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The retina, whose basic cellular structure is highly conserved across vertebrates, constitutes an accessible system for studying the central nervous system. In recent years, single-cell RNA sequencing studies have uncovered cellular diversity in the retina of a variety of species, providing new insights on retinal evolution and development. However, similar data in cartilaginous fishes, the sister group to all other extant jawed vertebrates, are still lacking. Here, we present a single-nucleus RNA sequencing atlas of the postnatal retina of the catshark Scyliorhinus canicula, consisting of the expression profiles for 17,438 individual cells from three female, juvenile catshark specimens. Unsupervised clustering revealed 22 distinct cell types comprising all major retinal cell classes, as well as retinal progenitor cells (whose presence reflects the persistence of proliferative activity in postnatal stages in sharks) and oligodendrocytes. Thus, our dataset serves as a foundation for further studies on the development and function of the catshark retina. Moreover, integration of our atlas with data from other species will allow for a better understanding of vertebrate retinal evolution., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates. Immunohistochemical techniques with specific antibodies against GABA or against its synthesizing enzyme, glutamic acid decarboxylase (GAD) allowed characterizing GABAergic neurons and fibers in the CNS. However, studies on the CNS distribution of GABAergic neurons and fibers of bony fishes are scant and were done in teleost species. With the aim of understanding the early evolution of this system in bony vertebrates, we analyzed the distribution of GABA-immunoreactive (-ir) and GAD-ir neurons and fibers in the CNS of a basal ray-finned fish, the Siberian sturgeon (Chondrostei, Acipenseriformes), using immunohistochemical techniques. Our results revealed the presence and distribution of GABA/GAD-ir cells in different regions of the CNS such as olfactory bulbs, pallium and subpallium, hypothalamus, thalamus, pretectum, optic tectum, tegmentum, cerebellum, central grey, octavolateralis area, vagal lobe, rhombencephalic reticular areas, and the spinal cord. Abundant GABAergic innervation was observed in most brain regions, and GABAergic fibers were very abundant in the hypothalamic floor along the hypothalamo-hypophyseal tract and neurohypophysis. In addition, GABA-ir cerebrospinal fluid-contacting cells were observed in the alar and basal hypothalamus, saccus vasculosus, and spinal cord central canal. The distribution of GABAergic systems in the sturgeon brain shows numerous similarities to that observed in lampreys, but also to those of teleosts and tetrapods., (© 2024 Wiley Periodicals LLC.)

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.

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 retina., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hernández-Núñez, Robledo, Mayeur, Mazan, Sánchez, Adrio, Barreiro-Iglesias and Candal.)

The turbot, Scophthalmus maximus, belongs to the flatfishes (order Pleuronectiformes), which display substantial asymmetry of the olfactory organs and forebrain. Sox genes code for SRY-related HMG domain-bearing transcription factors involved in various developmental processes. Group B1 Sox genes as Sox2 and Sox19 appear to play major roles in neural development. Here, we characterized by in situ hybridization the developmental expression of Sox2 and Sox19 genes in metamorphic and postmetamorphic specimens and young adults of both sexes. Expression of S. maximus Sox2 (Sm-Sox2) and Sm-Sox19 mRNAs was detected in ependymal cells of different regions of the telencephalon, preoptic region, hypothalamus, and thalamus at all stages investigated. Sm-Sox2 expression but not Sm-Sox19 occurred in neurons located in particular regions such as the dorsal nucleus of the ventral telencephalon, the medial zone of the dorsal telencephalon, preoptic area and hypothalamus. Although Sm-Sox2 and Sm-Sox19 are expressed differentially in gonads, no sex differences in their expression were observed between male and female forebrains. We also investigated the topographical relation between Sox expression and cell proliferation using series double immunostained for a radial glial marker (BLBP) and cell proliferation marker (PCNA). Sm-Sox2 and Sm-Sox19 were strongly expressed in ependymal cells located in neurogenic niches revealed by the BLBP and PCNA immunostaining. Comparison with other teleosts indicates similar expression of Sox2 and Sox19 in the telencephalon, supporting conserved roles for both genes in teleost brains., (© 2017 Wiley Periodicals, Inc.)

Regulation of bone development, growth, and remodeling traditionally has been thought to depend on endocrine and autocrine/paracrine modulators. Recently, however, brain-derived signals have emerged as key regulators of bone metabolism, although their mechanisms of action have been poorly understood. We reveal the existence of an ancient parathyroid hormone (Pth)4 in zebrafish that was secondarily lost in the eutherian mammals' lineage, including humans, and that is specifically expressed in neurons of the hypothalamus and appears to be a central neural regulator of bone development and mineral homeostasis. Transgenic fish lines enabled mapping of axonal projections leading from the hypothalamus to the brainstem and spinal cord. Targeted laser ablation demonstrated an essential role for of pth4-expressing neurons in larval bone mineralization. Moreover, we show that Runx2 is a direct regulator of pth4 expression and that Pth4 can activate cAMP signaling mediated by Pth receptors. Finally, gain-of-function experiments show that Pth4 can alter calcium/phosphorus levels and affect expression of genes involved in phosphate homeostasis. Based on our discovery and characterization of Pth4, we propose a model for evolution of bone homeostasis in the context of the vertebrate transition from an aquatic to a terrestrial lifestyle.-Suarez-Bregua, P., Torres-Nuñez, E., Saxena, A., Guerreiro, P., Braasch, I., Prober, D. A., Moran, P., Cerda-Reverter, J. M., Du, S. J., Adrio, F., Power, D. M., Canario, A. V. M., Postlethwait, J. H., Bronner, M E., Cañestro, C., Rotllant, J. Pth4, an ancient parathyroid hormone lost in eutherian mammals, reveals a new brain-to-bone signaling pathway., (© FASEB.)

Estrogens affect brain development of vertebrates, not only by impacting activity and morphology of existing circuits, but also by modulating embryonic and adult neurogenesis. The issue is complex as estrogens can not only originate from peripheral tissues, but also be locally produced within the brain itself due to local aromatization of androgens. In this respect, teleost fishes are quite unique because aromatase is expressed exclusively in radial glial cells, which represent pluripotent cells in the brain of all vertebrates. Expression of aromatase in the brain of fish is also strongly stimulated by estrogens and some androgens. This creates a very intriguing positive auto-regulatory loop leading to dramatic aromatase expression in sexually mature fish with elevated levels of circulating steroids. Looking at the effects of estrogens or anti-estrogens in the brain of adult zebrafish showed that estrogens inhibit rather than stimulate cell proliferation and newborn cell migration. The functional meaning of these observations is still unclear, but these data suggest that the brain of fish is experiencing constant remodeling under the influence of circulating steroids and brain-derived neurosteroids, possibly permitting a diversification of sexual strategies, notably hermaphroditism. Recent data in frogs indicate that aromatase expression is limited to neurons and do not concern radial glial cells. Thus, until now, there is no other example of vertebrates in which radial progenitors express aromatase. This raises the question of when and why these new features were gained and what are their adaptive benefits. This article is part of a Special Issue entitled: Nuclear receptors in animal development., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The cerebellum is recognized as an evolutionary innovation of jawed vertebrates, whose most primitive group is represented by the chondrichthyans, or cartilaginous fishes. A comprehensive knowledge of cerebellar connections in these fishes might shed light on the basal organization of the cerebellar system. Although the organization of the precerebellar system is known in adults, developmental studies are essential for understanding the origin and evolution of precerebellar nuclei. In the present work we performed a developmental study of cerebellar connections in embryos and juveniles of an advanced shark species, Scyliorhinus canicula, by application of tract tracing in combination with immunohistochemical techniques. Main precerebellar cell populations were located in the diencephalon (pretectum and thalamus), mesencephalon (reticular formation and nucleus ruber), rhombencephalon (cerebellar nucleus, reticular formation, and inferior olive), and spinal cord (ventral horn). The order of arrival of cerebellar afferent projections throughout development revealed a common pattern with other jawed vertebrates, which was helpful for comparison of stages of cerebellar development. The neurochemical study of the inferior olive and other precerebellar nuclei revealed many shared features with other gnathostomes. Furthermore, because many precerebellar nuclei originate from rhombic lips, the first analysis of neuronal migrations from these lips was performed with markers of neuroblasts. The shared features of development and organization of precerebellar connections observed between sharks and amniotes suggest that their basic pattern was established early in gnathostome evolution., (Copyright © 2013 Wiley Periodicals, Inc.)

The glycinergic cell populations in the brain of the lesser spotted dogfish were studied by a glycine immunofluorescence method. Numerous glycine-immunoreactive (Gly-ir) neurons were observed in different brain nuclei. In the telencephalon, Gly-ir cells were observed in the olfactory bulb, telencephalic hemispheres, and preoptic region. In the hypothalamus, cerebrospinal fluid-contacting Gly-ir neurons were observed in the lateral and posterior recess nuclei. Coronet cells of the saccus vasculosus were Gly-ir. In the diencephalon, Gly-ir neurons were observed in the prethalamus and pretectum. In the midbrain, both the optic tectum and lateral mesencephalic nucleus contained numerous Gly-ir neurons. In the cerebellum, many Golgi cells were Gly-ir. In the rhombencephalon, Gly-ir cells were observed in the medial and ventral octavolateral nuclei, vagal lobe, visceromotor nuclei, and reticular formation, including the inferior raphe nucleus. In the spinal cord, some neurons of the marginal nucleus and some cells of the dorsal and ventral horns were Gly-ir. Comparison of dogfish Gly-ir cell populations with those reported for the sea lamprey, Siberian sturgeon, and zebrafish revealed some shared features but also notable differences. For example, Gly-ir cells were observed in the dogfish cerebellum, unlike the case in the Siberian sturgeon and zebrafish, whereas the absence of Gly-ir neurons in the isthmus is shared by all these species, except for lampreys. Gly-ir populations in the dogfish hypothalamus and telencephalon are notable in comparison with those of the other jawed vertebrates investigated to date. Together, these results reveal a complex and divergent evolution of glycinergic systems in the major groups of fishes., (Copyright © 2013 Wiley Periodicals, Inc.)

We used a Tg(glyt2:gfp) transgenic zebrafish expressing the green fluorescent protein (GFP) under control of the glycine transporter 2 (GLYT2) regulatory sequences to study for the first time the glycinergic neurons in the brain of an adult teleost. We also performed in situ hybridization using a GLYT2 probe and glycine immunohistochemistry. This study was combined with biocytin tract tracing from the spinal cord to reveal descending glycinergic pathways. A few groups of GFP(+) /GLYT2(-) cells were observed in the midbrain and forebrain, including numerous pinealocytes. Conversely, a small nucleus of the midbrain tegmentum was GLYT2(+) but GFP(-) . Most of the GFP(+) and GLYT2(+) neurons were observed in the rhombencephalon and spinal cord, and a portion of these cells showed double GLYT2/GFP labeling. In the hindbrain, GFP/GLYT2(+) populations were observed in the medial octavolateral nucleus; the secondary, magnocellular, and descending octaval nuclei; the viscerosensory lobes; and reticular populations distributed from trigeminal to vagal levels. No glycinergic cells were observed in the cerebellum. Tract tracing revealed three conspicuous pairs of GFP/GLYT2(+) reticular neurons projecting to the spinal cord. In the spinal cord, GFP/GLYT2(+) cells were observed in the dorsal and ventral horns. GFP(+) fibers were observed from the olfactory bulbs to the spinal cord, although their density varied among regions. The Mauthner neurons received very rich GFP(+) innervation, mainly around the axon cap. Comparison of the zebrafish glycinergic system with the glycinergic systems of other adult vertebrates reveals shared patterns but also divergent traits in the evolution of this system., (Copyright © 2012 Wiley Periodicals, Inc.)

Glycine and γ-aminobutyric acid (GABA) are the main inhibitory neurotransmitters in the central nervous system (CNS) of vertebrates. Studies on the distribution of glycinergic neurons and fibers have been carried out mainly in rodents and lampreys. With the aim of discovering more about the early evolution of this system in vertebrates, we analyzed the distribution of glycine-immunoreactive (Gly-ir) neurons and fibers in the CNS of a basal ray-finned fish, the Siberian sturgeon (Chondrostei, Acipenseriformes), by use of immunohistochemical techniques. We also compared the distribution of glycine and GABA by the use of double-immunofluorescence techniques and confocal microscopy. Our results revealed the presence of Gly-ir cells in different regions of the CNS, such as olfactory bulbs, preoptic area, hypothalamus, thalamus, pretectum, optic tectum, tegmentum and rostral spinal cord, although most of the Gly-ir cells and the most intensely immunoreactive cells were located in the rhombencephalon, mainly in the octavolateral area and reticular formation. In addition, coronet cells of the basal hypothalamus and saccus vasculosus were Gly-ir. Glycinergic fibers coursed along most brain regions and were more abundant in the thalamus, hypothalamus, optic tectum, tegmentum, isthmic region, and basal rhombencephalon. The Mauthner cell perikaryon was richly innervated by Gly-ir boutons, as reported for teleosts. With regard to the colocalization of glycine and GABA, double-immunoreactive cells were located mainly in the rhombencephalon. The results enable us to conclude that the distribution of glycine in the sturgeon brain is more similar to that observed in lampreys than that observed in mammals., (Copyright © 2011 Wiley-Liss, Inc.)

Somatostatin (SOM) is a neuropeptide that is widely distributed in the central nervous system of vertebrates. Two isoforms of somatostatin (SS1 and SS2) have been characterized in sturgeon and in situ hybridisation studies in the sturgeon brain have demonstrated that mRNAs of the two somatostatin precursors (PSS1 and PSS2) are differentially expressed in neurons [Trabucchi, M., Tostivint, H., Lihrmann, I., Sollars, C., Vallarino, M., Dores, R.M., Vaudry, H., 2002. Polygenic expression of somatostatin in the sturgeon Acipenser transmontanus: molecular cloning and distribution of the mRNAs encoding two somatostatin precursors. J. Comp. Neurol. 443, 332-345.]. However, neither the morphology of somatostatinergic neurons nor the patterns of innervation have yet been characterized. To gain further insight into the evolution of this system in primitive bony fishes, we studied the distribution of somatostatin-immunoreactive (SOM-ir) cells and fibres in the brain of the Siberian sturgeon (Acipenser baeri). Most SOM-ir cells were found in the preoptic area and hypothalamus and abundant SOM-ir fibres coursed along the hypothalamic floor towards the median eminence, suggesting a hypophysiotrophic role for SOM in sturgeon. In addition, SOM-ir cells and fibres were observed in extrahypothalamic regions such as the telencephalon thalamus, rhombencephalon and spinal cord, which also suggests neuromodulatory and/or neurotransmitter functions for this peptide. Overall there was a good correlation between the distribution of SOM-ir neurons throughout the brain of A. baeri and that of PSS1 mRNA in Acipenser transmontanus. Comparative analysis of the results with those obtained in other groups of fishes and tetrapods indicates that widespread distribution of this peptide in the brain is shared by early vertebrate lines and that the general organization of the somatostatinergic systems has been well-conserved during evolution.

The saccus vasculosus (SV) is a circumventricular organ of the hypothalamus of many jawed fishes whose functions have not yet been clarified. It is a vascularized neuroepithelium that consists of coronet cells, cerebrospinal fluid-contacting (CSF-c) neurons and supporting cells. To assess the organization, development and evolution of the SV, the expression of glial fibrillary acidic protein (GFAP) and the neuronal markers gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD; the GABA synthesizing enzyme), neuropeptide Y (NPY), neurophysin II (NPH), tyrosine hydroxylase (TH; the rate-limiting catecholamine-synthesizing enzyme) and serotonin (5-HT), were investigated by immunohistochemistry in developing and adult sharks. Coronet cells showed GFAP immunoreactivity from embryos at stage 31 to adults, indicating a glial nature. GABAergic CSF-c neurons were evidenced just when the primordium of the SV becomes detectable (at stage 29). Double immunolabeling revealed colocalization of NPY and GAD in these cells. Some CSF-c cells showed TH immunoreactivity in postembryonic stages. Saccofugal GABAergic fibers formed a defined SV tract from the stage 30 and scattered neurosecretory (NPH-immunoreactive) and monoaminergic (5-HT- and TH-immunoreactive) saccopetal fibers were first detected at stages 31 and 32, respectively. The early differentiation of GABAergic neurons and the presence of a conspicuous GABAergic saccofugal system are shared by elasmobranch and teleosts (trout), suggesting that GABA plays a key function in the SV circuitry. Monoaminergic structures have not been reported in the SV of bony fishes, and were probably acquired secondarily in sharks. The existence of saccopetal monoaminergic and neurosecretory fibers reveals reciprocal connections between the SV and hypothalamic structures which have not been previously detected in teleosts., (Copyright (c) 2007 S. Karger AG, Basel.)

We have studied the development of catecholaminergic (CA) neuronal groups in the brain of the dogfish Scyliorhinus canicula using immunohistochemistry to tyrosine hydroxylase (TH). The earliest TH-immunoreactive (THir) cells were detected in the primordia of the posterior tubercle and suprachiasmatic nuclei (PTN and SCN, respectively) of stage 26 embryos. At stage 28, THir cells were also seen extending between the SCN and the PTN at ventral thalamic levels. At stage 30, some THir cerebrospinal fluid-contacting neurons and migrated THir cells were found in the walls of the posterior recess, and a few weakly THir cells also appeared at the isthmus level (locus coeruleus) and in the caudal rhombencephalic tegmentum. At stage 31, further THir cell groups appeared in the synencephalon and midbrain (ventral tegmental area/substantia nigra, VTA/SN), and the rhombencephalon (viscerosensory and visceromotor columns). At stage 32, the first THir cells appeared in the pallium, the olfactory bulb and the preoptic area. THir cells are seen in the retina from stage 33. The developmental sequence of THir cell groups in dogfish appears to be rather similar to that described for teleosts, apart from the appearance of the VTA/SN and pallial cells, which lack in teleosts.

Galanin is a 29-amino acid peptide widely distributed in the central nervous system of vertebrates. The organization of galaninergic systems is well known in teleosts, the most advanced actinopterygians, but no data are available on primitive bony fish. To extend the evolutionary analysis of galaninergic systems we studied the distribution of galanin-like immunoreactive (GAL-ir) cells and fibers in the sturgeon brain, since chondrosteans are among the most primitive extant actinopterygians. Double-immunolabeling experiments were performed to compare the distribution of galanin with that of neurophysin, tyrosine hydroxylase, and serotonin. Numerous GAL-ir cells of cerebrospinal fluid-contacting (CSF-C) type were found in the ventral telencephalon, preoptic area, and in the tuberal and caudal hypothalamus. The distribution of GAL-ir elements in the sturgeon brain shows many similarities to that observed in other vertebrates, but also important differences, such as the abundance of GAL-ir CSF-C cells, which appear to be a primitive characteristic. GAL-ir neurons observed in the sturgeon telencephalic hemispheres perhaps represent the basic organization of common ancestors of bony fishes and tetrapods. In the preoptic-hypophyseal system, GAL-ir cells appeared to be related not only with neurophysin-expressing neurons (in the tuberal hypothalamus) but also with serotoninergic and catecholamines-synthesizing neurons (in preoptic and tuberal nuclei). Numerous GAL-ir fibers were observed in the median eminence and neural lobe of the hypophysis, indicating that galanin may play a role in the modulation of hypophyseal secretion. GAL-ir neurons were absent from the sturgeon brainstem, suggesting that their presence in other vertebrates could represent an evolutionary recent acquisition., ((c) 2005 Wiley-Liss, Inc.)

Teleost fish are known for exhibiting a high aromatase activity mainly due to the expression of the cyp19b gene, encoding aromatase B (AroB). Recent studies based on both in situ hybridization and immunohistochemistry have demonstrated in three different species that this activity is restricted to radial glial cells. In agreement with measurements of aromatase activity, such aromatase-expressing cells are more abundant in the telencephalon, preoptic area, and mediobasal hypothalamus, although positive cells are also found in the midbrain and hindbrain. Comparative distribution of AroB and estrogen receptor (ERalpha, ERbeta1, and ERbeta2) expression indicates that the preoptic region and hypothalamus are major target for locally produced estradiol (E2) which is likely involved in controlling expression of genes implicated in neuroendocrine regulations. However, AroB and ER have never been reported to be co-expressed in the same cells which is intriguing given that, at least in some species, AroB is strongly up-regulated by E2 itself in agreement with the presence of an estrogen-responsive element (ERE) in the proximal promoter of the cyp19b gene. In vivo data in zebrafish have shown that E2 up-regulates AroB only in radial glial cells. This is in agreement with in vitro transfection experiments indicating that this ERE is functional, but not sufficient, as the E2 regulation of AroB only occurs in glial cell contexts, suggesting a cooperation between ER and so far unidentified glial-specific factors. These data also suggest that radial glial cells may express low amounts of ER that escaped detection until now. The expression of AroB in radial cells, well known for their roles in neurogenesis and now considered as progenitor cells, suggests that local E2 production within these cells could influence the well-documented capacity of the brain of teleosts to grow during adulthood.

To obtain a better understanding of the evolution of the brain catecholaminergic systems of fishes, we have examined the distribution of catecholamine-synthesizing enzymes in two species of sturgeon (Acipenser baeri and Huso huso) using antibodies against tyrosine hydroxylase (TH) and dopamine-beta -hydroxylase (DBH; only analyzed in Acipenser). Both sturgeons showed TH-immunoreactive (THir) neurons widely distributed in most regions of the brain, the highest number of THir cells being located in the forebrain (olfactory bulb, preoptic area, and posterior tuberculum). THir cells were also seen in other forebrain areas (retrobulbar area, dorsal and ventral telencephalic areas, hypothalamus, ventral thalamus, pretectal area) and in the brainstem (locus coeruleus, viscerosensory area, caudal reticular formation, and area postrema). Immunoreactive fibers and varicosities showed a wide distribution, being particularly abundant in the diencephalon and mesencephalon. DBH-immunoreactive (DBHir) cells were observed in the anterior tuberal nucleus, where these cells were TH-negative, and in the locus coeruleus and the caudal rhombencephalon (vagal reticular formation), where the DBHir cells were also THir. DBHir fibers were scarce in the telencephalon and very abundant in the diencephalon, mesencephalon, and rhombencephalon. The comparative analysis of the catecholaminergic systems of chondrosteans and those observed in other groups of fishes and tetrapods indicate a similar organization of many nuclei, as well as characteristics that are probably primitive, such as the presence of a large number of forebrain catecholaminergic groups., (Copyright 2002 Wiley-Liss, Inc.)

Using choline acetyltransferase immunocytochemistry, we compared the cholinergic systems of the brains of four groups of fishes (lampreys, elasmobranchs, chondrosteans, and teleosts). Cholinergic nuclei were classified in four groups according to their distribution in vertebrates. The cranial motor nuclei and the habenulo-interpeduncular system were cholinergic in all vertebrates. The cholinergic nuclei of the isthmus of fishes showed many similarities with those of tetrapods. The magnocellular preoptic neurosecretory cells were cholinergic in most fishes, whereas in neurosecretory nuclei of tetrapods, cholinergic cells were only observed adjacent to the magnocellular cells. In the subpallium, cholinergic cells were observed in all fishes, with the exception of elasmobranchs, which suggests that they might be secondarily lost. In the pallium of fishes, cholinergic neurons were only observed in elasmobranchs. Because pallial cholinergic cells were only observed in lizard and mammals, they could have appeared several times during evolution. The same is suggested for the presence of cholinergic cells in the optic tectum of only a few vertebrate groups, including teleosts. This preliminary analysis enlarges our knowledge of the cholinergic systems of fishes, although more species and groups need to be studied to provide a more complete scenario of their evolution.

On the basis of labeling with an anti-gamma-aminobutyric acid (GABA) antibody, we report for the first time the presence and distribution of GABA-immunoreactive cells in the central and peripheral nervous system of amphioxus. In the nerve cord, there is a large dorsorostral group of cerebrospinal-fluid-contacting (CSFc) cells at the caudal end of the brain vesicle that gives rise to a large ventral commissure and neuropilar region. In the middle and caudal region of the brain, numerous commissural and CSFc neurons are situated below the region of large dorsal cells. In the spinal cord, several types of GABA-immunoreactive neurons of different size, appearance, and distribution were observed. In the dorsalmost region, very small commissural cells are scattered regularly along the cord. More ventrally in the cord, GABAergic neurons, both of commissural and CSFc cell types, form segmental groups, but scattered cells are observed throughout. These cells give rise to dense longitudinal fascicles of GABAergic fibers and to scattered commissural fibers. The caudal ampulla lacks GABAergic cells and fibers. Some of the fibers of the most rostral and caudal peripheral (sensory) nerves, as well as some sensory cells of the rostral and caudal epidermis, are GABA immunoreactive. The significance of these results for the understanding of the evolution of GABAergic systems of vertebrates is discussed.

The extrahypohysial projections of the neurosecretory preoptic nucleus (PON) of the electric ray were studied with the aid of an antibody against nitric oxide synthase (NOS). PON neurons were the only NOS-like-immunoreactive (NOS-ir) cells in the brain. These neurons gave rise to both hypophysial and extrahypophysial NOS-ir projections. Some fibres coursed from the PON to the neurointermediate lobe in the preoptic-hypophysial tract. Other NOS-ir fibres coursed either rostrally or caudally forming terminal fields in the telencephalon (subpallial region), diencephalon (preoptic nucleus, ventrolateral thalamus and posterior recess nucleus), tuberal region (area tegmentalis ventralis and substantia nigra), mesencephalon (lateral tegmentum), rhombencephalon (isthmal nucleus, vagal viscerosensory column and ventrolateral reticular area) and the spinal cord (intermediate horn). The possible involvement of the extrahypophysial PON projections in neuroregulation of visceral centres is discussed.