Pretectal neurons control hunting behaviour

[Abstract] 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.Royal Society; 101195/Z/13/

The organization of the zebrafish pallium from a hodological perspective

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG[Abstract] 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

Neurogranin-Like Immunoreactivity in the Zebrafish Brain During Development

[Abstract] 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.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Alba-González A. is recipient of a Predoctoral Fellowship from Xunta de Galicia (Grant number ED481A-2019/003)Xunta de Galicia; ED481A-2019/00

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

[Abstract] 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.Xunta de Galicia; ED481A-2019/00

Morphogenesis underlying the development of the everted teleost telencephalon

Background: Although the mechanisms underlying brain patterning and regionalization are very much conserved, the morphology of different brain regions is extraordinarily variable across vertebrate phylogeny. This is especially manifest in the telencephalon, where the most dramatic variation is seen between ray-finned fish, which have an everted telencephalon, and all other vertebrates, which have an evaginated telencephalon. The mechanisms that generate these distinct morphologies are not well understood. Results: Here we study the morphogenesis of the zebrafish telencephalon from 12 hours post fertilization (hpf) to 5 days post fertilization (dpf) by analyzing forebrain ventricle formation, evolving patterns of gene and transgene expression, neuronal organization, and fate mapping. Our results highlight two key events in telencephalon morphogenesis. First, the formation of a deep ventricular recess between telencephalon and diencephalon, the anterior intraencephalic sulcus (AIS), effectively creates a posterior ventricular wall to the telencephalic lobes. This process displaces the most posterior neuroepithelial territory of the telencephalon laterally. Second, as telencephalic growth and neurogenesis proceed between days 2 and 5 of development, the pallial region of the posterior ventricular wall of the telencephalon bulges into the dorsal aspect of the AIS. This brings the ventricular zone (VZ) into close apposition with the roof of the AIS to generate a narrow ventricular space and the thin tela choroidea (tc). As the pallial VZ expands, the tc also expands over the upper surface of the telencephalon. During this period, the major axis of growth and extension of the pallial VZ is along the anteroposterior axis. This second step effectively generates an everted telencephalon by 5 dpf. Conclusion: Our description of telencephalic morphogenesis challenges the conventional model that eversion is simply due to a laterally directed outfolding of the telencephalic neuroepithelium. This may have significant bearing on understanding the eventual organization of the adult fish telencephalon

Anatomy and Connectivity of the Torus Longitudinalis of the Adult Zebrafish

[Abstract] 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.IB was supported by a Sir Henry Dale Fellowship from the Royal Society and Wellcome Trust (101195/Z/13/Z) and a UCL Excellence Fellowship. This project was also funded by University of A CoruñaReino Unido. Royal Society; 101195/Z/13/

Manganese Overexposure Alters Neurogranin Expression and Causes Behavioral Deficits in Larval Zebrafish

Manganese (Mn), a cofactor for various enzyme classes, is an essential trace metal for all organisms. However, overexposure to Mn causes neurotoxicity. Here, we evaluated the effects of exposure to Mn chloride (MnCl2) on viability, morphology, synapse function (based on neurogranin expression) and behavior of zebrafish larvae. MnCl2 exposure from 2.5 h post fertilization led to reduced survival (60%) at 5 days post fertilization. Phenotypical changes affected body length, eye and olfactory organ size, and visual background adaptation. This was accompanied by a decrease in both the fluorescence intensity of neurogranin immunostaining and expression levels of the neurogranin-encoding genes nrgna and nrgnb, suggesting the presence of synaptic alterations. Furthermore, overexposure to MnCl2 resulted in larvae exhibiting postural defects, reduction in motor activity and impaired preference for light environments. Following the removal of MnCl2 from the fish water, zebrafish larvae recovered their pigmentation pattern and normalized their locomotor behavior, indicating that some aspects of Mn neurotoxicity are reversible. In summary, our results demonstrate that Mn overexposure leads to pronounced morphological alterations, changes in neurogranin expression and behavioral impairments in zebrafish larvae

Detection of Aeromonas Salmonicida Subsp. Salmonicida Infection in Zebrafish by Labelling Bacteria With Gfp and a Fluorescent Probe Based on the Siderophore Amonabactin

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG[Abstract] Zebrafish (Danio rerio) is an excellent model to study bacterial infections in fish and their treatment. We used zebrafish as a model of infection for Aeromonas salmonicida subsp. salmonicida (hereinafter A. salmonicida), the causative agent of fish furunculosis. The infection process of A. salmonicida was studied by immersion of zebrafish larvae in 2 different doses of the bacteria and the fish mortality was monitored for three days. The bacterium caused a high mortality (65 %) in zebrafish larvae only when they were exposed to a high bacterial concentration (107 bacterial cells/mL). To evaluate the use of fluorescence microscopy to follow A. salmonicida infection in vivo, two different fluorescent strains generated by labeling an A. salmonicida strain with either, the green fluorescent protein (GFP), or with a previously reported siderophore amonabactin-sulforhodamine B conjugate (AMB-SRB), were used. The distribution of both labeled bacterial strains in the larvae tissues was evaluated by conventional and confocal fluorescence microscopy. The fluorescent signal showed a greater intensity with the GFP-labeled bacteria, so it could be observed using conventional fluorescence microscopy. Since the AMB-SRB labeled bacteria showed a weaker signal, the larvae were imaged using a laser scanning confocal microscope after 48 h of exposure to the bacteria. Both fluorescent signals were mainly observed in the larvae digestive tract, suggesting that this is the main colonization route of zebrafish for waterborne A. salmonicida. This is the first report of the use of a siderophore-fluorophore conjugate to study a bacterial infection in fish. The use of a siderophore-fluorophore conjugate has the advantage that it is a specific marker and that does not require genetic manipulation of the bacteria.This work was supported by grants RTI2018-093634-B-C21/C22 and PID2021-122732OB-C22/C21 from MCIN/AEI/10.13039/501100011033/FEDER “A way to make Europe” (AEI, Spanish State Agency for Research and FEDER Programme from the European Union). Work in University of Santiago de Compostela and University of A Coruña was also supported by grants ED431C 2022/23 and ED431C 2022/39, respectively, from Xunta de Galicia. Funding for open access charge: Universidade da Coruña/CISUGXunta de Galicia; ED431C 2022/23Xunta de Galicia; ED431C 2022/3

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

[Abstract] 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.Ministerio de Ciencia e Innovación; SAF2015–70377Ministerio de Ciencia e Innovación; RTI2018–093493-B-I00Generalitat de Catalunya; SGR2014–1178Generalitat de Catalunya; SGR014–2016Instituto de Salud Carlos III; PI16/00267-R-Fede