Catalina Sueiro

New insights on Saccus vasculosus evolution: a developmental and immunohistochemical study in elasmobranchs.

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 γ-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.

Development of the cerebellar body in sharks: spatiotemporal relations of Pax6 expression, cell proliferation and differentiation.

We have studied the patterns of cell proliferation, regional organization and differentiation in the cerebellar body of embryos and juveniles of two shark species by immunohistochemistry with antibodies against proliferating cell nuclear antigen (PCNA), Pax6, reelin (RELN), GABA, glutamic acid decarboxylase (GAD) and calretinin (CR). The organization of Pax6-expressing cells was also studied by in situ hybridization. Our results reveal that a transient secondary matrix zone, the external germinal layer, is formed in sharks at early stages of cerebellar development and is the source of the earliest Pax6-expressing (granule) cells. Later in development, new granule Pax6-expressing cells arise from medial proliferation zones and accumulate medially in the granular eminences. The GABAergic components appear very early, and show clear regional differences. The medial proliferation zones remain active even in adults. Taken together, the proliferation and differentiation markers used in the present study highlight striking similarities during development between the cerebellar body of elasmobranchs and the cerebella of tetrapods. These results show the importance of elasmobranch models to reconstruct the evolutionary developmental history of the vertebrate cerebellum.

Tangentially migrating GABAergic cells of subpallial origin invade massively the pallium in developing sharks

We studied the development of the GABAergic system in the telencephalon of the dogfish Scyliorhinus canicula using GABA and glutamate decarboxylase (GAD) immunocytochemistry. The earliest GABA-expressing cells appeared in the basal telencephalon (subpallium) of stage 24 embryos. Shortly after, the subpallium showed abundant GABA-expressing neuroblasts near the meningeal surface or migrating radially in the neuroepithelium. The limit between the GABA-expressing region and the remainder of the telencephalon (pallium) was sharp and coincides with the pallial/subpallial boundary. At stage 28, GABA-expressing cells with the morphology of tangentially migrating cells (showing a thick growth cone-like leading process) migrate from a dome-shaped protrusion of the lateral subpallium and extended laterally and rostrodorsally into the pallium following either a superficial route or coursing periventricularly. At later stages, abundant GABA-expressing cells were seen in various pallial regions and strings of GABA-expressing cells, possibly migrating, were also noted. The colonization of the dogfish pallium by GABA-expressing cells, originating from the subpallium, is strongly reminiscent of the palliopetal tangential migrations of GABA-expressing cells demonstrated in the telencephalon of mammals and follows similar routes. These results strongly suggest that tangential migrations of GABA-expressing cells appeared very early in vertebrate forebrain evolution.

Distribution and development of glutamic acid decarboxylase immunoreactivity in the spinal cord of the dogfish Scyliorhinus canicula (elasmobranchs)

The adult distribution and development of γ‐aminobutyric acid (GABA)‐synthesizing cells and fibers in the spinal cord of the lesser spotted dogfish (Scyliorhinus canicula L.) was studied by means of immunohistochemistry using antibodies against glutamic acid decarboxylase (GAD). Complementary immunostaining with antibodies against GABA, tyrosine hydroxylase (TH), and HuC/HuD (members of the Hu/Elav family of RNA‐associated proteins) and staining with a reduced silver procedure (“en bloc” Bielschowski method), Nissl, and hematoxylin were also used. In adults, GAD‐immunoreactive (GAD‐ir) cells were observed in the ventral horns, in the spinal nucleus of the dorsal horn, at the base of the dorsal horns, and around the central canal, where some GAD‐ir cells were cerebrospinal fluid‐contacting (CSF‐c). In addition, a few GAD‐ir cells were observed in the lateral funiculus between the ventral horn and the marginal nucleus. The adult spinal cord was richly innervated by GAD‐ir fibers. Large numbers of GAD‐ir fibers and boutons were observed in the dorsal and ventral horns and also interstitially in the dorsal, lateral, and ventral funiculi. In addition, a rich GAD‐ir innervation was observed in the marginal nucleus of the spinal cord. In the embryonic spinal cord, GAD‐ir cells develop very early: The earliest cells were observed in the very thin mantle/marginal layer of stage 22 embryos in a short length of the spinal cord. At stages 25 and 26, several types of GAD‐ir cells (commissural and noncommissural) were distinguished, and two of these cells were of CSF‐c type. At stages 28, 30, and 31, the GAD‐ir populations exhibited a marked longitudinal columnar organization. Double‐immunolabeling experiments in embryos showed the presence of two different GAD‐ir CSF‐c cell populations, one ventral that is simultaneously TH‐ir and other more dorsal that is TH‐negative. By stage 33 (prehatching), GAD‐expressing cells are present in virtually all loci, as in adults, especially in the ventral horn and base of the dorsal horn. The present results for the lesser spotted dogfish suggest an important role for γ‐aminobutyric acid in sensory and motor circuits of the spinal cord. J. Comp. Neurol. 478:189–206, 2004.

Contributions of developmental studies in the dogfish Scyliorhinus canicula to the brain anatomy of elasmobranchs: insights on the basal ganglia.

The basic anatomy of the elasmobranch brain has been previously established after studying the organization of the different subdivisions in the adult brain. However, despite the relatively abundant immunohistochemical and hodologic studies performed in different species of sharks and skates, the organization of some brain subdivisions remains unclear. The present study focuses on some brain regions in which subdivisions established on the basis of anatomical data in adults remain controversial, such as the subpallium, mainly the striatal subdivision. Taking advantage of the great potential of the lesser spotted dogfish, Scyliorhinus canicula, as a model for developmental studies, we have characterized the subpallium throughout development and postembryonic stages by analyzing the distribution of immunomarkers for GABA, catecholamines, and neuropeptides, such as substance P. Moreover, we have analyzed the expression pattern of regulatory genes involved in the regionalization of the telencephalon, such as Dlx2, Nkx2.1, and Shh, and followed their derivatives throughout development in relation to the distribution of such neurochemical markers. For further characterization, we have also analyzed the patterns of innervation of the subpallium after applying tract-tracing techniques. Our observations may shed light on postulate equivalences of regions and nuclei among elasmobranchs and support homologies with other vertebrates.

Development of catecholaminergic systems in the spinal cord of the dogfish Scyliorhinus canicula (Elasmobranchs)

The development of catecholamine-synthesizing cells and fibers in the spinal cord of dogfish (Scyliorhinus canicula L.) was studied by means of immunohistochemistry using antibodies against tyrosine hydroxylase (TH). The only TH-immunoreactive (TH-ir) cells already present in the spinal cord of stage 26 embryos were of cerebrospinal fluid-contacting (CSF-c) type. These cells were the first catecholaminergic neurons of the dogfish CNS. The number of these TH-ir cells increased very considerably in later embryos and adult dogfish. In later embryos (stage 33; prehatching), faintly TH-ir non-CSF-contacting neurons were observed in the ventral horn throughout most of the spinal cord. In adult dogfish, some non-CSF-contacting TH-ir cells were observed ventral or lateral to the central canal. In the rostral spinal cord, the catecholaminergic neurons observed in dorsal regions were continuous with caudal rhombencephalic populations. Numerous TH-ir fibers were observed in the spinal cord of later embryos and in adults, both intrinsic and descending from the brain, innervating many regions of the cord including the dorsal and ventral horns. In addition, some TH-ir fibers innervated the marginal nucleus of the spinal cord. The early appearance of catecholaminergic cells and fibers in the embryonic spinal cord of the dogfish, and the large number of these elements observed in adults, suggests an important role for catecholamines through development and adulthood in sensory and motor functions.

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 γ‐aminobutyric acid (GABA), glutamic acid decarboxylase (GAD), and the GABAA receptor subunits δ and β2/β3, and with tract tracing methods. Two types of neuron were characterized: medium‐sized GABAergic neurons and small GABA‐negative granule cells. GABAA receptor subunit δ‐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. J. Comp. Neurol. 503:348–370, 2007.

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.

GABAergic system of the pineal organ of an elasmobranch (Scyliorhinus canicula): a developmental immunocytochemical study

The present immunocytochemical study provides evidence of a previously unrecognized, rich, γ-aminobutyric acid (GABA)-ergic innervation of the pineal organ in the dogfish (Scyliorhinus canicula). In this elasmobranch, the pineal primordium is initially detected at embryonic stage 24 and grows to form a long pineal tube by stage 28. Glutamic acid decarboxylase (GAD)-immunoreactive (-ir) fibers were first observed at stage 26, and by stage 28, thin GAD-ir fibers were detectable at the base of the pineal neuroepithelium. In pre-hatchling embryos, most fibers gave rise to GAD-ir boutons that were localized in the basal region of the neuroepithelium, although a smaller number of labeled terminals ascended to the pineal lumen. A few pale GAD-ir perikarya were observed within the pineal organ of stage 29 embryos, but GAD-ir perikarya were not observed at other developing stages or in adults. In contrast, GABA immunocytochemistry revealed the presence of GABAergic perikarya and fibers in the pineal organ of late stage embryos and adults. Although high densities of GABAergic cells were observed in the paracommissural pretectum, posterior tubercle, and tegmentum of dogfish embryos (regions previously demonstrated to contain pinealopetal cells), the presence of GABA-ir perikarya in the pineal organ strongly suggests that the rich GABAergic innervation of the elasmobranch pineal organ is intrinsic. This contrasts with the central origin of GABAergic fibers in the pineal gland of some mammals.

Temporal and spatial organization of tyrosine hydroxylase-immunoreactive cell groups in the embryonic brain of an elasmobranch, the lesser-spotted dogfish Scyliorhinus canicula.

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.