Spontaneous regeneration of cholecystokinergic reticulospinal axons after a complete spinal cord injury in sea lampreys.

In contrast to humans, lampreys spontaneously recover their swimming capacity after a complete spinal cord injury (SCI). This recovery process involves the regeneration of descending axons. Spontaneous axon regeneration in lampreys has been mainly studied in giant descending neurons. However, the regeneration of neurochemically distinct descending neuronal populations with small-caliber axons, as those found in mammals, has been less studied. Cholecystokinin (CCK) is a regulatory neuropeptide found in the brain and spinal cord that modulates several processes such as satiety, or locomotion. CCK shows high evolutionary conservation and is present in all vertebrate species. Work in lampreys has shown that all CCKergic spinal cord axons originate in a single neuronal population located in the caudal rhombencephalon. Here, we investigate the spontaneous regeneration of CCKergic descending axons in larval lampreys following a complete SCI. Using anti-CCK-8 immunofluorescence, confocal microscopy and lightning adaptive deconvolution, we demonstrate the partial regeneration of CCKergic axons (81% of the number of axonal profiles seen in controls) 10 weeks after the injury. Our data also revealed a preference for regeneration of CCKergic axons in lateral spinal cord regions. Regenerated CCKergic axons exhibit colocalization with synaptic vesicle marker SV2, indicative of functional synaptic connections. We also extracted swimming dynamics in injured animals by using DeepLabCut. Interestingly, the degree of CCKergic reinnervation correlated with improved swimming performance in injured animals, suggesting a potential role in locomotor recovery. These findings open avenues for further exploration into the role of specific neuropeptidergic systems in post-SCI spinal locomotor networks.

An in vivo drug screen in zebrafish reveals that cyclooxygenase 2-derived prostaglandin D2 promotes spinal cord neurogenesis.

The study of neurogenesis is essential to understanding fundamental developmental processes and for the development of cell replacement therapies for central nervous system disorders. Here, we designed an in vivo drug screening protocol in developing zebrafish to find new molecules and signalling pathways regulating neurogenesis in the ventral spinal cord. This unbiased drug screen revealed that 4 cyclooxygenase (COX) inhibitors reduced the generation of serotonergic interneurons in the developing spinal cord. These results fitted very nicely with available single-cell RNAseq data revealing that floor plate cells show differential expression of 1 of the 2 COX2 zebrafish genes (ptgs2a). Indeed, several selective COX2 inhibitors and two different morpholinos against ptgs2a reduced the number of serotonergic neurons in the ventral spinal cord and led to locomotor deficits. Single-cell RNAseq data and different pharmacological manipulations further revealed that COX2-floor plate-derived prostaglandin D2 promotes neurogenesis in the developing spinal cord by promoting mitotic activity in progenitor cells. Rescue experiments using a phosphodiesterase-4 inhibitor suggest that intracellular changes in cAMP levels underlie the effects of COX inhibitors on neurogenesis and locomotion. Our study provides compelling in vivo evidence showing that prostaglandin signalling promotes neurogenesis in the ventral spinal cord.

A lamprey neural cell type atlas illuminates the origins of the vertebrate brain.

The vertebrate brain emerged more than ~500 million years ago in common evolutionary ancestors. To systematically trace its cellular and molecular origins, we established a spatially resolved cell type atlas of the entire brain of the sea lamprey-a jawless species whose phylogenetic position affords the reconstruction of ancestral vertebrate traits-based on extensive single-cell RNA-seq and in situ sequencing data. Comparisons of this atlas to neural data from the mouse and other jawed vertebrates unveiled various shared features that enabled the reconstruction of cell types, tissue structures and gene expression programs of the ancestral vertebrate brain. However, our analyses also revealed key tissues and cell types that arose later in evolution. For example, the ancestral brain was probably devoid of cerebellar cell types and oligodendrocytes (myelinating cells); our data suggest that the latter emerged from astrocyte-like evolutionary precursors in the jawed vertebrate lineage. Altogether, our work illuminates the cellular and molecular architecture of the ancestral vertebrate brain and provides a foundation for exploring its diversification during evolution.

Organization of the corticotropin-releasing hormone and corticotropin-releasing hormone-binding protein systems in the central nervous system of the sea lamprey Petromyzon marinus.

The expression of the corticotropin-releasing hormone (PmCRH) and the CRH-binding protein (PmCRHBP) mRNAs was studied by in situ hybridization in the brain of prolarvae, larvae, and adults of the sea lamprey Petromyzon marinus. We also generated an antibody against the PmCRH mature peptide to study the distribution of PmCRH-immunoreactive cells and fibers. PmCRH immunohistochemistry was combined with antityrosine hydroxylase immunohistochemistry, PmCRHBP in situ hybridization, or neurobiotin transport from the spinal cord. The most numerous PmCRH-expressing cells were observed in the magnocellular preoptic nucleus-paraventricular nucleus and in the superior and medial rhombencephalic reticular formation. PmCRH expression was more extended in adults than in larvae, and some cell populations were mainly (olfactory bulb) or only (striatum, ventral hypothalamus, prethalamus) observed in adults. The preopto-paraventricular fibers form conspicuous tracts coursing toward the neurohypophysis, but many immunoreactive fibers were also observed coursing in many other brain regions. Brain descending fibers in the spinal cord mainly come from cells located in the isthmus and in the medial rhombencephalic reticular nucleus. The distribution of PmCRHBP-expressing neurons was different from that of PmCRH cells, with cells mainly present in the septum, striatum, preoptic region, tuberal hypothalamus, pretectum, pineal complex, isthmus, reticular formation, and spinal cord. Again, expression in adults was more extended than in larvae. PmCRH- and PmCRHBP-expressing cells are different, excluding colocalization of these substances in the same neuron. Present findings reveal a complex CRH/CRHBP system in the brain of the oldest extant vertebrate group, the agnathans, which shows similarities but important divergences with that of mammals.

Full regeneration of descending corticotropin-releasing hormone axons after a complete spinal cord injury in lampreys.

Sea lampreys are a vertebrate model of interest for the study of spontaneous axon regeneration after spinal cord injury (SCI). Axon regeneration research in lampreys has focused on the study of giant descending neurons, but less so on neurochemically-distinct descending neuronal populations with small caliber axons. Corticotropin-releasing hormone (CRH) is a neuropeptide that regulates the stress response or locomotion. CRH is also a neuropeptide of interest in the SCI context because descending CRHergic projections from the Barrington's nucleus control micturition behavior in mammals. Recent work from our group revealed that in sea lampreys the CRHergic innervation of the spinal cord is only of descending origin. Thus, the lack of intrinsic CRH spinal cord neurons provides the opportunity to analyze the regeneration of this descending system by using immunofluorescence methods. Here, we used an antibody against the sea lamprey mature CRH peptide, confocal microscopy, lightning adaptive deconvolution, and ImageJ to analyze the regenerative capacity of the descending CRH-immunoreactive (-ir) axons of larval sea lampreys after a complete SCI at the level of the fifth gill. At 10 weeks post-lesion, when behavioral analyses showed that injured animals had recovered normal appearing locomotion, our results revealed a full recovery of the number of CRH-ir profiles (axons) at the level of the sixth gill. Thus, the CRH descending axons of lampreys fully regenerate after a complete SCI. Our study provides a new model to study spontaneous and successful axonal regeneration in a specific neuronal type with small caliber axons by using simple immunohistochemical methods.

A nop56 Zebrafish Loss-of-Function Model Exhibits a Severe Neurodegenerative Phenotype.

NOP56 belongs to a C/D box small nucleolar ribonucleoprotein complex that is in charge of cleavage and modification of precursor ribosomal RNAs and assembly of the 60S ribosomal subunit. An intronic expansion in NOP56 gene causes Spinocerebellar Ataxia type 36, a typical late-onset autosomal dominant ataxia. Although vertebrate animal models were created for the intronic expansion, none was studied for the loss of function of NOP56. We studied a zebrafish loss-of-function model of the nop56 gene which shows 70% homology with the human gene. We observed a severe neurodegenerative phenotype in nop56 mutants, characterized mainly by absence of cerebellum, reduced numbers of spinal cord neurons, high levels of apoptosis in the central nervous system (CNS) and impaired movement, resulting in death before 7 days post-fertilization. Gene expression of genes related to C/D box complex, balance and CNS development was impaired in nop56 mutants. In our study, we characterized the first NOP56 loss-of-function vertebrate model, which is important to further understand the role of NOP56 in CNS function and development.

Activity-regulated growth of motoneurons at the neuromuscular junction is mediated by NADPH oxidases.

Neurons respond to changes in the levels of activity they experience in a variety of ways, including structural changes at pre- and postsynaptic terminals. An essential plasticity signal required for such activity-regulated structural adjustments are reactive oxygen species (ROS). To identify sources of activity-regulated ROS required for structural plasticity in vivo we used the Drosophila larval neuromuscular junction as a highly tractable experimental model system. For adjustments of presynaptic motor terminals, we found a requirement for both NADPH oxidases, Nox and dual oxidase (Duox), that are encoded in the Drosophila genome. This contrasts with the postsynaptic dendrites from which Nox is excluded. NADPH oxidases generate ROS to the extracellular space. Here, we show that two aquaporins, Bib and Drip, are necessary ROS conduits in the presynaptic motoneuron for activity regulated, NADPH oxidase dependent changes in presynaptic motoneuron terminal growth. Our data further suggest that different aspects of neuronal activity-regulated structural changes might be regulated by different ROS sources: changes in bouton number require both NADPH oxidases, while activity-regulated changes in the number of active zones might be modulated by other sources of ROS. Overall, our results show NADPH oxidases as important enzymes for mediating activity-regulated plasticity adjustments in neurons.

Expression of Kisspeptin 1 in the Brain of the Adult Sea Lamprey Petromyzon marinus.

Kisspeptin peptides play major roles in the regulation of reproduction and puberty onset in mammals. While most mammals only have one kisspeptin gene, other jawed vertebrates present two or three genes. Recent data also revealed the presence of two genes in lampreys (jawless vertebrates). However, apart from gene sequence data, there is almost no information on the kisspeptinergic system of lampreys. Here, we report phylogenetic and cluster-based analyses showing that the duplication of the ancestral kisspeptin gene occurred before the separation of jawless and jawed vertebrates. We also studied the expression of the kisspeptin transcripts in the brain of post-metamorphic juveniles and upstream migrating adult sea lampreys. Our in situ hybridization results revealed expression of kisspeptin 1 in hypothalamic neurons, which indicates that the hypothalamic expression of kisspeptins is an ancestral character in vertebrates. We also observed the presence of kisspeptin 1 expressing neurons in the paratubercular (posterior tubercle) nucleus of the diencephalon. This is the first description of the presence of kisspeptin 1 expressing neurons in this brain region in any vertebrate. We did not detect expression of kisspeptin 2 in the juvenile or adult sea lamprey brain with in situ hybridization. Our data provides an anatomical basis to study the role of kisspeptin 1 in the hypothalamic-pituitary system of lampreys and the contribution of diencephalic kisspeptinergic neurons to different circuits of the lamprey brain.

Expression of Urocortin 3 mRNA in the Central Nervous System of the Sea Lamprey Petromyzon marinus.

In this study, we analyzed the organization of urocortin 3 (Ucn3)-expressing neuronal populations in the brain of the adult sea lamprey by means of in situ hybridization. We also studied the brain of larval sea lampreys to establish whether this prosocial neuropeptide is expressed differentially in two widely different phases of the sea lamprey life cycle. In adult sea lampreys, Ucn3 transcript expression was observed in neurons of the striatum, prethalamus, nucleus of the medial longitudinal fascicle, torus semicircularis, isthmic reticular formation, interpeduncular nucleus, posterior rhombencephalic reticular formation and nucleus of the solitary tract. Interestingly, in larval sea lampreys, only three regions showed Ucn3 expression, namely the prethalamus, the nucleus of the medial longitudinal fascicle and the posterior rhombencephalic reticular formation. A comparison with distributions of Ucn3 in other vertebrates revealed poor conservation of Ucn3 expression during vertebrate evolution. The large qualitative differences in Ucn3 expression observed between larval and adult phases suggest that the maturation of neuroregulatory circuits in the striatum, torus semicircularis and hindbrain chemosensory systems is closely related to profound life-style changes occurring after the transformation from larval to adult life.

Zebrafish Models of Autosomal Recessive Ataxias.

Autosomal recessive ataxias are much less well studied than autosomal dominant ataxias and there are no clearly defined systems to classify them. Autosomal recessive ataxias, which are characterized by neuronal and multisystemic features, have significant overlapping symptoms with other complex multisystemic recessive disorders. The generation of animal models of neurodegenerative disorders increases our knowledge of their cellular and molecular mechanisms and helps in the search for new therapies. Among animal models, the zebrafish, which shares 70% of its genome with humans, offer the advantages of being small in size and demonstrating rapid development, making them optimal for high throughput drug and genetic screening. Furthermore, embryo and larval transparency allows to visualize cellular processes and central nervous system development in vivo. In this review, we discuss the contributions of zebrafish models to the study of autosomal recessive ataxias characteristic phenotypes, behavior, and gene function, in addition to commenting on possible treatments found in these models. Most of the zebrafish models generated to date recapitulate the main features of recessive ataxias.

Zebrafish Models of Autosomal Dominant Ataxias.

Hereditary dominant ataxias are a heterogeneous group of neurodegenerative conditions causing cerebellar dysfunction and characterized by progressive motor incoordination. Despite many efforts put into the study of these diseases, there are no effective treatments yet. Zebrafish models are widely used to characterize neuronal disorders due to its conserved vertebrate genetics that easily support genetic edition and their optic transparency that allows observing the intact CNS and its connections. In addition, its small size and external fertilization help to develop high throughput assays of candidate drugs. Here, we discuss the contributions of zebrafish models to the study of dominant ataxias defining phenotypes, genetic function, behavior and possible treatments. In addition, we review the zebrafish models created for X-linked repeat expansion diseases X-fragile/fragile-X tremor ataxia. Most of the models reviewed here presented neuronal damage and locomotor deficits. However, there is a generalized lack of zebrafish adult heterozygous models and there are no knock-in zebrafish models available for these diseases. The models created for dominant ataxias helped to elucidate gene function and mechanisms that cause neuronal damage. In the future, the application of new genetic edition techniques would help to develop more accurate zebrafish models of dominant ataxias.

Developmentally-programmed cellular senescence is conserved and widespread in zebrafish.

Cellular senescence is considered a stress response imposing a stable cell cycle arrest to restrict the growth of damaged cells. More recently however, cellular senescence was identified during mouse embryo development at particular structures during specific periods of time. This programmed cell senescence has been proposed to serve developmental and morphogenetic functions and to potentially represent an evolutionary origin of senescence. Cellular senescence has also been described to take place during bird (chick and quail) and amphibian (xenopus and axoltl) development. Fish however, have been described to show a very narrow and restricted pattern of developmental cell senescence. Here we carried out a detailed characterization of senescence during zebrafish development and found it to be conserved and widespread. Apart from yolk and cloaca, previously described structures, we also identified senescence in the developing central nervous system, intestine, liver, pronephric ducts, and crystalline. Interestingly, senescence at these developing structures disappeared upon treatment with senolytic compound ABT-263, supporting their senescent identity and opening the possibility of studying the contribution of this process to development. In summary, our findings extend the description of developmentally-programmed cell senescence to lower vertebrates contributing to the notion of the relevance of this process for embryo development.

Inhibition of Gamma-Secretase Promotes Axon Regeneration After a Complete Spinal Cord Injury.

In a recent study, we showed that GABA and baclofen (a GABAB receptor agonist) inhibit caspase activation and promote axon regeneration in descending neurons of the sea lamprey brainstem after a complete spinal cord injury (Romaus-Sanjurjo et al., 2018a). Now, we repeated these treatments and performed 2 independent Illumina RNA-Sequencing studies in the brainstems of control and GABA or baclofen treated animals. GABA treated larval sea lampreys with their controls were analyzed 29 days after a complete spinal cord injury and baclofen treated larvae with their controls 9 days after the injury. One of the most significantly downregulated genes after both treatments was a HES gene (HESB). HES proteins are transcription factors that are key mediators of the Notch signaling pathway and gamma-secretase activity is crucial for the activation of this pathway. So, based on the RNA-Seq results we subsequently treated spinal cord injured larval sea lampreys with a novel gamma-secretase inhibitor (PF-3804014). This treatment also reduced the expression of HESB in the brainstem and significantly enhanced the regeneration of individually identifiable descending neurons after a complete spinal cord injury. Our results show that gamma-secretase could be a novel target to promote axon regeneration after nervous system injuries.

Differential expression of five prosomatostatin genes in the central nervous system of the catshark Scyliorhinus canicula.

Five prosomatostatin genes (PSST1, PSST2, PSST3, PSST5, and PSST6) have been recently identified in elasmobranchs (Tostivint et al., General and Comparative Endocrinology, 2019, 279, 139-147). In order to gain insight into the contribution of each somatostatin to specific nervous systems circuits and behaviors in this important jawed vertebrate group, we studied the distribution of neurons expressing PSST mRNAs in the central nervous system (CNS) of Scyliorhinus canicula using in situ hybridization. Additionally, we combined in situ hybridization with tyrosine hydroxylase (TH) immunochemistry for better characterization of PSST1 and PSST6 expressing populations. We observed differential expression of PSST1 and PSST6, which are the most widely expressed PSST transcripts, in cell populations of many CNS regions, including the pallium, subpallium, hypothalamus, diencephalon, optic tectum, midbrain tegmentum, and rhombencephalon. Interestingly, numerous small pallial neurons express PSST1 and PSST6, although in different populations judging from the colocalization of TH immunoreactivity and PSST6 expression but not with PSST1. We observed expression of PSST1 in cerebrospinal fluid-contacting (CSF-c) neurons of the hypothalamic paraventricular organ and the central canal of the spinal cord. Unlike PSST1 and PSST6, PSST2, and PSST3 are only expressed in cells of the hypothalamus and in some hindbrain lateral reticular neurons, and PSST5 in cells of the region of the entopeduncular nucleus. Comparative data of brain expression of PSST genes indicate that the somatostatinergic system of sharks is the most complex reported in any fish.

Taurine Promotes Axonal Regeneration after a Complete Spinal Cord Injury in Lampreys.

Taurine is one of the most abundant free amino acids in the brain. It is well known that taurine protects the brain from further damage after a traumatic event. However, only a few ex vivo studies have looked at the possible role of taurine in the regulation of axon regeneration after injury. Here, we aimed to reveal the possible role for taurine in the modulation of axonal regeneration following a complete spinal cord injury (SCI) using lampreys as an animal model. The brainstem of lampreys contains several individually identifiable descending neurons that differ greatly in their capacity for axonal regeneration after SCI. This offers a convenient model to promote or inhibit axonal regrowth in the same in vivo preparation. First, we carried out high performance liquid chromatography experiments to measure taurine levels in the spinal cord following SCI. Our results revealed a statistically significant increase in taurine levels 4 weeks post-lesion, which suggested that taurine might have a positive effect on axonal regrowth. Based on these results, we decided to apply an acute taurine treatment at the site of injury to study its effect on axon regeneration. Results from these experiments show that an acute taurine treatment enhances axonal regeneration following SCI in lampreys. This offers a novel way to try to promote axon regeneration after nervous system injuries in mammalian models.

Cell senescence contributes to tissue regeneration in zebrafish.

Cellular senescence is a stress response that limits the proliferation of damaged cells by establishing a permanent cell cycle arrest. Different stimuli can trigger senescence but excessive production or impaired clearance of these cells can lead to their accumulation during aging with deleterious effects. Despite this potential negative side of cell senescence, its physiological role as a pro-regenerative and morphogenetic force has emerged recently after the identification of programmed cell senescence during embryogenesis and during wound healing and limb regeneration. Here, we explored the conservation of tissue injury-induced senescence in a model of complex regeneration, the zebrafish. Fin amputation in adult fish led to the appearance of senescent cells at the site of damage, and their removal impaired tissue regeneration. Despite many conceptual similarities, this tissue repair response is different from developmental senescence. Our results lend support to the notion that cell senescence is a positive response promoting tissue repair and homeostasis.

Galanin in an Agnathan: Precursor Identification and Localisation of Expression in the Brain of the Sea Lamprey Petromyzon marinus.

Galanin is a neuropeptide that is widely expressed in the mammalian brain, where it regulates many physiological processes, including feeding and nociception. Galanin has been characterized extensively in jawed vertebrates (gnathostomes), but little is known about the galanin system in the most ancient extant vertebrate class, the jawless vertebrates or agnathans. Here, we identified and cloned a cDNA encoding the sea lamprey (Petromyzon marinus) galanin precursor (PmGalP). Sequence analysis revealed that PmGalP gives rise to two neuropeptides that are similar to gnathostome galanins and galanin message-associated peptides. Using mRNA in situ hybridization, the distribution of PmGalP-expressing neurons was mapped in the brain of larval and adult sea lampreys. This revealed PmGalP-expressing neurons in the septum, preoptic region, striatum, hypothalamus, prethalamus, and displaced cells in lateral areas of the telencephalon and diencephalon. In adults, the laterally migrated PmGalP-expressing neurons are observed in an area that extends from the ventral pallium to the lateral hypothalamus and prethalamus. The striatal and laterally migrated PmGalP-expressing cells of the telencephalon were not observed in larvae. Comparison with studies on jawed vertebrates reveals that the presence of septal and hypothalamic galanin-expressing neuronal populations is highly conserved in vertebrates. However, compared to mammals, there is a more restricted pattern of expression of the galanin transcript in the brain of lampreys. This work provides important new information on the early evolution of the galanin system in vertebrates and provides a genetic and neuroanatomical basis for functional analyses of the galanin system in lampreys.

Serotonin inhibits axonal regeneration of identifiable descending neurons after a complete spinal cord injury in lampreys.

Classical neurotransmitters are mainly known for their roles as neuromodulators, but they also play important roles in the control of developmental and regenerative processes. Here, we used the lamprey model of spinal cord injury to study the effect of serotonin in axon regeneration at the level of individually identifiable descending neurons. Pharmacological and genetic manipulations after a complete spinal cord injury showed that endogenous serotonin inhibits axonal regeneration in identifiable descending neurons through the activation of serotonin 1A receptors and a subsequent decrease in cyclic adenosine monophosphate (cAMP) levels. RNA sequencing revealed that changes in the expression of genes that control axonal guidance could be a key factor determining the serotonin effects during regeneration. This study provides new targets of interest for research in non-regenerating mammalian models of traumatic central nervous system injuries and extends the known roles of serotonin signalling during neuronal regeneration. This article has an associated First Person interview with the first author of the paper.