Fourier Motion Processing in the Optic Tectum and Pretectum of the Zebrafish Larva

In the presence of moving visual stimuli, the majority of animals follow the Fourier motion energy (luminance), independently of other stimulus features (edges, contrast, etc.). While the behavioral response to Fourier motion has been studied in the past, how Fourier motion is represented and processed by sensory brain areas remains elusive. Here, we investigated how visual moving stimuli with or without the first Fourier component (square-wave signal or missing fundamental signal) are represented in the main visual regions of the zebrafish brain. First, we monitored the larva's optokinetic response (OKR) induced by square-wave and missing fundamental signals. Then, we used two-photon microscopy and GCaMP6f zebrafish larvae to monitor neuronal circuit dynamics in the optic tectum and the pretectum. We observed that both the optic tectum and the pretectum circuits responded to the square-wave gratings. However, only the pretectum responded specifically to the direction of the missing-fundamental signal. In addition, a group of neurons in the pretectum responded to the direction of the behavioral output (OKR), independently of the type of stimulus presented. Our results suggest that the optic tectum responds to the different features of the stimulus (e.g., contrast, spatial frequency, direction, etc.), but does not respond to the direction of motion if the motion information is not coherent (e.g., the luminance and the edges and contrast in the missing-fundamental signal). On the other hand, the pretectum mainly responds to the motion of the stimulus based on the Fourier energy.

Affordable and effective optokinetic response methods to assess visual acuity and contrast sensitivity in larval to juvenile zebrafish

The optokinetic response (OKR) is an effective behavioural assay to investigate functional vision in zebrafish. The rapid and widespread use of gene editing, drug screening and environmental modulation technologies has resulted in a broader need for visual neuroscience researchers to access affordable and more sensitive OKR, contrast sensitivity (CS) and visual acuity (VA) assays. Here, we demonstrate how 2D- and 3D-printed, striped patterns or drums coupled with a motorised base and microscope provide a simple, cost-effective but efficient means to assay OKR, CS and VA in larval-juvenile zebrafish. In wild-type, five days post-fertilisation (dpf) zebrafish, the 2D or 3D set-ups of 0.02 cycles per degree (cpd) (standard OKR stimulus) and 100% black-white contrast evoked equivalent responses of 24.2±3.9 or 21.8±3.9 saccades per minute, respectively. Furthermore, although the OKR number was significantly reduced compared to the 0.02 cpd drum (p<0.0001), 0.06 and 0.2 cpd drums elicited equivalent responses with both set-ups. Notably, standard OKRs varied with time of day; peak responses of 29.8±7 saccades per minute occurred in the early afternoon with significantly reduced responses occurring in the early morning or late afternoon (18.5±3 and 18.4±4.5 saccades per minute, respectively). A customised series of 2D printed drums enabled analysis of VA and CS in 5-21 dpf zebrafish. The saccadic frequency in VA assays was inversely proportional to age and spatial frequency and in CS assays was inversely proportional to age and directly proportional to contrast of the stimulus. OKR, VA and CS of zebrafish larvae can be efficiently measured using 2D- or 3D-printed striped drums. For data consistency the luminance of the OKR light source, the time of day when the analysis is performed, and the order of presentation of VA and CS drums must be considered. These simple methods allow effective and more sensitive analysis of functional vision in zebrafish.

An inducible rodent glaucoma model that exhibits gradual sustained increase in intraocular pressure with distinct inner retina and optic nerve inflammation

Glaucoma is a chronic and progressive neurodegenerative disease of the optic nerve resulting in loss of retinal ganglion cells (RGCs) and vision. The most prominent glaucoma risk factor is increased intraocular pressure (IOP), and most models focus on reproducing this aspect to study disease mechanisms and targets. Yet, current models result in IOP profiles that often do not resemble clinical glaucoma. Here we introduce a new model that results in a gradual and sustained IOP increase over time. This approach modifies a circumlimbal suture method, taking care to make the sutures ‘snug’ instead of tight, without inducing an initial IOP spike. This approach did not immediately affect IOPs, but generated gradual ocular hypertension (gOHT) as the sutures tighten over time, in comparison to loosely sutured control eyes (CON), resulting in an average 12.6 mmHg increase in IOP at 17 weeks (p < 0.001). Corresponding characterization revealed relevant retinal and optic nerve pathology, such as thinning of the retinal nerve fiber layer, decreased optokinetic response, RGC loss, and optic nerve head remodeling. Yet, angles remained open, with no evidence of inflammation. Corresponding biochemical profiling indicated significant increases in TGF-β2 and 3, and IL-1 family cytokines in gOHT optic nerve tissues compared to CON, with accompanying microglial reactivity, consistent with active tissue injury and repair mechanisms. Remarkably, this signature was absent from optic nerves following acute ocular hypertension (aOHT) associated with intentionally tightened sutures, although the resulting RGC loss was similar in both methods. These results suggest that the pattern of IOP change has an important impact on underlying pathophysiology.

A mutant wfs1 zebrafish model of Wolfram syndrome manifesting visual dysfunction and developmental delay

Wolfram syndrome (WS) is an ultra-rare progressive neurodegenerative disorder defined by early-onset diabetes mellitus and optic atrophy. The majority of patients harbour recessive mutations in the WFS1 gene, which encodes for Wolframin, a transmembrane endoplasmic reticulum protein. There is limited availability of human ocular and brain tissues, and there are few animal models for WS that replicate the neuropathology and clinical phenotype seen in this disorder. We, therefore, characterised two wfs1 zebrafish knockout models harbouring nonsense wfs1a and wfs1b mutations. Both homozygous mutant wfs1a−/− and wfs1b−/− embryos showed significant morphological abnormalities in early development. The wfs1b−/− zebrafish exhibited a more pronounced neurodegenerative phenotype with delayed neuronal development, progressive loss of retinal ganglion cells and clear evidence of visual dysfunction on functional testing. At 12 months of age, wfs1b−/− zebrafish had a significantly lower RGC density per 100 μm2 (mean ± standard deviation; 19 ± 1.7) compared with wild-type (WT) zebrafish (25 ± 2.3, p < 0.001). The optokinetic response for wfs1b−/− zebrafish was significantly reduced at 8 and 16 rpm testing speeds at both 4 and 12 months of age compared with WT zebrafish. An upregulation of the unfolded protein response was observed in mutant zebrafish indicative of increased endoplasmic reticulum stress. Mutant wfs1b−/− zebrafish exhibit some of the key features seen in patients with WS, providing a versatile and cost-effective in vivo model that can be used to further investigate the underlying pathophysiology of WS and potential therapeutic interventions.

Affordable and effective optokinetic response methods to assess visual acuity and contrast sensitivity in larval to juvenile zebrafish

The optokinetic response (OKR) is an effective behavioural assay to investigate functional vision in zebrafish. The rapid and widespread use of gene editing, drug screening and environmental modulation technologies has resulted in a broader need for visual neuroscience researchers to access affordable and more sensitive OKR, contrast sensitivity (CS) and visual acuity (VA) assays. Here, we demonstrate how 2D- and 3D-printed, striped patterns or drums coupled with a motorised base and microscope provide a simple, cost-effective but efficient means to assay OKR, CS and VA in larval-juvenile zebrafish. In wild-type, five days post-fertilisation (dpf) zebrafish, the 2D or 3D drums printed with the standard OKR stimulus of 0.02 cycles per degree (cpd), 100% black-white contrast evoked equivalent responses of 24.2 or 21.8 saccades per minute, respectively. Furthermore, although the OKR number was significantly reduced compared to the 0.02 cpd drum (p<0.0001), the 2D and 3D drums evoked equivalent responses with the 0.06 and 0.2 cpd drums. Notably, standard OKRs varied with time of day; peak responses of 29.8 saccades per minute occurred in the early afternoon with significantly reduced responses occurring in the early morning or late afternoon (18.5 and 18.4 saccades per minute, respectively). A customised series of 2D printed drums enabled analysis of VA and CS in 5-21 dpf zebrafish. The saccadic frequency in VA and CS assays was inversely proportional to age, spatial frequency and contrast of the stimulus. OKR, VA and CS of zebrafish larvae can be efficiently measured using 2D- or 3D-printed striped drums. For data consistency the luminance of the OKR light source, the time of day when the analysis is performed, and the order of presentation of VA and CS drums must be considered. These simple methods allow effective and more sensitive analysis of functional vision in zebrafish.

Behavioural red-light sensitivity in fish according to the optomotor response

Various procedures have been adopted to investigate spectral sensitivity of animals, e.g. absorption spectra of visual pigments, electroretinography, optokinetic response, optomotor response (OMR) and phototaxis. The use of these techniques has led to various conclusions about animal vision. However, visual sensitivity should be evaluated consistently for a reliable comparison. In this study, we retrieved behavioural data of several fish species using a single OMR procedure and compared their sensitivities to near-infrared light. Besides cavefish that lack eyes, some species were not appropriate for the OMR test because they either stayed still or changed swimming direction frequently. Eight of 13 fish species tested were OMR positive. Detailed analyses using medaka, goldfish, zebrafish, guppy, stickleback and cichlid revealed that all the fish were sensitive to light at a wavelength greater than or equal to 750 nm, where the threshold wavelengths varied from 750 to 880 nm. Fish opsin repertoire affected the perception of red light. By contrast, the copy number of long-wavelength-sensitive ( LWS ) genes did not necessarily improve red-light sensitivity. While the duplication of LWS and other cone opsin genes that has occurred extensively during fish evolution might not aid increasing spectral sensitivity, it may provide some other advantageous ophthalmic function, such as enhanced spectral discrimination.

Circuit Organization Underlying Optic Flow Processing in Zebrafish

Animals’ self-motion generates a drifting movement of the visual scene in the entire field of view called optic flow. Animals use the sensation of optic flow to estimate their own movements and accordingly adjust their body posture and position and stabilize the direction of gaze. In zebrafish and other vertebrates, optic flow typically drives the optokinetic response (OKR) and optomotor response (OMR). Recent functional imaging studies in larval zebrafish have identified the pretectum as a primary center for optic flow processing. In contrast to the view that the pretectum acts as a relay station of direction-selective retinal inputs, pretectal neurons respond to much more complex visual features relevant to behavior, such as spatially and temporally integrated optic flow information. Furthermore, optic flow signals, as well as motor signals, are represented in the cerebellum in a region-specific manner. Here we review recent findings on the circuit organization that underlies the optic flow processing driving OKR and OMR.

Kdm3b haploinsufficiency impairs the consolidation of cerebellum-dependent motor memory in mice

Histone modifications are a key mechanism underlying the epigenetic regulation of gene expression, which is critically involved in the consolidation of multiple forms of memory. However, the roles of histone modifications in cerebellum-dependent motor learning and memory are not well understood. To test whether changes in histone methylation are involved in cerebellar learning, we used heterozygous Kdm3b knockout (Kdm3b+/−) mice, which show reduced lysine 9 on histone 3 (H3K9) demethylase activity. H3K9 di-methylation is significantly increased selectively in the granule cell layer of the cerebellum of Kdm3b+/− mice. In the cerebellum-dependent optokinetic response (OKR) learning, Kdm3b+/− mice show deficits in memory consolidation, whereas they are normal in basal oculomotor performance and OKR acquisition. In addition, RNA-seq analyses revealed that the expression levels of several plasticity-related genes were altered in the mutant cerebellum. Our study suggests that active regulation of histone methylation is critical for the consolidation of cerebellar motor memory.

A distributed saccade-associated network encodes high velocity conjugate and monocular eye movements in the zebrafish hindbrain

Saccades are rapid eye movements that redirect gaze. Their magnitudes and directions are tightly controlled by the oculomotor system, which is capable of generating conjugate, monocular, convergent and divergent saccades. Recent studies suggest a mainly monocular control of saccades in mammals, although the development of binocular control and the interaction of different functional populations is less well understood. For zebrafish, a well-established model in sensorimotor research, the nature of binocular control in this key oculomotor behavior is unknown. Here, we use the optokinetic response and calcium imaging to characterize how the developing zebrafish oculomotor system encodes the diverse repertoire of saccades. We find that neurons with phasic saccade-associated activity (putative burst neurons) are most frequent in dorsal regions of the hindbrain and show elements of both monocular and binocular encoding, revealing a mix of the response types originally hypothesized by Helmholtz and Hering. Additionally, we observed a certain degree of behavior-specific recruitment in individual neurons. Surprisingly, calcium activity is only weakly tuned to saccade size. Instead, saccade size is apparently controlled by a push–pull mechanism of opposing burst neuron populations. Our study reveals the basic layout of a developing vertebrate saccade system and provides a perspective into the evolution of the oculomotor system.

Spherical arena reveals optokinetic response tuning to stimulus location, size, and frequency across entire visual field of larval zebrafish

Many animals have large visual fields, and sensory circuits may sample those regions of visual space most relevant to behaviours such as gaze stabilisation and hunting. Despite this, relatively small displays are often used in vision neuroscience. To sample stimulus locations across most of the visual field, we built a spherical stimulus arena with 14,848 independently controllable LEDs. We measured the optokinetic response gain of immobilised zebrafish larvae to stimuli of different steradian size and visual field locations. We find that the two eyes are less yoked than previously thought and that spatial frequency tuning is similar across visual field positions. However, zebrafish react most strongly to lateral, nearly equatorial stimuli, consistent with previously reported spatial densities of red, green and blue photoreceptors. Upside-down experiments suggest further extra-retinal processing. Our results demonstrate that motion vision circuits in zebrafish are anisotropic, and preferentially monitor areas with putative behavioural relevance.