María Jesús Manso

Calretinin immunoreactivity in the brain of the zebrafish, Danio rerio: distribution and comparison with some neuropeptides and neurotransmitter-synthesizing enzymes. I. Olfactory organ and forebrain.

The distribution of calretinin (CR) in the forebrain and the olfactory system of the adult zebrafish was studied by using immunocytochemical techniques. Previous studies in trout forebrain have indicated that CR‐immunoreactive neurons acquire this phenotype rather early in development (Castro et al., J. Comp. Neurol. 467:254–269, 2003 ). Thus, precise knowledge of CR‐expressing neuronal populations in adult zebrafish may help to decipher late stages of forebrain morphogenesis. For analysis of some forebrain nuclei and regions, CR distribution was compared with that of various ancillary markers: choline acetyltransferase, glutamic acid decarboxylase, tyrosine hydroxylase, neuropeptide Y, thyrotropin‐releasing hormone, and galanin. The results reveal that calretinin is a specific marker of olfactory receptor neurons and of various neuronal populations distributed throughout the telencephalon and diencephalon. In addition, CR immunocytochemistry revealed characteristic patterns of fibers and neuropil in several telencephalic and diencephalic regions, indicating that it is a useful marker for characterizing a number of neural centers, pathways, and neuronal subpopulations in the zebrafish forebrain. Some ancillary markers also showed a distinctive distribution in pallial and subpallial regions, revealing additional aspects of forebrain organization. Comparison of the distribution of CR observed in the forebrain of zebrafish with that reported in other teleosts revealed a number of similarities and also some interesting differences. This indicates that various neuronal populations have maintained the CR phenotype in widely divergent teleost lines and suggests that CR studies may prove very useful for comparative analysis. J. Comp. Neurol. 494:435–459, 2006.

Differential bulbar and extrabulbar projections of diverse olfactory receptor neuron populations in the adult zebrafish (Danio rerio).

Immunohistochemical methods were used to characterize the expression of two calcium‐binding proteins, calretinin (CR) and S100, in the olfactory rosette of the adult zebrafish. These proteins are expressed in different sets of sensory neurons, and together represent a large proportion of these cells. Double immunofluorescence for CR and Gαolf protein, and CR immunoelectron microscopy, indicated that most CR‐immunoreactive (ir) cells were ciliary neurons. Differential S100‐ and CR‐ir projections to glomerular fields of the olfactory bulb were also observed, although these projections overlap in some glomeruli. Application of the carbocyanine dye DiI to either S100‐ir or CR‐ir glomerular regions led to labeling of cells mostly similar to S100‐ir and CR‐ir neurons, respectively. Instead, these bulbar regions project to similar telencephalic targets. On the other hand, antibodies against keyhole limpet hemocyanin (KLH)‐stained numerous sensory cells in the olfactory rosette, including cells that were CR‐ and S100‐negative. This antiserum also stained most primary bulbar projections and revealed extrabulbar olfactory primary projections coursing to the ventral area of the telencephalon through the medial olfactory tract. This extrabulbar projection was confirmed by tract‐tracing with DiI. A loose association of this extrabulbar primary olfactory projection and the catecholaminergic populations of the ventral area was also observed with double tyrosine hydroxylase/KLH‐like immunohistochemistry. Comparison between KLH‐like‐ir pathways and the structures revealed by FMRFamide immunohistochemistry (a marker of terminal ganglion cells and fibers) indicated that the KLH‐like‐ir extrabulbar projection was different from the terminal nerve system. The significance of the extrabulbar olfactory projection of zebrafish is discussed. J. Comp. Neurol. 519:247‐276, 2011.

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.

Calretinin immunoreactivity in the brain of the zebrafish, Danio rerio: Distribution and comparison with some neuropeptides and neurotransmitter-synthesizing enzymes. II. Midbrain, hindbrain, and rostral spinal cord

The distribution of calretinin (CR) in the brainstem and rostral spinal cord of the adult zebrafish was studied by using immunocytochemical techniques. For analysis of some brainstem nuclei and regions, CR distribution was compared with that of complementary markers (choline acetyltransferase, glutamic acid decarboxylase, tyrosine hydroxylase, neuropeptide Y). The results reveal that CR is a marker of various neuronal populations distributed throughout the brainstem, including numerous cells in the optic tectum, torus semicircularis, secondary gustatory nucleus, reticular formation, somatomotor column, gustatory lobes, octavolateral area, and inferior olive, as well as of characteristic tracts of fibers and neuropil. These results indicate that CR may prove useful for characterizing a number of neuronal subpopulations in zebrafish. Comparison of the distribution of CR observed in the brainstem of zebrafish with that reported in an advanced teleost (the gray mullet) revealed a number of similarities, and also some interesting differences. Our results indicate that many brainstem neuronal populations have maintained the CR phenotype in widely divergent teleost lines, so CR studies may prove very useful for comparative analysis. J. Comp. Neurol. 494:792–814, 2006.

Distribution and development of calretinin-like immunoreactivity in the telencephalon of the brown trout, Salmo trutta fario.

Immunocytochemical techniques were used to investigate the distribution of calretinin (CR) in the telencephalon of adult and developing brown trout (Salmo trutta fario L.). Previous immunoblotting analysis of trout brain extracts with a CR antibody revealed a single protein band of 29 kDa, similar to that observed in rat brain extracts. In the forebrain of adult trout, CR immunoreactivity was distributed in well‐defined cell groups, which allowed us to analyze the CR‐immunoreactive (ir) neuronal populations in terms of their respective regions of origin. Our results show that the CR‐ir populations of the dorsal and ventral telencephalon are differentially distributed along the rostrocaudal axis, indicating the existence of four main populations of pallial origin and several ventral (subpallial) populations. A highly specific pattern of innervation by CR‐ir fibers of different telencephalic regions was observed from alevins to adults. The first CR‐ir cell groups of the telencephalic hemispheres were observed in the ventral telencephalic area and preoptic region of 7–8‐mm embryos. In later embryos and in alevins, further CR‐ir cell groups appeared in the ventral and dorsal telencephalic areas, showing a dorsoventrally banded pattern at precommissural levels. Study of CR expression provided new criteria for understanding the organization of the telencephalon of trout, and hence of teleosts. J. Comp. Neurol. 467:254–269, 2003.

Distribution of thyrotropin-releasing hormone (TRH) immunoreactivity in the brain of the zebrafish (Danio rerio).

The distribution of thyrotropin‐releasing hormone (TRH) in the brain of the adult zebrafish was studied with immunohistochemical techniques. In the telencephalon, abundant TRH‐immunoreactive (TRHir) neurons were observed in the central, ventral, and supra‐ and postcommissural regions of the ventral telencephalic area. In the diencephalon, TRHir neurons were observed in the anterior parvocellular preoptic nucleus, the suprachiasmatic nucleus, the lateral hypothalamic nucleus, the rostral parts of the anterior tuberal nucleus and torus lateralis, and the posterior tuberal nucleus. Some TRHir neurons were also observed in the central posterior thalamic nucleus and in the habenula. The mesencephalon contained TRHir cells in the rostrodorsal tegmentum, the Edinger‐Westphal nucleus, the torus semicircularis, and the nucleus of the lateral lemniscus. Further TRHir neurons were observed in the interpeduncular nucleus. In the rhombencephalon, TRHir cells were observed in the nucleus isthmi and the locus coeruleus, rostrally, and in the vagal lobe and vagal motor nucleus, caudally. In the forebrain, TRHir fibers were abundant in several regions, including the medial and caudodorsal parts of the dorsal telencephalic area, the ventral and commissural parts of the ventral telencephalic area, the preoptic area, the posterior tubercle, the anterior tuberal nucleus, and the posterior hypothalamic lobe. The dorsal thalamus exhibited moderate TRHir innervation. In the mesencephalon, the optic tectum received a rich TRHir innervation between the periventricular gray zone and the stratum griseum centrale. A conspicuous TRHir longitudinal tract traversed the tegmentum and extended to the rhombencephalon. The medial and lateral mesencephalic reticular areas and the interpeduncular nucleus were richly innervated by TRHir fibers. In the rhombencephalon, the secondary gustatory nucleus received abundant TRHir fibers. TRHir fibers moderately innervated the ventrolateral and ventromedial reticular area and richly innervated the vagal lobe and Cajals commissural nucleus. Some TRHir fibers coursed in the lateral funiculus of the spinal cord. Some TRHir amacrine cells were observed in the retina. The wide distribution of TRHir neurons and fibers observed in the zebrafish brain suggests that TRH plays different roles. These results in the adult zebrafish reveal a number of differences with respect to the TRHir systems reported in other adult teleosts but were similar to those found during late developmental stages of trout (Díaz et al., 2001 ). J. Comp. Neurol. 450:45–60, 2002.

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

Abstract 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.

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. J. Comp. Neurol. 440:43–64, 2001.

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. J. Comp. Neurol. 429:299–320, 2001.

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.