Optokinetic response in D. melanogaster reveals the nature of common repellent odorants.

Animals' ability to orient and navigate relies on selecting an appropriate motor response based on the perception and integration of the environmental information. This is the case, for instance, of the optokinetic response (OKR) in Drosophila melanogaster, where optic flow visual stimulation modulates head movements. Despite a large body of literature on the OKR, there is still a limited understanding, in flies, of the impact on OKR of concomitant, and potentially conflicting, inputs. To evaluate the impact of this multimodal integration, we combined in D. melanogaster, while flying in a tethered condition, the optic flow stimulation leading to OKR with the simultaneous presentation of olfactory cues, based on repellent or masking compounds typically used against noxious insect species. First, this approach allowed us to directly quantify the effect of several substances and of their concentration on the dynamics of the flies' OKR in response to moving gratings by evaluating the number of saccades and the velocity of the slow phase. Subsequently, this analysis was capable of easily revealing the actual effect, i.e. masking vs. repellent, of the compound tested. In conclusion, we show that D. melanogaster, a cost-affordable species, represents a viable option for studying the effects of several compounds on the navigational abilities of insects.

A 3D-Printed and Freely Available Device to Measure the Zebrafish Optokinetic Response Before and After Injury.

Zebrafish eyes are anatomically similar to humans and have a higher percentage of cone photoreceptors more akin to humans than most rodent models, making them a beneficial model organism for studying vision. However, zebrafish are different in that they can regenerate their optic nerve after injury, which most other animals cannot. Vision in zebrafish and many other vertebrate animals, including humans, can be accessed using the optokinetic response (OKR), which is an innate eye movement that occurs when tracking an object. Because fish cannot use an eye chart, we utilize the OKR that is present in virtually all vertebrates to determine if a zebrafish has vision. To this end, we have developed an inexpensive OKR setup that uses 3D-printed and off-the-shelf parts. This setup has been designed and used by undergraduate researchers and is also scalable to a classroom laboratory setup. We demonstrate that this setup is fully functional for assessing the OKR, and we use it to illustrate the return of the OKR following optic nerve injury in adult zebrafish.

Plastic vasomotion entrainment.

The presence of global synchronization of vasomotion induced by oscillating visual stimuli was identified in the mouse brain. Endogenous autofluorescence was used and the vessel 'shadow' was quantified to evaluate the magnitude of the frequency-locked vasomotion. This method allows vasomotion to be easily quantified in non-transgenic wild-type mice using either the wide-field macro-zoom microscopy or the deep-brain fiber photometry methods. Vertical stripes horizontally oscillating at a low temporal frequency (0.25 Hz) were presented to the awake mouse, and oscillatory vasomotion locked to the temporal frequency of the visual stimulation was induced not only in the primary visual cortex but across a wide surface area of the cortex and the cerebellum. The visually induced vasomotion adapted to a wide range of stimulation parameters. Repeated trials of the visual stimulus presentations resulted in the plastic entrainment of vasomotion. Horizontally oscillating visual stimulus is known to induce horizontal optokinetic response (HOKR). The amplitude of the eye movement is known to increase with repeated training sessions, and the flocculus region of the cerebellum is known to be essential for this learning to occur. Here, we show a strong correlation between the average HOKR performance gain and the vasomotion entrainment magnitude in the cerebellar flocculus. Therefore, the plasticity of vasomotion and neuronal circuits appeared to occur in parallel. Efficient energy delivery by the entrained vasomotion may contribute to meeting the energy demand for increased coordinated neuronal activity and the subsequent neuronal circuit reorganization.

Early life stress aggravates disease pathogenesis in mice with experimental autoimmune encephalomyelitis: Support for a two-hit hypothesis of multiple sclerosis etiology.

Vision problems are one of the earliest diagnosed symptoms of multiple sclerosis (MS). The onset and progression of vision loss and the underlying pathogenesis in MS may be influenced by cumulative psychophysiological stress. Here, we used a two-hit model of stress in female mice to determine if early life stress (ELS, the first hit) influences the response to an immunization that induces experimental autoimmune encephalomyelitis (EAE, the second hit) later in life. We hypothesized that ELS caused by animal transportation from a vendor during early postnatal development represents a co-factor which can exacerbate the clinical severity of EAE. Indeed, adult EAE mice with a history of ELS displayed more severe clinical signs and delayed recovery compared to non-stressed EAE mice. ELS also diminished visual acuity measured by optokinetic responses, as well as locomotion and exploratory behaviours in EAE mice. Notably, ELS accelerated vision loss and caused earlier onset of visual impairments in EAE. Exacerbated functional impairments in stressed EAE mice were highly correlated with circulating corticosterone levels. The findings show that the progression of induced EAE in adulthood can be significantly impacted by adverse early life experiences. These observations emphasize the importance of comprehensive behavioural testing, including non-motor functions, to enhance the translational value of preclinical animal models of MS. Moreover, shipment stress of laboratory animals should be considered a necessary variable in preclinical MS research. The consideration of cumulative lifetime stresses provides a new perspective of MS pathogenesis within a personalized medicine framework.

Glial modulation of the parallel memory formation.

Actions from glial cells could affect the readiness and efficacy of learning and memory. Using a mouse cerebellar-dependent horizontal optokinetic response motor learning paradigm, short-term memory (STM) formation during the online training period and long-term memory (LTM) formation during the offline rest period were studied. A large variability of online and offline learning efficacies was found. The early bloomers with booming STM often had a suppressed LTM formation and late bloomers with no apparent acute training effect often exhibited boosted offline learning performance. Anion channels containing LRRC8A are known to release glutamate. Conditional knockout of LRRC8A specifically in astrocytes including cerebellar Bergmann glia resulted in a complete loss of STM formation while the LTM formation during the rest period remained. Optogenetic manipulation of glial activity by channelrhodopsin-2 or archaerhodopsin-T (ArchT) during the online training resulted in enhancement or suppression of STM formation, respectively. STM and LTM are likely to be triggered simultaneously during online training, but LTM is expressed later during the offline period. STM appears to be volatile and the achievement during the online training is not handed over to LTM. In addition, we found that glial ArchT photoactivation during the rest period resulted in the augmentation of LTM formation. These data suggest that STM formation and LTM formation are parallel separate processes. Strategies to weigh more on the STM or the LTM could depend on the actions of the glial cells.

A quantitative approach to study the adaptation of rhythmic eye movements and the resulting tonic eye deviation in larval zebrafish.

To optimize performance during vital tasks, animals are capable of tuning rhythmic neural signals that drive repetitive behaviors, such as motor reflexes under constant sensory stimuli. In the oculomotor system, animals track the moving image during slow phases while repetitively resetting the eye position from the eccentricity during quick phases. During optokinetic response (OKR), larval zebrafish occasionally show a delayed quick phase; thus, the eyes remain tonically deviated from the center. In this study, we scrutinized OKR in larval zebrafish under a broad range of stimulus velocities to determine the parametric property of the quick-phase delay. A prolonged stimulation revealed that the slow-phase (SP) duration-the interval between two quick phases-was tuned increasingly over time toward a homeostatic range, regardless of stimulus velocity. Attributed to this rhythm control, larval zebrafish exhibited a tonic eye deviation following slow phases, which was especially pronounced when tracking a fast stimulus over an extended time period. In addition to the SP duration, the fixation duration between spontaneous saccades in darkness also revealed a similar adaptive property after the prolonged optokinetic stimulation. Our results provide a quantitative description of the adaptation of rhythmic eye movements in developing animals and pave the way for potential animal models for eye movement disorders.

Analysis of Visual Recovery After Optic Nerve Crush in Adult Zebrafish.

Zebrafish can successfully regenerate axons after optic nerve crush (ONC). Here, we describe two different behavioral tests to map visual recovery: the dorsal light reflex (DLR) test and the optokinetic response (OKR) test. The DLR is based on the tendency of fish to orient their back to a light source, and it can be tested by rotating a flashlight around the dorsolateral axis of the animal or by measuring the angle between the left/right body axis and the horizon. The OKR, in contrast, consists of reflexive eye movements triggered by motion in the visual field of the subject and is measured by placing the fish in a drum on which rotating black-and-white stripes are projected.

Quantifying visual acuity of Terrapene carolina triunguis utilizing optokinetic response.

OBJECTIVE: Quantify visual acuity of a semi-aquatic turtle species, Terrapene carolina triunguis (three-toed box turtles), utilizing optokinetic response/reflex/nystagmus. METHODS: Ten turtles were placed on a custom platform and an image of white and black gratings of varying sized widths was projected onto a white screen in front of the turtles. Optokinetic response (OKR) was observed through a web camera as well as physically by an observer. The grating sizes were decreased until OKR could not be elicited. The size of grating was recorded and was used to calculate visual acuity. RESULTS: The average visual acuity was 0.26 cycle per degree (cpd) with the highest recorded value being 0.43 cpd and lowest being 0.15 cpd. Neither sex nor weight were significantly associated with visual acuity (p = .24 and .98, respectively). CONCLUSIONS: Visual acuity of these semi-aquatic turtles was much lower than previously reported visual acuities of aquatic turtles. This could be due to differences in refractive indices between water and air, behavioral adaptations for the different species and/or method of obtaining visual acuity.

Azole-Induced Color Vision Deficiency Associated with Thyroid Hormone Signaling: An Integrated In Vivo, In Vitro, and In Silico Study.

Azoles that are used in pesticides, pharmaceuticals, and personal care products can have toxic effects on fish. However, there is no information regarding azole-induced visual disorder associated with thyroid disruption. We evaluated changes in retinal morphology, optokinetic response, transcript abundance of the genes involved in color perception and hypothalamic-pituitary-thyroid (HPT) axis, and thyroid hormone (TH) levels in zebrafish larvae exposed to common azoles, such as climbazole (CBZ, 0.1 and 10 µg/L) and triadimefon (TDF, 50 and 500 µg/L), at environmentally relevant and predicted worst-case environmental concentrations. Subsequently, the effect of azoles on TH-dependent GH3 cell proliferation and thyroid receptor (TR)-regulated transcriptional activity, as well as the in silico binding affinity between azoles and TR isoforms, was investigated. Azole exposure decreased cell densities of the ganglion cell layer, inner nuclear layer, and photoreceptor layer. Zebrafish larvae exposed to environmentally relevant concentrations of CBZ and TDF showed a decrease in optokinetic response to green-white and red-white stripes but not blue-white stripes, consistent with disturbance in the corresponding opsin gene expression. Azole exposure also reduced triiodothyronine levels and concomitantly increased HPT-related gene expression. Molecular docking analysis combined with in vitro TR-mediated transactivation and dual-luciferase reporter assays demonstrated that CBZ and TDF exhibited TR antagonism. These results are comparable to those obtained from a known TR antagonist, namely, TR antagonist 1, as a positive control. Therefore, damage to specific color perception by azoles appears to result from lowered TH signaling, indicating the potential threat of environmental TH disruptors to the visual function of fish.

Out of water in the dark: Plasticity in visual structures and function in an amphibious fish.

Many fishes encounter periods of prolonged darkness within their lifetime, yet the consequences for the visual system are poorly understood. We used an amphibious fish (Kryptolebias marmoratus) that occupies dark terrestrial environments during seasonal droughts to test whether exposure to prolonged darkness diminishes visual performance owing to reduced optic tectum (OT) size and/or neurogenesis. We performed a 3-week acclimation with a 2 ×$\times $ 2 factorial design, in which fish were either acclimated to a 12 h:12 h or 0 h:24 h light:dark photoperiod in water or in air. We found that water-exposed fish had poorer visual acuity when acclimated to the dark, while air-acclimated fish had poorer visual acuity regardless of photoperiod. The ability of K. marmoratus to capture aerial prey from water followed a similar trend, suggesting that good vision is important for hunting effectively. Changes in visual acuity did not result from changes in OT size, but air-acclimated fish had 37% fewer proliferating cells in the OT than water-acclimated fish. As K. marmoratus are unable to eat on land, reducing cell proliferation in the OT may serve as a mechanism to reduce maintenance costs associated with the visual system. Overall, we suggest that prolonged darkness and air exposure can impair vision in K. marmoratus, and that changes in visual performance may be mediated, in part, by OT neurogenesis. More broadly, we show that plastic changes to the visual system of fishes can have potential consequences for organismal performance and fitness.

A robust receptive field code for optic flow detection and decomposition during self-motion.

The perception of optic flow is essential for any visually guided behavior of a moving animal. To mechanistically predict behavior and understand the emergence of self-motion perception in vertebrate brains, it is essential to systematically characterize the motion receptive fields (RFs) of optic-flow-processing neurons. Here, we present the fine-scale RFs of thousands of motion-sensitive neurons studied in the diencephalon and the midbrain of zebrafish. We found neurons that serve as linear filters and robustly encode directional and speed information of translation-induced optic flow. These neurons are topographically arranged in pretectum according to translation direction. The unambiguous encoding of translation enables the decomposition of translational and rotational self-motion information from mixed optic flow. In behavioral experiments, we successfully demonstrated the predicted decomposition in the optokinetic and optomotor responses. Together, our study reveals the algorithm and the neural implementation for self-motion estimation in a vertebrate visual system.

Efficacy of Tricaine (MS-222) and Hypothermia as Anesthetic Agents for Blocking Sensorimotor Responses in Larval Zebrafish.

Tricaine, or MS-222, is the most commonly used chemical anesthetic in zebrafish research. It is thought to act via blocking voltage-gated sodium channels, though its mechanism of action, particularly at the neuronal level, is not yet fully understood. Here, we first characterized the effects of tricaine on both body balance and touch responses in freely swimming animals, before determining its effect on the neural activity underlying the optokinetic response at the level of motion perception, sensorimotor signaling and the generation of behavior in immobilized animals. We found that the standard dose for larvae (168 mg/L) induced loss of righting reflex within 30 seconds, which then recovered within 3 minutes. Optokinetic behavior recovered within 15 minutes. Calcium imaging showed that tricaine interferes with optokinetic behavior by interruption of the signals between the pretectum and hindbrain. The motion sensitivity indices of identified sensory neurons were unchanged in larvae exposed to tricaine, though fewer such neurons were detected, leaving a small population of active sensory neurons. We then compared tricaine with gradual cooling, a potential non-chemical alternative method of anesthesia. While neuronal tuning appeared to be affected in a similar manner during gradual cooling, gradual cooling induced a surge in calcium levels in both the pretectum and hindbrain. This calcium surge, alongside a drop in heartrate, is potentially associated with harmful changes in physiology and suggests that tricaine is a better anesthetic agent than gradual cooling for zebrafish laboratory research.

Relationship between miR-203a inhibition and oil-induced toxicity in early life stage zebrafish (Danio rerio).

Dysregulation of microRNA (miRNA, miR) by environmental stressors influences the transcription of mRNA which may impair organism development and/or lead to adverse physiological outcomes. Early studies evaluating the effects of oil on developmental toxicity in early life stages of fish showed that reductions in expression of miR-203a were associated with enhanced expression of downstream mRNAs that predicted altered eye development, cardiovascular disease, and improper fin development. To better understand the effects of miR-203a inhibition as an outcome of oil-induced toxicity in early life stage (ELS) fish, embryonic zebrafish were injected with an miR-203a inhibitor or treated with 3.5 µM phenanthrene (Phe) as a positive control for morphological alterations of cardiovascular and eye development caused by oil. Embryos treated with Phe had diminished levels of miR-203a at 7 and 72 h after injection. Embryos treated with the miR-203a inhibitor and Phe exhibited a reduced heart rate by 48 h post fertilization (hpf), with an increased incidence of developmental deformities (including pericardial edema, altered eye development, and spinal deformities) and reduced caudal fin length by 72 hpf. There were significant reductions in lens and eye diameters in 120 hpf miR-203a-inhibitor and Phe-treated fish, as well as a significantly reduced number of eye saccades, determined by an optokinetic response (OKR) behavioral assay. The expression of vegfa, which is an important activator during neovascularization, was significantly upregulated in embryos receiving miR-203a inhibitor injections by 7 and 72 hpf with increased trends in vegfa expression in 72 hpf larvae treated with Phe. There were decreasing trends in crx, neurod1, and pde6h expression by 72 hpf in miR-203a inhibitor and Phe treatments, which are involved in photoreceptor function in developing eyes and regulated by miR-203a. These results suggest that an inhibition of miR-203a in ELS fish exhibits an oil-induced toxic response that is consistent with Phe treatment and specifically impacts retinal, cardiac, and fin development in ELS fish.

Toxic effects of triclocarban on larval zebrafish: A focus on visual dysfunction.

Triclocarban (TCC) is considered an endocrine disruptor and shows antagonist activity on thyroid receptors. In view of the report that thyroid hormone signaling mediates retinal cone photoreceptor specification, we hypothesize that TCC could impair visual function, which is vital to wildlife. In order to verify our hypothesis, we assessed alteration in the retinal structure (retinal layer thickness and cell density), visually-mediated behavior, cone and rod opsin gene expression, and photoreceptor immunostaining in zebrafish larvae exposed to TCC at environmentally realistic concentrations (0.16 ± 0.005 µg/L, L-group) and one-fifth of the median lethal concentrations (25.4 ± 1.02 µg/L, H-group). Significant decrease in eye size, ganglion cell density, optokinetic response, and phototactic response can be observed in the L-group, while the thickness of outer nuclear layer, where the cell bodies of cone and rod cells are located, was significantly reduced with the down-regulation of critical opsin gene (opn1sw2, opn1mw1, opn1mw3, opn1lw1, opn1lw2, and rho) expression and rhodopsin immunofluorescence in the H-group. It should be noted that TCC could affect the sensitivity of zebrafish larvae to red and green light according to the results of behavioral and opsin gene expression analysis. These findings provide the first evidence to support our hypothesis that the visual system, a novel toxicological target, is affected by TCC. Consequently, we urgently call for a more in-depth exploration of TCC-induced ocular toxicity to aquatic organisms and even to humans.

Seeing in the swamp: hydrogen sulfide inhibits eye metabolism and visual acuity in a sulfide-tolerant fish.

In fish, vision may be impaired when eye tissue is in direct contact with environmental conditions that limit aerobic ATP production. We hypothesized that the visual acuity of fishes exposed to hydrogen sulfide (H2S)-rich water would be altered owing to changes in cytochrome c oxidase (COX) activity. Using the H2S-tolerant mangrove rivulus (Kryptolebias marmoratus), we showed that a 10 min exposure to greater than or equal to 200 µM of H2S impaired visual acuity and COX activity in the eye. Visual acuity and COX activity were restored in fish allowed to recover in H2S-free water for up to 1 h. Since K. marmoratus are found in mangrove pools with H2S concentrations exceeding 1000 µM, visual impairment may impact predator avoidance, navigation and foraging behaviour in the wild.

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.

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.

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.

Visual Function is Gradually Restored During Retina Regeneration in Adult Zebrafish.

In comparison to mammals, zebrafish are able to regenerate many organs and tissues, including the central nervous system (CNS). Within the CNS-derived neural retina, light lesions result in a loss of photoreceptors and the subsequent activation of Müller glia, the retinal stem cells. Müller glia-derived progenitors differentiate and eventually restore the anatomical tissue architecture within 4 weeks. However, little is known about how light lesions impair vision functionally, as well as how and to what extent visual function is restored during the course of regeneration, in particular in adult animals. Here, we applied quantitative behavioral assays to assess restoration of visual function during homeostasis and regeneration in adult zebrafish. We developed a novel vision-dependent social preference test, and show that vision is massively impaired early after lesion, but is restored to pre-lesion levels within 7 days after lesion. Furthermore, we employed a quantitative optokinetic response assay with different degrees of difficulty, similar to vision tests in humans. We found that vision for easy conditions with high contrast and low level of detail, as well as color vision, was restored around 7-10 days post lesion. Vision under more demanding conditions, with low contrast and high level of detail, was regained only later from 14 days post lesion onwards. Taken together, we conclude that vision based on contrast sensitivity, spatial resolution and the perception of colors is restored after light lesion in adult zebrafish in a gradual manner.