Zebrafish Tectum Research Articles

Decreased GABA levels during development result in increased connectivity in the larval zebrafish tectum.

γ-aminobutyric acid (GABA) is an abundant neurotransmitter that plays multiple roles in the vertebrate central nervous system (CNS). In the early developing CNS, GABAergic signaling acts to depolarize cells. It mediates several aspects of neural development, including cell proliferation, neuronal migration, neurite growth, and synapse formation, as well as the development of critical periods. Later in CNS development, GABAergic signaling acts in an inhibitory manner when it becomes the predominant inhibitory neurotransmitter in the brain. This behavior switch occurs due to changes in chloride/cation transporter expression. Abnormalities of GABAergic signaling appear to underlie several human neurological conditions, including seizure disorders. However, the impact of reduced GABAergic signaling on brain development has been challenging to study in mammals. Here we take advantage of zebrafish and light sheet imaging to assess the impact of reduced GABAergic signaling on the functional circuitry in the larval zebrafish optic tectum. Zebrafish have three gad genes: two gad1 paralogs known as gad1a and gad1b, and gad2. The gad1b and gad2 genes are expressed in the developing optic tectum. Null mutations in gad1b significantly reduce GABA levels in the brain and increase electrophysiological activity in the optic tectum. Fast light sheet imaging of genetically encoded calcium indicator (GCaMP)-expressing gab1b null larval zebrafish revealed patterns of neural activity that were different than either gad1b-normal larvae or gad1b-normal larvae acutely exposed to pentylenetetrazole (PTZ). These results demonstrate that reduced GABAergic signaling during development increases functional connectivity and concomitantly hyper-synchronization of neuronal networks.

Neuro-protective effects of increased O-GlcNAcylation by glucosamine in an optic tectum traumatic brain injury model of adult zebrafish.

This study investigated the behavioral and molecular changes in the telencephalon following needle stab-induced injury in the optic tectum of adult zebrafish. At 3 days post-injury (dpi), there was noticeable structural damage to brain tissue and reduced neuronal proliferation in the telencephalon that persisted until 30 dpi. Neurobehavioral deficits observed at 3 dpi included decreased exploratory and social activities and impaired learning and memory (L/M) functions; all of these resolved by 7 dpi. The injury led to a reduction in telencephalic phosphorylated cAMP response element-binding protein and O-GlcNAcylation, both of which were restored by 30 dpi. There was an increase in GFAP expression and nuclear translocation of NF-κB p65 at 3 dpi, which were not restored by 30 dpi. The injury caused decreased O-GlcNAc transferase and increased O-GlcNAcase levels at 3 dpi, normalizing by 30 dpi. Glucosamine (GlcN) treatment at 3 dpi significantly restored O-GlcNAcylation levels and L/M function, also reducing GFAP activation. Glucose treatment recovered L/M function by 7 dpi, but inhibition of the hexosamine biosynthetic pathway by 6-diazo-5-oxo-L-norleucine blocked this recovery. These findings suggest that the O-GlcNAc pathway is a potential therapeutic target for addressing L/M impairment following traumatic brain injury in zebrafish.

Circadian regulation of developmental synaptogenesis via the hypocretinergic system

The circadian clock orchestrates a wide variety of physiological and behavioral processes, enabling animals to adapt to daily environmental changes, particularly the day-night cycle. However, the circadian clock’s role in the developmental processes remains unclear. Here, we employ the in vivo long-term time-lapse imaging of retinotectal synapses in the optic tectum of larval zebrafish and reveal that synaptogenesis, a fundamental developmental process for neural circuit formation, exhibits circadian rhythm. This rhythmicity arises primarily from the synapse formation rather than elimination and requires the hypocretinergic neural system. Disruption of this synaptogenic rhythm, by impairing either the circadian clock or the hypocretinergic system, affects the arrangement of the retinotectal synapses on axon arbors and the refinement of the postsynaptic tectal neuron’s receptive field. Thus, our findings demonstrate that the developmental synaptogenesis is under hypocretin-dependent circadian regulation, suggesting an important role of the circadian clock in neural development.

A genetic labeling system to study dendritic spine development in zebrafish models of neurodevelopmental disorders.

ABSTRACTDendritic spines are the principal site of excitatory synapse formation in the human brain. Several neurodevelopmental disorders cause spines to develop abnormally, resulting in altered spine number and morphology. Although spine development has been thoroughly characterized in the mammalian brain, spines are not unique to mammals. We have developed a genetic system in zebrafish to enable high-resolution in vivo imaging of spine dynamics during larval development. Although spiny neurons are rare in the larval zebrafish, pyramidal neurons (PyrNs) of the zebrafish tectum form an apical dendrite containing a dense array of dendritic spines. To characterize dendritic spine development, we performed mosaic genetic labeling of individual PyrNs labeled by an id2b:gal4 transgene. Our findings identify a developmental period during which PyrN dendrite growth is concurrent with spine formation. Throughout this period, motile, transient filopodia gradually transform into stable spines containing postsynaptic specializations. The utility of this system to study neurodevelopmental disorders was validated by examining spine development in fmr1 mutant zebrafish, a model of fragile X syndrome. PyrNs in fmr1 mutants exhibited pronounced defects in dendrite growth and spine stabilization. Taken together, these findings establish a genetic labeling system to study dendritic spine development in larval zebrafish. In the future, this system could be combined with high-throughput screening approaches to identify genes and drug targets that regulate spine formation.

Time-Dependent Monitoring of Dopamine in the Brain of Live Embryonic Zebrafish Using Electrochemically Pretreated Carbon Fiber Microelectrodes.

Neurotransmitters are involved in functions related to signaling, stress response, and pathological disorder development, and thus, their real-time monitoring at the site of production is important for observing the changes related to these disorders. Here, we demonstrate the first time-dependent quantification of dopamine in the brains of live zebrafish embryos using electrochemically pretreated carbon fiber microelectrodes (CFMEs) utilizing differential pulse voltammetry as the measurement technique. The pretreatment of the CFMEs in 0.1 M NaOH held at a potential of +1.0 V for 600 s improves the sensitivity toward dopamine and allows for reliable measurements in low ionic strength media. We demonstrate the measurement of extracellular dopamine concentrations in the zebrafish brain during late embryogenesis. The extracellular dopamine concentration in the tectum of zebrafish varies between 200 and 400 nM. The conventional pharmacological manipulation of neurotransmitter levels in the brain demonstrates the selective detection of dopamine at the implantation site. Exposure to the dopamine transporter inhibitor nomifensine induces an increase in extracellular dopamine from 201.9 (±34.9) nM to 352.2 (±20.0) nM, while exposure to the norepinephrine transporter inhibitor desipramine does not lead to a significant modulation of the measured signal. Furthermore, we report the quantitative assessment of the catecholamine stress response of embryos to tricaine, an anesthetic frequently used in zebrafish assays. Exposure to tricaine induces a short-lived increase in brain dopamine from 198.6 (±15.7) nM to a maximum of 278.8 (±14.0) nM. Thus, in vivo electrochemistry can detect real-time changes in zebrafish neurochemical physiology resulting from drug exposure.

Stab Wound Injury Model of the Adult Optic Tectum using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity.

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

Electrophysiological and pharmacological characterization of spreading depolarization in the adult zebrafish tectum.

Spreading depolarization (SD) is a slowly propagating wave of neuronal and glial depolarization. A growing number of studies show that SD and SD-like phenomena play a role in neurological disorders such as migraine, stroke, and traumatic brain injury. Despite the clinical importance of SD, its underlying molecular and cellular mechanisms remain elusive, possibly because of insufficient animal model allowing genetic manipulation. Such a model would also allow high-throughput screening for SD-suppressing drug development. To address this, we developed a novel experimental system to study SD using zebrafish. Electrophysiological recordings in the immobilized adult zebrafish revealed that increasing extracellular potassium concentration elicited SD with a large and long-lasting negative shift of direct current (DC) potential in the optic tectum. It also reduced the oscillatory activity in the extracellular field potential and increased the expression of the immediate early gene c-fos. Pharmacological blocking of the N-methyl-d-aspartate (NMDA) glutamate receptor attenuated the propagation of SD, suggesting that glutamatergic neurotransmission mediated tectal SD in zebrafish. Our analyses revealed that the zebrafish tectum and rodent cortex had similar SD kinetics. The current study provides electrophysiological and pharmacological evidence that zebrafish SD and mammal SD are comparable. This zebrafish SD model is suitable for genetic manipulation and cost-effective high-throughput screening. It could pave the way to novel diagnostic and therapeutic methods applicable to SD-associated neurological disorders.NEW & NOTEWORTHY Previous studies have implicated spreading depolarization (SD) in stroke and migraine. Here, we demonstrate SD, for the first time, in the adult zebrafish tectum showing waveform kinetics, c-fos expression, and attenuation by N-methyl-d-aspartate glutamate receptor blocker as observed in the rodent cortex. Since the zebrafish is an animal model amenable to genetic manipulation and chemical screening, this result could pave the way to novel diagnostic and therapeutic methods applicable to SD-associated neurological disorders.

Differential Regenerative Capacity of the Optic Tectum of Adult Medaka and Zebrafish.

Zebrafish have superior regenerative capacity in the central nervous system (CNS) compared to mammals. In contrast, medaka were shown to have low regenerative capacity in the adult heart and larval retina, despite the well-documented high tissue regenerative ability of teleosts. Nevertheless, medaka and zebrafish share similar brain structures and biological features to those of mammals. Hence, this study aimed to compare the neural stem cell (NSC) responses and regenerative capacity in the optic tectum of adult medaka and zebrafish after stab wound injury. Limited neuronal differentiation was observed in the injured medaka, though the proliferation of radial glia (RG) was induced in response to tectum injury. Moreover, the expression of the pro-regenerative transcriptional factors ascl1a and oct4 was not enhanced in the injured medaka, unlike in zebrafish, whereas expression of sox2 and stat3 was upregulated in both fish models. Of note, glial scar-like structures composed of GFAP+ radial fibers were observed in the injured area of medaka at 14 days post injury (dpi). Altogether, these findings suggest that the adult medaka brain has low regenerative capacity with limited neuronal generation and scar formation. Hence, medaka represent an attractive model for investigating and evaluating critical factors for brain regeneration.

Spontaneous and evoked activity patterns diverge over development.

The immature brain is highly spontaneously active. Over development this activity must be integrated with emerging patterns of stimulus-evoked activity, but little is known about how this occurs. Here we investigated this question by recording spontaneous and evoked neural activity in the larval zebrafish tectum from 4 to 15 days post-fertilisation. Correlations within spontaneous and evoked activity epochs were comparable over development, and their neural assemblies refined in similar ways. However, both the similarity between evoked and spontaneous assemblies, and also the geometric distance between spontaneous and evoked patterns, decreased over development. At all stages of development, evoked activity was of higher dimension than spontaneous activity. Thus, spontaneous and evoked activity do not converge over development in this system, and these results do not support the hypothesis that spontaneous activity evolves to form a Bayesian prior for evoked activity.

Functional, molecular and morphological heterogeneity of superficial interneurons in the larval zebrafish tectum.

The superficial interneurons, SINs, of the zebrafish tectum, have been implicated in a range of visual functions, including size discrimination, directional selectivity, and looming-evoked escape. This raises the question if SIN subpopulations, despite their morphological similarities and shared anatomical position in the retinotectal processing stream, carry out diverse, task-specific functions in visual processing, or if they have simple tuning properties in common. Here we have further characterized the SINs through functional imaging, electrophysiological recordings, and neurotransmitter typing in two transgenic lines, the widely used Gal4s1156t and the recently reported LCRRH2-RH2-2:GFP. We found that about a third of the SINs strongly responded to changes in whole-field light levels, with a strong preference for OFF over ON stimuli. Interestingly, individual SINs were selectively tuned to a diverse range of narrow luminance decrements. Overall responses to whole-field luminance steps did not vary with the position of the SIN cell body along the depth of the tectal neuropil or with the orientation of its neurites. We ruled out the possibility that intrinsic photosensitivity of Gal4s1156t+ SINs contribute to the measured visual responses. We found that, while most SINs express GABAergic markers, a substantial minority express an excitatory neuronal marker, the vesicular glutamate transporter, expanding the possible roles of SIN function in the tectal circuitry. In conclusion, SINs represent a molecularly, morphologically, and functionally heterogeneous class of interneurons, with subpopulations that detect a range of specific visual features, to which we have now added narrow luminance decrements.

Parallel Channels for Motion Feature Extraction in the Pretectum and Tectum of Larval Zebrafish

Non-cortical visual areas in vertebrate brains extract relevant stimulus features, such as motion, object size, and location, to support diverse behavioral tasks. The optic tectum and pretectum, two primary visual areas in zebrafish, are involved in motion processing, and yet their differential neural representation of behaviorally relevant visual features is unclear. Here, we characterize receptive fields (RFs) of motion-sensitive neurons in the diencephalon and midbrain. We show that RFs of many pretectal neurons are large and sample the lower visual field, whereas RFs of tectal neurons are mostly small-size selective and sample the upper nasal visual field more densely. Furthermore, optomotor swimming can reliably be evoked by presenting forward motion in the lower temporal visual field alone, matching the lower visual field bias of the pretectum. Thus, tectum and pretectum extract different visual features from distinct regions of visual space, which is likely a result of their adaptations to hunting and optomotor behavior, respectively.

Phenotypic Characterization of Larval Zebrafish (Danio rerio) with Partial Knockdown of the cacna1a Gene

The CACNA1A gene encodes the pore-forming α1 subunit of voltage-gated P/Q type Ca2+ channels (Cav2.1). Mutations in this gene, among others, have been described in patients and rodents suffering from absence seizures and episodic ataxia type 2 with/without concomitant seizures. In this study, we aimed for the first time to assess phenotypic and behavioral alterations in larval zebrafish with partial cacna1aa knockdown, placing special emphasis on changes in epileptiform-like electrographic discharges in larval brains. Whole-mount in situ hybridization analysis revealed expression of cacna1aa in the optic tectum and medulla oblongata of larval zebrafish at 4 and 5 days post-fertilization. Next, microinjection of two antisense morpholino oligomers (individually or in combination) targeting all splice variants of cacna1aa into fertilized zebrafish eggs resulted in dose-dependent mortality and decreased or absent touch response. Over 90% knockdown of cacna1aa on protein level induced epileptiform-like discharges in the optic tectum of larval zebrafish brains. Incubation of morphants with antiseizure drugs (sodium valproate, ethosuximide, lamotrigine, topiramate) significantly decreased the number and, in some cases, cumulative duration of epileptiform-like discharges. In this context, sodium valproate seemed to be the least effective. Carbamazepine did not affect the number and duration of epileptiform-like discharges. Altogether, our data indicate that cacna1aa loss-of-function zebrafish may be considered a new model of absence epilepsy and may prove useful both for the investigation of Cacna1a-mediated epileptogenesis and for in vivo drug screening.

Bayesian inference of neuronal assemblies.

In many areas of the brain, both spontaneous and stimulus-evoked activity can manifest as synchronous activation of neuronal assemblies. The characterization of assembly structure and dynamics provides important insights into how brain computations are distributed across neural networks. The proliferation of experimental techniques for recording the activity of neuronal assemblies calls for a comprehensive statistical method to describe, analyze and characterize these high dimensional datasets. The performance of existing methods for defining assemblies is sensitive to noise and stochasticity in neuronal firing patterns and assembly heterogeneity. To address these problems, we introduce a generative hierarchical model of synchronous activity to describe the organization of neurons into assemblies. Unlike existing methods, our analysis provides a simultaneous estimation of assembly composition, dynamics and within-assembly statistical features, such as the levels of activity, noise and assembly synchrony. We have used our method to characterize population activity throughout the tectum of larval zebrafish, allowing us to make statistical inference on the spatiotemporal organization of tectal assemblies, their composition and the logic of their interactions. We have also applied our method to functional imaging and neuropixels recordings from the mouse, allowing us to relate the activity of identified assemblies to specific behaviours such as running or changes in pupil diameter.

Parallel Channels for Motion Feature Extraction in the Pretectum and Tectum of Larval Zebrafish

Non-cortical visual areas in vertebrate brains extract different stimulus features, such as motion, object size and location, to support behavioural tasks. The optic tectum and pretectum, two primary visual areas, are thought to fulfil complementary biological functions in zebrafish to support prey capture and optomotor stabilisation behaviour. However, the adaptations of these brain areas to behaviourally relevant stimulus statistics are unknown. Here, we used calcium imaging to characterize the receptive fields of 1,926 motion-sensitive neurons in diencephalon and midbrain. We show that many caudal pretectal neurons have large receptive fields (RFs), whereas RFs of tectal neurons are smaller and mostly size-selective. RF centres of large-size RF neurons in the pretectum are predominantly located in the lower visual field, while tectal neurons sample the upper-nasal visual field more densely. This tectal visual field sampling matches the expected prey item locations, suggesting that the tectal magnification of the upper-nasal visual field might be an adaptation to hunting behaviour. Finally, we probed optomotor responsiveness and found that even relatively small stimuli drive optomotor swimming, if presented in the lower-temporal visual field, suggesting that the pretectum preferably samples information from this region on the ground to inform optomotor behaviour. Our characterization of the parallel processing channels for non-cortical motion feature extraction provides a basis for further investigation into the sensorimotor transformations of the zebrafish brain and its adaptations to habitat and lifestyle.

Role of Reelin in cell positioning in the cerebellum and the cerebellum-like structure in zebrafish

The cerebellum and the cerebellum-like structure in the mesencephalic tectum in zebrafish contain multiple cell types, including principal cells (i.e., Purkinje cells and type I neurons) and granule cells, that form neural circuits in which the principal cells receive and integrate inputs from granule cells and other neurons. It is largely unknown how these cells are positioned and how neural circuits form. While Reelin signaling is known to play an important role in cell positioning in the mammalian brain, its role in the formation of other vertebrate brains remains elusive. Here we found that zebrafish with mutations in Reelin or in the Reelin-signaling molecules Vldlr or Dab1a exhibited ectopic Purkinje cells, eurydendroid cells (projection neurons), and Bergmann glial cells in the cerebellum, and ectopic type I neurons in the tectum. The ectopic Purkinje cells and type I neurons received aberrant afferent fibers in these mutants. In wild-type zebrafish, reelin transcripts were detected in the internal granule cell layer, while Reelin protein was localized to the superficial layer of the cerebellum and the tectum. Laser ablation of the granule cell axons perturbed the localization of Reelin, and the mutation of both kif5aa and kif5ba, which encode major kinesin I components in the granule cells, disrupted the elongation of granule cell axons and the Reelin distribution. Our findings suggest that in zebrafish, (1) Reelin is transported from the granule cell soma to the superficial layer by axonal transport; (2) Reelin controls the migration of neurons and glial cells from the ventricular zone; and (3) Purkinje cells and type I neurons attract afferent axons during the formation of the cerebellum and the cerebellum-like structure.

Selective processing of all rotational and translational optic flow directions in the zebrafish pretectum and tectum

BackgroundThe processing of optic flow in the pretectum/accessory optic system allows animals to stabilize retinal images by executing compensatory optokinetic and optomotor behavior. The success of this behavior depends on the integration of information from both eyes to unequivocally identify all possible translational or rotational directions of motion. However, it is still unknown whether the precise direction of ego-motion is already identified in the zebrafish pretectum or later in downstream premotor areas.ResultsHere, we show that the zebrafish pretectum and tectum each contain four populations of motion-sensitive direction-selective (DS) neurons, with each population encoding a different preferred direction upon monocular stimulation. In contrast, binocular stimulation revealed the existence of pretectal and tectal neurons that are specifically tuned to only one of the many possible combinations of monocular motion, suggesting that further downstream sensory processing might not be needed to instruct appropriate optokinetic and optomotor behavior.ConclusionOur results suggest that local, task-specific pretectal circuits process DS retinal inputs and carry out the binocular sensory computations necessary for optokinetic and optomotor behavior.

Topography of a Visuomotor Transformation

The brain converts perceptual information into appropriate patterns of muscle activity depending on the categorization and localization of sensory cues. Sensorimotor information might either be encoded by distributed networks or by "labeled lines" connecting sensory channels to dedicated behavioral pathways. Here we investigate, in the context of natural behavior, how the tectum of larval zebrafish can inform downstream premotor areas. Optogenetic mapping revealed a tectal motor map underlying locomotor maneuvers for escape and approach. Single-cell reconstructions and high-resolution functional imaging showed that two spatially segregated and uncrossed descending axon tracts selectively transmit approach and escape signals to the hindbrain. Moreover, the approach pathway conveys information about retinotopic target coordinates to specific premotor ensembles via spatially ordered axonal projections. This topographic organization supports a tectum-generated space code sufficient to steer orienting movements. We conclude that specific labeled lines guide object-directed behavior in the larval zebrafish brain.

Involvement of sonic hedgehog and notch signaling in regenerative neurogenesis in adult zebrafish optic tectum after stab injury.

Unlike humans and other mammals, adult zebrafish have the superior capability to recover from central nervous system (CNS) injury. We previously found that proliferation of radial glia (RG) is induced in response to stab injury in optic tectum and that new neurons are generated from RG after stab injury. However, molecular mechanisms which regulate proliferation and differentiation of RG are not well known. In the present study, we investigated Shh and Notch signaling as potential mechanisms regulating regeneration in the optic tectum of adult zebrafish. We used Shh reporter fish and confirmed that canonical Shh signaling is activated specifically in RG after stab injury. Moreover, we have shown that Shh signaling promotes RG proliferation and suppresses their differentiation into neurons after stab injury. In contrast, Notch signaling was down-regulated after stab injury, indicated by the decrease in the expression level of her4 and her6, a target gene of Notch signaling. We also found that inhibition of Notch signaling after stab injury induced more proliferative RG, but that inhibition of Notch signaling inhibited generation of newborn neurons from RG after stab injury. These results suggest that high level of Notch signaling keeps RG quiescent and that appropriate level of Notch signaling is required for generation of newborn neurons from RG. Under physiological condition, activation of Shh signaling or inhibition of Notch signaling also induced RG proliferation. In adult optic tectum of zebrafish, canonical Shh signaling and Notch signaling play important roles in proliferation and differentiation of RG in physiological and regenerative conditions.

Imaging Neuronal Activity in the Optic Tectum of Late Stage Larval Zebrafish

The zebrafish is an established model to study the development and function of visual neuronal circuits in vivo, largely due to their optical accessibility at embryonic and larval stages. In the past decade multiple experimental paradigms have been developed to study visually-driven behaviours, particularly those regulated by the optic tectum, the main visual centre in lower vertebrates. With few exceptions these techniques are limited to young larvae (7–9 days post-fertilisation, dpf). However, many forms of visually-driven behaviour, such as shoaling, emerge at later developmental stages. Consequently, there is a need for an experimental paradigm to image the visual system in zebrafish larvae beyond 9 dpf. Here, we show that using NBT:GCaMP3 line allows for imaging neuronal activity in the optic tectum in late stage larvae until at least 21 dpf. Utilising this line, we have characterised the receptive field properties of tectal neurons of the 2–3 weeks old fish in the cell bodies and the neuropil. The NBT:GCaMP3 line provides a complementary approach and additional opportunities to study neuronal activity in late stage zebrafish larvae.

Wnt signaling regulates proliferation and differentiation of radial glia in regenerative processes after stab injury in the optic tectum of adult zebrafish.

Zebrafish have superior abilities to generate new neurons in the adult brain and to regenerate brain tissue after brain injury compared with mammals. There exist two types of neural stem cells (NSCs): neuroepithelial-like stem cells (NE) and radial glia (RG) in the optic tectum. We established an optic tectum stab injury model to analyze the function of NSCs in the regenerative condition and confirmed that the injury induced the proliferation of RG, but not NE and that the proliferated RG differentiated into new neurons after the injury. We then analyzed the involvement of Wnt signaling after the injury, using a Wnt reporter line in which canonical Wnt signaling activation induced GFP expression and confirmed that GFP expression was induced specifically in RG after the injury. We also analyzed the expression level of genes related to Wnt signaling, and confirmed that endogenous Wnt antagonist dkk1b expression was significantly decreased after the injury. We observed that Wnt signal inhibitor IWR1 treatment suppressed the proliferation and differentiation of RG after the injury, suggesting that up-regulation of Wnt signaling in RG after the stab injury was required for optic tectum regeneration. We also confirmed that Wnt activation by treatment with GSK3β inhibitor BIO in uninjured zebrafish induced proliferation of RG in the optic tectum. This optic tectum stab injury model is useful for the study of the molecular mechanisms of brain regeneration and analysis of the RG functions in physiological and regenerative conditions.