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.

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.

Regulation of the apical extension morphogenesis tunes the mechanosensory response of microvilliated neurons.

Multiple types of microvilliated sensory cells exhibit an apical extension thought to be instrumental in the detection of sensory cues. The investigation of the mechanisms underlying morphogenesis of sensory apparatus is critical to understand the biology of sensation. Most of what we currently know comes from the study of the hair bundle of the inner ear sensory cells, but morphogenesis and function of other sensory microvilliated apical extensions remain poorly understood. We focused on spinal sensory neurons that contact the cerebrospinal fluid (CSF) through the projection of a microvilliated apical process in the central canal, referred to as cerebrospinal fluid-contacting neurons (CSF-cNs). CSF-cNs respond to pH and osmolarity changes as well as mechanical stimuli associated with changes of flow and tail bending. In vivo time-lapse imaging in zebrafish embryos revealed that CSF-cNs are atypical neurons that do not lose their apical attachment and form a ring of actin at the apical junctional complexes (AJCs) that they retain during differentiation. We show that the actin-based protrusions constituting the microvilliated apical extension arise and elongate from this ring of actin, and we identify candidate molecular factors underlying every step of CSF-cN morphogenesis. We demonstrate that Crumbs 1 (Crb1), Myosin 3b (Myo3b), and Espin orchestrate the morphogenesis of CSF-cN apical extension. Using calcium imaging in crb1 and espin mutants, we further show that the size of the apical extension modulates the amplitude of CSF-cN sensory response to bending of the spinal cord. Based on our results, we propose that the apical actin ring could be a common site of initiation of actin-based protrusions in microvilliated sensory cells. Furthermore, our work provides a set of actors underlying actin-based protrusion elongation shared by different sensory cell types and highlights the critical role of the apical extension shape in sensory detection.

Light on a sensory interface linking the cerebrospinal fluid to motor circuits in vertebrates.

The cerebrospinal fluid (CSF) is circulating around the entire central nervous system (CNS). The main function of the CSF has been thought to insure the global homeostasis of the CNS. Recent evidence indicates that the CSF also dynamically conveys signals modulating the development and the activity of the nervous system. The later observation implies that cues from the CSF could act on neurons in the brain and the spinal cord via bordering receptor cells. Candidate neurons to enable such modulation are the cerebrospinal fluid-contacting neurons (CSF-cNs) that are located precisely at the interface between the CSF and neuronal circuits. The atypical apical extension of CSF-cNs bears a cluster of microvilli bathing in the CSF indicating putative sensory or secretory roles in relation with the CSF. In the brainstem and spinal cord, CSF-cNs have been described in over two hundred species by Kolmer and Agduhr, suggesting an important function within the spinal cord. However, the lack of specific markers and the difficulty to access CSF-cNs hampered their physiological investigation. The transient receptor potential channel PKD2L1 is a specific marker of spinal CSF-cNs in vertebrate species. The transparency of zebrafish at early stages eases the functional characterization of pkd2l1+ CSF-cNs. Recent studies demonstrate that spinal CSF-cNs detect spinal curvature via the channel PKD2L1 and modulate locomotion and posture by projecting onto spinal interneurons and motor neurons in vivo. In vitro recordings demonstrated that spinal CSF-cNs are sensing pH variations mainly through ASIC channels, in combination with PKD2L1. Altogether, neurons contacting the CSF appear as a novel sensory modality enabling the detection of mechanical and chemical stimuli from the CSF and modulating the excitability of spinal circuits underlying locomotion and posture.

Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway.

The habenulo-interpeduncular pathway, a highly conserved cholinergic system, has emerged as a valuable model to study left-right asymmetry in the brain. In larval zebrafish, the bilaterally paired dorsal habenular nuclei (dHb) exhibit prominent left-right differences in their organization, gene expression, and connectivity, but their cholinergic nature was unclear. Through the discovery of a duplicated cholinergic gene locus, we now show that choline acetyltransferase and vesicular acetylcholine transporter homologs are preferentially expressed in the right dHb of larval zebrafish. Genes encoding the nicotinic acetylcholine receptor subunits α2 and ß4 are transcribed in the target interpeduncular nucleus (IPN), suggesting that the asymmetrical cholinergic pathway is functional. To confirm this, we activated channelrhodopsin-2 specifically in the larval dHb and performed whole-cell patch-clamp recording of IPN neurons. The response to optogenetic or electrical stimulation of the right dHb consisted of an initial fast glutamatergic excitatory postsynaptic current followed by a slow-rising cholinergic current. In adult zebrafish, the dHb are divided into discrete cholinergic and peptidergic subnuclei that differ in size between the left and right sides of the brain. After exposing adults to nicotine, fos expression was activated in subregions of the IPN enriched for specific nicotinic acetylcholine receptor subunits. Our studies of the newly identified cholinergic gene locus resolve the neurotransmitter identity of the zebrafish habenular nuclei and reveal functional asymmetry in a major cholinergic neuromodulatory pathway of the vertebrate brain.

Optogenetic dissection of a behavioural module in the vertebrate spinal cord.

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.

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.

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.

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.

[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.

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.

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.

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.

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.

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.

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.

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.

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.