21 per 100,000 population or 1:4,756. Excluding Usher and Bardet-Biedl syndromes, the prevalence is 1:5,193. Estimated birth incidence of persons who will become affected with non-syndrome retinitis pigmentosa is 1:3,544. Incidence of newly diagnosed cases per year is about six per 1,000,000 population. Among kindreds, 16 of 85 (19%) were autosomal dominant, 55 of 85 (65%) autosomal recessive or isolated cases, seven of 85 (8%) X-linked recessive, and seven of 85 (8%) not classified by mode of transmission.
Ganglion cells of the terminal nerve in goldfish are located in the olfactory nerve and bulb and send peripheral processes
into the olfactory epithelium and central processes to the supracommissural nuclei of the telencephalon as well as to the
retina. Correlations between terminal nerve projections and neurobehavioral studies suggest that the terminal nerve mediates
responses to sex pheromones.
In cichlid, poecilid and centrarchid fishes luteinizing hormone releasing hormone (LHRH)-immunoreactive neurons are found in a cell group (nucleus olfactoretinalis) located at the transition between the ventral telencephalon and olfactory bulb. Processes of these neurons project to the contralateral retina, traveling along the border between the internal plexiform and internal nuclear layer, and probably terminating on amacrine or bipolar cells. Horseradish peroxidase (HRP) injected into the eye or optic nerve is transported retrogradely in the optic nerve to the contralateral nucleus olfactoretinalis where neuronal perikarya are labeled. Labeled processes leave this nucleus in a rostral direction and terminate in the olfactory bulb. The nucleus olfactoretinalis is present only in fishes, such as cichlids, poecilids and centrarchids, in which the olfactory bulbs border directly the telencephalic hemispheres. In cyprinid, silurid and notopterid fishes, in which the olfactory bulbs lie beneath the olfactory epithelium and are connected to the telencephalon via olfactory stalks, the nucleus olfactoretinalis or a comparable arrangement of LHRH-immunoreactive neurons is lacking. After retrograde transport of HRP in the optic nerve of these fishes no labeling of neurons in the telencephalon occurred. It is proposed that the nucleus olfactoretinalis anatomically and functionally interconnects and integrates parts of the olfactory and optic systems.
The visual properties of zebrafish tectal cells have been studied with a variety of stimulation routines. These include illumination of the whole receptive field and of the surround, use of moving edges, very small spots, bars of varying orientations, moving spots with varying direction and speeds, growing discs, and pairs of spots whose presentation varies in position and sequence. A number of properties correlate with the classification scheme set forth in the preceding paper. Type B cells, unlike other types, are insensitive to moving stimuli. Experiments involving surround stimulation show that type S cells have inhibitory surrounds while those of type I do not. Type I cells, however, exhibit several properties which are consistent with an intratectal delayed inhibitory mechanism operating within the receptive field. These properties include the response to moving edges and growing stimuli, and the dependence of response duration on the size of a flashed stimulus. Various explanations of these properties are considered, and a specific model is proposed which states that cells of type I receive inhibitory input from neighbouring tectal cells of the same physiological type. The properties involved may be of direct importance in the visual behavior of the fish.
The zebrafish optic tectum is anatomically similar to those of goldfish and other teleosts, both in its laminar structure and the morphology of intrinsic neurons as studied with Golgi stains. We have applied standard electrophysiological techniques to study the visual properties of tectal cells, utilizing a computer system for stimulus control and data recording. All tectal cells have very large receptive fields, averaging 25-39 degrees in linear dimensions. Retinal receptive fields are smaller, averaging 7-13 degrees. In many cases the receptive fields of tectal cells, but never of retinal cells, consist of two parts (main field and accessory field) separated by tens of degrees. The two parts are differentially adapted by background illumination, accessory fields becoming unresponsive under lit conditions while main fields do not. This may reflect separate retinal input channels. Four types of tectal cells are described, which differ in their spontaneous activity in the dark and response to stationary spots. Type I are not spontaneously active in the dark, but respond phasically at response to ON and OFF. Type T are tonically active and give more prolonged phasic responses to ON and OFF. They may also have pure-inhibitory receptive fields in which spot ON suppresses the spontaneous firing with no phasic excitation. Type S are also silent in the dark, but give sustained firing as long as a spot is ON in the receptive field. Cells of type B fire spontaneously in bursts; the burst rate may be raised or lowered by stationary spots, but there is no phasic response. Each of the four physiological types is found to occur among the cells of the periventricular layer, all of which share a stereotyped overall morphology. Tectal cells do not exhibit spatially separated ON and OFF areas or orientation specificity.