Pilar Molist

Distribution of choline acetyltransferase immunoreactivity in the brain of an elasmobranch, the lesser spotted dogfish (Scyliorhinus canicula).

Although the distribution of cholinergic cells is remarkably similar across the vertebrate species, no data are available on more primitive species, such as cartilaginous fishes. To extend the evolutionary analysis of the cholinergic systems, we studied the distribution of cholinergic neurons in the brain and rostral spinal cord of Scyliorhinus canicula by immunocytochemistry using an antibody against the enzyme choline acetyltransferase (ChAT). Western blot analysis of brain extracts of dogfish, sturgeon, trout, and rat showed that this antibody recognized similar bands in the four species. Putative cholinergic neurons were observed in most brain regions, including the telencephalon, diencephalon, cerebellum, and brainstem. In the retrobulbar region and superficial dorsal pallium of the telencephalon, numerous small pallial cells were ChAT‐like immunoreactive. In addition, tufted cells of the olfactory bulb and some cells in the lateral pallium showed faint immunoreactivity. In the preoptic‐hypothalamic region, ChAT‐immunoreactive (ChAT‐ir) cells were found in the preoptic nucleus, the vascular organ of the terminal lamina, and a small population in the caudal tuber. In the epithalamus, the pineal photoreceptors were intensely positive. Many cells of the habenula were faintly ChAT‐ir, but the neuropil of the interpeduncular nucleus showed intense ChAT immunoreactivity. In the pretectal region, ChAT‐ir cells were observed only in the superficial pretectal nucleus. In the brainstem, the somatomotor and branchiomotor nuclei, the octavolateral efferent nucleus, and a cell group just rostral to the Edinger‐Westphal (EW) nucleus contained ChAT‐ir neurons. In addition, the trigeminal mesencephalic nucleus, the nucleus G of the isthmus, some locus coeruleus cells, and some cell populations of the vestibular nuclei and of the electroreceptive nucleus of the octavolateral region exhibited ChAT immunoreactivity. In the reticular areas of the brainstem, the nucleus of the medial longitudinal fascicle, many reticular neurons of the rhombencephalon, and cells of the nucleus of the lateral funiculus were immunoreactive to this antibody. In the cerebellum, Golgi cells of the granule cell layer and some cells of the cerebellar nucleus were also ChAT‐ir. In the rostral spinal cord, ChAT immunoreactivity was observed in cells of the motor column, the dorsal horn, the marginal nucleus (a putative stretch‐receptor organ), and in interstitial cells of the ventral funiculus. These results demonstrate for the first time that cholinergic neurons are distributed widely in the central nervous system of elasmobranchs and that their cholinergic systems have evolved several characteristics that are unique to this group. J. Comp. Neurol. 420:139–170, 2000.

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 serotoninergic system in the central nervous system of a shark, the lesser spotted dogfish Scyliorhinus canicula.

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. J. Comp. Neurol. 511:804–831, 2008.

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.

Organization of cholinergic systems in the brain of different fish groups: a comparative analysis

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.

A DiI-tracing study of the neural connections of the pineal organ in two elasmobranchs (Scyliorhinus canicula and Raja montagui) suggests a pineal projection to the midbrain GnRH-immunoreactive nucleus

Abstract. The pineal organ of elasmobranchs is an elongated photoreceptive organ. In order to investigate the afferent and efferent connections of the pineal organ of two elasmobranchs, the skate (Raja montagui) and the dogfish (Scyliorhinus canicula), a fluorescent carbocyanine (DiI) was applied to the pineal organ of paraformaldehyde-fixed brains. This application strongly labeled the pineal tract, which formed extensive bilateral projections. In both species, the pinealofugal fibers coursed to the dorsomedial thalamus, the medial pretectal area, the posterior tubercle, and the medial mesencephalic tegmentum and branched profusely in these areas. Application of DiI to the pineal organ also labeled occasional perikarya in the dorsomedial thalamus, posterior commissural region, posterior tubercle, and mesencephalic tegmentum. A comparison of these results with those of immunocytochemical analyses of the dogfish brain with an anti-salmon gonadotropin-releasing hormone (sGnRH) antiserum revealed a close topographical relation between the pineal projections and the midbrain sGnRH-immunoreactive (ir) nucleus, the only structure in the dogfish brain that contained sGnRHir neurons. This and the widespread distribution of sGnRHir fibers in the brain suggest that the midbrain sGnRHir nucleus is a part of the secondary pineal pathways and may be involved in light-mediated pineal regulation of brain function. Although GnRH distribution has not been studied in the skate, a midbrain GnRHir nucleus has been identified in three other elasmobranchs, including a skate relative. The probable existence of direct pineal projections to the GnRHir midbrain nucleus in elasmobranchs and other anamniotes is discussed.

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.

Marginal Cells in the Spinal Cord of Four Elasmobranchs (Torpedo marmorata, T. torpedo, Raja undulata and Scyliorhinus canicula): Evidence for Homology with Lamprey lntraspinal Stretch Receptor Neurons

This study reports for the first time the presence of marginal cells, probably with stretch receptor function, in the spinal cord of four elasmobranch species, two electric rays, Torpedo marmorata and T. torpedo, the skate Raja undulata and the dogfish Scyliorhinus canicula. In all four species, the marginal cells were located close to the lateroventral surface of the cord and possessed thick dendrites which formed part of characteristic glomerular structures. In vitro retrograde labelling of the spinal cord of the dogfish with horseradish peroxidase (HRP) showed that some of these cells have contralateral projections. Ultrastructural study of normal and retrograde HRP‐labelled material showed that the glomerular dendrites of marginal cells give rise to numerous fingerlike structures and are associated with a rich plexus of nerve terminals. Characteristically, these dendrites contain numerous mitochondria. Immunocytochemical studies revealed a rich plexus of somatostatin‐ and GABA‐immunoreactive fibres in the glomeruli. These results strongly suggest that the marginal cells of the elasmobranch spinal cord are stretch receptors homologous to lamprey edge cells and to the marginal nucleus cells of the spinal cord of urodeles and snakes. We discuss the possible role of these cells in the modulation of swimming movements.

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.