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Chondrychthyans (cartilaginous fishes) are key to understanding the ancestral gnathostome condition since they provide an outgroup to sarcopterygians and actinopterygians. To gain comparative knowledge about the development of the vertebrate serotoninergic systems, we studied by immunohistochemistry the origin, spatiotemporal organization, and migration patterns of serotonin-containing neurons and the growth of axonal pathways in the central nervous system of a shark, the lesser spotted dogfish. Hindbrain serotonin-immunoreactive cells arose close to the floor plate and most populations migrated ventrally and mediolaterally to form the various raphe and reticular groups. The order of appearance of serotoninergic populations in the rhombencephalon and spinal cord (first the superior groups and then the inferior and spinal populations) roughly matched with that reported in other vertebrates but important differences were noted in the formation of prosencephalic groups in fishes. In addition to preoptic and hypothalamic areas, serotoninergic cerebrospinal fluid-contacting cells were observed in the isthmus (raphe dorsalis anterioris). Transient serotonin-immunoreactive cells were noted in the pineal organ, habenula, and pretectum. Further, we provide a revised anatomical framework for reticular and raphe serotoninergic populations considering their origin and segmental organization. Two distinct phases of development of the serotoninergic innervation were distinguished, that of the formation of the main axonal pathways and that of the branching of fibers. The development of main serotoninergic ascending pathways in dogfish was notably similar to that described in mammals. Our findings suggest the conservation of developmental patterns in serotoninergic systems and enhance the importance of elasmobranchs for understanding the early evolution of this system in vertebrates.

The adult distribution and development of gamma-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 gamma-aminobutyric acid in sensory and motor circuits of the spinal cord.

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.