Distribution and development of FMRFamide-like immunoreactive neuronal systems in the brain of the brown trout, Salmo trutta fario.

The distribution of Phe-Met-Arg-Phe-amide (FMRFamide) peptide-immunoreactive (FMRF-ir) cells and fibers in the terminal nerve and central nervous system was investigated in developing stages and adults of the brown trout, Salmo trutta fario. The first FMRF-ir neurons appeared in the terminal nerve system of 8-mm embryos in and below the olfactory placode. In the brain, FMRF-ir neurons were first observed in the rostral hypothalamus, primordial hypothalamic lobe, mesencephalic laminar nucleus, and locus coeruleus of 12- to 13 -m embryos. After hatching, FMRF-ir cells appeared in the lateral part of the ventral telencephalic area and the anterior tuberal nucleus. In adult trout, FMRF-ir cells were observed in all these areas. The number of FMRF-ir neurons increased markedly in some of these populations during development. Dense innervation by FMRF-ir fibers was observed in the dorsal and lateral parts of the dorsal telencephalic area, and in the ventral telencephalic area, the lateral preoptic area, the medial hypothalamic and posterior tubercle regions, midbrain tegmentum and rhombencephalic reticular areas, the central gray, the superior raphe nucleus, the secondary visceral nucleus, the vagal nuclei, and the area postrema. Fairly rich FMRF-ir innervation was also observed in the optic tectum and some parts of the torus semicircularis. The saccus vasculosus and hypophysis received a moderate amount of FMRF-ir fibers. Innervation of most of these regions appeared either in late alevins or fry, although FMRF-ir fibers in the preoptic area, hypothalamus, and reticular areas appeared in embryos. Comparative analysis of the complex innervation pattern observed in the brain of trout suggests that FMRF is involved in a variety of functions, like the FMRF family of peptides in mammals.

Presence and development of thyrotropin-releasing hormone-immunoreactive amacrine cells in the retina of a teleost, the brown trout (Salmo trutta fario).

The presence of thyrotropin-releasing hormone-immunoreactive (TRHir) amacrine cells is described for the first time in the retina of a teleost. These amacrine cells were mostly located in the inner nuclear layer, with occasional perikarya in the ganglion cell layer. Their processes formed a conspicuous plexus at the level of the ganglion cell perikarya. The TRHir amacrine cells appeared in posthatching stages, with the total number in retinas of juveniles approximately four times the number of cells in adults. Two types of TRHir cells, large and small, can be distinguished in developing stages, small cells outnumbering large cells. The TRHir cells of adults appears mainly to correspond to large, multistratified amacrine cells of developing stages. The possibility of transient expression of TRH in small amacrine cells during development is discussed.

Development of thyrotropin-releasing hormone immunoreactivity in the brain of the brown trout Salmo trutta fario.

The development and adult distribution of thyrotropin-releasing hormone-immunoreactive (TRHir) neurons in the brain of the brown trout, Salmo trutta fario, was studied with the streptavidin-biotin immunohistochemical method. Study of embryos, alevin, and juveniles revealed groups of TRHir neurons in the mesencephalon and rhombencephalon that have not been noted previously in adult teleosts. The earliest TRHir cells observed were those of the trigeminal motor nucleus, which expressed this substance only in embryos and alevins. In the forebrain, early-arising TRH populations were observed in the supra- and postcommissural region of the ventral telencephalic area, the anterior parvocellular preoptic nucleus, the organon vasculosum laminae terminalis, the magnocellular preoptic nucleus, the suprachiasmatic nucleus, and the posterior tuberal nucleus. TRHir cells of the olfactory bulb, abundant in the adult, appeared later. A small TRHir neuronal population was transiently observed in the habenula of alevins and juveniles. The laminar nucleus of the mesencephalon contained a small population of TRH cells in alevins and juveniles. In the isthmus, TRH was observed in cells of the interpeduncular nucleus, the nucleus isthmi, the dorsolateral tegmental nucleus, the superior reticular nucleus, and the central gray, although perikarya were TRHir only in alevin and/or juvenile stages. Some vagal motoneurons were TRHir from the late embryo stage onward. TRHir fibers were abundant in several forebrain regions of alevins and juveniles, including the medial region of the dorsal telencephalic area, the ventral telencephalic area and commissural region, the preoptic neuropil, the posterior tubercle, the anterior tuberal nucleus, and the posterior hypothalamic lobe. TRHir fibers invaded the neurohypophysis in early alevins, and their number increased subsequently to adulthood. The parvocellular superficial pretectal nucleus and the optic tectum received a rich TRHir innervation from juvenile stages onward. The interpeduncular nucleus and the secondary gustatory nucleus contained many TRHir fibers. In the rhombencephalon, TRHir fibers were scarce, except in the ventrolateral regions and the inferior olive. The distribution of TRHir fibers suggests that they were mainly related to hypophysiotropic and visceral centers, although the presence of TRH in centers related to the visual system indicates that TRH also plays other roles in the brain. We discuss the possibility that the strong expression of TRH in the embryonic trigeminal motoneurons plays a role in head morphogenesis.

Development of immunoreactivity to neuropeptide Y in the brain of brown trout (Salmo trutta fario).

The development of neuropeptide Y-immunoreactive (NPY-ir) neurons in the brain of the brown trout, Salmo trutta fario, was studied by using the streptavidin-biotin immunohistochemical method. Almost all NPY-ir neurons found in the brain of adults already appeared in embryonic stages. The earliest NPY-ir neurons were observed in the laminar nucleus, the locus coeruleus, and the vagal region of 9-mm-long embryos. In the lateral area of the ventral telencephalon, habenula, hypothalamus, optic tectum, and saccus vasculosus, NPY-ir cells appeared shortly after (embryos 12-14 mm in length). The finding of NPY-ir cells in the saccus vasculosus and the vagal region expand the NPY-ir structures known in teleosts. Among the regions of the trout brain most richly innervated by NPY-ir fibers are the hypothalamus, the isthmus, and the complex of the nucleus of the solitary tract/area postrema, suggesting a correlation of NPY with visceral functions. Two patterns of development of NPY-ir populations were observed: Some populations showed a lifetime increase in cell number, whereas, in other populations, cell number was established early in development or even diminished in adulthood. These developmental patterns were compared with those found in other studies of teleosts and with those found in other vertebrates. J. Comp. Neurol. 414:13-32, 1999.

Expression of a low-molecular-weight (10 kDa) calcium binding protein in glial cells of the brain of the trout (Teleostei).

Calcium-binding proteins of the EF-hand family are widely distributed in the vertebrate central nervous system. In the present study of the trout brain, immunocytochemistry with a monoclonal antibody against chick gut calbindin-28k and a polyclonal antibody against bovine S100 protein specifically stained ependymocytes and radial glia cells with identical patterns. Western blot analysis of trout brain extracts with the antibodies to S100 and calbindin stained the same low-molecular-weight (10 kDa) protein band. In rat brain extracts, however, the monoclonal antibody to calbindin recognized a major protein band with molecular weight corresponding to that of calbindin-28k. This indicates that the trout protein is a new calcium-binding-like (calbindin-like) molecule that is immunologically related to both S100 and calbindin. Immunocytochemical studies of the trout brain using the antibodies to CaB and S100 showed that ependymocytes were stained in most ventricular regions, except in a few specialized ependymal areas of the ventral telencephalon, epithalamus, hypothalamus (including the paraventricular organ and saccus vasculosus) and brain stem. Immunocytochemistry also indicated the presence of calbindin-like protein in radial glia cells of several regions of the brain (thalamus, pretectal region, optic tectum, and rhombencephalon). Differences in immunoreactivity between neighbouring ependymal areas suggest that this protein may be a useful marker of different territories. All immunoreactive glial cells were nicotin-adenin-dinucleotide-phosphate diaphorase-positive, although this enzymohistochemical reaction is not specific for these glial cells since it reveals oligodendrocytes and some neurons. Immunoreactivity appears at different developmental stages in the different brain regions, with a broadly caudorostral gradient, suggesting that the expression of this protein is developmentally regulated. Comparison of the distribution of the calbindin-like protein with that of glial acidic fibrillary protein indicates that calbindin-like immunocytochemistry is a specific technique for revealing radial glia and ependymocytes in the trout.

Octavolateral neurons projecting to the middle and posterior rhombencephalic reticular nuclei of larval lamprey: a retrograde horseradish peroxidase labeling study.

The octavolateral area of lampreys, which receives primary fibers from the octaval and lateral line nerves, is involved in the premotor organization of body movements through secondary projections to the reticular formation. Here, the typology of neurons of the three octavolateral nuclei (ventral, medial, and dorsal) that putatively project to the middle and posterior rhombencephalic reticular nuclei were studied by retrograde transport of horseradish peroxidase (HRP) applied to these reticular nuclei. Several types of neurons were labeled in the ventral nucleus, both ipsilateral and contralateral to the site of HRP application. Some of these neurons showed a rather simple morphology (octavomotor neurons, monopolar cells), but most had more- or less-branched dendrites that were associated with one, or several, fields of terminal fibers in the octavolateral area. Unlike those of the ventral nucleus, labeled neurons of the medial nucleus were homogeneous in appearance (mostly pear-shaped). The dorsal nucleus was scarcely developed in larvae, as judged from the very simple and small labeled cells. The presence of terminal or "en-passant" boutons of secondary octavolateral fibers in the reticular area and the commissural nature of these fibers were also investigated by means of application of HRP or indocarbocyanine dye to the octavolateral nuclei. In addition, neurons of other alar plate nuclei that were labeled by the HRP application to the reticular nuclei (trigeminal descending root nucleus and solitary nucleus) were also characterized. The functional significance of these results is discussed.

The neuronal system of the saccus vasculosus of trout (Salmo trutta fario and Oncorhynchus mykiss): an immunocytochemical and nerve tracing study.

The neuronal system of the saccus vasculosus of two species of trout was studied with immunocytochemical methods and carboindocyanine-dye (DiI) tract-tracing. The cerebrospinal-fluid-contacting neurons of the saccus were immunoreactive for gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD), and neuropeptide Y (NPY). Immunostaining of alternate sections of the saccus vasculosus of fry with anti-GAD and anti-NPY indicated that these substances were colocalized. The tractus sacci vasculosi and the neuropil of the nucleus sacci vasculosi were also immunoreactive to these substances. The GABA, GAD, and neuropeptide Y immunoreactivity of the saccus vasculosus system appeared early in trout ontogeny. After applying DiI to various levels of the tractus sacci vasculosi of adult trout, we observed massive bilateral saccular projections to the nucleus sacci vasculosi and could follow the course of the sacco-thalamic tract. This tract extended in the subependymal region of the thalamus rostral to the nucleus sacci vasculosi and split into two small tracts that reached the subhabenular-preoptic region. Sacco-thalamic fibers formed extensive periependymal plexuses along their trajectory. Interestingly, no clear evidence of the existence of a saccopetal system was obtained. On the basis of these results, we postulate that the saccus vasculosus system modulates the function of centers of the posterior tubercle and periventricular thalamus.

Neurons of the olfactory organ projecting to the caudal telencephalon and hypothalamus: a carbocyanine-dye labelling study in the brown trout (Teleostei).

The caudal extrabulbar projections and their neurons of origin in the trout were studied after carbocyanine-dye (DiI) labelling in either the olfactory organ or the caudal telencephalon. DiI application to the caudal telencephalon labelled bipolar neurons in the olfactory epithelium, where they were sparsely distributed throughout the olfactory lamellae. Labelled fibres ran scattered in the olfactory nerve without forming bundles. DiI application to the olfactory organ labelled extrabulbar projections to the ventral telencephalon, preoptic region and tuberal hypothalamus. These results confirm that primary sensory fibres running in the medial olfactory tract of trout have an olfactory origin.

Ontogeny of somatostatin-immunoreactive systems in the brain of the brown trout (Teleostei).

The development of somatostatin-immunoreactive neurons and fibres was studied, using immunocytochemistry, in the brain of the brown trout. Somatostatinergic perikarya were found in many regions including several telencephalic areas, the preoptic nucleus, anterior tuberal and lateral tuberal nuclei, the lateral recess nucleus, dorsal tuberal nucleus, the pre- and pseudoglomerular nuclei, central thalamic nucleus, optic tectum, interpeduncular nucleus, several isthmal and reticular nuclei and the solitary fascicle nucleus. The ventrolateral area of the telencephalon and the nucleus lateralis tuberis are the first immunoreactive nuclei to appear in ontogeny, and cells of some telencephalic areas and of the lateral optic recess nucleus, the latest. Somatostatin-immunoreactive fibre tracts innervate the hypophysis and different regions of the brain. The most richly innervated areas in adults are the dorsolateral telencephalic area and the organon vasculosum laminae terminalis. Two patterns of production of somatostatinergic cells were observed: that of populations in which cell numbers increase over the lifetime of the fish, and that of populations whose cell number is established early in development or even diminishes in adulthood. These results provide interesting contrasts to those previously reported in birds and mammals.

The structure and development of dopaminergic interplexiform cells in the retina of the brown trout, Salmo trutta fario: a tyrosine hydroxylase immunocytochemical study.

The organisation and development of the dopaminergic (DA) system in the retina of the adult brown trout were studied with tyrosine hydroxylase immunocytochemical techniques. Adult DA cells are rather homogeneous in appearance and possess thick dendritic processes running to the ganglion cell layer and thinner axonal processes which run to the horizontal cell layer, where they form a rich plexus of varicose fibres closely associated with the surface of these cells. Contact of DA fibres with photoreceptor processes was not observed. We therefore consider this DA population to consist mainly of interplexiform cells. These cells appear late in development, being first observed in prehatching (16 mm) embryos (after photoreceptors have begun to differentiate). DA cells increased in number throughout the fry and juvenile stages, but even in the largest juveniles studied (30-35 mm) the size of the DA cell population was only about 20% of that in adults. DA cells appear to arise in the marginal retina. In developing stages (embryos and fry) only inner nuclear layer processes were observed, the horizontal cell layer DA plexus appearing late in development (28 mm juveniles).

Primary olfactory fibres project to the ventral telencephalon and preoptic region in trout (Salmo trutta): a developmental immunocytochemical study.

We studied the development of the primary olfactory system of a teleost, the brown trout, with the aims of clarifying whether the caudal projection pertains to the olfactory or to the terminal nerve system, of identifying the brain regions receiving this projection, and of investigating its possible functional significance. As olfactory markers (OMs) we used two polyclonal antibodies (to substance P and to alpha-melanocyte-stimulating hormone) that were found to label the olfactory projection strongly after preadsortion of the antibody with the corresponding antigen (OMs), and as a terminal nerve marker we used an antiserum to FMRF-amide peptide. OM labelling was observed in both perikarya and axons of olfactory neurons. In adults, olfactory neurons projected not only to olfactory glomeruli in the olfactory bulb but also, as has been reported previously, to more caudal targets in the forebrain through the medial olfactory tract. Our results show that these targets include the ventral and commissural nuclei of the area ventralis telencephali, the periventricular preoptic region, and the organum vasculosum laminae terminalis. Glomeruli were not observed before hatching, and the extrabulbar olfactory projections appear late in development. Extensive periventricular preoptic olfactory plexuses and olfactory innervation of the organum vasculosum laminae terminalis did not appear until adulthood. The cells of the ganglion nervus terminalis, which form ganglionic groups along the olfactory nerves, were not stained with these olfactory markers at any developmental stage studied, nor was the medial olfactory tract FMRP-amide peptide immunoreactive. Our results thus confirm the existence of primary olfactory projections to extrabulbar targets in trout. The target regions identified in this study are implicated in sexual behaviour: We discuss the related possibility that, in teleosts, these extrabulbar olfactory projections (rather than projections of the terminal nerve, as is widely held) are the primary mediators of neuroendocrine response to pheromones.

Distribution of substance P-like immunoreactivity in the brain of the elasmobranch Scyliorhinus canicula.

Immunohistochemical methods were used to study the distribution of substance P in the brain of the small-spotted dogfish (Scyliorhinus canicula). Substance P-like immunoreactive (SP-IR) cell bodies and fibers were widely distributed. In the telencephalon, sparse populations of SP-IR neurons are present in the olfactory bulbs, pallium, and subpallium. In the subpallium numerous SP-IR boutons form unusual coats ("pericellular appositions") on SP-immunonegative neurons. In the diencephalon numerous SP-IR cerebrospinal fluid-contacting neurons are present in the preoptic recess organ and organon vasculosum hypothalami. Numerous SP-IR fibers also run in the hypothalamus, although no immunoreactivity was observed in the habenulo-interpeduncular system. A terminal field of SP-IR fibers is present in the median eminence. In the mesencephalic tegmentum, SP-IR neurons were observed in the Edinger-Westphal nucleus. SP-IR fibers are present at high density in the basal tegmentum, forming a conspicuous tract. In the hindbrain, numerous SP-IR fibers were observed in the isthmal region, the trigeminal descending root, the visceral sensory area and commissural nucleus, and the visceromotor column. SP-IR fibers occur at high density in the substantia gelatinosa of the rostral spinal cord.

Golgi study of the telencephalon of the small-spotted dogfish Scyliorhinus canicula L.

The telencephalon of the small-spotted dogfish, Scyliorhinus canicula L., was examined by Nissl and Golgi-aldehyde techniques. On the basis of differences in perikaryal and dendritic morphology and size, several cell types were distinguished in pallial and subpallial regions, most of them reported here for the first time in elasmobranchs. In the pallium, the pallium dorsalis is the richest in cell types (eight types of neurons), whereas the neuron population of the pallium medialis is the most homogeneous. Dendrites of most neuron types in the pallium are smooth or sparsely thorny. Interestingly, the pallium dorsalis and pallium lateralis contain a type of primitive pyramidal cell characterized by the dense appearance of its thorny dendrites. In the subpallium, the area superficialis basalis contains a heterogeneous population (six types of neurons): large radial cells are the most characteristic cell type. Dendrites of these cell types are smooth or sparsely thorny. The cell populations of the nucleus N are roughly similar to those of the area superficialis basalis, but they lack the large radial cells characteristic of this area. The area centralis subpallialis and striatum consist of populations of small neurons. The regio septalis contains a rather homogeneous population of small cells. The populations in the nucleus entopeduncularis and the nucleus interstitialis of the basal forebrain bundle are the least varied and consist of large radial cells and bipolar cells similar to those of the area superficialis basalis. This investigation reveals important differences in cytoarchitecture that should be useful in the interpretation of immunocytochemical, tracing, and electrophysiological studies of the telencephalon of elasmobranchs.

Distribution and development of catecholaminergic neurons in the brain of the brown trout. A tyrosine hydroxylase immunohistochemical study.

We report the distribution of tyrosine hydroxylase immunoreactive (TH-ir) neurons in the brain of adult and developing trout. In the adult brain TH-ir neurons were observed in the telencephalon (in the olfactory bulbs and the ventral telencephalic region), the diencephalon (in both the parvicellular and magnocellular portions of the preoptic nucleus, the suprachiasmatic nucleus, the ventromedial thalamic nucleus, the posterior tuberal nucleus, the organon vasculosum hypothalami, the lateral recess nucleus and the dorsal periventricular region of the medial lobe recess), the pretectal region (nucleus pretectalis periventricularis), the isthmal region (locus coeruleus) and the medulla oblongata (in the area postrema, nucleus solitarius, the reticular area of the vagal region, and the reticular nucleus of the octaval region). The earliest TH-ir nuclei were observed in 10- to 11-min embryos in the posterior tuberal nucleus, locus coeruleus, nucleus solitarius and vagal reticular neurons. In the olfactory bulb, nucleus ventromedialis thalami and octaval reticular area, TH-ir cells a latter appeared at a later stage in development. In fry, juveniles and adults cerebrospinal fluid-contacting TH-ir cells were observed in circumventricular organs. The evolution of these TH-ir neuronal populations during development is described, and we discuss their significance for both comparative and developmental analysis.

Distribution of types of neurons in the optic tectum of the small-spotted dogfish, Scyliorhinus canicula L. A Golgi study.

The distribution of neuronal types in the six layers of the optic tectum of the small-spotted dogfish, Scyliorhinus canicula, was studied using Golgi methods. The eight types of neurons have a variable distribution, some of them represented in all six tectal layers. Most neurons of the optic tectum have a radial orientation, and a small portion are disposed horizontally. The relative ratios of the different neuronal types in the six layers indicate that about 80% of the tectal neurons are intermingled with the optic nerve axon terminals in the superficial layers of the tectum. These results corroborate previous reports that the tectum of this selachian species is more specialized than formerly supposed, in spite of the differences in degree of neuropil lamination with other non-mammalian vertebrates.

Specialized presynaptic dendrites in the stratum cellulare externum of the optic tectum of an elasmobranch, Scyliorhinus canicula L.

Electron microscopy of the stratum cellulare externum of the optic tectum of an elasmobranch revealed the presence of two types of presynaptic dendrites in the neuropil as well as axo-dendritic synapses. In the dendro-dendritic or dendro-axonic synapses, the presynaptic process was a beaded dendrite. These findings support the view that the synaptic organization of the tectum in elasmobranchs is basically similar to that of higher vertebrates, rather than the classical opinion that it is less highly organized.

The optic tectum of the dogfish Scyliorhinus canicula L.: a Golgi study.

The optic tectum of the dogfish Scyliorhinus canicula L. was studied by using the methods of Nissl, reduced silver nitrate, Golgi-aldehyde, and Golgi-Cox. Six layers and eight types of neurons were recognized. These cell types are not restricted to one layer; in fact some are found in all six tectal layers. The types of cells found are A) monopolar, B) triangular, C) radial bipolar, D) horizontal fusiform, E) large tectal, F) small tectal, G) pyriform, and H) stellate cells. In at least six of the cell types a series of dendritic specializations can be observed, such as spines in the form of "drumsticks" and thin varicose appendages, similar to those reported previously in the optic tecta of amphibians and teleosts. The optic tectum of the dogfish shows a degree of complexity comparable to that of amphibians and teleosts.