Telencephalic eversion in embryos and early larvae of four teleost species.

The telencephalon of ray-finned fishes undergoes eversion, which is very different to the evagination that occurs in most other vertebrates. Ventricle morphogenesis is key to build an everted telencephalon. Thus, here we use the apical marker zona occludens 1 to understand ventricle morphology, extension of the tela choroidea and the eversion process during early telencephalon development of four teleost species: giant danio (Devario aequipinnatus), blind cavefish (Astyanax mexicanus), medaka (Oryzias latipes), and paradise fish (Macroposus opercularis). In addition, by using immunohistochemistry against tubulin and calcium-binding proteins, we analyze the general morphology of the telencephalon, showing changes in the location and extension of the olfactory bulb and other telencephalic regions from 2 to 5 days of development. We also analyze the impact of abnormal eye and telencephalon morphogenesis on eversion, showing that cyclops mutants do undergo eversion despite very dramatic abnormal eye morphology. We discuss how the formation of the telencephalic ventricle in teleost fish, with its characteristic shape, is a crucial event during eversion.

Effects of Zinc Oxide Nanoparticle Exposure on Human Glial Cells and Zebrafish Embryos.

Zinc oxide nanoparticles (ZnO NPs) are among the most widely used nanomaterials. They have multiple applications in cosmetics, textiles, paints, electronics and, recently, also in biomedicine. This extensive use of ZnO NPs notably increases the probability that both humans and wildlife are subjected to undesirable effects. Despite being among the most studied NPs from a toxicological point of view, much remains unknown about their ecotoxicological effects or how they may affect specific cell types, such as cells of the central nervous system. The main objective of this work was to investigate the effects of ZnO NPs on human glial cells and zebrafish embryo development and to explore the role of the released Zn2+ ions in these effects. The effects on cell viability on human A172 glial cells were assessed with an MTT assay and morphological analysis. The potential acute and developmental toxicity was assessed employing zebrafish (Danio rerio) embryos. To determine the role of Zn2+ ions in the in vitro and in vivo observed effects, we measured their release from ZnO NPs with flame atomic absorption spectrometry. Then, cells and zebrafish embryos were treated with a water-soluble salt (zinc sulfate) at concentrations that equal the number of Zn2+ ions released by the tested concentrations of ZnO NPs. Exposure to ZnO NPs induced morphological alterations and a significant decrease in cell viability depending on the concentration and duration of treatment, even after removing the overestimation due to NP interference. Although there were no signs of acute toxicity in zebrafish embryos, a decrease in hatching was detected after exposure to the highest ZnO NP concentrations tested. The ability of ZnO NPs to release Zn2+ ions into the medium in a concentration-dependent manner was confirmed. Zn2+ ions did not seem entirely responsible for the effects observed in the glial cells, but they were likely responsible for the decrease in zebrafish hatching rate. The results obtained in this work contribute to the knowledge of the toxicological potential of ZnO NPs.

Severe neurometabolic phenotype in npc1 -/- zebrafish with a C-terminal mutation.

Niemann Pick disease type C (NPC) is an autosomal recessive neurodegenerative lysosomal disorder characterized by an accumulation of lipids in different organs. Clinical manifestations can start at any age and include hepatosplenomegaly, intellectual impairment, and cerebellar ataxia. NPC1 is the most common causal gene, with over 460 different mutations with heterogeneous pathological consequences. We generated a zebrafish NPC1 model by CRISPR/Cas9 carrying a homozygous mutation in exon 22, which encodes the end of the cysteine-rich luminal loop of the protein. This is the first zebrafish model with a mutation in this gene region, which is frequently involved in the human disease. We observed a high lethality in npc1 mutants, with all larvae dying before reaching the adult stage. Npc1 mutant larvae were smaller than wild type (wt) and their motor function was impaired. We observed vacuolar aggregations positive to cholesterol and sphingomyelin staining in the liver, intestine, renal tubules and cerebral gray matter of mutant larvae. RNAseq comparison between npc1 mutants and controls showed 284 differentially expressed genes, including genes with functions in neurodevelopment, lipid exchange and metabolism, muscle contraction, cytoskeleton, angiogenesis, and hematopoiesis. Lipidomic analysis revealed significant reduction of cholesteryl esters and increase of sphingomyelin in the mutants. Compared to previously available zebrafish models, our model seems to recapitulate better the early onset forms of the NPC disease. Thus, this new model of NPC will allow future research in the cellular and molecular causes/consequences of the disease and on the search for new treatments.

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.

Neurogranin-like immunoreactivity in the zebrafish brain during development.

Neurogranin (Nrgn) is a neural protein that is enriched in the cerebral cortex and is involved in synaptic plasticity via its interaction with calmodulin. Recently we reported its expression in the brain of the adult zebrafish (Alba-González et al. J Comp Neurol 530:1569-1587, 2022). In this study we analyze the development of Nrgn-like immunoreactivity (Nrgn-like-ir) in the brain and sensory structures of zebrafish embryos and larvae, using whole mounts and sections. First Nrgn-like positive neurons appeared by 2 day post-fertilization (dpf) in restricted areas of the brain, mostly in the pallium, epiphysis and hindbrain. Nrgn-like populations increased noticeably by 3 dpf, reaching an adult-like pattern in 6 dpf. Most Nrgn-like positive neurons were observed in the olfactory organ, retina (most ganglion cells, some amacrine and bipolar cells), pallium, lateral hypothalamus, thalamus, optic tectum, torus semicircularis, octavolateralis area, and viscerosensory column. Immunoreactivity was also observed in axonal tracts originating in Nrgn-like neuronal populations, namely, the projection of Nrgn-like immunopositive primary olfactory fibers to olfactory glomeruli, that of Nrgn-like positive pallial cells to the hypothalamus, the Nrgn-like-ir optic nerve to the pretectum and optic tectum, the Nrgn-like immunolabeled lateral hypothalamus to the contralateral region via the horizontal commissure, the octavolateralis area to the midbrain via the lateral lemniscus, and the viscerosensory column to the dorsal isthmus via the secondary gustatory tract. The late expression of Nrgn in zebrafish neurons is probably related to functional maturation of higher brain centers, as reported in the mammalian telencephalon. The analysis of Nrgn expression in the zebrafish brain suggests that it may be a useful marker for specific neuronal circuitries.

A Structural Atlas of the Developing Zebrafish Telencephalon Based on Spatially-Restricted Transgene Expression.

Zebrafish telencephalon acquires an everted morphology by a two-step process that occurs from 1 to 5 days post-fertilization (dpf). Little is known about how this process affects the positioning of discrete telencephalic cell populations, hindering our understanding of how eversion impacts telencephalic structural organization. In this study, we characterize the neurochemistry, cycle state and morphology of an EGFP positive (+) cell population in the telencephalon of Et(gata2:EGFP)bi105 transgenic fish during eversion and up to 20dpf. We map the transgene insertion to the early-growth-response-gene-3 (egr3) locus and show that EGFP expression recapitulates endogenous egr3 expression throughout much of the pallial telencephalon. Using the gata2:EGFP bi105 transgene, in combination with other well-characterized transgenes and structural markers, we track the development of various cell populations in the zebrafish telencephalon as it undergoes the morphological changes underlying eversion. These datasets were registered to reference brains to form an atlas of telencephalic development at key stages of the eversion process (1dpf, 2dpf, and 5dpf) and compared to expression in adulthood. Finally, we registered gata2:EGFPbi105 expression to the Zebrafish Brain Browser 6dpf reference brain (ZBB, see Marquart et al., 2015, 2017; Tabor et al., 2019), to allow comparison of this expression pattern with anatomical data already in ZBB.

Distribution of neurogranin-like immunoreactivity in the brain and sensory organs of the adult zebrafish.

We studied the expression of neurogranin in the brain and some sensory organs (barbel taste buds, olfactory organs, and retina) of adult zebrafish. Database analysis shows zebrafish has two paralog neurogranin genes (nrgna and nrgnb) that translate into three peptides with a conserved IQ domain, as in mammals. Western blots of zebrafish brain extracts using an anti-neurogranin antiserum revealed three separate bands, confirming the presence of three neurogranin peptides. Immunohistochemistry shows neurogranin-like expression in the brain and sensory organs (taste buds, neuromasts and olfactory epithelium), not being able to discern its three different peptides. In the retina, the most conspicuous positive cells were bipolar neurons. In the brain, immunopositive neurons were observed in all major regions (pallium, subpallium, preoptic area, hypothalamus, diencephalon, mesencephalon and rhombencephalon, including the cerebellum), a more extended distribution than in mammals. Interestingly, dendrites, cell bodies and axon terminals of some neurons were immunopositive, thus zebrafish neurogranins may play presynaptic and postsynaptic roles. Most positive neurons were found in primary sensory centers (viscerosensory column and medial octavolateral nucleus) and integrative centers (pallium, subpallium, optic tectum and cerebellum), which have complex synaptic circuitry. However, we also observed expression in areas not related to sensory or integrative functions, such as in cerebrospinal fluid-contacting cells associated with the hypothalamic recesses, which exhibited high neurogranin-like immunoreactivity. Together, these results reveal important differences with the patterns reported in mammals, suggesting divergent evolution from the common ancestor.

The organization of the zebrafish pallium from a hodological perspective.

We studied the connections (connectome) of the adult zebrafish pallium using carbocyanine dye tracing and ancillary anatomical methods. The everted zebrafish pallium (dorsal telencephalic area, D) is composed of several major zones (medial, lateral, dorsal, central, anterior, and posterior) distinguishable by their topography, cytoarchitecture, immunohistochemistry, and genoarchitecture. Our comprehensive study reveals poor interconnectivity between these pallial areas, especially between medial (Dm), lateral/dorsal (Dl, Dd), and posterior (Dp) regions. This suggests that the zebrafish pallium has dedicated modules for different neural processes. Pallial connections with extrapallial regions also show compartmental organization. Major extratelencephalic afferents come from preglomerular nuclei (to Dl, Dd, and Dm), posterior tuberal nucleus (to Dm), and lateral recess nucleus (to Dl). The subpallial (ventral, V) zones dorsal Vv, Vd, and Vs, considered homologues of the striatum, amygdala, and pallidum, are mainly afferent to Dl/Dd and Dp. Regarding the efferent pathways, they also appear characteristic of each pallial region. Rostral Dm projects to the dorsal entopeduncular nucleus. Dp is interconnected with the olfactory bulbs. The central region (Dc) defined here receives mainly projections from Dl-Dd and projects toward the pretectum and optic tectum, connections, which help to delimiting Dc. The connectome of the adult pallium revealed here complements extant studies on the neuroanatomical organization of the brain, and may be useful for neurogenetic studies performed during early stages of development. The connectome of the zebrafish pallium was also compared with the pallial connections reported in other teleosts, a large group showing high pallial diversity.

Half-Sandwich Ru(p-cymene) Compounds with Diphosphanes: In Vitro and In Vivo Evaluation As Potential Anticancer Metallodrugs.

Ruthenium(II) complexes are currently considered attractive alternatives to the widely used platinum-based drugs. We present herein the synthesis and characterization of half-sandwich ruthenium compounds formulated as [Ru(p-cymene)(L)Cl][CF3SO3] (L = 1,1-bis(methylenediphenylphosphano)ethylene, 1; L = 1,1-bis(diphenylphosphano)ethylene, 2), which were characterized by elemental analysis, mass spectrometry, 1H and 31P{1H} NMR, UV-vis and IR spectroscopy, conductivity measurements and cyclic voltammetry. The molecular structures for both complexes were determined by single-crystal X-ray diffraction. Their cytotoxic activity was evaluated using the MTT assay against human tumor cells, namely ovarian (A2780) and breast (MCF7 and MDA-MB-231). Both complexes were active against breast adenocarcinoma cells, with complex 1 exhibiting a quite remarkable cytotoxicity in the submicromolar range. Interestingly, at concentrations equivalent to the IC50 values in the MCF7 cancer cells, complexes 1 and 2 presented lower cytotoxicity in normal human primary fibroblasts. The antiproliferative effects of 1 and 2 in MCF7 cells might be associated with the induction of reactive oxygen species (ROS), leading to a combined cell death mechanism via apoptosis and autophagy. Despite the fact that in vitro a partial intercalation between complexes and DNA was observed, no MCF7 cell cycle delay or arrest was observed, indicating that DNA might not be a direct target. Complexes 1 and 2 both exhibited a moderate to strong interaction with human serum albumin, suggesting that protein targets may be involved in their mode of action. Their acute toxicity was evaluated in the zebrafish model. Complex 1 (the most toxic of the two) exhibited a lethal toxicity LC50 value about 1 order of magnitude higher than any IC50 concentrations found for the cancer cell models used, highlighting its therapeutic relevance as a drug candidate in cancer chemotherapy.

Anatomy and Connectivity of the Torus Longitudinalis of the Adult Zebrafish.

This study describes the cytoarchitecture of the torus longitudinalis (TL) in adult zebrafish by using light and electron microscopy, as well as its main connections as revealed by DiI tract tracing. In addition, by using high resolution confocal imaging followed by digital tracing, we describe the morphology of tectal pyramidal cells (type I cells) that are GFP positive in the transgenic line Tg(1.4dlx5a-dlx6a:GFP)ot1. The TL consists of numerous small and medium-sized neurons located in a longitudinal eminence attached to the medial optic tectum. A small proportion of these neurons are GABAergic. The neuropil shows three types of synaptic terminals and numerous dendrites. Tracing experiments revealed that the main efference of the TL is formed of parallel-like fibers that course within the marginal layer of the optic tectum. A toral projection to the thalamic nucleus rostrolateralis is also observed. Afferents to the TL come from visual and cerebellum-related nuclei in the pretectum, namely the central, intercalated and the paracommissural pretectal nuclei, as well as from the subvalvular nucleus in the isthmus. Additional afferents to the TL may come from the cerebellum but their origins could not be confirmed. The tectal afferent projection to the TL originates from cells similar to the type X cells described in other cyprinids. Tectal pyramidal neurons show round or piriform cell bodies, with spiny apical dendritic trees in the marginal layer. This anatomical study provides a basis for future functional and developmental studies focused on this cerebellum-like circuit in zebrafish.

Comparison of zebrafish and mice knockouts for Megalencephalic Leukoencephalopathy proteins indicates that GlialCAM/MLC1 forms a functional unit.

BACKGROUND: Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a rare type of leukodystrophy characterized by astrocyte and myelin vacuolization, epilepsy and early-onset macrocephaly. MLC is caused by mutations in MLC1 or GLIALCAM, coding for two membrane proteins with an unknown function that form a complex specifically expressed in astrocytes at cell-cell junctions. Recent studies in Mlc1-/- or Glialcam-/- mice and mlc1-/- zebrafish have shown that MLC1 regulates glial surface levels of GlialCAM in vivo and that GlialCAM is also required for MLC1 expression and localization at cell-cell junctions. METHODS: We have generated and analysed glialcama-/- zebrafish. We also generated zebrafish glialcama-/- mlc1-/- and mice double KO for both genes and performed magnetic resonance imaging, histological studies and biochemical analyses. RESULTS: glialcama-/- shows megalencephaly and increased fluid accumulation. In both zebrafish and mice, this phenotype is not aggravated by additional elimination of mlc1. Unlike mice, mlc1 protein expression and localization are unaltered in glialcama-/- zebrafish, possibly because there is an up-regulation of mlc1 mRNA. In line with these results, MLC1 overexpressed in Glialcam-/- mouse primary astrocytes is located at cell-cell junctions. CONCLUSIONS: This work indicates that the two proteins involved in the pathogenesis of MLC, GlialCAM and MLC1, form a functional unit, and thus, that loss-of-function mutations in these genes cause leukodystrophy through a common pathway.

Pretectal neurons control hunting behaviour.

For many species, hunting is an innate behaviour that is crucial for survival, yet the circuits that control predatory action sequences are poorly understood. We used larval zebrafish to identify a population of pretectal neurons that control hunting. By combining calcium imaging with a virtual hunting assay, we identified a discrete pretectal region that is selectively active when animals initiate hunting. Targeted genetic labelling allowed us to examine the function and morphology of individual cells and identify two classes of pretectal neuron that project to ipsilateral optic tectum or the contralateral tegmentum. Optogenetic stimulation of single neurons of either class was able to induce sustained hunting sequences, in the absence of prey. Furthermore, laser ablation of these neurons impaired prey-catching and prevented induction of hunting by optogenetic stimulation of the anterior-ventral tectum. We propose that this specific population of pretectal neurons functions as a command system to induce predatory behaviour.

RuII( p-cymene) Compounds as Effective and Selective Anticancer Candidates with No Toxicity in Vivo.

Ruthenium(II) complexes are currently considered a viable alternative to the widely used platinum complexes as efficient anticancer agents. We herein present the synthesis and characterization of half-sandwich ruthenium compounds with the general formula [Ru( p-cymene)(L-N,N)Cl][CF3SO3] (L = 3,6-di-2-pyridyl-1,2,4,5-tetrazine (1) 6,7-dimethyl-2,3-bis(pyridin-2-yl)quinoxaline (2)), which have been synthesized by substitution reactions from the precursor dimer [Ru( p-cymene)(Cl)(µ-Cl)]2 and were characterized by elemental analysis, mass spectrometry, 1H NMR, UV-vis, and IR spectroscopy, conductivity measurements, and cyclic voltammetry. The molecular structure for complex 2 was determined by single-crystal X-ray diffraction. The cytotoxic activity of these compounds was evaluated against human tumor cells, namely ovarian carcinoma A2780 and breast MCF7 and MDAMB231 adenocarcinoma cells, and against normal primary fibroblasts. Whereas the cytotoxic activity of 1 is moderate, IC50 values found for 2 are among the lowest previously reported for Ru( p-cymene) complexes. Both compounds present no cytotoxic effect in normal human primary fibroblasts when they are used at the IC50 concentration in A2780 and MCF7 cancer cells. Their antiproliferative capacity is associated with a combined mechanism of apoptosis and autophagy. A strong interaction with DNA was observed for both with a binding constant value of the same magnitude as that of the classical intercalator [Ru(phen)2(dppz)]2+. Both complexes bind to human serum albumin with moderate to strong affinity, with conditional binding constants (log Kb) of 4.88 for complex 2 and 5.18 for complex 1 in 2% DMSO/10 mM Hepes pH7.0 medium. The acute toxicity was evaluated in zebrafish embryo model using the fish embryo acute toxicity test (FET). Remarkably, our results show that compounds 1 and 2 are not toxic/lethal even at extremely high concentrations. The novel compounds reported herein are highly relevant antitumor metallodrug candidates, given their in vitro cytotoxicity toward cancer cells and the lack of in vivo toxicity.

Neural connections of the pretectum in zebrafish (Danio rerio).

The pretectum is a complex region of the caudal diencephalon which in adult zebrafish comprises both retinorecipient (parvocellular superficial, central, intercalated, paracommissural, and periventricular) and non-retinorecipient (magnocellular superficial, posterior, and accessory) pretectal nuclei distributed from periventricular to superficial regions. We conducted a comprehensive study of the connections of pretectal nuclei by using neuronal tracing with fluorescent carbocyanine dyes. This study reveals specialization of efferent connections of the various pretectal nuclei, with nuclei projecting to the optic tectum (paracommissural, central, and periventricular pretectal nuclei), the torus longitudinalis and the cerebellar corpus (paracommissural, central, and intercalated pretectal nuclei), the lateral hypothalamus (magnocellular superficial, posterior, and central pretectal nuclei), and the tegmental regions (accessory and superficial pretectal nuclei). With regard to major central afferents to the pretectum, we observed projections from the telencephalon to the paracommissural and central pretectal nuclei, from the optic tectum to the paracommissural, central, accessory and parvocellular superficial pretectal nuclei, from the cerebellum to the paracommissural and periventricular pretectal nuclei and from the nucleus isthmi to the parvocellular superficial and accessory pretectal nuclei. The parvocellular superficial pretectal nucleus sends conspicuous projections to the contralateral magnocellular superficial pretectal nucleus. The composite figure of results reveals large differences in connections of neighbor pretectal nuclei, indicating high degree of nuclear specialization. Our results will have important bearings in functional studies that analyze the relationship between specific circuits and behaviors in zebrafish. Comparison with results available in other species also reveals differences in the organization and connections of the pretectum in vertebrates.

Gustatory and general visceral centers and their connections in the brain of adult zebrafish: a carbocyanine dye tract-tracing study.

The central connections of the gustatory/general visceral system of the adult zebrafish (Danio rerio) were examined by means of carbocyanine dye tracing. Main primary gustatory centers (facial and vagal lobes) received sensory projections from the facial and vagal nerves, respectively. The vagal nerve also projects to the commissural nucleus of Cajal, a general visceral sensory center. These primary centers mainly project on a prominent secondary gustatory and general visceral nucleus (SGN/V) located in the isthmic region. Secondary projections on the SGN/V were topographically organized, those of the facial lobe mainly ending medially to those of the vagal lobe, and those from the commissural nucleus ventrolaterally. Descending facial lobe projections to the medial funicular nucleus were also noted. Ascending fibers originating from the SGN/V mainly projected to the posterior thalamic nucleus and the lateral hypothalamus (lateral torus, lateral recess nucleus, hypothalamic inferior lobe diffuse nucleus) and an intermediate cell- and fiber-rich region termed here the tertiary gustatory nucleus proper, but not to a nucleus formerly considered as the zebrafish tertiary gustatory nucleus. The posterior thalamic nucleus, tertiary gustatory nucleus proper, and nucleus of the lateral recess gave rise to descending projections to the SGN/V and the vagal lobe. The connectivity between diencephalic gustatory centers and the telencephalon was also investigated. The present results showed that the gustatory connections of the adult zebrafish are rather similar to those reported in other cyprinids, excepting the tertiary gustatory nucleus. Similarities between the gustatory systems of zebrafish and other fishes are also discussed. J. Comp. Neurol. 525:333-362, 2017. © 2016 Wiley Periodicals, Inc.

Afferent Connectivity of the Zebrafish Habenulae.

The habenulae are bilateral nuclei located in the dorsal diencephalon that are conserved across vertebrates. Here we describe the main afferents to the habenulae in larval and adult zebrafish. We observe afferents from the subpallium, nucleus rostrolateralis, posterior tuberculum, posterior hypothalamic lobe, median raphe; we also see asymmetric afferents from olfactory bulb to the right habenula, and from the parapineal to the left habenula. In addition, we find afferents from a ventrolateral telencephalic nucleus that neurochemical and hodological data identify as the ventral entopeduncular nucleus (vENT), confirming and extending observations of Amo et al. (2014). Fate map and marker studies suggest that vENT originates from the diencephalic prethalamic eminence and extends into the lateral telencephalon from 48 to 120 hour post-fertilization (hpf). No afferents to the habenula were observed from the dorsal entopeduncular nucleus (dENT). Consequently, we confirm that the vENT (and not the dENT) should be considered as the entopeduncular nucleus "proper" in zebrafish. Furthermore, comparison with data in other vertebrates suggests that the vENT is a conserved basal ganglia nucleus, being homologous to the entopeduncular nucleus of mammals (internal segment of the globus pallidus of primates) by both embryonic origin and projections, as previously suggested by Amo et al. (2014).

Megalencephalic leukoencephalopathy with subcortical cysts protein 1 regulates glial surface localization of GLIALCAM from fish to humans.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by myelin vacuolization and caused by mutations in MLC1 or GLIALCAM. Patients with recessive mutations in either MLC1 or GLIALCAM show the same clinical phenotype. It has been shown that GLIALCAM is necessary for the correct targeting of MLC1 to the membrane at cell junctions, but its own localization was independent of MLC1 in vitro. However, recent studies in Mlc1(-/-) mice have shown that GlialCAM is mislocalized in glial cells. In order to investigate whether the relationship between Mlc1 and GlialCAM is species-specific, we first identified MLC-related genes in zebrafish and generated an mlc1(-/-) zebrafish. We have characterized mlc1(-/-) zebrafish both functionally and histologically and compared the phenotype with that of the Mlc1(-/-) mice. In mlc1(-/-) zebrafish, as in Mlc1(-/-) mice, Glialcam is mislocalized. Re-examination of a brain biopsy from an MLC patient indicates that GLIALCAM is also mislocalized in Bergmann glia in the cerebellum. In vitro, impaired localization of GlialCAM was observed in astrocyte cultures from Mlc1(-/-) mouse only in the presence of elevated potassium levels, which mimics neuronal activity. In summary, here we demonstrate an evolutionary conserved role for MLC1 in regulating glial surface levels of GLIALCAM, and this interrelationship explains why patients with mutations in either gene (MLC1 or GLIALCAM) share the same clinical phenotype.

Immunohistochemical distribution of calretinin and calbindin (D-28k) in the brain of the cladistian Polypterus senegalus.

Polypteriform fishes are believed to be basal to other living ray-finned bony fishes, and they may be useful for providing information of the neural organization that existed in the brain of the earliest ray-finned fishes. The calcium-binding proteins calretinin (CR) and calbindin-D28k (CB) have been widely used to characterize neuronal populations in vertebrate brains. Here, the distribution of the immunoreactivity against CR and CB was investigated in the olfactory organ and brain of Polypterus senegalus and compared to the distribution of these molecules in other ray-finned fishes. In general, CB-immunoreactive (ir) neurons were less abundant than CR-ir cells. CR immunohistochemistry revealed segregation of CR-ir olfactory receptor neurons in the olfactory mucosa and their bulbar projections. Our results confirmed important differences between pallial regions in terms of CR immunoreactivity of cell populations and afferent fibers. In the habenula, these calcium-binding proteins revealed right-left asymmetry of habenular subpopulations and segregation of their interpeduncular projections. CR immunohistochemistry distinguished among some thalamic, pretectal, and posterior tubercle-derived populations. Abundant CR-ir populations were observed in the midbrain, including the tectum. CR immunoreactivity was also useful for characterizing a putative secondary gustatory/visceral nucleus in the isthmus, and for distinguishing territories in the primary viscerosensory column and octavolateral region. Comparison of the data obtained within a segmental neuromeric context indicates that some CB-ir and CR-ir populations in polypteriform fishes are shared with other ray-finned fishes, but other positive structures appear to have evolved following the separation between polypterids and other ray-finned fishes.