Automated synapse-level reconstruction of neural circuits in the larval zebrafish brain.

Dense reconstruction of synaptic connectivity requires high-resolution electron microscopy images of entire brains and tools to efficiently trace neuronal wires across the volume. To generate such a resource, we sectioned and imaged a larval zebrafish brain by serial block-face electron microscopy at a voxel size of 14 × 14 × 25 nm3. We segmented the resulting dataset with the flood-filling network algorithm, automated the detection of chemical synapses and validated the results by comparisons to transmission electron microscopic images and light-microscopic reconstructions. Neurons and their connections are stored in the form of a queryable and expandable digital address book. We reconstructed a network of 208 neurons involved in visual motion processing, most of them located in the pretectum, which had been functionally characterized in the same specimen by two-photon calcium imaging. Moreover, we mapped all 407 presynaptic and postsynaptic partners of two superficial interneurons in the tectum. The resource developed here serves as a foundation for synaptic-resolution circuit analyses in the zebrafish nervous system.

Optogenetic precision toolkit to reveal form, function and connectivity of single neurons.

All-optical methods enable the control and monitoring of neuronal activity with minimal perturbation of the system. Although imaging and optogenetic manipulations can be performed at cellular resolution, the morphology of single cells in a dense neuronal population has often remained unresolvable. Here we describe in detail two recently established optogenetic protocols for systematic description of function and morphology of single neurons in zebrafish. First, the Optobow toolbox allows unbiased mapping of excitatory functional connectivity. Second, the FuGIMA technique enables selective labeling and anatomical tracing of neurons that are responsive to a given sensory stimulus or correlated with a specific behavior. Both strategies can be genetically targeted to a neuronal population of choice using the Gal4/UAS system. As these in vivo approaches are non-invasive, we envision useful applications for the study of neuronal structure, function and connectivity during development and behavior.

Spontaneous activity in whisker-innervating region of neonatal mouse trigeminal ganglion.

Spontaneous activity during the early postnatal period is thought to be crucial for the establishment of mature neural circuits. It remains unclear if the peripheral structure of the developing somatosensory system exhibits spontaneous activity, similar to that observed in the retina and cochlea of developing mammals. By establishing an ex vivo calcium imaging system, here we found that neurons in the whisker-innervating region of the trigeminal ganglion (TG) of neonatal mice generate spontaneous activity. A small percentage of neurons showed some obvious correlated activity, and these neurons were mostly located close to one another. TG spontaneous activity was majorly exhibited by medium-to-large diameter neurons, a characteristic of mechanosensory neurons, and was blocked by chelation of extracellular calcium. Moreover, this activity was diminished by the adult stage. Spontaneous activity in the TG during the first postnatal week could be a source of spontaneous activity observed in the neonatal mouse barrel cortex.

Cath6, a bHLH atonal family proneural gene, negatively regulates neuronal differentiation in the retina.

Basic helix-loop-helix (bHLH) transcription factors play important roles in cell type specification and differentiation during the development of the nervous system. In this study, we identified a chicken homolog of Atonal 8/ath6 (Cath6) and examined its role in the developing retina. Unlike other Atonal-family proneural genes that induce neuronal differentiation, Cath6 was expressed in stem cell-like progenitor cells in the marginal region of the retina, and its overexpression inhibited neuronal differentiation. A Cath6 fused with a VP16 transactivation domain recapitulated the inhibitory effect of Cath6 on neuronal differentiation, indicating that Cath6 functions as a transcription activator. These results demonstrate that Cath6 constitutes a unique member of the Atonal-family of genes in that it acts as a negative regulator of neuronal differentiation.

[Brain Mechanisms for Optical Illusions: From Motion Aftereffect in the Zebrafish].

An optical illusion represents a perception that deviates from the actual visual information of the visualized scene and is considered to be generated by an error in visual processing in the brain. The mechanisms by which optical illusions are generated have attracted the interest of scientists for many years. In this review, I focus on a vertebrate model, the zebrafish, and discuss how research using zebrafish has uncovered the mechanisms underlying optical illusions, and how optical illusions serve as a tool to help us understand how normal visual processing works in the brain.

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.

An Optical Illusion Pinpoints an Essential Circuit Node for Global Motion Processing.

Direction-selective (DS) neurons compute the direction of motion in a visual scene. Brain-wide imaging in larval zebrafish has revealed hundreds of DS neurons scattered throughout the brain. However, the exact population that causally drives motion-dependent behaviors-e.g., compensatory eye and body movements-remains largely unknown. To identify the behaviorally relevant population of DS neurons, here we employ the motion aftereffect (MAE), which causes the well-known "waterfall illusion." Together with region-specific optogenetic manipulations and cellular-resolution functional imaging, we found that MAE-responsive neurons represent merely a fraction of the entire population of DS cells in larval zebrafish. They are spatially clustered in a nucleus in the ventral lateral pretectal area and are necessary and sufficient to steer the entire cycle of optokinetic eye movements. Thus, our illusion-based behavioral paradigm, combined with optical imaging and optogenetics, identified key circuit elements of global motion processing in the vertebrate brain.

A calibrated optogenetic toolbox of stable zebrafish opsin lines.

Optogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. Here, we developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. Next, we performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking. We observed substantial variation in efficacy, associated with differences in both opsin expression level and photocurrent characteristics, and identified conditions for optimal use of the most efficient opsins. Overall, our calibrated optogenetic toolkit will facilitate the design of controlled optogenetic circuit manipulations.

Neuronal Architecture of a Visual Center that Processes Optic Flow.

Animals use global image motion cues to actively stabilize their position by compensatory movements. Neurons in the zebrafish pretectum distinguish different optic flow patterns, e.g., rotation and translation, to drive appropriate behaviors. Combining functional imaging and morphological reconstruction of single cells, we revealed critical neuroanatomical features of this sensorimotor transformation. Terminals of direction-selective retinal ganglion cells (DS-RGCs) are located within the pretectal retinal arborization field 5 (AF5), where they meet dendrites of pretectal neurons with simple tuning to monocular optic flow. Translation-selective neurons, which respond selectively to optic flow in the same direction for both eyes, are intermingled with these simple cells but do not receive inputs from DS-RGCs. Mutually exclusive populations of pretectal projection neurons innervate either the reticular formation or the cerebellum, which in turn control motor responses. We posit that local computations in a defined pretectal circuit transform optic flow signals into neural commands driving optomotor behavior. VIDEO ABSTRACT.

A Cellular-Resolution Atlas of the Larval Zebrafish Brain.

Understanding brain-wide neuronal dynamics requires a detailed map of the underlying circuit architecture. We built an interactive cellular-resolution atlas of the zebrafish brain at 6 days post-fertilization (dpf) based on the reconstructions of over 2,000 individually GFP-labeled neurons. We clustered our dataset in "morphotypes," establishing a unique database of quantitatively described neuronal morphologies together with their spatial coordinates in vivo. Over 100 transgene expression patterns were imaged separately and co-registered with the single-neuron atlas. By annotating 72 non-overlapping brain regions, we generated from our dataset an inter-areal wiring diagram of the larval brain, which serves as ground truth for synapse-scale, electron microscopic reconstructions. Interrogating our atlas by "virtual tract tracing" has already revealed previously unknown wiring principles in the tectum and the cerebellum. In conclusion, we present here an evolving computational resource and visualization tool, which will be essential to map function to structure in a vertebrate brain. VIDEO ABSTRACT.

Wnt signaling in retinal stem cells and regeneration.

In the vertebrate retina, stem cells with prolonged proliferative capacities reside in the most peripheral region, the ciliary marginal zone (CMZ), and they persist even after the functional eye has formed. These stem cells contribute to the formation of the retinal structures during the postnatal period in vivo, or can expand as neurospheres in vitro. Despite the wealth of anatomical descriptions of the characteristics of CMZ cells, molecular mechanisms for their specification or maintenance have long been uncharacterized. Recent studies provide evidence that certain secreted signaling molecules act as key regulators at multiple steps during these processes. In this review, we discuss the molecular basis for the regulation of retinal stem cells and their related cell types, especially focusing on the role of Wnt signaling.

Genetic targeting and anatomical registration of neuronal populations in the zebrafish brain with a new set of BAC transgenic tools.

Genetic access to small, reproducible sets of neurons is key to an understanding of the functional wiring of the brain. Here we report the generation of a new Gal4- and Cre-driver resource for zebrafish neurobiology. Candidate genes, including cell type-specific transcription factors, neurotransmitter-synthesizing enzymes and neuropeptides, were selected according to their expression patterns in small and unique subsets of neurons from diverse brain regions. BAC recombineering, followed by Tol2 transgenesis, was used to generate driver lines that label neuronal populations in patterns that, to a large but variable extent, recapitulate the endogenous gene expression. We used image registration to characterize, compare, and digitally superimpose the labeling patterns from our newly generated transgenic lines. This analysis revealed highly restricted and mutually exclusive tissue distributions, with striking resolution of layered brain regions such as the tectum or the rhombencephalon. We further show that a combination of Gal4 and Cre transgenes allows intersectional expression of a fluorescent reporter in regions where the expression of the two drivers overlaps. Taken together, our study offers new tools for functional studies of specific neural circuits in zebrafish.

Estrogens Suppress a Behavioral Phenotype in Zebrafish Mutants of the Autism Risk Gene, CNTNAP2.

Autism spectrum disorders (ASDs) are a group of devastating neurodevelopmental syndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD risk genes, the mechanisms underlying ASDs remain unknown. Homozygous loss-of-function mutations in Contactin Associated Protein-like 2 (CNTNAP2) are strongly linked to ASDs. Here we investigate the function of Cntnap2 and undertake pharmacological screens to identify phenotypic suppressors. We find that zebrafish cntnap2 mutants display GABAergic deficits, particularly in the forebrain, and sensitivity to drug-induced seizures. High-throughput behavioral profiling identifies nighttime hyperactivity in cntnap2 mutants, while pharmacological testing reveals dysregulation of GABAergic and glutamatergic systems. Finally, we find that estrogen receptor agonists elicit a behavioral fingerprint anti-correlative to that of cntnap2 mutants and show that the phytoestrogen biochanin A specifically reverses the mutant behavioral phenotype. These results identify estrogenic compounds as phenotypic suppressors and illuminate novel pharmacological pathways with relevance to autism.

Functional architecture of an optic flow-responsive area that drives horizontal eye movements in zebrafish.

Animals respond to whole-field visual motion with compensatory eye and body movements in order to stabilize both their gaze and position with respect to their surroundings. In zebrafish, rotational stimuli need to be distinguished from translational stimuli to drive the optokinetic and the optomotor responses, respectively. Here, we systematically characterize the neural circuits responsible for these operations using a combination of optogenetic manipulation and in vivo calcium imaging during optic flow stimulation. By recording the activity of thousands of neurons within the area pretectalis (APT), we find four bilateral pairs of clusters that process horizontal whole-field motion and functionally classify eleven prominent neuron types with highly selective response profiles. APT neurons are prevalently direction selective, either monocularly or binocularly driven, and hierarchically organized to distinguish between rotational and translational optic flow. Our data predict a wiring diagram of a neural circuit tailored to drive behavior that compensates for self-motion.

Ectopic Mitf in the embryonic chick retina by co-transfection of ß-catenin and Otx2.

PURPOSE: Development of the retinal pigment epithelium (RPE) is controlled by intrinsic and extrinsic regulators including orthodenticle homeobox 2 (Otx2) and the Wnt/ß-catenin pathway, respectively. Otx2 and ß-catenin are necessary for the expression of the RPE key regulator microphthalmia-associated transcription factor (Mitf); however, neither factor is sufficient to promote Mitf expression in vivo. The study was conducted to determine whether Otx2 and ß-catenin act in a combinatorial manner and tested whether co-expression in the presumptive chick retina induces ectopic Mitf expression. METHODS: The sufficiency of Wnt/ß-catenin activation and/or Otx2 expression to induce RPE-specific gene expression was examined in chick optic vesicle explant cultures or in the presumptive neural retina using in ovo-electroporation. Luciferase assays were used to examine the transactivation potentials of Otx2 and ß-catenin on the Mitf-D enhancer and autoregulation of the Mitf-D and Otx2T0 enhancers. RESULTS: In optic vesicles explant cultures, RPE-specific gene expression was activated by lithium chloride, a Wnt/ß-catenin agonist. However, in vivo, Mitf was induced only in the presumptive retina if both ß-catenin and Otx2 are co-expressed. Furthermore, both Mitf and Otx2 can autoregulate their own enhancers in vitro. CONCLUSIONS: The present study provides evidence that ß-catenin and Otx2 are sufficient, at least in part, to convert retinal progenitor cells into presumptive RPE cells expressing Mitf. Otx2 may act as a competence factor that allows RPE specification in concert with additional RPE-promoting factors such as ß-catenin.

Hairy1 acts as a node downstream of Wnt signaling to maintain retinal stem cell-like progenitor cells in the chick ciliary marginal zone.

In the vertebrate retina, stem cell-like progenitor cells are maintained in a distinct region called the ciliary marginal zone (CMZ). Canonical Wnt signaling regulates the maintenance of the progenitor cells in the CMZ. However, its downstream molecular mechanisms have remained largely unclear. Here, we show that chick Hairy1, an established Notch signaling effector, mediates the Wnt-dependent maintenance of CMZ progenitor cells in chicken. Interestingly, unlike other developmental contexts in which Hes gene expression is regulated by Notch signaling, Hairy1 expression in the CMZ is regulated by Wnt signaling. Hairy1 is necessary and sufficient for the expression of a set of molecular markers characteristic of the CMZ, and Wnt2b fails to induce CMZ markers when Hairy1 activity is inhibited. Furthermore, microarray analysis identifies multiple Wnt-responsive transcription factors that activate Hairy1 expression. We thus propose that Hairy1 functions as a node downstream of Wnt signaling to maintain progenitor cells in the chick CMZ.

Wnt2b inhibits differentiation of retinal progenitor cells in the absence of Notch activity by downregulating the expression of proneural genes.

During the development of the central nervous system, cell proliferation and differentiation are precisely regulated. In the vertebrate eye, progenitor cells located in the marginal-most region of the neural retina continue to proliferate for a much longer period compared to the ones in the central retina, thus showing stem-cell-like properties. Wnt2b is expressed in the anterior rim of the optic vesicles, and has been shown to control differentiation of the progenitor cells in the marginal retina. In this paper, we show that stable overexpression of Wnt2b in retinal explants inhibited cellular differentiation and induced continuous growth of the tissue. Notably, Wnt2b maintained the undifferentiated progenitor cells in the explants even under the conditions where Notch signaling was blocked. Wnt2b downregulated the expression of multiple proneural bHLH genes as well as Notch. In addition, expression of Cath5 under the control of an exogenous promoter suppressed the negative effect of Wnt2b on neuronal differentiation. Importantly, Wnt2b inhibited neuronal differentiation independently of cell cycle progression. We propose that Wnt2b maintains the naive state of marginal progenitor cells by attenuating the expression of both proneural and neurogenic genes, thus preventing those cells from launching out into the differentiation cascade regulated by proneural genes and Notch.

Wnt2b controls retinal cell differentiation at the ciliary marginal zone.

The ciliary marginal zone of the vertebrate retina contains undifferentiated progenitor cells that continue to proliferate and add new neurons and glia peripherally during the embryonic stages - even after the formation of a functional retina. To understand the molecular mechanism that controls the prolonged progenitor cell proliferation in the ciliary marginal zone, we employed a candidate molecule approach, focusing on Wnt2b (formerly know as Wnt13), which is expressed in the marginal most tip of the retina. Frizzled 4 and 5, seven-pass transmembrane Wnt receptors, were expressed in the peripheral and central part of the retina, respectively. LEF1, a downstream Wnt signaling component, was expressed at high levels in the ciliary marginal zone with expression gradually decreasing towards the central retina. The LEF1-expressing region, which is where Wnt signaling is supposedly activated, expressed a set of molecular markers that are characteristic of the progenitor cells in the ciliary marginal zone. Overexpression of Wnt2b by use of in ovo electroporation in the central retina inhibited neuronal differentiation and induced the progenitor cell markers. Blocking of the Wnt downstream signaling pathway by a dominant-negative LEF1 inhibited proliferation of the cells in the marginal area, which resulted in their premature neuronal differentiation. The progenitor cells in the ciliary marginal zone differentiated into all the neuronal and glial cell types when cultured in vitro, and they proliferated for a longer period than did centrally located progenitor cells that underwent a limited number of cell divisions. In addition, the proliferation of these progenitor cells was promoted in the presence of Wnt2b. These results suggest that Wnt2b functions to maintain undifferentiated progenitor cells in the ciliary marginal zone, and thus serves as a putative stem cell factor in the retina.