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

Use of vivo-morpholinos for gene knockdown in the postnatal shark retina

Work in the catshark Scyliorhinus canicula has shown that the evolutionary origin of postnatal neurogenesis in vertebrates is earlier than previously thought. Thus, the catshark can serve as a model of interest to understand postnatal neurogenic processes and their evolution in vertebrates. One of the best characterized neurogenic niches of the catshark CNS is found in the peripheral region of the retina. Unfortunately, the lack of genetic tools in sharks limits the possibilities to deepen in the study of genes involved in the neurogenic process. Here, we report a method for gene knockdown in the juvenile catshark retina based on the use of Vivo-Morpholinos. To establish the method, we designed Vivo-Morpholinos against the proliferation marker PCNA. We first evaluated the possible toxicity of 3 different intraocular administration regimes. After this optimization step, we show that a single intraocular injection of the PCNA Vivo-Morpholino decreases the expression of PCNA in the peripheral retina, which leads to reduced mitotic activity in this region. This method will help in deciphering the role of other genes potentially involved in postnatal neurogenesis in this animal modelGrant PID 2020-115121 GB-I00 funded by MCIN/AEI/10.13039/501100011033 to A. Barreiro-Iglesias. Grant ED 431C 2021/18 funded by Xunta de Galicia to E. CandalS

Data on the recovery of glycinergic neurons afterspinal cord injury in lampreys

We used immunohistochemical methods to quantify changes inthe number of glycine-immunoreactive neurons of the dorsome-dial, lateral and cerebrospinalfluid contacting cell populations ofthe spinal cord of larval sea lampreys after a complete spinal cordinjury. The data presented here are quantifications of the numberof glycine-immunoreactive neurons located in the rostral andcaudal stumps of the spinal cord and the corresponding statisticalanalyses. These data show that, glycine immunoreactivity is lost inglycinergic neurons immediately after injury and that the numberof glycine-immunoreactive neurons is recovered in the followingtwo weeks. These data are useful for researchers investigatingdeterminants that underlie the spontaneous recovery of locomo-tion following spinal injuries in regenerating animal models, andfor analysing the role of glycinergic neurons in spinal cord repairafter an injuryGrant sponsors: FEDER/Ministerio de Ciencia, Innovación y Universidades - Agencia Estatal deInvestigación (Grant number: BFU-2017-87079-P)S

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

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.Grant PID2020-115121GB-I00 funded by MCIN/AEI/10.13039/501100011033 to A. Barreiro-Iglesias and L. Sánchez. Grant ED 431C 2021/18 funded by Xunta de Galicia. The European Molecular Biology Organization (EMBO) granted a long-term EMBO fellowship to D. Sobrido-Cameán (ALTF 62-2021).S

Cloning of the GABAB Receptor Subunits B1 and B2 and their Expression in the Central Nervous System of the Adult Sea Lamprey

In vertebrates, γ-aminobutyric acid (GABA) is the main inhibitory transmitter in the central nervous system (CNS) acting through ionotropic (GABAA) and metabotropic (GABAB) receptors. The GABAB receptor produces a slow inhibition since it activates second messenger systems through the binding and activation of guanine nucleotide-binding proteins [G-protein-coupled receptors (GPCRs)]. Lampreys are a key reference to understand molecular evolution in vertebrates. The importance of the GABAB receptor for the modulation of the circuits controlling locomotion and other behaviors has been shown in pharmacological/physiological studies in lampreys. However, there is no data about the sequence of the GABAB subunits or their expression in the CNS of lampreys. Our aim was to identify the sea lamprey GABAB1 and GABAB2 transcripts and study their expression in the CNS of adults. We cloned two partial sequences corresponding to the GABAB1 and GABAB2 cDNAs of the sea lamprey as confirmed by sequence analysis and comparison with known GABAB sequences of other vertebrates. In phylogenetic analyses, the sea lamprey GABAB sequences clustered together with GABABs sequences of vertebrates and emerged as an outgroup to all gnathostome sequences. We observed a broad and overlapping expression of both transcripts in the entire CNS. Expression was mainly observed in neuronal somas of the periventricular regions including the identified reticulospinal cells. No expression was observed in identifiable fibers. Comparison of our results with those reported in other vertebrates indicates that a broad and overlapping expression of the GABAB subunits in the CNS is a conserved character shared by agnathans and gnathostomesS

Decline in Constitutive Proliferative Activity in the Zebrafish Retina with Ageing

Supplementary Materials The following are available online at https://www.mdpi.com/article/10.3390/ijms222111715/s1. Table S1: Studies demonstrating the presence of proliferating cells in the juvenile/adult retina of different teleost species. File S1: Mean ± S.E.M. data and data on statistical multiple comparisons related to graphs shown in Figure 2; File S2: Mean ± S.E.M. data and data on statistical multiple comparisons related to graphs shown in Figure 3.It is largely assumed that the teleost retina shows continuous and active proliferative and neurogenic activity throughout life. However, when delving into the teleost literature, one finds that assumptions about a highly active and continuous proliferation in the adult retina are based on studies in which proliferation was not quantified in a comparative way at the different life stages or was mainly studied in juveniles/young adults. Here, we performed a systematic and comparative study of the constitutive proliferative activity of the retina from early developing (2 days post-fertilisation) to aged (up to 3–4 years post-fertilisation) zebrafish. The mitotic activity and cell cycle progression were analysed by using immunofluorescence against pH3 and PCNA, respectively. We observed a decline in the cell proliferation in the retina with ageing despite the occurrence of a wave of secondary proliferation during sexual maturation. During this wave of secondary proliferation, the distribution of proliferating and mitotic cells changes from the inner to the outer nuclear layer in the central retina. Importantly, in aged zebrafish, there is a virtual disappearance of mitotic activity. Our results showing a decline in the proliferative activity of the zebrafish retina with ageing are of crucial importance since it is generally assumed that the fish retina has continuous proliferative activity throughout life.This research was funded by Ministerio de Economía Industria y Competitividad (to E.C.), grant number BFU-2017-89861-P; Ministerio de Ciencia e Innovación-Agencia Estatal de Investigación (to A.B.-I.), grant number PID2020-115121GB-I00; Xunta de Galicia (to E.C.), grant number ED431C 2021/18; Xunta de Galicia (to I.H.-N.), grant number ED 481 A2018 216. “The APC was funded by the Xunta de Galicia”. Grants were partially financed by the European Social Fund.S

Loss of active neurogenesis in the adult shark retina

Neurogenesis is the process by which progenitor cells generate new neurons. As development progresses neurogenesis becomes restricted to discrete neurogenic niches, where it persists during postnatal life. The retina of teleost fishes is thought to proliferate and produce new cells throughout life. Whether this capacity may be an ancestral characteristic of gnathostome vertebrates is completely unknown. Cartilaginous fishes occupy a key phylogenetic position to infer ancestral states fixed prior to the gnathostome radiation. Previous work from our group revealed that the juvenile retina of the catshark Scyliorhinus canicula, a cartilaginous fish, shows active proliferation and neurogenesis. Here, we compared the morphology and proliferative status of the retina in catshark juveniles and adults. Histological and immunohistochemical analyses revealed an important reduction in the size of the peripheral retina (where progenitor cells are mainly located), a decrease in the thickness of the inner nuclear layer (INL), an increase in the thickness of the inner plexiform layer and a decrease in the cell density in the INL and in the ganglion cell layer in adults. Contrary to what has been reported in teleost fish, mitotic activity in the catshark retina was virtually absent after sexual maturation. Based on these results, we carried out RNA-Sequencing (RNA-Seq) analyses comparing the retinal transcriptome of juveniles and adults, which revealed a statistically significant decrease in the expression of many genes involved in cell proliferation and neurogenesis in adult catsharks. Our RNA-Seq data provides an excellent resource to identify new signaling pathways controlling neurogenesis in the vertebrate retinaFunded by the Ministerio de Economía Industria y Competitividad (to EC; grant number BFU-2017-89861-P) and Xunta de Galicia Predoctoral Fellowship (to IH-N; grant number ED 481 A 2018 216). Both grants were partially financed by the European Social FundS

Full Anatomical Recovery of the Dopaminergic System after a Complete Spinal Cord Injury in Lampreys

Following a spinal injury, lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by the regeneration of descending axons from the brain and the production of new neurons in the spinal cord. Here, we aimed to analyse the changes in the dopaminergic system of the sea lamprey after a complete spinal transection by studying the changes in dopaminergic cell numbers and dopaminergic innervation in the spinal cord. Changes in the expression of the D2 receptor were also studied. We report the full anatomical regeneration of the dopaminergic system after an initial decrease in the number of dopaminergic cells and fibres. Numbers of dopaminergic cells were recovered rostrally and caudally to the site of injury. Quantification of dopaminergic profiles revealed the full recovery of the dopaminergic innervation of the spinal cord rostral and caudal to the site of injury. Interestingly, no changes in the expression of the D2 receptor were observed at time points in which a reduced dopaminergic innervation of the spinal cord was observed. Our observations reveal that in lampreys a spinal cord injury is followed by the full anatomical recovery of the dopaminergic system.This work was funded by the Spanish Ministry of Science and Innovation, Grant no. BFU2010–17174, to María Celina Rodicio. Sonia Gómez-Fernández and Antón Barreiro-Iglesias were supported by predoctoral and postdoctoral grants, respectively, from the Xunta de Galicia (Galicia, Spain)S

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 injuriesGrant sponsors: FEDER/Ministerio de Ciencia, Innovación y Universidades – Agencia Estatal de Investigación (Grant number: BFU-2017-87079-P) and the Xunta de Galicia (Grant number: ED431C 2018/28). DR was supported by the BBSRC Institute Strategic Programme Grants to the Roslin Institute (BB/P013732/1, BB/P013740/1, and BB/P013759/1)S