Biomedical subjects

Stephan C F Neuhauss

Publications and source records attributed to Stephan C F Neuhauss.

At least 19 recordsLinked to original sources

PubMed2006

Oculomotor instabilities in zebrafish mutant belladonna: a behavioral model for congenital nystagmus caused by axonal misrouting.

A large fraction of homozygous zebrafish mutant belladonna (bel) larvae display a reversed optokinetic response (OKR) that correlates with failure of the retinal ganglion cells to cross the midline and form the optic chiasm. Some of these achiasmatic mutants display strong spontaneous eye oscillations (SOs) in the absence of motion in the surround. The presentation of a stationary grating was necessary and sufficient to evoke SO. Both OKR reversal and SO depend on vision and are contrast sensitive. We built a quantitative model derived from bel fwd (forward) eye behaviors. To mimic the achiasmatic condition, we reversed the sign of the retinal slip velocity in the model, thereby successfully reproducing both reversed OKR and SO. On the basis of the OKR data, and with the support of the quantitative model, we hypothesize that the reversed OKR and the SO can be completely attributed to RGC misrouting. The strong resemblance between the SO and congenital nystagmus (CN) seen in humans with defective retinotectal projections implies that CN, of so far unknown etiology, may be directly caused by a projection defect.

Animals↗

PubMed2006

Mutations in the tight-junction gene claudin 19 (CLDN19) are associated with renal magnesium wasting, renal failure, and severe ocular involvement.

Claudins are major components of tight junctions and contribute to the epithelial-barrier function by restricting free diffusion of solutes through the paracellular pathway. We have mapped a new locus for recessive renal magnesium loss on chromosome 1p34.2 and have identified mutations in CLDN19, a member of the claudin multigene family, in patients affected by hypomagnesemia, renal failure, and severe ocular abnormalities. CLDN19 encodes the tight-junction protein claudin-19, and we demonstrate high expression of CLDN19 in renal tubules and the retina. The identified mutations interfere severely with either cell-membrane trafficking or the assembly of the claudin-19 protein. The identification of CLDN19 mutations in patients with chronic renal failure and severe visual impairment supports the fundamental role of claudin-19 for normal renal tubular function and undisturbed organization and development of the retina.

Adolescent↗

PubMed2006

Time course and development of light adaptation processes in the outer zebrafish retina.

Retinomotor movements are morphological changes in the outer retina in response to changing light conditions. They can be separated into two components: Migration of pigment granules within the microvilli of the retinal pigment epithelium (RPE) and positional changes in photoreceptor cells. These positional changes optimize exposure of the cone and rod photoreceptors to light. The aim of this study was to analyze both the time course of retinomotor movements in the adult zebrafish and the maturation of these processes in the developing fish. We show that retinomotor movements are used as a dark/light adaptation mechanism in zebrafish. In adult zebrafish, melanin granules of the RPE migrate with constant speed and reach the fully light adapted (LA) state approximately after 1 h. In contrast, about two thirds of double cone outer segment movements are finished in 5 min, and are fully completed in 10 to 20 min. During development there are three crucial stages leading to mature retinomotor movements in response to light: at 5 dpf (days post fertilization) the migration of pigment granules begins, at 20 dpf the pigment granules condense in the apical part of the RPE microvilli, and at 28 dpf, concomitant with the functional maturation of rods, the double cones contract as in adult retinas.

Adaptation, Ocular↗

PubMed2006

Genetic identification of AChE as a positive modulator of addiction to the psychostimulant D-amphetamine in zebrafish.

Addiction is a complex maladaptive behavior involving alterations in several neurotransmitter networks. In mammals, psychostimulants trigger elevated extracellular levels of dopamine, which can be modulated by central cholinergic transmission. Which elements of the cholinergic system might be targeted for drug addiction therapies remains unknown. The rewarding properties of drugs of abuse are central for the development of addictive behavior and are most commonly measured by means of the conditioned place preference (CPP) paradigm. We demonstrate here that adult zebrafish show robust CPP induced by the psychostimulant D-amphetamine. We further show that this behavior is dramatically reduced upon genetic impairment of acetylcholinesterase (AChE) function in ache/+ mutants, without involvement of concomitant defects in exploratory activity, learning, and visual performance. Our observations demonstrate that the cholinergic system modulates drug-induced reward in zebrafish, and identify genetically AChE as a promising target for systemic therapies against addiction to psychostimulants. More generally, they validate the zebrafish model to study the effect of developmental mutations on the molecular neurobiology of addiction in vertebrates.

Acetylcholine↗

PubMed2006

Nyctalopin is essential for synaptic transmission in the cone dominated zebrafish retina.

The first synapse in the vertebrate visual system is the photoreceptor synapse between rod and cone photoreceptors and the second-order bipolar cells. Although mutations in the nyctalopin gene (NYX) in humans lead to congenital stationary night blindness (CSNB1), affecting synaptic transmission between both types of photoreceptors and ON-bipolar cells, the function of nyctalopin in cone-dominant animal models has not been studied. Because the larval zebrafish retina is cone-dominant, we isolated the zebrafish nyx ortholog and raised a polyclonal antibody against the protein. Nyctalopin is expressed postsynaptically in both synaptic layers of the retina. Functional disruption via morpholino antisense injection leads to characteristic defects in the electroretinogram and defects in visual contrast sensitivity. We therefore demonstrated that nyctalopin plays a similar role in retinal synapse function in the cone pathway as in the rod pathway, thereby creating a genetic model for CSNB1 and its effects on cone vision.

Animals↗

PubMed2006

The Zebrafish fade out mutant: a novel genetic model for Hermansky-Pudlak syndrome.

PURPOSE: To characterize retinal morphology and visual system function in the zebrafish mutant fade out (fad) and to establish the mutant as a lower vertebrate model for Hermansky-Pudlak syndrome (HPS). METHODS: Retinal morphology of fad larvae was examined between 3 and 9 days postfertilization (dpf) by standard histology, transmission electron microscopy, and immunohistochemistry examination. Apoptotic cells were visualized by TdT-mediated dUTP nick-end labeling (TUNEL) staining. Visual system function was probed by electroretinography and behavioral assessment by optokinetic response measurements. Blood clotting was evaluated by time to occlusion testing of blood vessels as an arterial thrombosis assay. The chromosomal location of fad was determined by simple sequence-length polymorphism mapping. Genomic fragments of candidate genes were cloned by standard molecular techniques and mapped to the zebrafish genome by radiation hybrid mapping. RESULTS: Mutant fad larvae are hypopigmented and show structural defects in the outer retina. Melanosomes of these larvae in the retinal pigment epithelium are hypopigmented, generally smaller, and progressively reduced in number compared to nonmutant larvae. Progressive microvilli protrusions into the photoreceptor cell layer are not detectable, and photoreceptor outer segments get shorter and are misaligned. Photoreceptors subsequently undergo apoptosis, with a peak of cell death at 6 dpf. Electrical responses of the retina and visual performance are severely reduced. Blood clotting is prolonged in mutant fad larvae. Genomic mapping of fad reveals distinct genomic positions of the mutant gene from known human HPS genes. CONCLUSIONS: The fad mutant shows syndromic defects in pigmentation, outer retinal structure and function, and blood clotting. This syndrome is characteristic of Hermansky-Pudlak syndrome (HPS), making fad a novel genetic model of HPS. The gene does not cosegregate with the known human HPS genes, suggesting a novel molecular cause of HPS.

Animals↗

PubMed2006

belladonna/(Ihx2) is required for neural patterning and midline axon guidance in the zebrafish forebrain.

Some of the earliest axon pathways to form in the vertebrate forebrain are established as commissural and retinal axons cross the midline of the diencephalon and telencephalon. To better understand axon guidance in the forebrain, we characterized the zebrafish belladonna (bel) mutation, which disrupts commissural and retinal axon guidance in the forebrain. Using a positional cloning strategy, we determined that the bel locus encodes zebrafish Lhx2, a lim-homeodomain transcription factor expressed in the brain, eye and fin buds. We show that bel(Ihx2) function is required for patterning in the ventral forebrain and eye, and that loss of bel function leads to alterations in regulatory gene expression, perturbations in axon guidance factors, and the absence of an optic chiasm and forebrain commissures. Our analysis reveals new roles for Ihx2 in midline axon guidance, forebrain patterning and eye morphogenesis.

Amino Acid Sequence↗

PubMed2005

A mutation in the silver gene leads to defects in melanosome biogenesis and alterations in the visual system in the zebrafish mutant fading vision.

Forward genetic screens have been instrumental in defining molecular components of visual function. The zebrafish mutant fading vision (fdv) has been identified in such a screen due to defects in vision accompanied by hypopigmentation in the retinal pigment epithelium (RPE) and body melanocytes. The RPE forms the outer most layer of the retina, and its function is essential for vision. In fdv mutant larvae, the outer segments of photoreceptors are strongly reduced in length or absent due to defects in RPE cells. Ultrastructural analysis of RPE cells reveals dramatic cellular changes such as an absence of microvilli and vesicular inclusions. The retinoid profile is altered as judged by biochemical analysis, arguing for a partial block in visual pigment regeneration. Surprisingly, homozygous fdv vision mutants survive to adulthood and show, despite a persistence of the hypopigmentation, a partial recovery of retinal morphology. By positional cloning and subsequent morpholino knock-down, we identified a mutation in the silver gene as the molecular defect underlying the fdv phenotype. The Silver protein is required for intralumenal fibril formation in melanosomes by amylogenic cleavage. Our data reveal an unexpected link between melanosome biogenesis and the visual system, undetectable in cell culture.

Amino Acid Sequence↗

PubMed2005

Knockdown of cone-specific kinase GRK7 in larval zebrafish leads to impaired cone response recovery and delayed dark adaptation.

Phosphorylation of rhodopsin by rhodopsin kinase GRK1 is an important desensitization mechanism in scotopic vision. For cone vision GRK1 is not essential. However, cone opsin is phosphorylated following light stimulation. In cone-dominant animals as well as in humans, but not in rodents, GRK7, a cone-specific homolog of GRK1, has been identified in cone outer segments. To investigate the function of GRK7 in vivo, we cloned two orthologs of grk7 in zebrafish and knocked down gene expression of grk7a in zebrafish larvae by morpholino antisense nucleotides. Photoresponse recovery in Grk7a-deficient larvae was delayed in electroretinographic measurements, and temporal contrast sensitivity was reduced, particularly under bright-light conditions. These results show that function of a cone-specific kinase is essential for cone vision in the zebrafish retina and argue that pigment bleaching and spontaneous decay alone are not sufficient for light adaptation and rapid cone response inactivation.

Animals↗

PubMed2005

Fish possess multiple copies of fgfrl1, the gene for a novel FGF receptor.

FGFRL1 is a novel FGF receptor that lacks the intracellular tyrosine kinase domain. While mammals, including man and mouse, possess a single copy of the FGFRL1 gene, fish have at least two copies, fgfrl1a and fgfrl1b. In zebrafish, both genes are located on chromosome 14, separated by about 10 cM. The two genes show a similar expression pattern in several zebrafish tissues, although the expression of fgfrl1b appears to be weaker than that of fgfrl1a. A clear difference is observed in the ovary of Fugu rubripes, which expresses fgfrl1a but not fgfrl1b. It is therefore possible that subfunctionalization has played a role in maintaining the two fgfrl1 genes during the evolution of fish. In human beings, the FGFRL1 gene is located on chromosome 4, adjacent to the SPON2, CTBP1 and MEAEA genes. These genes are also found adjacent to the fgfrl1a gene of Fugu, suggesting that FGFRL1, SPON2, CTBP1 and MEAEA were preserved as a coherent block during the evolution of Fugu and man.

Amino Acid Sequence↗

PubMed2005

Duplicated genes with split functions: independent roles of protocadherin15 orthologues in zebrafish hearing and vision.

In the sensory receptors of both the eye and the ear, specialized apical structures have evolved to detect environmental stimuli such as light and sound. Despite the morphological divergence of these specialized structures and differing transduction mechanisms, the receptors appear to rely in part on a shared group of genes for function. For example, mutations in Usher (USH) genes cause a syndrome of visual and acoustic-vestibular deficits in humans. Several of the affected genes have been identified, including the USH1F gene, which encodes protocadherin 15 (PCDH15). Pcdh15 mutant mice also have both auditory and vestibular defects, although visual defects are not evident. Here we show that zebrafish have two closely related pcdh15 genes that are required for receptor-cell function and morphology in the eye or ear. Mutations in pcdh15a cause deafness and vestibular dysfunction, presumably because hair bundles of inner-ear receptors are splayed. Vision, however, is not affected in pcdh15a mutants. By contrast, reduction of pcdh15b activity using antisense morpholino oligonucleotides causes a visual defect. Optokinetic and electroretinogram responses are reduced in pcdh15b morpholino-injected larvae. In electron micrographs, morphant photoreceptor outer segments are improperly arranged, positioned perpendicular to the retinal pigment epithelium and are clumped together. Our results suggest that both cadherins act within their respective transduction organelles: Pcdh15a is necessary for integrity of the stereociliary bundle, whereas Pcdh15b is required for alignment and interdigitation of photoreceptor outer segments with the pigment epithelium. We conclude that after a duplication of pcdh15, one gene retained an essential function in the ear and the other in the eye.

Animals↗

PubMed2005

Contrast sensitivity, spatial and temporal tuning of the larval zebrafish optokinetic response.

PURPOSE: To characterize the quantitative properties of the optokinetic response (OKR) in zebrafish larvae as a tool to test visual performance in genetically modified larvae. METHODS: Horizontal OKR was triggered in 5-day-old zebrafish larvae by stimulation with projected computer-generated gratings of varying contrast, angular velocity, temporal and spatial frequency, and brightness. Eye movements were analyzed by a custom-made eye tracker based on image analysis. RESULTS: The gain of the OKR slow phase was dependent on angular velocity, spatial frequency, and contrast of a moving grating, but largely independent on brightness. Eye velocity was a logarithmically linear function of grating contrast with a slope of approximately 0.8 per log unit contrast. CONCLUSIONS: The OKR of the larval zebrafish is not scaled for stimulus contrast and spatial frequency. These properties make the OKR a valuable tool to quantify behavioral visual performance such as visual acuity, contrast sensitivity, and light adaptation. This behavioral paradigm will be useful for analyzing visual performance in mutant and gene-knockdown larval zebrafish.

Animals↗

PubMed2004

An inexpensive device for non-invasive electroretinography in small aquatic vertebrates.

Electroretinographic (ERG) method records a sum field potential of the retina in response to light. It mainly arises in the outer retina and is used as a non-invasive measure in both animal experiments and the clinic. Since it is a comprehensive method to assess outer retinal function, it is becoming increasingly useful in genetic studies of vision. Here we present a simple in-house built setup to measure ERGs of aquatic vertebrates. We have used this setup to efficiently and reliably measure intact larvae of zebrafish (Danio rerio), Medaka fish (Oryzias latipes), and Xenopus laevis tadpoles. By slight modification of the setup, we were also able to measure adult zebrafish and Medaka, demonstrating the general versatility of the setup. We picked these organisms since they are increasingly used to study visual function with genetic means. This setup is easily built and will be particularly useful for laboratories setting up ERG measurements as a complement to their genetic studies.

Adaptation, Physiological↗

PubMed2003

Synaptic plasticity and functionality at the cone terminal of the developing zebrafish retina.

Previous studies have analyzed photoreceptor development, some inner retina cell types, and specific neurotransmitters in the zebrafish retina. However, only minor attention has been paid to the morphology of the synaptic connection between photoreceptors and second order neurons even though it represents the transition from the light sensitive receptor to the neuronal network of the visual system. Here, we describe the appearance and differentiation of pre- and postsynaptic elements at cone synapses in the developing zebrafish retina together with the maturation of the directly connecting second order neurons and a dopaminergic third order feedback-neuron from the inner retina. Zebrafish larvae were examined at developmental stages from 2 to 7dpf (days postfertilization) and in the adult. Synaptic maturation at the photoreceptor terminals was examined with antibodies against synapse associated proteins. The appearance of synaptic plasticity at the so-called spinule-type synapses between cones and horizontal cells was assessed by electron microscopy, and the maturation of photoreceptor downstream connection was identified by immunocytochemistry for GluR4 (AMPA-type glutamate receptor subunit), protein kinase beta(1) (mixed rod-cone bipolar cells), and tyrosine hydroxylase (dopaminergic interplexiform cells). We found that developing zebrafish retinas possess first synaptic structures at the cone terminal as early as 3.5dpf. Morphological maturation of these synapses at 3.5-4dpf, together with the presence of synapse associated proteins at 2.5dpf and the maturation of second order neurons by 5dpf, indicate functional synaptic connectivity and plasticity between the cones and their second order neurons already at 5dpf. However, the mere number of spinules and ribbons at 7dpf still remains below the adult values, indicating that synaptic functionality of the zebrafish retina is not entirely completed at this stage of development.

Animals↗

PubMed2003

Behavioral genetic approaches to visual system development and function in zebrafish.

The zebrafish is a recent vertebrate model system that shows great potential for a genetic analysis of behavior. Early development is extraordinarily rapid, so that larvae already display a range of behaviors 5 days after fertilization. In particular the visual system develops precociously, supporting a number of visually mediated behaviors in the larva. This provides the opportunity to use these visually mediated behaviors to screen chemically mutagenized strains for defects in vision. Larval optokinetic and optomotor responses have already been successfully employed to screen for mutant strains with defects in the visual system. In the adult zebrafish a visually mediated escape response has proved useful for screening for dominant mutations of the visual system. Here, I summarize visually mediated behaviors of both larval and adult zebrafish and their applicability for genetic screens, and present, the approaches and results of visual behavior carried out to date.

Animals↗

PubMed2003

Retinal defects in the zebrafish bleached mutant.

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.

Animals↗

PubMed2003

Retinal function and morphology in two zebrafish models of oculo-renal syndromes.

We characterized visual system defects in two recessive zebrafish mutants oval and elipsa. These mutants share the syndromic phenotype of outer retinal dystrophy in conjunction with cystic renal disorder. We tested the function of the larval visual system in a behavioural assay, eliciting optokinetic eye movements by high-contrast motion stimulation while recording eye movements in parallel. Visual stimulation did not elicit eye movements in mutant larvae, while spontaneous eye movements could be observed. The retina proved to be unresponsive to light using electroretinography, indicative of a defect in the outer retina. Histological analysis of mutant retinas revealed progressive degeneration of photoreceptors, initiated in central retinal locations and spreading to more peripheral regions with increasing age. The inner retina remains unaffected by the mutation. Photoreceptors display cell type-specific immunoreactivity prior to apoptotic cell death, arguing for a dystrophic defect. Genomic mapping employing simple sequence-length polymorphisms located both mutations on different regions of zebrafish linkage group 9. These mutants may serve as accessible animal models of human outer retinal dystrophies, including oculo-renal diseases, and show the general usefulness of a behavioural genetic approach to study visual system development in the model vertebrate zebrafish.

Animals↗

PubMed2003

Double cone dystrophy and RPE degeneration in the retina of the zebrafish gnn mutant.

PURPOSE: To characterize morphologic alterations in the retina of the visual mutant zebrafish gantenbein (gnn) and to examine whether these alterations correlate with those present in human hereditary eye diseases. METHODS: The gnn mutant was isolated by behavioral and macroscopic screening. Retinas of gnn zebrafish larvae were examined at different developmental stages from 2 to 9 days postfertilization (dpf) by standard histologic staining techniques and by immunocytochemistry. Ultrastructural alterations were examined by electron microscopy. The genetic map position of the induced mutation was identified by mapping with two candidate primer pairs on single larvae. RESULTS: The gnn mutant exhibited shortened outer photoreceptor segments and altered RPE morphology. In the photoreceptor layer of the mutant, the total number of lectin-labeled cones was reduced in all developmental stages from 2 to 7 dpf, whereas the amount of rhodopsin-positive cells remained at the wild-type (WT) level. Labeling with zebrafish opsin antibodies revealed dystrophic red cones at 5 dpf, whereas the morphology of all other cone types was largely unaffected. Electron microscopy unveiled electron-dense deposits between the discs of the double cone outer segments. In addition, the onset of progressive RPE degeneration was observed at this stage of development. At later stages, all cone types and the RPE became degenerative. The morphology of distinct second-order neurons remained largely unaffected by the mutation. The gnn mutation was located approximately 4.3 cM from the simple sequence length polymorphism (SSLP) marker Z15453 on linkage group 16. CONCLUSIONS: In gnn mutant zebrafish, cones, and especially red cones, are dystrophic in early retinal development. Subsequent to this cone dystrophy, the RPE becomes dysfunctional and starts to degenerate in later stages of development. Thus, the early developmental morphology of gnn exhibits similarities to cone dystrophies most commonly seen in age-related macular degeneration (AMD) among humans, whereas the later stages of degeneration in gnn resemble RPE alterations in retinitis pigmentosa (RP) in humans. The gnn zebrafish mutant may therefore be a useful model for examining the possible interplay and connection between cone dystrophy and RPE degeneration.

Animals↗