Sensory inputs are subjected to modulation by central neural networks involved in controlling movements. It has been shown that serotonin (5-HT) modulates sensory transmission. This study examines in lampreys the effects of 5-HT on sensory transmission to brainstem reticulospinal (RS) neurons and the distribution of 5-HT cells that innervate RS cells. Cells were recorded intracellularly in the in vitro isolated brainstem of larval lampreys. Trigeminal nerve stimulation elicited disynaptic excitatory responses in RS neurons, and bath application of 5-HT reduced the response amplitude with maximum effect at 10 mum. Local ejection of 5-HT either onto the RS cells or onto the relay cells decreased sensory-evoked excitatory postsynaptic potentials (EPSPs) in RS cells. The monosynaptic EPSPs elicited from stimulation of the relay cells were also reduced by 5-HT. The reduction was maintained after blocking either N-methyl-d-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. The local ejection of glutamate over RS cells elicited excitatory responses that were only slightly depressed by 5-HT. In addition, 5-HT increased the threshold for eliciting sustained depolarizations in response to trigeminal nerve stimulation but did not prevent them. Combined 5-HT immunofluorescence with axonal tracing revealed that the 5-HT innervation of RS neurons of the middle rhombencephalic reticular nucleus comes mainly from neurons in the isthmic region, but also from neurons located in the pretectum and caudal rhombencephalon. Our results indicate that 5-HT modulates sensory transmission to lamprey brainstem RS cells.

Genetically targeted light-activated ion channels and pumps make it possible to determine the role of specific neurons in neuronal circuits, information processing and behavior. We developed a K+-selective ionotropic glutamate receptor that reversibly inhibits neuronal activity in response to light in dissociated neurons and brain slice and also reversibly suppresses behavior in zebrafish. The receptor is a chimera of the pore region of a K+-selective bacterial glutamate receptor and the ligand-binding domain of a light-gated mammalian kainate receptor. This hyperpolarizing light-gated channel, HyLighter, is turned on by a brief light pulse at one wavelength and turned off by a pulse at a second wavelength. The control is obtained at moderate intensity. After optical activation, the photocurrent and optical silencing of activity persists in the dark for extended periods. The low light requirement and bi-stability of HyLighter represent advantages for the dissection of neural circuitry.

Urotensin II (UII) is an evolutionarily conserved neuropeptide initially isolated from teleost fish on the basis of its smooth muscle-contracting activity. Subsequent studies have demonstrated the occurrence of several UII-related peptides (URPs), such that the UII family is now known to include four paralogue genes called UII, URP, URP1 and URP2. These genes probably arose through the two rounds of whole genome duplication that occurred during early vertebrate evolution. URP has been identified both in tetrapods and teleosts. In contrast, URP1 and URP2 have only been observed in ray-finned and cartilaginous fishes, suggesting that both genes were lost in the tetrapod lineage. In the present study, the distribution of urp1 mRNA compared to urp2 mRNA is reported in the central nervous system of zebrafish. In the spinal cord, urp1 and urp2 mRNAs were mainly colocalized in the same cells. These cells were also shown to be GABAergic and express the gene encoding the polycystic kidney disease 2-like 1 (pkd2l1) channel, indicating that they likely correspond to cerebrospinal fluid-contacting neurons. In the hindbrain, urp1-expressing cells were found in the intermediate reticular formation and the glossopharyngeal-vagal motor nerve nuclei. We also showed that synthetic URP1 and URP2 were able to induce intracellular calcium mobilization in human UII receptor (hUT)-transfected CHO cells with similar potencies (pEC50=7.99 and 7.52, respectively) albeit at slightly lower potencies than human UII and mammalian URP (pEC50=9.44 and 8.61, respectively). The functional redundancy of URP1 and URP2 as well as the colocalization of their mRNAs in the spinal cord suggest the robustness of this peptidic system and its physiological importance in zebrafish.

The pattern of blood flow has long been thought to play a significant role in vascular morphogenesis, yet the flow-sensing mechanism that is involved at early embryonic stages, when flow forces are low, remains unclear. It has been proposed that endothelial cells use primary cilia to sense flow, but this has never been tested in vivo. Here we show, by noninvasive, high-resolution imaging of live zebrafish embryos, that endothelial cilia progressively deflect at the onset of blood flow and that the deflection angle correlates with calcium levels in endothelial cells. We demonstrate that alterations in shear stress, ciliogenesis, or expression of the calcium channel PKD2 impair the endothelial calcium level and both increase and perturb vascular morphogenesis. Altogether, these results demonstrate that endothelial cilia constitute a highly sensitive structure that permits the detection of low shear forces during vascular morphogenesis.

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.

Locomotion occurs sporadically and needs to be started, maintained, and stopped. The neural substrate underlying the activation of locomotion is partly known, but little is known about mechanisms involved in termination of locomotion. Recently, reticulospinal neurons (stop cells) were found to play a crucial role in stopping locomotion in the lamprey: their activation halts ongoing locomotion and their inactivation slows down the termination process. Intracellular recordings of these cells revealed a distinct activity pattern, with a burst of action potentials at the beginning of a locomotor bout and one at the end (termination burst). The termination burst was shown to be time linked to the end of locomotion, but the mechanisms by which it is triggered have remained unknown. We studied this in larval sea lampreys (Petromyzon marinus; the sex of the animals was not taken into account). We found that the mesencephalic locomotor region (MLR), which is known to initiate and control locomotion, stops ongoing locomotion by providing synaptic inputs that trigger the termination burst in stop cells. When locomotion is elicited by MLR stimulation, a second MLR stimulation stops the locomotor bout if it is of lower intensity than the initial stimulation. This occurs for MLR-induced, sensory-evoked, and spontaneous locomotion. Furthermore, we show that glutamatergic and, most likely, monosynaptic projections from the MLR activate stop cells during locomotion. Therefore, activation of the MLR not only initiates locomotion, but can also control the end of a locomotor bout. These results provide new insights onto the neural mechanisms responsible for stopping locomotion.SIGNIFICANCE STATEMENT The mesencephalic locomotor region (MLR) is a brainstem region well known to initiate and control locomotion. Since its discovery in cats in the 1960s, the MLR has been identified in all vertebrate species tested from lampreys to humans. We now demonstrate that stimulation of the MLR not only activates locomotion, but can also stop it. This is achieved through a descending glutamatergic signal, most likely monosynaptic, from the MLR to the reticular formation that activates reticulospinal stop cells. Together, our findings have uncovered a neural mechanism for stopping locomotion and bring new insights into the function of the MLR.

Molecules present in an animal's environment can indicate the presence of predators, food, or sexual partners and consequently, induce migratory, reproductive, foraging, or escape behaviors. Three sensory systems, the olfactory, gustatory, and solitary chemosensory cell (SCC) systems detect chemical stimuli in vertebrates. While a great deal of research has focused on the olfactory and gustatory system over the years, it is only recently that significant attention has been devoted to the SCC system. The SCCs are microvillous cells that were first discovered on the skin of fish, and later in amphibians, reptiles, and mammals. Lampreys also possess SCCs that are particularly numerous on cutaneous papillae. However, little is known regarding their precise distribution, innervation, and function. Here, we show that sea lampreys (Petromyzon marinus L.) have cutaneous papillae located around the oral disk, nostril, gill pores, and on the dorsal fins and that SCCs are particularly numerous on these papillae. Tract-tracing experiments demonstrated that the oral and nasal papillae are innervated by the trigeminal nerve, the gill pore papillae are innervated by branchial nerves, and the dorsal fin papillae are innervated by spinal nerves. We also characterized the response profile of gill pore papillae to some chemicals and showed that trout-derived chemicals, amino acids, and a bile acid produced potent responses. Together with a companion study (Suntres et al., Journal of Comparative Neurology, this issue), our results provide new insights on the function and evolution of the SCC system in vertebrates.

Experience influences behavior, but little is known about how experience is encoded in the brain, and how changes in neural activity are implemented at a network level to improve performance. Here we investigate how differences in experience impact brain circuitry and behavior in larval zebrafish prey capture. We find that experience of live prey compared to inert food increases capture success by boosting capture initiation. In response to live prey, animals with and without prior experience of live prey show activity in visual areas (pretectum and optic tectum) and motor areas (cerebellum and hindbrain), with similar visual area retinotopic maps of prey position. However, prey-experienced animals more readily initiate capture in response to visual area activity and have greater visually-evoked activity in two forebrain areas: the telencephalon and habenula. Consequently, disruption of habenular neurons reduces capture performance in prey-experienced fish. Together, our results suggest that experience of prey strengthens prey-associated visual drive to the forebrain, and that this lowers the threshold for prey-associated visual activity to trigger activity in motor areas, thereby improving capture performance.

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.

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.

Organ development depends on the integration of coordinated long-range communication between cells. The cerebrospinal fluid composition and flow properties regulate several aspects of central nervous system development, including progenitor proliferation, neurogenesis, and migration [1-3]. One understudied component of the cerebrospinal fluid, described over a century ago in vertebrates, is the Reissner fiber. This extracellular thread forming early in development results from the assembly of the SCO-spondin protein in the third and fourth brain ventricles and central canal of the spinal cord [4]. Up to now, the function of the Reissner fiber has remained elusive, partly due to the lack of genetic invalidation models [4]. Here, by mutating the scospondin gene, we demonstrate that the Reissner fiber is critical for the morphogenesis of a straight posterior body axis. In zebrafish mutants where the Reissner fiber is lost, ciliogenesis and cerebrospinal fluid flow are intact but body axis morphogenesis is impaired. Our results also explain the frequently observed phenotype that mutant embryos with defective cilia exhibit defects in body axis curvature. Here, we reveal that these mutants systematically fail to assemble the Reissner fiber. We show that cilia promote the formation of the Reissner fiber and that the fiber is necessary for proper body axis morphogenesis. Our study sets the stage for future investigations of the mechanisms linking the Reissner fiber to the control of body axis curvature during vertebrate development.

Circulation of the cerebrospinal fluid (CSF) contributes to body axis formation and brain development. Here, we investigated the unexplained origins of the CSF flow bidirectionality in the central canal of the spinal cord of 30 hpf zebrafish embryos and its impact on development. Experiments combined with modeling and simulations demonstrate that the CSF flow is generated locally by caudally-polarized motile cilia along the ventral wall of the central canal. The closed geometry of the canal imposes the average flow rate to be null, explaining the reported bidirectionality. We also demonstrate that at this early stage, motile cilia ensure the proper formation of the central canal. Furthermore, we demonstrate that the bidirectional flow accelerates the transport of particles in the CSF via a coupled convective-diffusive transport process. Our study demonstrates that cilia activity combined with muscle contractions sustain the long-range transport of extracellular lipidic particles, enabling embryonic growth.

The choroid plexus (ChP) epithelium is a source of secreted signaling factors in cerebrospinal fluid (CSF) and a key barrier between blood and brain. Here, we develop imaging tools to interrogate these functions in adult lateral ventricle ChP in whole-mount explants and in awake mice. By imaging epithelial cells in intact ChP explants, we observed calcium activity and secretory events that increased in frequency following delivery of serotonergic agonists. Using chronic two-photon imaging in awake mice, we observed spontaneous subcellular calcium events as well as strong agonist-evoked calcium activation and cytoplasmic secretion into CSF. Three-dimensional imaging of motility and mobility of multiple types of ChP immune cells at baseline and following immune challenge or focal injury revealed a range of surveillance and defensive behaviors. Together, these tools should help illuminate the diverse functions of this understudied body-brain interface.

In the vertebrate spinal cord, cerebrospinal fluid-contacting neurons (CSF-cNs) are GABAergic neurons whose functions are only beginning to unfold. Recent evidence indicates that CSF-cNs detect local spinal bending and relay this mechanosensory feedback information to motor circuits, yet many CSF-cN targets remain unknown. Using optogenetics, patterned illumination, and in vivo electrophysiology, we show here that CSF-cNs provide somatic inhibition to fast motor neurons and excitatory sensory interneurons involved in the escape circuit. Ventral CSF-cNs respond to longitudinal spinal contractions and induce large inhibitory postsynaptic currents (IPSCs) sufficient to silence spiking of their targets. Upon repetitive stimulation, these IPSCs promptly depress, enabling the mechanosensory response to the first bend to be the most effective. When CSF-cNs are silenced, postural control is compromised, resulting in rollovers during escapes. Altogether, our data demonstrate how GABAergic sensory neurons provide powerful inhibitory feedback to the escape circuit to maintain balance during active locomotion.

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.

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

In lampreys, respiration consists of a fast and a slow rhythm. This study was aimed at characterizing both anatomically and physiologically the brainstem regions involved in generating the two rhythms. The fast rhythm generator has been located by us and others in the rostral hindbrain, rostro-lateral to the trigeminal motor nucleus. More recently, this was challenged by researchers reporting that the fast rhythm generator was located more rostrally and dorsomedially, in a region corresponding to the mesencephalic locomotor region. These contradictory observations made us re-examine the location of the fast rhythm generator using anatomical lesions and physiological recordings. We now confirm that the fast respiratory rhythm generator is in the rostro-lateral hindbrain as originally described. The slow rhythm generator has received less attention. Previous studies suggested that it was composed of bilateral, interconnected rhythm generating regions located in the caudal hindbrain, with ascending projections to the fast rhythm generator. We used anatomical and physiological approaches to locate neurons that could be part of this slow rhythm generator. Combinations of unilateral injections of anatomical tracers, one in the fast rhythm generator area and another in the lateral tegmentum of the caudal hindbrain, were performed to label candidate neurons on the non-injected side of the lateral tegmentum. We found a population of neurons extending from the facial to the caudal vagal motor nuclei, with no clear clustering in the cell distribution. We examined the effects of stimulating different portions of the labeled population on the respiratory activity. The rostro-caudal extent of the population was arbitrarily divided in three portions that were each stimulated electrically or chemically. Stimulation of either of the three sites triggered bursts of discharge characteristic of the slow rhythm, whereas inactivating any of them stopped the slow rhythm. Substance P injected locally in the lateral tegmentum accelerated the slow respiratory rhythm in a caudal hindbrain preparation. Our results show that the fast respiratory rhythm generator consists mostly of a population of neurons rostro-lateral to the trigeminal motor nucleus, whereas the slow rhythm generator is distributed in the lateral tegmentum of the caudal hindbrain.

There is a high demand for 3D multiphoton imaging in neuroscience and other fields but scanning in axial direction presents technical challenges. We developed a focusing technique based on a remote movable mirror that is conjugate to the specimen plane and translated by a voice coil motor. We constructed cost-effective z-scanning modules from off-the-shelf components that can be mounted onto standard multiphoton laser scanning microscopes to extend scan patterns from 2D to 3D. Systems were designed for large objectives and provide high resolution, high speed and a large z-scan range (>300 μm). We used these systems for 3D multiphoton calcium imaging in the adult zebrafish brain and measured odor-evoked activity patterns across >1500 neurons with single-neuron resolution and high signal-to-noise ratio.

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.

Defects in cerebrospinal fluid (CSF) flow may contribute to idiopathic scoliosis. However, the mechanisms underlying detection of CSF flow in the central canal of the spinal cord are unknown. Here we demonstrate that CSF flows bidirectionally along the antero-posterior axis in the central canal of zebrafish embryos. In the cfap298tm304 mutant, reduction of cilia motility slows transport posteriorly down the central canal and abolishes spontaneous activity of CSF-contacting neurons (CSF-cNs). Loss of the sensory Pkd2l1 channel nearly abolishes CSF-cN calcium activity and single channel opening. Recording from isolated CSF-cNs in vitro, we show that CSF-cNs are mechanosensory and require Pkd2l1 to respond to pressure. Additionally, adult pkd2l1 mutant zebrafish develop an exaggerated spine curvature, reminiscent of kyphosis in humans. These results indicate that CSF-cNs are mechanosensory cells whose Pkd2l1-driven spontaneous activity reflects CSF flow in vivo. Furthermore, Pkd2l1 in CSF-cNs contributes to maintenance of natural curvature of the spine.