Position-independent functional refinement within the vagus motor topographic map.

Motor neurons in the central nervous system often lie in a continuous topographic map, where neurons that innervate different body parts are spatially intermingled. This is the case for the efferent neurons of the vagus nerve, which innervate diverse muscle and organ targets in the head and viscera for brain-body communication. It remains elusive how neighboring motor neurons with different fixed peripheral axon targets develop the separate somatodendritic (input) connectivity they need to generate spatially precise body control. Here we show that vagus motor neurons in the zebrafish indeed generate spatially appropriate peripheral responses to focal sensory stimulation even when they are transplanted into ectopic positions within the topographic map, indicating that circuit refinement occurs after the establishment of coarse topography. Refinement depends on motor neuron synaptic transmission, suggesting that an experience-dependent periphery-to-brain feedback mechanism establishes specific input connectivity amongst intermingled motor populations.

Functional and ultrastructural analysis of reafferent mechanosensation in larval zebrafish.

All animals need to differentiate between exafferent stimuli, which are caused by the environment, and reafferent stimuli, which are caused by their own movement. In the case of mechanosensation in aquatic animals, the exafferent inputs are water vibrations in the animal's proximity, which need to be distinguishable from the reafferent inputs arising from fluid drag due to locomotion. Both of these inputs are detected by the lateral line, a collection of mechanosensory organs distributed along the surface of the body. In this study, we characterize in detail how hair cells-the receptor cells of the lateral line-in zebrafish larvae discriminate between such reafferent and exafferent signals. Using dye labeling of the lateral line nerve, we visualize two parallel descending inputs that can influence lateral line sensitivity. We combine functional imaging with ultra-structural EM circuit reconstruction to show that cholinergic signals originating from the hindbrain transmit efference copies (copies of the motor command that cancel out self-generated reafferent stimulation during locomotion) and that dopaminergic signals from the hypothalamus may have a role in threshold modulation, both in response to locomotion and salient stimuli. We further gain direct mechanistic insight into the core components of this circuit by loss-of-function perturbations using targeted ablations and gene knockouts. We propose that this simple circuit is the core implementation of mechanosensory reafferent suppression in these young animals and that it might form the first instantiation of state-dependent modulation found at later stages in development.

Long-Range Optogenetic Control of Axon Guidance Overcomes Developmental Boundaries and Defects.

Axons connect neurons together, establishing the wiring architecture of neuronal networks. Axonal connectivity is largely built during embryonic development through highly constrained processes of axon guidance, which have been extensively studied. However, the inability to control axon guidance, and thus neuronal network architecture, has limited investigation of how axonal connections influence subsequent development and function of neuronal networks. Here, we use zebrafish motor neurons expressing a photoactivatable Rac1 to co-opt endogenous growth cone guidance machinery to precisely and non-invasively direct axon growth using light. Axons can be guided over large distances, within complex environments of living organisms, overriding competing endogenous signals and redirecting axons across potent repulsive barriers to construct novel circuitry. Notably, genetic axon guidance defects can be rescued, restoring functional connectivity. These data demonstrate that intrinsic growth cone guidance machinery can be co-opted to non-invasively build new connectivity, allowing investigation of neural network dynamics in intact living organisms.

Sensorimotor Transformations in the Zebrafish Auditory System.

Organisms use their sensory systems to acquire information from their environment and integrate this information to produce relevant behaviors. Nevertheless, how sensory information is converted into adequate motor patterns in the brain remains an open question. Here, we addressed this question using two-photon and light-sheet calcium imaging in intact, behaving zebrafish larvae. We monitored neural activity elicited by auditory stimuli while simultaneously recording tail movements. We observed a spatial organization of neural activity according to four different response profiles (frequency tuning curves), suggesting a low-dimensional representation of frequency information, maintained throughout the development of the larvae. Low frequencies (150-450 Hz) were locally processed in the hindbrain and elicited motor behaviors. In contrast, higher frequencies (900-1,000 Hz) rarely induced motor behaviors and were also represented in the midbrain. Finally, we found that the sensorimotor transformations in the zebrafish auditory system are a continuous and gradual process that involves the temporal integration of the sensory response in order to generate a motor behavior.

Functional Integration of Newborn Neurons in the Zebrafish Optic Tectum.

Neurogenesis persists during adulthood in restricted parts of the vertebrate brain. In the optic tectum (OT) of the zebrafish larva, newborn neurons are continuously added and contribute to visual information processing. Recent studies have started to describe the functional development and fate of newborn neurons in the OT. Like the mammalian brain, newborn neurons in the OT require sensory inputs for their integration into local networks and survival. Recent findings suggest that the functional development of newborn neurons requires both activity-dependent and hard-wired mechanisms for proper circuit integration. Here, we review these findings and argue that the study of neurogenesis in non-mammalian species will help elucidate the general mechanisms of circuit assembly following neurogenesis.

Functional Interactions between Newborn and Mature Neurons Leading to Integration into Established Neuronal Circuits.

From development up to adulthood, the vertebrate brain is continuously supplied with newborn neurons that integrate into established mature circuits. However, how this process is coordinated during development remains unclear. Using two-photon imaging, GCaMP5 transgenic zebrafish larvae, and sparse electroporation in the larva's optic tectum, we monitored spontaneous and induced activity of large neuronal populations containing newborn and functionally mature neurons. We observed that the maturation of newborn neurons is a 4-day process. Initially, newborn neurons showed undeveloped dendritic arbors, no neurotransmitter identity, and were unresponsive to visual stimulation, although they displayed spontaneous calcium transients. Later on, newborn-labeled neurons began to respond to visual stimuli but in a very variable manner. At the end of the maturation period, newborn-labeled neurons exhibited visual tuning curves (spatial receptive fields and direction selectivity) and spontaneous correlated activity with neighboring functionally mature neurons. At this developmental stage, newborn-labeled neurons presented complex dendritic arbors and neurotransmitter identity (excitatory or inhibitory). Removal of retinal inputs significantly perturbed the integration of newborn neurons into the functionally mature tectal network. Our results provide a comprehensive description of the maturation of newborn neurons during development and shed light on potential mechanisms underlying their integration into a functionally mature neuronal circuit.

An integrated calcium imaging processing toolbox for the analysis of neuronal population dynamics.

The development of new imaging and optogenetics techniques to study the dynamics of large neuronal circuits is generating datasets of unprecedented volume and complexity, demanding the development of appropriate analysis tools. We present a comprehensive computational workflow for the analysis of neuronal population calcium dynamics. The toolbox includes newly developed algorithms and interactive tools for image pre-processing and segmentation, estimation of significant single-neuron single-trial signals, mapping event-related neuronal responses, detection of activity-correlated neuronal clusters, exploration of population dynamics, and analysis of clusters' features against surrogate control datasets. The modules are integrated in a modular and versatile processing pipeline, adaptable to different needs. The clustering module is capable of detecting flexible, dynamically activated neuronal assemblies, consistent with the distributed population coding of the brain. We demonstrate the suitability of the toolbox for a variety of calcium imaging datasets. The toolbox open-source code, a step-by-step tutorial and a case study dataset are available at https://github.com/zebrain-lab/Toolbox-Romano-et-al.

The Emergence of the Spatial Structure of Tectal Spontaneous Activity Is Independent of Visual Inputs.

The brain is spontaneously active, even in the absence of sensory stimulation. The functionally mature zebrafish optic tectum shows spontaneous activity patterns reflecting a functional connectivity adapted for the circuit's functional role and predictive of behavior. However, neither the emergence of these patterns during development nor the role of retinal inputs in their maturation has been characterized. Using two-photon calcium imaging, we analyzed spontaneous activity in intact and enucleated zebrafish larvae throughout tectum development. At the onset of retinotectal connections, intact larvae showed major changes in the spatiotemporal structure of spontaneous activity. Although the absence of retinal inputs had a significant impact on the development of the temporal structure, the tectum was still capable of developing a spatial structure associated with the circuit's functional roles and predictive of behavior. We conclude that neither visual experience nor intrinsic retinal activity is essential for the emergence of a spatially structured functional circuit.

Sustained Rhythmic Brain Activity Underlies Visual Motion Perception in Zebrafish.

Following moving visual stimuli (conditioning stimuli, CS), many organisms perceive, in the absence of physical stimuli, illusory motion in the opposite direction. This phenomenon is known as the motion aftereffect (MAE). Here, we use MAE as a tool to study the neuronal basis of visual motion perception in zebrafish larvae. Using zebrafish eye movements as an indicator of visual motion perception, we find that larvae perceive MAE. Blocking eye movements using optogenetics during CS presentation did not affect MAE, but tectal ablation significantly weakened it. Using two-photon calcium imaging of behaving GCaMP3 larvae, we find post-stimulation sustained rhythmic activity among direction-selective tectal neurons associated with the perception of MAE. In addition, tectal neurons tuned to the CS direction habituated, but neurons in the retina did not. Finally, a model based on competition between direction-selective neurons reproduced MAE, suggesting a neuronal circuit capable of generating perception of visual motion.

Elucidating molecular phenotypes caused by the SORL1 Alzheimer's disease genetic risk factor using human induced pluripotent stem cells.

Predisposition to sporadic Alzheimer's disease (SAD) involves interactions between a person's unique combination of genetic variants and the environment. The molecular effect of these variants may be subtle and difficult to analyze with standard in vitro or in vivo models. Here we used hIPSCs to examine genetic variation in the SORL1 gene and possible contributions to SAD-related phenotypes in human neurons. We found that human neurons carrying SORL1 variants associated with an increased SAD risk show a reduced response to treatment with BDNF, at the level of both SORL1 expression and APP processing. shRNA knockdown of SORL1 demonstrates that the differences in BDNF-induced APP processing between genotypes are dependent on SORL1 expression. We propose that the variation in SORL1 expression induction by BDNF is modulated by common genetic variants and can explain how genetic variation in this one locus can contribute to an individual's risk of developing SAD.