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.

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.

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.

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 torus semicircularis in the adult Zebrafish.

The torus semicircularis (TS) of teleosts is a key midbrain center of the lateral line and acoustic sensory systems. To characterize the TS in adult zebrafish, we studied their connections using the carbocyanine tracers applied to the TS and to other related nuclei and tracts. Two main TS nuclei, central and ventrolateral, were differentiable by their afferent connections. From central TS, (TSc) numerous toropetal cells were labeled bilaterally in several primary octaval nuclei (anterior, magnocellular, descending, and posterior octaval nuclei), in the secondary octaval nucleus, in the caudal octavolateralis nucleus, and in the perilemniscular region. In the midbrain, numerous toropetal cells were labeled in the contralateral TSc. In the diencephalon, toropetal cells labeled from the TSc were observed ipsilaterally in the medial prethalamic nucleus and the periventricular posterior tubercle nucleus. TSc toropetal neurons were also labeled bilaterally in the hypothalamic anterior tuberal nucleus (ATN) and ipsilaterally in the parvicellular preoptic nucleus but not in the telencephalon. Tracer application to the medial octavolateralis nucleus revealed contralateral projections to the ventrolateral TS (TSvl), whereas tracer application to the secondary octaval nucleus labeled fibers bilaterally in TSc and neurons in rostral TSc. The TSc sends ascending fibers to the ipsilateral lateral preglomerular region that, in turn, projects to the pallium. Application of DiI to the optic tectum labeled cells and fibers in the TSvl, whereas application of DiI to the ATN labeled cells and fibers in the TSc. These results reveal that the TSvl and TSc are mainly related with the mechanosensory lateral line and acoustic centers, respectively, and that they show different higher order connections.

Gene-activated hyaluronic acid-based cryogels for cartilage tissue engineering.

Articular cartilage injuries are very frequent lesions that if left untreated may degenerate into osteoarthritis. Gene transfer to mesenchymal stem cells (MSCs) provides a powerful approach to treat these lesions by promoting their chondrogenic differentiation into the appropriate cartilage phenotype. Non-viral vectors constitute the safest gene transfer tools, as they avoid important concerns of viral systems including immunogenicity and insertional mutagenesis. However, non-viral gene transfer usually led to lower transfection efficiencies when compared with their viral counterparts. Biomaterial-guided gene delivery has emerged as a promising alternative to increase non-viral gene transfer efficiency by achieving sustained delivery of the candidate gene into cellular microenvironment. In the present study, we designed hyaluronic acid-based gene-activated cryogels (HACGs) encapsulating a novel formulation of non-viral vectors based on niosomes (P80PX) to promote MSCs in situ transfection. The developed HACG P80PX systems showed suitable physicochemical properties to promote MSCs in situ transfection with very low cytotoxicity. Incorporation of a plasmid encoding for the transcription factor SOX9 (psox9) into HACG P80PX systems led to an effective MSCs chondrogenic differentiation with reduced expression of fibrocartilage and hypertrophic markers. The capacity of the developed systems to restore cartilage extracellular matrix was further confirmed in an ex vivo model of chondral defect.

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.

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.

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.

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.

Afferent and efferent connections of the pineal organ in the European sea bass Dicentrarchus labrax: a carbocyanine dye tract-tracing study.

The pineal organ of fish is a photosensitive structure that receives light information from the environment and transduces it into hormonal (rhythmic melatonin secretion) and neural (efferent projections/neurotransmitters) signals. In this study, we focused on this neural output. Thus, we performed a tract-tracing study using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), a fluorescent carbocyanine dye, in order to elucidate the efferent and afferent connections of the pineal organ in the European sea bass. The axonal transport of DiI revealed extensive bilateral projections in the sea bass brain. The efferent projections of the sea bass pineal organ reach the habenula, ventral thalamus, periventricular pretectum, central pretectal area, posterior tubercle and medial and dorsal tegmental areas. In addition, in this study we also examined the pinealopetal system in sea bass. This analysis demonstrated that the sea bass pineal organ receives central projections from neurons located, to a large extent, in brain areas innervated by pineal efferent projections, i.e. the thalamic eminence, habenula, ventral thalamus, dorsal thalamus, periventricular pretectum, posterior commissure, posterior tubercle and medial tegmental area. This study is the first description of pinealofugal projections in a representative of Perciformes, which constitutes a derived order within teleosts. Moreover, it represents the first evidence for the presence of pinealopetal neurons in the brain of a teleost species. Our findings, together with the analysis of retinal connections, represent a step forward in the understanding of the integration of photoperiodic signals into the central nervous system of sea bass.

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.

Calretinin-immunoreactive systems in the cerebellum and cerebellum-related lateral-line medullary nuclei of an elasmobranch, Scyliorhinus canicula.

Calretinin immunohistochemistry was used to study the organization of some cerebellar structures and lateral line medullary nuclei of an elasmobranch, the lesser-spotted dogfish Scyliorhinus canicula. In the cerebellar molecular layer, stellate cells are strongly calretinin-immunoreactive (CR-ir). Perikarya and dendrites of Purkinje cells are contacted by numerous stellate cell small CR-ir boutons. Some Purkinje cell perikarya are contacted by CR-ir climbing fibers forming complex axo-somatic contacts. In the granular layer, numerous CR-ir mossy fibers exhibited large swellings. Notable differences in density and diameter of mossy fibers are observed between the auricles and cerebellar body. Thin beaded CR-ir fibers are also present in the granular layer of the body. The lateral line nuclei of the octavolateralis region are comprised of a molecular-like cerebellar crest that covers the dorsal (electroreceptive) and the medial octavolateralis nuclei (mechanoreceptive). The cerebellar crest exhibited numerous CR-ir stellate cells. In the dorsal octavolateralis nucleus, the presence of conspicuous CR-ir cells and neuropil closely associated to the region of primary fiber terminals distinguishes it clearly from the medial nucleus, revealing major differences between the electroreceptive and mechanoreceptive primary nuclei of elasmobranchs. Moreover, CR distribution in the dogfish cerebellum showed interesting differences with those reported in cerebella of other vertebrates, indicating a high variability of cerebellar CR expression in phylogeny.

The organization of the pretectal nuclei in the trout: a revision based on experimental holodogical studies.

The neuronal tracer DiI was applied to different brain centers of the rainbow trout in order to study the connections of pretectal nuclei. Our results showed that some pretectal nuclei receive a direct projection from the contralateral retina: the parvocellular superficial pretectal nucleus, the central pretectal nucleus, the intermediate pretectal nucleus and the ventral accessory optic nucleus. In turn, the central pretectal, the intermediate pretectal and the ventral accessory optic nuclei, together with the paracommissural nucleus, project to the cerebellum and the torus longitudinalis. The magnocellular superficial pretectal nucleus does not receive retinal projections, but receives ipsilateral projections from the optic tectum and the mesencephalic tegmentum. In turn, it projects to the ipsilateral oculomotor nucleus and lateral nucleus of the valvula. The posterior pretectal nucleus and the parvocellular superficial pretectal nucleus receive afferents from the ipsilateral nucleus isthmi. The posterior pretectal nucleus projects to the inferior hypothalamic lobe. Our results reveal a conspicuous projection from the ipsilateral parvocellular superficial pretectal nucleus to the contralateral one and also to the contralateral posterior prectectal nucleus, not reported in previous experimental studies of teleosts. Pretectal centers appear to integrate visual/optic-related centers mainly with the hypothalamus and the cerebellum. The organization of the trout pretectum was compared with the pretectal organization patterns proposed in various teleosts.

Immunocytochemical study of calretinin and calbindin D-28K expression in the retina of three cartilaginous fishes and a cladistian (Polypterus).

The distribution of two calcium-binding proteins, calbindin D-28K (CB) and calretinin (CR) was studied in the retina of a cladistian, Polypterus senegalus, and three cartilaginous fishes (Scyliorhinus canicula, Raja undulata and Torpedo marmorata). Western blot analysis of brain extracts revealed the lack of cross-reactivity of the used antibodies. In Polypterus, CB and CR immunoreactivities were observed in some amacrine and ganglion cells, but scarce cells showed CR/CB colocalization. Furthermore, CR immunoreactivity was present in a number of displaced bipolar cells and in some putative displaced ganglion cells, whereas CB immunoreactivity was found in some cones. No positive retinal structure was observed with the CB antibody used in cartilaginous fishes. Instead, CR was expressed in some amacrine, horizontal and ganglion cells of the dogfish and skate and, in some ganglion cells of the electric ray. The comparative analysis suggests, (1) the presence of CB-positive photoreceptor cells in the retina of cladistians seems to be apomorphic (in jawed fishes) in contrast with the plesiomorphic condition of this character in land vertebrates; (2) the presence of CR in amacrine and ganglion cells is a conserved feature along vertebrate phylogeny, whereas its variable expression in bipolar and horizontal cells represents a derived character; (3) the absence of CB in horizontal cells in cladistians could represent a derived character; and (4) the presence of CR displaced bipolar and putative displaced ganglion cells in Polypterus is shared with basal groups of actinopterygians.

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.

Organization of the torus longitudinalis in the rainbow trout (Oncorhynchus mykiss): an immunohistochemical study of the GABAergic system and a DiI tract-tracing study.

The torus longitudinalis (TL) is a tectum-associated structure of actinopterygian fishes. The organization of the TL of rainbow trout was studied with Nissl staining, Golgi methods, immunocytochemistry with antibodies to gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD), and the GABA(A) receptor subunits delta and beta2/beta 3, and with tract tracing methods. Two types of neuron were characterized: medium-sized GABAergic neurons and small GABA-negative granule cells. GABA(A) receptor subunit delta-like immunoreactivity delineated two different TL regions, ventrolateral and central. Small GABAergic cells were also observed in marginal and periventricular strata of the optic tectum. These results indicate the presence of local GABAergic inhibitory circuits in the TL system. For tract-tracing, a lipophilic dye (DiI) was applied to the TL and to presumed toropetal nuclei or toral targets. Toropetal neurons were observed in the optic tectum, in pretectal (central, intermediate, and paracommissural) nuclei, in the subvalvular nucleus, and associated with the pretectocerebellar tract. Torofugal fibers were numerous in the stratum marginale of the optic tectum. Toropetal pretectal nuclei also project to the cerebellum, and a few TL cells project to the cerebellar corpus. The pyramidal cells of the trout tectum were also studied by Golgi methods and local DiI labeling. The connections of trout TL revealed here were more similar to those recently reported in carp and holocentrids (Ito et al. [2003] J. Comp. Neurol. 457:202-211; Xue et al. [2003] J. Comp. Neurol. 462:194-212), than to those reported in earlier studies. However, important differences in organization of toropetal nuclei were noted between salmonids and these other teleosts.

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.

Topography and connections of the telencephalon in a chondrostean, Acipenser baeri: an experimental study.

Sturgeons belong to an ancient group of the extant actinopterygian fishes. Accordingly, the study of their brain connections is important to understand brain evolution in the line leading to teleosts. We examined the topography and connections of the various telencephalic regions of the Siberian sturgeon (Acipenser baeri). The telencephalic regions were characterized on the basis of acetylcholinesterase histochemistry and calbindin-D28k and calretinin immunohistochemistry. The telencephalic connections were investigated by using the fluorescent dye DiI (1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate) in fixed brains. Application of DiI to different areas of the pallial (dorsal) regions of the telencephalic lobes showed that they have mostly intratelencephalic connections. A posterior pallial region is characterized by its similar hodology to that of the posterior zone of the teleosts dorsal telencephalon and those described in other ancient groups. Extratelencephalic connections of the pallium are scarce, although a few afferent and efferent connections with the diencephalon, mesencephalon, and rostral rhombencephalon were observed. DiI application to subpallial regions showed both intratelencephalic connections and connections with different brain regions. Afferents to the subpallium originate from the olfactory bulbs, preoptic area, thalamus, posterior tuberculum, hypothalamus, secondary gustatory nucleus, and raphe nuclei. Some of these connections are quite similar to those described for other vertebrates.

Afferent and efferent connections of the cerebellum of a salmonid, the rainbow trout (Oncorhynchus mykiss): a tract-tracing study.

The connections of the cerebellum of the rainbow trout were studied by experimental methods. The pretectal paracommissural nucleus has reciprocal connections with the cerebellum. Three additional pretectal nuclei project to both the corpus and valvula cerebelli, and seem to receive cerebellar afferents. A large number of cells of the lateral nucleus of the valvula project to wide regions of the cerebellum, including the valvula, the corpus, the granular eminences, and the caudal lobe, whereas the contralateral inferior olive and scattered reticular cells project only to the corpus and valvula cerebelli. Afferents to the corpus were also observed from the ventral tegmental nucleus, the "paraisthmic nucleus," the perilemniscal nucleus, the central gray, and the octavolateral area. Valvular afferents were also observed from the torus semicircularis and the midbrain tegmental areas. In most cases of cerebellar application, labeled fibers were seen in the thalamus, the pretectum, the torus longitudinalis and torus semicircularis, the nucleus of the medial longitudinal fascicle, and midbrain and rhombencephalic reticular areas. From the corpus cerebelli some fibers also project to the posterior tubercle and the hypothalamus. Moreover, the granular eminences project to the cerebellar crest. DiI application to most of the areas showing labeled fibers after cerebellar tracer application led to the labeling of characteristic eurydendroid cells, mainly in the valvula cerebelli and the caudal lobe. A few putative eurydendroid cells were labeled from the octavolateralis regions. These results in a teleost with a generalized brain indicate several differences with respect to the cerebellar connections reported in other teleost fishes that have specialized brains.