Visual acuity in larval zebrafish: behavior and histology

BACKGROUND: Visual acuity, the ability of the visual system to distinguish two separate objects at a given angular distance, is influenced by the optical and neuronal properties of the visual system. Although many factors may contribute, the ultimate limit is photoreceptor spacing. In general, at least one unstimulated photoreceptor flanked by two stimulated ones is needed to perceive two objects as separate. This critical interval is also referred to as the Nyquist frequency and is according to the Shannon sampling theorem the highest spatial frequency where a pattern can be faithfully transmitted. We measured visual acuity in a behavioral experiment and compared the data to the physical limit given by photoreceptor spacing in zebrafish larvae. RESULTS: We determined visual acuity by using the optokinetic response (OKR), reflexive eye movements in response to whole field movements of the visual scene. By altering the spatial frequency we determined the visual acuity at approximately 0.16 cycles/degree (cpd) (minimum separable angle = 3.1 degrees ). On histological sections we measured the retinal magnification factor and the distance between double cones, that are thought to mediate motion perception. These measurements set the physical limit at 0.24 cpd (2.1 degrees ). CONCLUSION: The maximal spatial information as limited by photoreceptor spacing can not be fully utilized in a motion dependent visual behavior, arguing that the larval zebrafish visual system has not matured enough to optimally translate visual information into behavior. Nevertheless behavioral acuity is remarkable close to its maximal value, given the immature state of young zebrafish larvae

Analysis of the activity-deprived zebrafish mutant macho reveals an essential requirement of neuronal activity for the development of a fine-grained visuotopic map.

The formation of a retinotopic map is thought to involve an activity-independent molecular phase for early steps of both axon pathfinding and projection and a later phase in which cross talk between retinal ganglion cells (RGCs) and tectal neurons modifies and refines the neuronal connections. We report that the maturation of the retinotopic map in the zebrafish tectum involves activity-dependent processes. Zebrafish larvae mutant for the gene macho (mao) lack neuronal activity in RGCs and also display an enlarged retinotectal projection field but no significant increase in single axon length. This morphological defect can be phenocopied by raising larvae under TTX-induced neural impulse blockade. The effect of activity deprivation is dependent on the developmental stage. The projection phenotype in mao as well as in the TTX-treated larvae develops between 4 and 6 d post-fertilization (dpf), after complete tectal coverage is first achieved. Electrophysiological recordings of RGCs in wild-type and mao zebrafish larvae reveal a temporally regulated reduction of sodium current in the mutant between 5 and 6 dpf. This coincides with the time of the axonal projection shifting on the tectum to compensate for the disparate growth patterns of the retina and the tectum. Our genetic and physiological analyses suggest a model in which neuronal activity in RGCs is needed for the establishment of morphological plasticity

Impaired retinal differentiation and maintenance in zebrafish laminin mutants

PURPOSE: To characterize morphologic and physiological alterations in the retina of three laminin mutant zebrafish, bashful (bal, lama1), grumpy (gup, lamb1), and sleepy (sly, lamc1), which were identified in forward genetic screens and were found to be impaired in visual functions. METHODS: Mutant larvae were observed for defects in visual behavior by testing their optokinetic response (OKR). In addition, electroretinograms (ERG) were measured and retinal morphology was examined by standard histology, immunocytochemistry, TUNEL assay, and electron microscopy. RESULTS: Both, gup and sly showed no OKR at any light intensity tested, whereas bal embryos showed some remaining OKR behavior at more than 40% of contrast. Consistent with the OKR result, gup and sly did not show an ERG response at any light intensity tested, whereas bal mutants exhibited small a- and b-waves at high light intensities. All three laminin mutants showed altered ganglion cell layers, optic nerve fasciculations, and lens defects. Again, bal showed the least severe morphologic phenotype with no additional defects. In contrast, both, gup and sly, showed severe photoreceptor outer segment shortening and synapse alteration (floating ribbons) as well as increased cell death. CONCLUSIONS: Lamb1 and lamc1 chains play an important role in the morphogenesis of photoreceptors and their synapses. In contrast, lama1 is not involved in outer retina development

Reflective multi-immersion microscope objectives inspired by the Schmidt telescope

Imaging large, cleared samples requires microscope objectives that combine a large field of view (FOV) with a long working distance (WD) and a high numerical aperture (NA). Ideally, such objectives should be compatible with a wide range of immersion media, which is challenging to achieve with conventional lens-based objective designs. Here we introduce the multi-immersion 'Schmidt objective' consisting of a spherical mirror and an aspherical correction plate as a solution to this problem. We demonstrate that a multi-photon variant of the Schmidt objective is compatible with all homogeneous immersion media and achieves an NA of 1.08 at a refractive index of 1.56, 1.1-mm FOV and 11-mm WD. We highlight its versatility by imaging cleared samples in various media ranging from air and water to benzyl alcohol/benzyl benzoate, dibenzyl ether and ethyl cinnamate and by imaging of neuronal activity in larval zebrafish in vivo. In principle, the concept can be extended to any imaging modality, including wide-field, confocal and light-sheet microscopy

Elevated photic response is followed by a rapid decay and depressed state in ictogenic networks

Objective: The switch between nonseizure and seizure states involves profound alterations in network excitability and synchrony. In this study, we aimed to identify and compare features of neural excitability and dynamics across multiple zebrafish seizure and epilepsy models. Methods: Inspired by video-electroencephalographic recordings in patients, we developed a framework to study spontaneous and photically evoked neural and locomotor activity in zebrafish larvae, by combining high-throughput behavioral tracking and whole-brain in vivo two-photon calcium imaging. Results: Our setup allowed us to dissect behavioral and physiological features that are divergent or convergent across multiple models. We observed that spontaneous locomotor and neural activity exhibit great diversity across models. Nonetheless, during photic stimulation, hyperexcitability and rapid response dynamics were well conserved across multiple models, highlighting the reliability of photically evoked activity for high-throughput assays. Intriguingly, in several models, we observed that the initial elevated photic response is often followed by rapid decay of neural activity and a prominent depressed state. Elevated photic response and following depressed state in seizure-prone networks are significantly reduced by the antiseizure medication valproic acid. Finally, rapid decay and depression of neural activity following photic stimulation temporally overlap with slow recruitment of astroglial calcium signals that are enhanced in seizure-prone networks. Significance: We argue that fast decay of neural activity and depressed states following photic response are likely due to homeostatic mechanisms triggered by excessive neural activity. An improved understanding of the interplay between elevated and depressed excitability states might suggest tailored epilepsy therapies. Keywords: astroglia; calcium imaging; depressed state; elevated state; epilepsy; high-throughput behavior; hyperexcitability; photic stimulation; seizure; zebrafis

Illusionary Self-Motion Perception in Zebrafish

Zebrafish mutant belladonna (bel) carries a mutation in the lhx2 gene (encoding a Lim domain homeobox transcription factor) that results in a defect in retinotectal axon pathfinding, which can lead to uncrossed optic nerves failing to form an optic chiasm. Here, we report on a novel swimming behavior of the bel mutants, best described as looping. Together with two previously reported oculomotor instabilities that have been related to achiasmatic bel mutants, reversed optokinetic response (OKR) and congenital nystagmus (CN, involuntary conjugate oscillations of both eyes), looping opens a door to study the influence of visual input and eye movements on postural balance. Our result shows that looping correlates perfectly with reversed OKR and CN and is vision-dependent and contrast sensitive. CN precedes looping and the direction of the CN slow phase is predictive of the looping direction, but is absent during looping. Therefore, looping may be triggered by CN in bel. Moreover, looping in wild-type fish can also be evoked by whole-field motion, suggesting that looping in a bel mutant larvae is a result of self-motion perception. In contrary to previous hypotheses, our findings indicate that postural control in vertebrates relies on both direct visual input (afference signal) and eye-movement-related signals (efference copy or reafference signal)

Rpgrip1 is required for rod outer segment development and ciliary protein trafficking in zebrafish

The authors would like to thank the Royal Society of London, the National Eye Research Centre, the Visual Research Trust, Fight for Sight, the W.H. Ross Foundation, the Rosetrees Trust, and the Glasgow Children’s Hospital Charity for supporting this work. This work was also supported by the Deanship of Scientific Research at King Saud University for funding this research (Research Project) grant number ‘RGP – VPP – 219’.Mutations in the RPGR-interacting protein 1 (RPGRIP1) gene cause recessive Leber congenital amaurosis (LCA), juvenile retinitis pigmentosa (RP) and cone-rod dystrophy. RPGRIP1 interacts with other retinal disease-causing proteins and has been proposed to have a role in ciliary protein transport; however, its function remains elusive. Here, we describe a new zebrafish model carrying a nonsense mutation in the rpgrip1 gene. Rpgrip1homozygous mutants do not form rod outer segments and display mislocalization of rhodopsin, suggesting a role for RPGRIP1 in rhodopsin-bearing vesicle trafficking. Furthermore, Rab8, the key regulator of rhodopsin ciliary trafficking, was mislocalized in photoreceptor cells of rpgrip1 mutants. The degeneration of rod cells is early onset, followed by the death of cone cells. These phenotypes are similar to that observed in LCA and juvenile RP patients. Our data indicate RPGRIP1 is necessary for rod outer segment development through regulating ciliary protein trafficking. The rpgrip1 mutant zebrafish may provide a platform for developing therapeutic treatments for RP patients.Publisher PDFPeer reviewe

Novel Expression Patterns of Metabotropic Glutamate Receptor 6 in the Zebrafish Nervous System

The metabotropic glutamate receptor 6 (mGluR6 or GRM6) belongs to the class III of the metabotropic glutamate receptor family. It is the only known mGluR that mediates direct synaptic transmission in the nervous system and is thought to mediate the ON-response in the ON-pathway of the vertebrate retina. Phylogenetic and gene structure analysis indicated that the zebrafish genome harbours two mglur6 paralogs, mglur6a and mglur6b. Besides expression in the inner nuclear layer and distinct regions in the brain, both mglur6 paralogs are expressed in ganglion cells of the retina, an expression pattern which can also be observed in the downstream effector molecules gnaoa and gnaob. This unexpected expression pattern is consistent with immunohistological labeling using a peptide antibody specific for the mGluR6b paralog. These expression patterns contradict the existing view that mGluR6 is solely located on ON-bipolar cells where it functions in signal transmission. Consistent with expression in ON-bipolar cells, we report a decreased b-wave amplitude in the electroretinogram after morpholino-based downregulation of mGluR6b, showing a function in the ON response. Our data suggest more widespread functions of mGluR6 mediated signaling in the central nervous system, possibly including sign reversing synapses in the inner retina

Functional MRI in Awake Unrestrained Dogs

Because of dogs' prolonged evolution with humans, many of the canine cognitive skills are thought to represent a selection of traits that make dogs particularly sensitive to human cues. But how does the dog mind actually work? To develop a methodology to answer this question, we trained two dogs to remain motionless for the duration required to collect quality fMRI images by using positive reinforcement without sedation or physical restraints. The task was designed to determine which brain circuits differentially respond to human hand signals denoting the presence or absence of a food reward. Head motion within trials was less than 1 mm. Consistent with prior reinforcement learning literature, we observed caudate activation in both dogs in response to the hand signal denoting reward versus no-reward

Phenylthiourea Specifically Reduces Zebrafish Eye Size

Phenylthiourea (PTU) is commonly used for inhibiting melanization of zebrafish embryos. In this study, the standard treatment with 0.2 mM PTU was demonstrated to specifically reduce eye size in larval fish starting at three days post-fertilization. This effect is likely the result of a reduction in retinal and lens size of PTU-treated eyes and is not related to melanization inhibition. This is because the eye size of tyr, a genetic mutant of tyrosinase whose activity is inhibited in PTU treatment, was not reduced. As PTU contains a thiocarbamide group which is presented in many goitrogens, suppressing thyroid hormone production is a possible mechanism by which PTU treatment may reduce eye size. Despite the fact that thyroxine level was found to be reduced in PTU-treated larvae, thyroid hormone supplements did not rescue the eye size reduction. Instead, treating embryos with six goitrogens, including inhibitors of thyroid peroxidase (TPO) and sodium-iodide symporter (NIS), suggested an alternative possibility. Specifically, three TPO inhibitors, including those that do not possess thiocarbamide, specifically reduced eye size; whereas none of the NIS inhibitors could elicit this effect. These observations indicate that TPO inhibition rather than a general suppression of thyroid hormone synthesis is likely the underlying cause of PTU-induced eye size reduction. Furthermore, the tissue-specific effect of PTU treatment might be mediated by an eye-specific TPO expression. Compared with treatment with other tyrosinase inhibitors or bleaching to remove melanization, PTU treatment remains the most effective approach. Thus, one should use caution when interpreting results that are obtained from PTU-treated embryos