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International audience; Reelin, a glycoprotein of the extracellular matrix, has been the focus of several studies over the years, mostly for its role in cell migration. Here we report the role of this molecule and of its downstream pathways in post-mitotic neurons and how they contribute to neural circuit assembly, refinement and function. Accumulating evidence has pointed at a major role for Reelin in axonal guidance, synaptogenesis and dendritic spine formation. In particular new evidence points at a direct role in axonal targeting and refinement at the target site. In addition, recent advances highlight new functions of Reelin in the modulation of synaptic activity, plasticity and behavior and it directly regulates GABA receptors expression and stability. We discuss these findings in the context of neurodevelopmental disorders. Highlights  Reelin signaling is required for axonal targeting and refinement at the target site.  Reelin signaling regulates dendritogenesis and spine formation in mature neurons.  Reelin modulates GABA receptors expression and stability, pre-and-post-synaptically.  Reelin is important for hippocampal integrity, synaptic plasticity and behavior.

Promoting the regeneration or survival of retinal ganglion cells (RGCs) is one focus of regenerative medicine. Homeobox Barhl transcription factors might be instrumental in these processes. In mammals, only barhl2 is expressed in the retina and is required for both subtype identity acquisition of amacrine cells and for the survival of RGCs downstream of Atoh7, a transcription factor necessary for RGC genesis. The underlying mechanisms of this dual role of Barhl2 in mammals have remained elusive. Whole genome duplication in the teleost lineage generated the barhl1a and barhl2 paralogues. In the Zebrafish retina, Barhl2 functions as a determinant of subsets of amacrine cells lineally related to RGCs independently of Atoh7. In contrast, barhl1a expression depends on Atoh7 but its expression dynamics and function have not been studied. Here we describe for the first time a Barhl1a reporter line in vivo showing that barhl1a turns on exclusively in subsets of RGCs and their post-mitotic precursors. We also show transient expression of barhl1a:GFP in diencephalic neurons extending their axonal projections as part of the post-optic commissure, at the time of optic chiasm formation. This work sets the ground for future studies on RGC subtype identity, axonal projections and genetic specification of Barhl1a-positive RGCs and commissural neurons.

Atkins et al. identify a new role for Fidgetin-like 1 in motor axon navigation via its regulation of bidirectional axonal transport. They show that Fidgetin-like 1 binds Kif1bβ and the opposed polarity-directed motor dynein/dynactin in a molecular complex and controls circuit wiring by reducing dynein velocity in developing motor axons., Neuronal connectivity relies on molecular motor-based axonal transport of diverse cargoes. Yet the precise players and regulatory mechanisms orchestrating such trafficking events remain largely unknown. We here report the ATPase Fignl1 as a novel regulator of bidirectional transport during axon navigation. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we showed that Fignl1 binds the kinesin Kif1bβ and the dynein/dynactin adaptor Bicaudal D-1 (Bicd1) in a molecular complex including the dynactin subunit dynactin 1. Fignl1 colocalized with Kif1bβ and showed bidirectional mobility in zebrafish axons. Notably, Kif1bβ and Fignl1 loss of function similarly altered zebrafish motor axon pathfinding and increased dynein-based transport velocity of Rab3 vesicles in these navigating axons, pinpointing Fignl1/Kif1bβ as a dynein speed limiter complex. Accordingly, disrupting dynein/dynactin activity or Bicd1/Fignl1 interaction induced motor axon pathfinding defects characteristic of Fignl1 gain or loss of function, respectively. Finally, pharmacological inhibition of dynein activity partially rescued the axon pathfinding defects of Fignl1-depleted larvae. Together, our results identify Fignl1 as a key dynein regulator required for motor circuit wiring.

Reelin (Reln) is a large extracellular glycoprotein that has an important role in brain patterning. During development Reln coordinates the radial migration of postmitotic cortical neurons, cerebellar and hippocampal neurons, whereas it promotes dendrite maturation, synaptogenesis, synaptic transmission, plasticity and neurotransmitter release in the postnatal and adult brain. Genetic studies of human patients have demonstrated an association between the RELN locus and autism spectrum disorder, schizophrenia, bipolar disorder and Alzheimer’s disease. In this study we have characterised the behavioural phenotype of reelin (reln) mutant zebrafish, as well as two canonical signalling pathway targets DAB adaptor protein 1a (dab1a) and the very low density lipoprotein receptor (vldlr). Zebrafish reln-/- mutants display a selective reduction in preference for social novelty that is not observed in dab1a-/- or vldlr-/- mutant lines. They also exhibit an increase in 5-HT signalling in the hindbrain that parallels but does not underpin the alteration in social preference. These results suggest that zebrafish reln-/- mutants can be used to model some aspects of human diseases in which changes to Reln signalling alter social behaviour.

Throughout vertebrates, cerebrospinal fluid-contacting neurons (CSF-cNs) are ciliated cells surrounding the central canal in the ventral spinal cord. Their contribution to modulate locomotion remains undetermined. Recently, we have shown CSF-cNs modulate locomotion by directly projecting onto the locomotor central pattern generators (CPGs), but the sensory modality these cells convey to spinal circuits and their relevance to innate locomotion remain elusive. Here, we demonstrate in vivo that CSF-cNs form an intraspinal mechanosensory organ that detects spinal bending. By performing calcium imaging in moving animals, we show that CSF-cNs respond to both passive and active bending of the spinal cord. In mutants for the channel Pkd2l1, CSF-cNs lose their response to bending and animals show a selective reduction of tail beat frequency, confirming the central role of this feedback loop for optimizing locomotion. Altogether, our study reveals that CSF-cNs constitute a mechanosensory organ operating during locomotion to modulate spinal CPGs., CSF-contacting neurons are known to project to locomotor CPGs although their relevance to active locomotion is unclear. Here, the authors show that these cells constitute a mechanosensory organ relying on PKD2L1 channels to detect spinal cord curvature and modulate locomotor frequency in freely moving animals.

Escape behaviors deliver organisms away from imminent catastrophe. Here, we characterize behavioral responses of freely swimming larval zebrafish to looming visual stimuli simulating predators. We report that the visual system alone can recruit lateralized, rapid escape motor programs, similar to those elicited by mechanosensory modalities. Two-photon calcium imaging of retino-recipient midbrain regions isolated the optic tectum as an important center processing looming stimuli, with ensemble activity encoding the critical image size determining escape latency. Furthermore, we describe activity in retinal ganglion cell terminals and superficial inhibitory interneurons in the tectum during looming and propose a model for how temporal dynamics in tectal periventricular neurons might arise from computations between these two fundamental constituents. Finally, laser ablations of hindbrain circuitry confirmed that visual and mechanosensory modalities share the same premotor output network. Together, we establish a circuit for the processing of aversive stimuli in the context of an innate visual behavior.

Understanding how distributed neuronal circuits integrate sensory information and generate behavior is a central goal of neuroscience. However, it has been difficult to study neuronal networks at single-cell resolution across the entire adult brain in vertebrates because of their size and opacity. We address this challenge here by introducing the fish Danionella translucida to neuroscience as a potential model organism. This teleost remains small and transparent even in adulthood, when neural circuits and behavior have matured. Despite having the smallest known adult vertebrate brain, D. translucida displays a rich set of complex behaviors, including courtship, shoaling, schooling, and acoustic communication. In order to carry out optical measurements and perturbations of neural activity with genetically encoded tools, we established CRISPR-Cas9 genome editing and Tol2 transgenesis techniques. These features make D. translucida a promising model organism for the study of adult vertebrate brain function at single-cell resolution.

A conserved organizational and functional principle of neural networks is the segregation of axon-dendritic synaptic connections into laminae. Here we report that targeting of synaptic laminae by retinal ganglion cell (RGC) arbors in the vertebrate visual system is regulated by a signaling system relying on target-derived Reelin and VLDLR/Dab1a on the projecting neurons. Furthermore, we find that Reelin is distributed as a gradient on the target tissue and stabilized by heparan sulfate proteoglycans (HSPGs) in the extracellular matrix (ECM). Through genetic manipulations, we show that this Reelin gradient is important for laminar targeting and that it is attractive for RGC axons. Finally, we suggest a comprehensive model of synaptic lamina formation in which attractive Reelin counter-balances repulsive Slit1, thereby guiding RGC axons toward single synaptic laminae. We establish a mechanism that may represent a general principle for neural network assembly in vertebrate species and across different brain areas.

Axon ensheathment by specialized glial cells is an important process for fast propagation of action potentials. The rapid electrical conduction along myelinated axons is mainly due to its saltatory nature characterized by the accumulation of ion channels at the nodes of Ranvier. However, how these ion channels are transported and anchored along axons is not fully understood. We have identified N-myc downstream-regulated gene 4, ndrg4, as a novel factor that regulates sodium channel clustering in zebrafish. Analysis of chimeric larvae indicates that ndrg4 functions autonomously within neurons for sodium channel clustering at the nodes. Molecular analysis of ndrg4 mutants shows that expression of snap25 and nsf are sharply decreased, revealing a role of ndrg4 in controlling vesicle exocytosis. This uncovers a previously unknown function of ndrg4 in regulating vesicle docking and nodes of Ranvier organization, at least through its ability to finely tune the expression of the t-SNARE/NSF machinery., Author Summary Myelination is an important process that enables fast propagation of action potential along the axons. Schwann cells (SCs) are the specialized glial cells that ensure the ensheathment of the corresponding axons in the Peripheral Nervous System. In order to do so, SCs and axons need to communicate to organize the myelinating segments and the clustering of sodium channels at the nodes of Ranvier. We have investigated the early events of myelination in the zebrafish embryo. We here identify ndrg4 as a novel neuronal factor essential for sodium channel clustering at the nodes. Immuno-labeling analysis show defective vesicle patterning along the axons of ndrg4 mutants, while timelapse experiments monitoring the presence and the transport of these vesicles reveal a normal behavior. Molecular analysis unravels a novel function of ndrg4 in controlling the expression of the t-SNARE/NSF machinery required for vesicle docking and release. However, inhibiting specifically regulated synaptic vesicle release does not lead to sodium channel clustering defects. We thus propose that ndrg4 can regulate this process, at least partially, through its ability to regulate the expression of key components of the t-SNARE/NSF machinery, responsible for clustering of sodium channels along myelinated axons.

Expression of halorhodopsin (NpHR), a light-driven microbial chloride pump, enables optical control of membrane potential and reversible silencing of targeted neurons. We generated transgenic zebrafish expressing enhanced NpHR under control of the Gal4/UAS system. Electrophysiological recordings showed that eNpHR stimulation effectively suppressed spiking of single neurons in vivo. Applying light through thin optic fibers positioned above the head of a semi-restrained zebrafish larva enabled us to target groups of neurons and to simultaneously test the effect of their silencing on behavior. The photostimulated volume of the zebrafish brain could be marked by subsequent photoconversion of co-expressed Kaede or Dendra. These techniques were used to localize swim command circuitry to a small hindbrain region, just rostral to the commissura infima Halleri . The kinetics of the hindbrain-generated swim command was investigated by combined and separate photo-activation of NpHR and Channelrhodopsin-2 (ChR2), a light-gated cation channel, in the same neurons. Together this “optogenetic toolkit” allows loss-of-function and gain-of-function analyses of neural circuitry at high spatial and temporal resolution in a behaving vertebrate.

Locomotion relies on neural networks called central pattern generators (CPGs) that generate periodic motor commands for rhythmic movements. In vertebrates, the excitatory synaptic drive for inducing the spinal CPG can originate from either supraspinal glutamatergic inputs or from within the spinal cord. Here we identify a spinal input to the CPG that drives spontaneous locomotion using a combination of intersectional gene expression and optogenetics in zebrafish larvae. The photo-stimulation of one specific cell type was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. This neuron is the Kolmer-Agduhr cell, which extends cilia into the central cerebrospinal-fluid-containing canal of the spinal cord and has an ipsilateral ascending axon that terminates in a series of consecutive segments. Genetically silencing Kolmer-Agduhr cells reduced the frequency of spontaneous free swimming, indicating that activity of Kolmer-Agduhr cells provides necessary tone for spontaneous forward swimming. Kolmer-Agduhr cells have been known for over 75 years, but their function has been mysterious. Our results reveal that during early development in zebrafish these cells provide a positive drive to the spinal CPG for spontaneous locomotion.

Development and function of highly polarized cells such as neurons depend on microtubule-associated intracellular transport, but little is known about contributions of specific molecular motors to the establishment of synaptic connections. In this study, we investigated the function of the Kinesin I heavy chain Kif5aa during retinotectal circuit formation in zebrafish. Targeted disruption of Kif5aa does not affect retinal ganglion cell differentiation, and retinal axons reach their topographically correct targets in the tectum, albeit with a delay. In vivo dynamic imaging showed that anterograde transport of mitochondria is impaired, as is synaptic transmission. Strikingly, disruption of presynaptic activity elicits upregulation of Neurotrophin-3 (Ntf3) in postsynaptic tectal cells. This in turn promotes exuberant branching of retinal axons by signaling through the TrkC receptor (Ntrk3). Thus, our study has uncovered an activity-dependent, retrograde signaling pathway that homeostatically controls axonal branching. DOI: http://dx.doi.org/10.7554/eLife.05061.001, eLife digest Different regions of a neuron have distinct structures and roles. For example, each neuron has a cable-like structure called the axon that extends out of the body of the cell and carries electrical signals away from the cell body. To pass these messages on to neighboring cells, branches on the axon form connections called synapses with other neurons. The axon lacks most of the cellular machinery needed to make proteins and other molecules that the cell needs to work correctly. Therefore, neurons must transport these materials from the cell body—where they are produced—down to the end of the axon. Specialized proteins called molecular motors carry this cargo down the axon along ‘tracks’ composed of filaments called microtubules. Auer, Xiao et al. have now used genetic techniques to disrupt the gene that encodes an important molecular motor, called Kif5A, in developing zebrafish larvae. The effects of this manipulation on the development of the zebrafish's visual system were then examined. When zebrafish are a few days old, neurons in the retina—the structure at the back of the eye that responds to light—extend axons into a region of the brain called the tectum. The formation of synapses between cells in the retina and the tectum provides a pathway that enables information to travel from the eye to the brain. Auer, Xiao et al. found that in larvae that lack Kif5A, axons from the retina enter the brain about a day later than they do in normal larvae. However, when these mutant axons arrive, they produce large numbers of branches, each with the potential to form multiple synapses with cells in the tectum. However, none of the resulting synapses appear to respond to visual stimuli, which is consistent with the fact that Kif5A mutant larvae are blind. Experiments to identify what triggers the excessive branching of retinal axons revealed that the mutant fish had elevated levels of a growth-promoting protein called neurotrophin-3 in cells in the tectum. This increased production of neurotrophin-3 was also observed when neuronal activity was blocked, for example by toxins. The lack of neuronal activity in retinal axons therefore seems to increase the production of neurotrophin-3, which in turn stimulates axonal branching. Future experiments could investigate the molecular signal that drives this increased production of neurotrophin-3, and how this is regulated during normal neuronal development. DOI: http://dx.doi.org/10.7554/eLife.05061.002

Neural circuits in the vertebrate retina extract the direction of object motion from visual scenes and convey this information to sensory brain areas, including the optic tectum. It is unclear how computational layers beyond the retina process directional inputs. Recent developmental and functional studies in the zebrafish larva, using minimally invasive optical imaging techniques, indicate that direction selectivity might be a genetically hardwired property of the zebrafish brain. Axons from specific direction-selective (DS) retinal ganglion cells (RGCs) appear to converge on distinct laminae in the superficial tectal neuropil where they serve as inputs to DS postsynaptic neurons of matching specificity. In addition, inhibitory recurrent circuits in the tectum might strengthen the DS response of tectal output neurons. Here we review these recent findings and discuss some controversies with a particular focus on the zebrafish tectum’s role in extracting directional features from moving visual scenes.

Summary Background Developing neural networks display spontaneous and correlated rhythmic bursts of action potentials that are essential for circuit refinement. In the spinal cord, it is poorly understood how correlated activity is acquired and how its emergence relates to the formation of the spinal central pattern generator (CPG), the circuit that mediates rhythmic behaviors like walking and swimming. It is also unknown whether early, uncorrelated activity is necessary for the formation of the coordinated CPG. Results Time-lapse imaging in the intact zebrafish embryo with the genetically encoded calcium indicator GCaMP3 revealed a rapid transition from slow, sporadic activity to fast, ipsilaterally correlated, and contralaterally anticorrelated activity, characteristic of the spinal CPG. Ipsilateral correlations were acquired through the coalescence of local microcircuits. Brief optical manipulation of activity with the light-driven pump halorhodopsin revealed that the transition to correlated activity was associated with a strengthening of ipsilateral connections, likely mediated by gap junctions. Contralateral antagonism increased in strength at the same time. The transition to coordinated activity was disrupted by long-term optical inhibition of sporadic activity in motoneurons and ventral longitudinal descending interneurons and resulted in more neurons exhibiting uncoordinated activity patterns at later time points. Conclusions These findings show that the CPG in the zebrafish spinal cord emerges directly from a sporadically active network as functional connectivity strengthens between local and then more distal neurons. These results also reveal that early, sporadic activity in a subset of ventral spinal neurons is required for the integration of maturing neurons into the coordinated CPG network.

Optogenetics has revolutionized the toolbox arsenal that neuroscientists now possess to investigate neuronal circuit function in intact and living animals. With a combination of light emitting ‘sensors’ and light activated ‘actuators’, we can monitor and control neuronal activity with minimal perturbation and unprecedented spatiotemporal resolution. Zebrafish neuronal circuits represent an ideal system to apply an optogenetic based analysis owing to its transparency, relatively small size and amenability to genetic manipulation. In this review, we describe some of the most recent advances in the development and applications of optogenetic sensors (i.e., genetically encoded calcium indicators and voltage sensors) and actuators (i.e., light activated ion channels and ion pumps). We focus mostly on the tools that have already been successfully applied in zebrafish and on those that show the greatest potential for the future. We also describe crucial technical aspects to implement optogenetics in zebrafish including strategies to drive a high level of transgene expression in defined neuronal populations, and recent optical advances that allow the precise spatiotemporal control of sample illumination.

Zebrafish became a model of choice for neurobiology because of the transparency of its brain and because of its amenability to genetic manipulation. In particular, at early stages of development the intact larva is an ideal system to apply optical techniques for deep imaging in the nervous system, as well as genetically encoded tools for targeting subsets of neurons and monitoring and manipulating their activity. For these applications,new genetically encoded optical tools, fluorescent sensors, and light-gated channels have been generated,creating the field of "optogenetics." It is now possible to monitor and control neuronal activity with minimal perturbation and unprecedented spatio-temporal resolution.We describe here the main achievements that have occurred in the last decade in imaging and manipulating neuronal activity in intact zebrafish larvae. We provide also examples of functional dissection of neuronal circuits achieved with the applications of these techniques in the visual and locomotor systems.

Small Is Attractive The optic tectum of zebrafish larvae is required for the detection, tracking, and capture of small, highly motile prey. Del Bene et al. (p. 669 ) applied a combination of optical, genetic, and pharmacological tools to investigate how neural circuits in the optic tectum filter out low-frequency visual information. Most tectal neurons were tuned to respond selectively to small, moving objects in the fish's visual environment and responded very poorly to large stimuli. This spatial filtering mechanism depended on the activity of a small population of GABAergic, inhibitory interneurons at the tectal surface. Inactivation or destruction of these interneurons removed the size selectivity of deeper neurons and the zebrafish lost their ability to catch prey.

The optokinetic response (OKR) to a visual stimulus moving at constant velocity consists of a series of two alternating components, a slow phase, during which the eyes follow the stimulus, and a quick phase, which resets the eyes to begin a new response cycle. The quick phases of the OKR resemble the saccades observed during free viewing. It is unclear to what extent the premotor circuitry underlying these two types of jerky, conjugate eye movements is conserved among vertebrates. Zebrafish (Danio rerio) larvae, broadly expressing halorhodopsin (NpHR) or channelrhodopsin-2 (ChR2) in most neurons, were used to map the location of neurons involved in this behavior. By blocking activity in localized groups of NpHR-expressing neurons with an optic fiber positioned above the head of the fish and by systematically varying the site of photostimulation, we discovered that activity in a small hindbrain area in rhombomere 5 was necessary for saccades to occur. Unilateral block of activity at this site affected behavior in a direction-specific manner. Inhibition of the right side suppressed rightward saccades of both eyes, while leaving leftward saccades unaffected, and vice versa. Photostimulation of this area in ChR2-transgenic fish was sufficient to trigger saccades that were precisely locked to the light pulses. These extra saccades could be induced both during free viewing and during the OKR, and were distinct in their kinetics from eye movements elicited by stimulating the abducens motor neurons. Zebrafishdouble indemnity(didy) mutants were identified in a chemical mutagenesis screen based on a defect in sustaining saccades during OKR. Positional cloning, molecular analysis, and electrophysiology revealed that thedidymutation disrupts the voltage-gated sodium channel Scn1lab (Nav1.lb). ChR2 photostimulation of the putative hindbrain saccade generator was able to fully reconstitute saccades in thedidymutant. Our studies demonstrate that an optogenetic approach is useful for targeted loss-of-function and gain-of-function manipulations of neural circuitry underlying eye movements in zebrafish and that the saccade-generating circuit in this species shares many of its properties with that in mammals.

The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.