Rohon-Beard Neurons Research Articles

Complete persistence of the primary somatosensory system in zebrafish

The somatosensory system detects peripheral stimuli that are translated into behaviors necessary for survival. Fishes and amphibians possess two somatosensory systems in the trunk: the primary somatosensory system, formed by the Rohon-Beard neurons, and the secondary somatosensory system, formed by the neural crest cell-derived neurons of the Dorsal Root Ganglia. Rohon-Beard neurons have been characterized as a transient population that mostly disappears during the first days of life and is functionally replaced by the Dorsal Root Ganglia. Here, I follow Rohon-Beard neurons in vivo and show that the entire repertoire remains present in zebrafish from 1-day post-fertilization until the juvenile stage, 15-days post-fertilization. These data indicate that zebrafish retain two complete somatosensory systems until at least a developmental stage when the animals display complex behavioral repertoires.

Rohon-beard neurons do not succumb to programmed cell death during zebrafish development

During neural development, sculpting of early formed circuits by cell death and synaptic pruning is necessary to generate a functional and efficient nervous system. This allows for the establishment of rudimentary circuits which necessitate early organism survival to later undergo subsequent refinement. These changes facilitate additional specificity to stimuli which can lead to increased behavioral complexity. In multiple species, Rohon-Beard neurons (RBs) are the earliest mechanosensory neurons specified and are critical in establishing a rudimentary motor response circuit. Sensory input from RBs gradually becomes redundant as dorsal root ganglion (DRG) neurons develop and integrate into motor circuits. Previous studies demonstrate that RBs undergo a dramatic wave of cell death concurrent with development of the DRG. However, contrary to these studies, we show that neurogenin1+ (ngn1) RBs do not undergo a widespread wave of programmed cell death during early zebrafish development and instead persist until at least 15 days post fertilization (dpf). Starting at 2 dpf, we also observed a dramatic medialization and shrinkage of ngn1+ RB somas along with a gradual downregulation of ngn1 in RBs. This alters a fundamental premise of early zebrafish neural development and opens new avenues to explore mechanisms of RB function, persistence, and circuit refinement.

Single-Cell Analysis of Rohon-Beard Neurons Implicates Fgf Signaling in Axon Maintenance and Cell Survival.

Peripheral sensory neurons are a critical part of the nervous system that transmit a multitude of sensory stimuli to the central nervous system. During larval and juvenile stages in zebrafish, this function is mediated by Rohon-Beard somatosensory neurons (RBs). RBs are optically accessible and amenable to experimental manipulation, making them a powerful system for mechanistic investigation of sensory neurons. Previous studies provided evidence that RBs fall into multiple subclasses; however, the number and molecular makeup of these potential RB subtypes have not been well defined. Using a single-cell RNA sequencing (scRNA-seq) approach, we demonstrate that larval RBs in zebrafish fall into three, largely nonoverlapping classes of neurons. We also show that RBs are molecularly distinct from trigeminal neurons in zebrafish. Cross-species transcriptional analysis indicates that one RB subclass is similar to a mammalian group of A-fiber sensory neurons. Another RB subclass is predicted to sense multiple modalities, including mechanical stimulation and chemical irritants. We leveraged our scRNA-seq data to determine that the fibroblast growth factor (Fgf) pathway is active in RBs. Pharmacological and genetic inhibition of this pathway led to defects in axon maintenance and RB cell death. Moreover, this can be phenocopied by treatment with dovitinib, an FDA-approved Fgf inhibitor with a common side effect of peripheral neuropathy. Importantly, dovitinib-mediated axon loss can be suppressed by loss of Sarm1, a positive regulator of neuronal cell death and axonal injury. This offers a molecular target for future clinical intervention to fight neurotoxic effects of this drug.

Unmyelinated sensory neurons use Neuregulin signals to promote myelination of interneurons in the CNS.

The signaling mechanisms neurons use to modulate myelination of circuits in the central nervous system (CNS) are only partly understood. Through analysis of isoform-specific neuregulin1 (nrg1) mutants in zebrafish, we demonstrate that nrg1 type II is an important regulator of myelination of two classes of spinal cord interneurons. Surprisingly, nrg1 type II expression is prominent in unmyelinated Rohon-Beard sensory neurons, whereas myelination of neighboring interneurons is reduced in nrg1 type II mutants. Cell-type-specific loss-of-function studies indicate that nrg1 type II is required in Rohon-Beard neurons to signal to other neurons, not oligodendrocytes, to modulate spinal cord myelination. Together, our data support a model in which unmyelinated neurons express Nrg1 type II proteins to regulate myelination of neighboring neurons, a mode of action that may coordinate the functions of unmyelinated and myelinated neurons in the CNS.

Endocannabinoid Receptor Expression in Early Zebrafish Development

The endocannabinoid system (ECS) is widely studied due to its interactions with cannabis and its role in modulating physiological responses. While most research has focused on the effects of cannabis on adult ECSs, recent studies have begun to investigate the role of the ECS in developing organisms. However, little is known about the spatial or temporal expression of these receptors during early development. This study combines reverse transcriptase-polymerase chain reaction (RT-PCR) with in situ hybridizations to compile a timeline of the developmental expression of six key cannabinoid receptors; cb1, cb2, trpv1, trpa1a, trpa1b, and gpr55 in zebrafish embryos, starting from as early as 6-h postfertilization (hpf) until 3 days pf (dpf). This time frame is roughly equivalent to 2–10 weeks in human embryonic development. All six genes were confirmed to be expressed within this time range and share similarities with human and rodent expression. Cb1 expression was first detected between 12 and 24 hpf in the retina and CNS, and its expression increased thereafter and was more evident in the olfactory bulb, tegmentum, hypothalamus, and gut. Cb2 expression was relatively high at the 6 and 24 hpf timepoints, as determined by RT-PCR but was undetectable at other times. Trpv1 was first detected at 1 dpf in the trigeminal (Tg) ganglia, Rohon-Beard neurons, and lateral line, and its expression increased in the first 3 dpf. Expression of trpa1a was first detected as late as 3 dpf in vagal (V) neurons, whereas trpa1b was first detected at 1 dpf associated with Tg, glossopharyngeal, and V ganglia. Expression of gpr55 was diffuse and widespread throughout the brain and head region but was undetectable elsewhere in the embryo. Thus, receptor expression was found to be enriched in the central nervous system and within sensory neurons. This work aims to serve as a foundation for further investigation on the role of cannabinoid and cannabinoid-interacting receptors in early embryonic development.

Single-cell RNA analysis identifies pre-migratory neural crest cells expressing markers of differentiated derivatives.

The neural crest is a migratory population of stem-like cells that contribute to multiple traits including the bones of the skull, peripheral nervous system, and pigment. How neural crest cells differentiate into diverse cell types is a fundamental question in the study of vertebrate biology. Here, we use single-cell RNA sequencing to characterize transcriptional changes associated with neural crest cell development in the zebrafish trunk during the early stages of migration. We show that neural crest cells are transcriptionally diverse and identify pre-migratory populations already expressing genes associated with differentiated derivatives, specifically in the xanthophore lineage. Further, we identify a population of Rohon-Beard neurons in the data. The data presented identify novel genetic markers for multiple trunk neural crest cell populations and Rohon-Beard neurons providing insight into previously uncharacterized genes critical for vertebrate development.

Microtubule organization of vertebrate sensory neurons in vivo

Dorsal root ganglion (DRG) neurons are the predominant cell type that innervates the vertebrate skin. They are typically described as pseudounipolar cells that have central and peripheral axons branching from a single root exiting the cell body. The peripheral axon travels within a nerve to the skin, where free sensory endings can emerge and branch into an arbor that receives and integrates information. In some immature vertebrates, DRG neurons are preceded by Rohon-Beard (RB) neurons. While the sensory endings of RB and DRG neurons function like dendrites, we use live imaging in zebrafish to show that they have axonal plus-end-out microtubule polarity at all stages of maturity. Moreover, we show both cell types have central and peripheral axons with plus-end-out polarity. Surprisingly, in DRG neurons these emerge separately from the cell body, and most cells never acquire the signature pseudounipolar morphology. Like another recently characterized cell type that has multiple plus-end-out neurites, ganglion cells in Nematostella, RB and DRG neurons maintain a somatic microtubule organizing center even when mature. In summary, we characterize key cellular and subcellular features of vertebrate sensory neurons as a foundation for understanding their function and maintenance.

Cover Image, Volume 529, Issue 7

The cover image is based on the Research Article Morphological and physiological properties of Rohon-Beard neurons along the zebrafish spinal cord by Melina E. Hale, Evdokia Menelaou, and Hilary Rose Katz, https://doi.org/10.1002/cne.25033.

Morphological and physiological properties of Rohon-Beard neurons along the zebrafish spinal cord.

Primary mechanosensory neurons play an important role in converting mechanical forces into the sense of touch. In zebrafish, Rohon-Beard (RB) neurons serve this role at embryonic and larval stages of development. Here we examine the morphology and physiology of RBs in larval zebrafish to better understand how mechanosensory stimuli are represented along the spinal cord. We report that the morphology of RB neurons differs along the rostrocaudal body axis. Rostral RB neurons arborize in the skin near the cell body whereas caudal cells arborize at a distance posterior to their cell body. Using a novel electrophysiological approach, we also found longitudinal differences in the mechanosensitivity and physiological properties of RB neurons. Rostral RB neurons respond to mechanical stimulations close to the soma and produce up to three spikes with increasing stimulus intensity, whereas caudal cells respond at more distal locations and can produce four or more spikes when the intensity of the mechanical stimulus increases. The mechanosensory properties of RB neurons are consistent with those of rapidly adapting mechanoreceptors and can signal the onset, offset and intensity of mechanical stimulation. This is the first report of the intensity encoding properties of RB neurons, where an increase in spike number and a decrease in spike latency are observed with increasing stimulation intensity. This study reveals an unappreciated complexity of the larval zebrafish mechanosensory system and demonstrates how differences in the morphological and physiological properties of RBs related to their rostrocaudal location can influence the signals that enter the spinal cord.

Dpysl2 (CRMP2) is required for the migration of facial branchiomotor neurons in the developing zebrafish embryo.

Dihydropyrimidinase-like family proteins (Dpysls) are relevant in several processes during nervous system development; among others, they are involved in axonal growth and cell migration. Dpysl2 (CRMP2) is the most studied member of this family; however, its role in vivo is still being investigated. Our previous studies in zebrafish showed the requirement of Dpysl2 for the proper positioning of caudal primary motor neurons and Rohon-Beard neurons in the spinal cord.In the present study, we show that Dpysl2 is necessary for the proper migration of facial branchiomotor neurons during early development in zebrafish. We generated a dpysl2 knock-out (KO) zebrafish mutant line and used different types of antisense morpholino oligonucleotides (AMO) to analyze the role of Dpysl2 in this process. Both dpysl2 KO mutants and morphants exhibited abnormalities in the migration of these neurons from rhombomers (r) 4 and 5 to 6 and 7. The facial branchiomotor neurons that were expected to be at r6 were still located at r4 and r5 hours after the migration process should have been completed. In addition, mutant phenotypes were rescued by injecting dpysl2 mRNA into the KO embryos. These results indicate that Dpysl2 is involved in the proper migration of facial branchiomotor neurons in developing zebrafish embryos.

Sensory neurons in the spinal cord of zebrafish and their local connectivity

Sensory neurons of the spinal cord provide critical information on the internal and external environment. We review recent work that has made significant strides in elucidating the structure and function of two such neurons, excitatory Rohon-Beard neurons and inhibitory cerebrospinal fluid contacting neurons. These studies provide newfound appreciation for the diversity within each of these cell types and for the high complexity of sensory inputs to motor circuits in the spinal cord. We highlight research that links sensory neurons and spinal circuits to behaviors, leveraging the zebrafish as a powerhouse system to interrogate whole, intact circuitry in a behaving animal.

Spatiotemporal expression of the cocaine- and amphetamine-regulated transcript-like (cart-like) gene during zebrafish embryogenesis

The cocaine- and amphetamine-regulated transcript (CART) genes are involved in the neural regulation of energy homeostasis; however, their developmental expressions and functions are not fully understood in vertebrates. We have identified a novel zebrafish cart-like gene that encodes a protein of 105 amino acids possessing sequence similarity to zebrafish and mammalian CART proteins. RT-PCR analysis revealed that the cart-like transcripts were maternally supplied and gradually decreased during the cleavage, blastula and gastrula stages; then, transcripts subsequently reaccumulated at the segmentation, pharyngula and hatching stages. Based on a whole-mount in situ hybridization analysis using an antisense cart-like RNA probe, we found that the cart-like transcript was predominantly expressed in both the Rohon-Beard neurons and trigeminal ganglia, suggesting the involvement of the cart-like gene in zebrafish neural development.

Muscarinic modulation of the Xenopus laevis tadpole spinal mechanosensory pathway

Muscarinic acetylcholine receptors (mAChRs) mediate effects of acetylcholine (ACh) in many systems, including those involved in spinal functions like locomotion. In Xenopus laevis tadpoles at two days old, a model vertebrate for motor control research, we investigated the role of mAChRs in the skin mechanosensory pathway. We found that mAChR activation by carbachol did not affect the sensory Rohon-Beard neuron properties. However, carbachol could hyperpolarise sensory interneurons and decrease their voltage responses to outward currents. Carbachol could increase the threshold for the mechanosensory pathway to start swimming, preventing the initiation of swimming at higher concentrations altogether. Recording from the sensory interneurons in carbachol showed that their spiking after skin stimulation was depressed. However, the general muscarinic antagonist atropine did not have a clear effect on the swimming threshold or the modulation of sensory interneuron membrane conductance. Our results suggest the skin mechanosensory pathway may be subject to muscarinic modulation in this simple vertebrate system.

Mechanosensory neurons control the timing of spinal microcircuit selection during locomotion.

Despite numerous physiological studies about reflexes in the spinal cord, the contribution of mechanosensory feedback to active locomotion and the nature of underlying spinal circuits remains elusive. Here we investigate how mechanosensory feedback shapes active locomotion in a genetic model organism exhibiting simple locomotion-the zebrafish larva. We show that mechanosensory feedback enhances the recruitment of motor pools during active locomotion. Furthermore, we demonstrate that inputs from mechanosensory neurons increase locomotor speed by prolonging fast swimming at the expense of slow swimming during stereotyped acoustic escape responses. This effect could be mediated by distinct mechanosensory neurons. In the spinal cord, we show that connections compatible with monosynaptic inputs from mechanosensory Rohon-Beard neurons onto ipsilateral V2a interneurons selectively recruited at high speed can contribute to the observed enhancement of speed. Altogether, our study reveals the basic principles and a circuit diagram enabling speed modulation by mechanosensory feedback in the vertebrate spinal cord.

The modulation of two motor behaviors by persistent sodium currents in Xenopus laevis tadpoles.

Persistent sodium currents (INaP) are common in neuronal circuitries and have been implicated in several diseases, such as amyotrophic lateral sclerosis (ALS) and epilepsy. However, the role of INaP in the regulation of specific behaviors is still poorly understood. In this study we have characterized INaP and investigated its role in the swimming and struggling behavior of Xenopus tadpoles. INaP was identified in three groups of neurons, namely, sensory Rohon-Beard neurons (RB neurons), descending interneurons (dINs), and non-dINs (neurons rhythmically active in swimming). All groups of neurons expressed INaP, but the currents differed in decay time constants, amplitudes, and the membrane potential at which INaP peaked. Low concentrations (1 µM) of the INaP blocker riluzole blocked INaP ~30% and decreased the excitability of the three neuron groups without affecting spike amplitudes or cellular input resistances. Riluzole reduced the number of rebound spikes in dINs and depressed repetitive firing in RB neurons and non-dINs. At the behavior level, riluzole at 1 µM shortened fictive swimming episodes. It also reduced the number of action potentials neurons fired on each struggling cycle. The results show that INaP may play important modulatory roles in motor behaviors.NEW & NOTEWORTHY We have characterized persistent sodium currents in three groups of spinal neurons and their role in shaping spiking activity in the Xenopus tadpole. We then attempted to evaluate the role of persistent sodium currents in regulating tadpole swimming and struggling motor outputs by using low concentrations of the persistent sodium current antagonist riluzole.

From neuron to behavior: Sensory‐motor coordination of zebrafish turning behavior

Recent development of optogenetics brought non-invasive neural activation in living organisms. Transparent zebrafish larva is one of the suitable animal models for this technique, which enables us to investigate neural circuits for behaviors based on a whole individual nervous system. In this article we review our recent finding that suggests sensory-motor coordination in larval zebrafish escape behavior. When water vibration stimulates mechanosensory Rohon-Beard (RB) neurons, intra-spinal reflex circuit launches contralateral trunk muscle contraction that makes rapid body curvature for turning. In addition, positional information of the stimulus is conveyed to supra-spinal circuits, and then regulates the curvature strength for appropriate escape pathway from the threat. Sensory-motor coordination is a fundamental feature to adapt behaviors to environment, and zebrafish larvae would be an excellent model for elucidating its neural backbones.

Position- and quantity-dependent responses in zebrafish turning behavior.

Neural reflexes are stereotypical automatic responses often modulated by both intrinsic and environmental factors. We report herein that zebrafish larval C-shaped turning is modulated by the stimulated position of Rohon-Beard (RB) neurons. Targeted stimulation of more anterior RB neurons produces larger trunk flexion, which anticipates adult escape behavior by coordinated turning toward the appropriate direction. We also demonstrated that turning laterality varies with the numbers of stimulated neurons. Multi-cell stimulation of RB neurons elicits contralateral turning, as seen in the touch response to physical contact, while minimum input from single-cell stimulation induces ipsilateral turning, a phenomenon not previously reported. This ipsilateral response, but not the contralateral one, is impaired by transecting the ascending neural tract known as the dorsolateral fascicule (DLF), indicating that two, distinct neural circuits trigger these two responses. Our results suggest that RB neurons transmit the position and quantity of sensory information, which are then processed separately to modulate behavioral strength and to select turning laterality.

Developmental suppression of schizophrenia-associated miR-137 alters sensorimotor function in zebrafish.

The neurodevelopmentally regulated microRNA miR-137 was strongly implicated as risk locus for schizophrenia in the most recent genome wide association study coordinated by the Psychiatric Genome Consortium (PGC). This molecule is highly conserved in vertebrates enabling the investigation of its function in the developing zebrafish. We utilized this model system to achieve overexpression and suppression of miR-137, both transiently and stably through transgenesis. While miR-137 overexpression was not associated with an observable specific phenotype, downregulation by antisense morpholino and/or transgenic expression of miR-sponge RNA induced significant impairment of both embryonic and larval touch-sensitivity without compromising overall anatomical development. We observed miR-137 expression and activity in sensory neurons including Rohon–Beard neurons and dorsal root ganglia, two neuronal cell types that confer touch-sensitivity in normal zebrafish, suggesting a role of these cell types in the observed phenotype. The lack of obvious anatomical or histological pathology in these cells, however, suggested that subtle axonal network defects or a change in synaptic function and neural connectivity might be responsible for the behavioral phenotype rather than a change in the cellular morphology or neuroanatomy.

The influence of magnetic field on the spatial orientation in zebrafish Danio rerio (Hamilton) and roach Rutilus rutilus (L.)

The influence of magnetic field on the spatial orientation in zebrafish Danio rerio (Hamilton) and roach Rutilus rutilus (L.)

Specification of sensory neurons occurs through diverse developmental programs functioning in the brain and spinal cord.

Vertebrates possess two populations of sensory neurons located within the central nervous system: Rohon-Beard (RB) and mesencephalic trigeminal nucleus (MTN) neurons. RB neurons are transient spinal cord neurons whilst MTN neurons are the proprioceptive cells that innervate the jaw muscles. It has been suggested that MTN and RB neurons share similarities and may have a common developmental program, but it is unclear how similar or different their development is. We have dissected RB and MTN neuronal specification in zebrafish. We find that RB and MTN neurons express a core set of genes indicative of sensory neurons, but find these are also expressed by adjacent diencephalic neurons. Unlike RB neurons, our evidence argues against a role for the neural crest during MTN development. We additionally find that neurogenin1 function is dispensable for MTN differentiation, unlike RB cells and all other sensory neurons. Finally, we demonstrate that, although Notch signalling is involved in RB development, it is not involved in the generation of MTN cells. Our work reveals fundamental differences between the development of MTN and RB neurons and suggests that these populations are non-homologous and thus have distinct developmental and, probably, evolutionary origins.