Uncovering multiscale structure in the variability of larval zebrafish navigation.

Animals chain movements into long-lived motor strategies, exhibiting variability across scales that reflects the interplay between internal states and environmental cues. To reveal structure in such variability, we build Markov models of movement sequences that bridges across time scales and enables a quantitative comparison of behavioral phenotypes among individuals. Applied to larval zebrafish responding to diverse sensory cues, we uncover a hierarchy of long-lived motor strategies, dominated by changes in orientation distinguishing cruising versus wandering strategies. Environmental cues induce preferences along these modes at the population level: while fish cruise in the light, they wander in response to aversive stimuli, or in search for appetitive prey. As our method encodes the behavioral dynamics of each individual fish in the transitions among coarse-grained motor strategies, we use it to uncover a hierarchical structure in the phenotypic variability that reflects exploration-exploitation trade-offs. Across a wide range of sensory cues, a major source of variation among fish is driven by prior and/or immediate exposure to prey that induces exploitation phenotypes. A large degree of variability that is not explained by environmental cues unravels motivational states that override the sensory context to induce contrasting exploration-exploitation phenotypes. Altogether, by extracting the timescales of motor strategies deployed during navigation, our approach exposes structure among individuals and reveals internal states tuned by prior experience.

Norepinephrine changes behavioral state via astroglial purinergic signaling.

Both neurons and glia communicate via diffusible neuromodulatory substances, but the substrates of computation in such neuromodulatory networks are unclear. During behavioral transitions in the larval zebrafish, the neuromodulator norepinephrine drives fast excitation and delayed inhibition of behavior and circuit activity. We find that the inhibitory arm of this feedforward motif is implemented by astroglial purinergic signaling. Neuromodulator imaging, behavioral pharmacology, and perturbations of neurons and astroglia reveal that norepinephrine triggers astroglial release of adenosine triphosphate, extracellular conversion into adenosine, and behavioral suppression through activation of hindbrain neuronal adenosine receptors. This work, along with a companion piece by Lefton and colleagues demonstrating an analogous pathway mediating the effect of norepinephrine on synaptic connectivity in mice, identifies a computational and behavioral role for an evolutionarily conserved astroglial purinergic signaling axis in norepinephrine-mediated behavioral and brain state transitions.

Uncovering multiscale structure in the variability of larval zebrafish navigation.

Animals chain movements into long-lived motor strategies, resulting in variability that ultimately reflects the interplay between internal states and environmental cues. To reveal structure in such variability, we build models that bridges across time scales that enable a quantitative comparison of behavioral phenotypes among individuals. Applied to larval zebrafish exposed to diverse sensory cues, we uncover a hierarchy of long-lived motor strategies, dominated by changes in orientation distinguishing cruising and wandering strategies. Environmental cues induce preferences along these modes at the population level: while fish cruise in the light, they wander in response to aversive (dark) stimuli or in search for prey. Our method enables us to encode the behavioral dynamics of each individual fish in the transitions among coarse-grained motor strategies. By doing so, we uncover a hierarchical structure to the phenotypic variability that corresponds to exploration-exploitation trade-offs. Within a wide range of sensory cues, a major source of variation among fish is driven by prior and immediate exposure to prey that induces exploitation phenotypes. However, a large degree of variability is unexplained by environmental cues, pointing to hidden states that override the sensory context to induce contrasting exploration-exploitation phenotypes. Altogether, our approach extracts the timescales of motor strategies deployed during navigation, exposing undiscovered structure among individuals and pointing to internal states tuned by prior experience.

SCO-spondin knockout mice exhibit small brain ventricles and mild spine deformation.

Reissner's fiber (RF) is an extracellular polymer comprising the large monomeric protein SCO-spondin (SSPO) secreted by the subcommissural organ (SCO) that extends through cerebrospinal fluid (CSF)-filled ventricles into the central canal of the spinal cord. In zebrafish, RF and CSF-contacting neurons (CSF-cNs) form an axial sensory system that detects spinal curvature, instructs morphogenesis of the body axis, and enables proper alignment of the spine. In mammalian models, RF has been implicated in CSF circulation. However, challenges in manipulating Sspo, an exceptionally large gene of 15,719 nucleotides, with traditional approaches has limited progress. Here, we generated a Sspo knockout mouse model using CRISPR/Cas9-mediated genome-editing. Sspo knockout mice lacked RF-positive material in the SCO and fibrillar condensates in the brain ventricles. Remarkably, Sspo knockout brain ventricle sizes were reduced compared to littermate controls. Minor defects in thoracic spine curvature were detected in Sspo knockouts, which did not alter basic motor behaviors tested. Altogether, our work in mouse demonstrates that SSPO and RF regulate ventricle size during development but only moderately impact spine geometry.

Design of mechanosensory feedback during undulatory locomotion to enhance speed and stability.

Undulatory locomotion relies on the propagation of a wave of excitation in the spinal cord leading to consequential activation of segmental skeletal muscles along the body. Although this process relies on self-generated oscillations of motor circuits in the spinal cord, mechanosensory feedback is crucial to entrain the underlying oscillatory activity and thereby, to enhance movement power and speed. This effect is achieved through directional projections of mechanosensory neurons either sensing stretching or compression of the trunk along the rostrocaudal axis. Different mechanosensory feedback pathways act in concert to shorten and fasten the excitatory wave propagating along the body. While inhibitory mechanosensory cells feedback inhibition on excitatory premotor interneurons and motor neurons, excitatory mechanosensory cells feedforward excitation to premotor excitatory interneurons. Together, diverse mechanosensory cells coordinate the activity of skeletal muscles controlling the head and tail to optimize speed and stabilize balance during fast locomotion.

The mesencephalic locomotor region recruits V2a reticulospinal neurons to drive forward locomotion in larval zebrafish.

The mesencephalic locomotor region (MLR) is a brain stem area whose stimulation triggers graded forward locomotion. How MLR neurons recruit downstream vsx2+ (V2a) reticulospinal neurons (RSNs) is poorly understood. Here, to overcome this challenge, we uncovered the locus of MLR in transparent larval zebrafish and show that the MLR locus is distinct from the nucleus of the medial longitudinal fasciculus. MLR stimulations reliably elicit forward locomotion of controlled duration and frequency. MLR neurons recruit V2a RSNs via projections onto somata in pontine and retropontine areas, and onto dendrites in the medulla. High-speed volumetric imaging of neuronal activity reveals that strongly MLR-coupled RSNs are active for steering or forward swimming, whereas weakly MLR-coupled medullary RSNs encode the duration and frequency of the forward component. Our study demonstrates how MLR neurons recruit specific V2a RSNs to control the kinematics of forward locomotion and suggests conservation of the motor functions of V2a RSNs across vertebrates.

The Reissner fiber under tension in vivo shows dynamic interaction with ciliated cells contacting the cerebrospinal fluid.

The Reissner fiber (RF) is an acellular thread positioned in the midline of the central canal that aggregates thanks to the beating of numerous cilia from ependymal radial glial cells (ERGs) generating flow in the central canal of the spinal cord. RF together with cerebrospinal fluid (CSF)-contacting neurons (CSF-cNs) form an axial sensory system detecting curvature. How RF, CSF-cNs and the multitude of motile cilia from ERGs interact in vivo appears critical for maintenance of RF and sensory functions of CSF-cNs to keep a straight body axis, but is not well-understood. Using in vivo imaging in larval zebrafish, we show that RF is under tension and resonates dorsoventrally. Focal RF ablations trigger retraction and relaxation of the fiber's cut ends, with larger retraction speeds for rostral ablations. We built a mechanical model that estimates RF stress diffusion coefficient D at 5 mm2/s and reveals that tension builds up rostrally along the fiber. After RF ablation, spontaneous CSF-cN activity decreased and ciliary motility changed, suggesting physical interactions between RF and cilia projecting into the central canal. We observed that motile cilia were caudally-tilted and frequently interacted with RF. We propose that the numerous ependymal motile monocilia contribute to RF's heterogenous tension via weak interactions. Our work demonstrates that under tension, the Reissner fiber dynamically interacts with motile cilia generating CSF flow and spinal sensory neurons.

Cerebrospinal fluid-contacting neurons: multimodal cells with diverse roles in the CNS.

The cerebrospinal fluid (CSF) is a complex solution that circulates around the CNS, and whose composition changes as a function of an animal's physiological state. Ciliated neurons that are bathed in the CSF - and thus referred to as CSF-contacting neurons (CSF-cNs) - are unusual polymodal interoceptive neurons. As chemoreceptors, CSF-cNs respond to variations in pH and osmolarity and to bacterial metabolites in the CSF. Their activation during infections of the CNS results in secretion of compounds to enhance host survival. As mechanosensory neurons, CSF-cNs operate together with an extracellular proteinaceous polymer known as the Reissner fibre to detect compression during spinal curvature. Once activated, CSF-cNs inhibit motor neurons, premotor excitatory neurons and command neurons to enhance movement speed and stabilize posture. At longer timescales, CSF-cNs instruct morphogenesis throughout life via the release of neuropeptides that act over long distances on skeletal muscle. Finally, recent evidence suggests that mouse CSF-cNs may act as neural stem cells in the spinal cord, inspiring new paths of investigation for repair after injury.

SCO-spondin knockout mice exhibit small brain ventricles and mild spine deformation.

Reissner's fiber (RF) is an extracellular polymer comprising the large monomeric protein SCO-spondin (SSPO) secreted by the subcommissural organ (SCO) that extends through cerebrospinal fluid (CSF)-filled ventricles into the central canal of the spinal cord. In zebrafish, RF and CSF-contacting neurons (CSF-cNs) form an axial sensory system that detects spinal curvature, instructs morphogenesis of the body axis, and enables proper alignment of the spine. In mammalian models, RF has been implicated in CSF circulation. However, challenges in manipulating Sspo , an exceptionally large gene of 15,719 nucleotides, with traditional approaches has limited progress. Here, we generated a Sspo knockout mouse model using CRISPR/Cas9-mediated genome-editing. Sspo knockout mice lacked RF-positive material in the SCO and fibrillar condensates in the brain ventricles. Remarkably, Sspo knockout brain ventricle sizes were reduced compared to littermate controls. Minor defects in thoracic spine curvature were detected in Sspo knockouts, which did not alter basic motor behaviors tested. Altogether, our work in mouse demonstrates that SSPO and RF regulate ventricle size during development but only moderately impact spine geometry.

Sensory Systems in the Peripheral and Central Nervous Systems Shape Host Response During Infections.

The function of sensory cells has been largely investigated in the field of neuroscience for how they report the physical and chemical changes of the environment ("exteroception") and of internal physiology ("interoception"). Investigations over the last century have largely focused on the morphological, electrical and receptor properties of sensory cells in the nervous system focusing on conscious perception of external cues or homeostatic regulation upon detection of internal cues. Research in the last decade has uncovered that sensory cells can often sense polymodal cues, such as mechanical, chemical, and/ or thermal. Furthermore, sensory cells in the peripheral as well as in the central nervous system can detect evidence associated with the invasion of pathogenic bacteria or viruses. The corresponding neuronal activation associated with the presence of pathogens can impact their classical functions within the nervous system and trigger the release of compounds modulating the response to intruders, either triggering pain to raise awareness, enhancing host defense or sometimes, aggravating the infection. This perspective brings to light the need for interdisciplinary training in immunology, microbiology and neuroscience for the next generation of investigators in this field.

[Polymodal sensory integration at the interface with the cerebrospinal fluid]. / Les neurones contactant le liquide cérébrospinal - Une intégration sensorielle polymodale.

The cerebrospinal fluid is the site of a sensory interface that allows interactions between the nervous system and cellular targets throughout the body. Sensory neurons in contact with the cerebrospinal fluid in the spinal cord respond to changes of its composition, in particular in the context of bacterial infections of the central nervous system. These cerebrospinal fluid-contacting neurons also form an axial mechanosensory system that detects spinal curvature through the coupling with a long a proteinaceous polymer under tension in the central canal called the Reissner fiber. Activated by compression of the body axis, neurons contacting the cerebrospinal fluid modulate motor circuits to increase the speed of movement and to stabilize posture. During development and aging, this sensory system aligns the body axis and spine via the release of peptides of the urotensin family that act at long range on their receptors expressed in skeletal muscles.

Title: Les neurones contactant le liquide cérébrospinal - Une intégration sensorielle polymodale. Abstract: Le liquide cérébrospinal est le siège d'une interface sensorielle qui permet des interactions entre le système nerveux et le reste du corps. Au centre de la moelle épinière, des neurones sensoriels en contact avec le liquide cérébrospinal détectent des changements de son contenu et s'activent lorsque sa composition change, en particulier lors d'une infection bactérienne du système nerveux central. Ces neurones de contact forment aussi un système mécanosensoriel axial détectant la courbure spinale grâce à un couplage avec la fibre de Reissner, un long polymère protéique situé dans le canal central. Sous l'effet d'une compression, les neurones de contact s'activent et modulent l'activité des circuits moteurs antérieurs au niveau du tronc cérébral et de la moelle épinière, contribuant à augmenter la vitesse du mouvement et à stabiliser la posture. Ce système sensoriel permet l'alignement de la colonne vertébrale en agissant sur sa structure via la libération de peptides de la famille de l'urotensine-II dont les récepteurs sont situés dans les muscles squelettiques.

Mechanosensory Control of Locomotion in Animals and Robots: Moving Forward.

While animals swim, crawl, walk, and fly with apparent ease, building robots capable of robust locomotion remains a significant challenge. In this review, we draw attention to mechanosensation-the sensing of mechanical forces generated within and outside the body-as a key sense that enables robust locomotion in animals. We discuss differences between mechanosensation in animals and current robots with respect to (1) the encoding properties and distribution of mechanosensors and (2) the integration and regulation of mechanosensory feedback. We argue that robotics would benefit greatly from a detailed understanding of these aspects in animals. To that end, we highlight promising experimental and engineering approaches to study mechanosensation, emphasizing the mutual benefits for biologists and engineers that emerge from moving forward together.

Loss of CSF-contacting neuron sensory function is associated with a hyper-kyphosis of the spine reminiscent of Scheuermann's disease.

Scheuermann's disease, also referred to as Scheuermann's kyphosis, is the second most frequent spine deformity occurring in humans after adolescent idiopathic scoliosis (AIS), both with an unclear etiology. Recent genetic studies in zebrafish unraveled new mechanisms linked to AIS, highlighting the role of the Reissner fiber, an acellular polymer bathing in the cerebrospinal fluid (CSF) in close proximity with ciliated cells and mechanosensory neurons lining the central canal of the spinal cord (CSF-cNs). However, while the Reissner fiber and ciliary beating have been linked to AIS-like phenotypes in zebrafish, the relevance of the sensory functions of CSF-cNs for human spine disorders remains unknown. Here, we show that the thoracic hyper-kyphosis of the spine previously reported in adult pkd2l1 mutant zebrafish, in which the mechanosensory function of CSF-cNs is likely defective, is restricted to the sagittal plane and is not associated with vertebral malformations. By applying orthopedic criteria to analyze the amplitude of the curvature at the apex of the kyphosis, the curve pattern, the sagittal balance and sex bias, we demonstrate that pkd2l1 knock-outs develop a phenotype reminiscent of Scheuermann's disease. Altogether our work consolidates the benefit of combining genetics and analysis of spine deformities in zebrafish to model idiopathic spine disorders in humans.

Unraveling the roles of cerebrospinal fluid-contacting neurons.

Sensory neurons previously shown to optimize speed and balance in fish by providing information about the curvature of the spine show similar morphology and connectivity in mice.

Granger causality analysis for calcium transients in neuronal networks, challenges and improvements.

One challenge in neuroscience is to understand how information flows between neurons in vivo to trigger specific behaviors. Granger causality (GC) has been proposed as a simple and effective measure for identifying dynamical interactions. At single-cell resolution however, GC analysis is rarely used compared to directionless correlation analysis. Here, we study the applicability of GC analysis for calcium imaging data in diverse contexts. We first show that despite underlying linearity assumptions, GC analysis successfully retrieves non-linear interactions in a synthetic network simulating intracellular calcium fluctuations of spiking neurons. We highlight the potential pitfalls of applying GC analysis on real in vivo calcium signals, and offer solutions regarding the choice of GC analysis parameters. We took advantage of calcium imaging datasets from motoneurons in embryonic zebrafish to show how the improved GC can retrieve true underlying information flow. Applied to the network of brainstem neurons of larval zebrafish, our pipeline reveals strong driver neurons in the locus of the mesencephalic locomotor region (MLR), driving target neurons matching expectations from anatomical and physiological studies. Altogether, this practical toolbox can be applied on in vivo population calcium signals to increase the selectivity of GC to infer flow of information across neurons.

CSF-contacting neurons respond to Streptococcus pneumoniae and promote host survival during central nervous system infection.

The pathogenic bacterium Streptococcus pneumoniae (S. pneumoniae) can invade the cerebrospinal fluid (CSF) and cause meningitis with devastating consequences. Whether and how sensory cells in the central nervous system (CNS) become activated during bacterial infection, as recently reported for the peripheral nervous system, is not known. We find that CSF infection by S. pneumoniae in larval zebrafish leads to changes in posture and behavior that are reminiscent of pneumococcal meningitis, including dorsal arching and epileptic-like seizures. We show that during infection, invasion of the CSF by S. pneumoniae massively activates in vivo sensory neurons contacting the CSF, referred to as "CSF-cNs" and previously shown to detect spinal curvature and to control posture, locomotion, and spine morphogenesis. We find that CSF-cNs express orphan bitter taste receptors and respond in vitro to bacterial supernatant and metabolites via massive calcium transients, similar to the ones observed in vivo during infection. Upon infection, CSF-cNs also upregulate the expression of numerous cytokines and complement components involved in innate immunity. Accordingly, we demonstrate, using cell-specific ablation and blockade of neurotransmission, that CSF-cN neurosecretion enhances survival of the host during S. pneumoniae infection. Finally, we show that CSF-cNs respond to various pathogenic bacteria causing meningitis in humans, as well as to the supernatant of cells infected by a neurotropic virus. Altogether, our work uncovers that central sensory neurons in the spinal cord, previously involved in postural control and morphogenesis, contribute as well to host survival by responding to the invasion of the CSF by pathogenic bacteria during meningitis.

Evolutionary divergence of locomotion in two related vertebrate species.

Locomotion exists in diverse forms in nature; however, little is known about how closely related species with similar neuronal circuitry can evolve different navigational strategies to explore their environments. Here, we investigate this question by comparing divergent swimming pattern in larval Danionella cerebrum (DC) and zebrafish (ZF). We show that DC displays long continuous swimming events when compared with the short burst-and-glide swimming in ZF. We reveal that mesencephalic locomotion maintenance neurons in the midbrain are sufficient to cause this increased swimming. Moreover, we propose that the availability of dissolved oxygen and timing of swim bladder inflation drive the observed differences in the swim pattern. Our findings uncover the neural substrate underlying the evolutionary divergence of locomotion and its adaptation to their environmental constraints.

A lexical approach for identifying behavioural action sequences.

Animals display characteristic behavioural patterns when performing a task, such as the spiraling of a soaring bird or the surge-and-cast of a male moth searching for a female. Identifying such recurring sequences occurring rarely in noisy behavioural data is key to understanding the behavioural response to a distributed stimulus in unrestrained animals. Existing models seek to describe the dynamics of behaviour or segment individual locomotor episodes rather than to identify the rare and transient sequences of locomotor episodes that make up the behavioural response. To fill this gap, we develop a lexical, hierarchical model of behaviour. We designed an unsupervised algorithm called "BASS" to efficiently identify and segment recurring behavioural action sequences transiently occurring in long behavioural recordings. When applied to navigating larval zebrafish, BASS extracts a dictionary of remarkably long, non-Markovian sequences consisting of repeats and mixtures of slow forward and turn bouts. Applied to a novel chemotaxis assay, BASS uncovers chemotactic strategies deployed by zebrafish to avoid aversive cues consisting of sequences of fast large-angle turns and burst swims. In a simulated dataset of soaring gliders climbing thermals, BASS finds the spiraling patterns characteristic of soaring behaviour. In both cases, BASS succeeds in identifying rare action sequences in the behaviour deployed by freely moving animals. BASS can be easily incorporated into the pipelines of existing behavioural analyses across diverse species, and even more broadly used as a generic algorithm for pattern recognition in low-dimensional sequential data.

A norepinephrine-dependent glial calcium wave travels in the spinal cord upon acoustovestibular stimuli.

Although calcium waves have been widely observed in glial cells, their occurrence in vivo during behavior remains less understood. Here, we investigated the recruitment of glial cells in the hindbrain and spinal cord after acousto-vestibular (AV) stimuli triggering escape responses using in vivo population calcium imaging in larval zebrafish. We observed that gap-junction-coupled spinal glial network exhibits large and homogenous calcium increases that rose in the rostral spinal cord and propagated bi-directionally toward the spinal cord and toward the hindbrain. Spinal glial calcium waves were driven by the recruitment of neurons and in particular, of noradrenergic signaling acting through α-adrenergic receptors. Noradrenergic neurons of the medulla-oblongata (NE-MO) were revealed in the vicinity of where the calcium wave started. NE-MO were recruited upon AV stimulation and sent dense axonal projections in the rostro-lateral spinal cord, suggesting these cells could trigger the glial wave to propagate down the spinal cord. Altogether, our results revealed that a simple AV stimulation is sufficient to recruit noradrenergic neurons in the brainstem that trigger in the rostral spinal cord two massive glial calcium waves, one traveling caudally in the spinal cord and another rostrally into the hindbrain.

Spinal sensory neurons project onto the hindbrain to stabilize posture and enhance locomotor speed.

In the spinal cord, cerebrospinal fluid-contacting neurons (CSF-cNs) are GABAergic interoceptive sensory neurons that detect spinal curvature via a functional coupling with the Reissner fiber. This mechanosensory system has recently been found to be involved in spine morphogenesis and postural control but the underlying mechanisms are not fully understood. In zebrafish, CSF-cNs project an ascending and ipsilateral axon reaching two to six segments away. Rostralmost CSF-cNs send their axons ipsilaterally into the hindbrain, a brain region containing motor nuclei and reticulospinal neurons (RSNs), which send descending motor commands to spinal circuits. Until now, the synaptic connectivity of CSF-cNs has only been investigated in the spinal cord, where they synapse onto motor neurons and premotor excitatory interneurons. The identity of CSF-cN targets in the hindbrain and the behavioral relevance of these sensory projections from the spinal cord to the hindbrain are unknown. Here, we provide anatomical and molecular evidence that rostralmost CSF-cNs synapse onto the axons of large RSNs including Mauthner cells and V2a neurons. Functional anatomy and optogenetically assisted mapping reveal that rostral CSF-cNs also synapse onto the soma and dendrites of cranial motor neurons innervating hypobranchial muscles. During acousto-vestibular evoked escape responses, ablation of rostralmost CSF-cNs results in a weaker escape response with a decreased C-bend amplitude, lower speed, and deficient postural control. Our study demonstrates that spinal sensory feedback enhances speed and stabilizes posture, and reveals a novel spinal gating mechanism acting on the output of descending commands sent from the hindbrain to the spinal cord.