Distribution of calretinin during development of the olfactory system in the brown trout, Salmo trutta fario: Comparison with other immunohistochemical markers.

Immunocytochemical techniques were used to investigate the appearance and distribution of calretinin in the olfactory system of developing and adult brown trout (Salmo trutta fario L.). The earliest calretinin-immunoreactive (CR-ir) cells were detected in the olfactory placode of 5-mm embryos. In 8-mm embryos, a CR-ir olfactory nerve was observed. The number of CR-ir olfactory receptor cells increased rapidly, and in fry and adults they were characterized by light and electron microscopy as pertaining to three morphological types of receptor cell, called microvillous, ciliated and rod-like cells or crypt cells. Comparisons of the cells labeled with CR and with more general olfactory markers (acetylated tubulin and keyhole limpet haemocyanin) in alevins and fry revealed that CR-ir cells represent only a subpopulation of olfactory receptor cells. Large cells located in the primordial mitral cell layer were the first CR-ir neuronal population of the olfactory bulbs and were observed in 7-mm embryos. These cells express high HuC/D immunoreactivity and were negative for glutamic acid decarboxylase and tyrosine hydroxylase. CR immunoreactivity diminished with development and most large cells of the mitral cell layer were CR-negative in fry. In later embryos and in alevins, CR-ir granule-like cells were observed in the olfactory bulbs. Comparisons of the terminal fields of primary olfactory fibers labeled with CR and with a more general olfactory marker in the olfactory bulbs of fry and adults revealed significant differences, with most glomeruli of the dorsomedial field receiving CR-negative olfactory fibers. These results suggest new criteria for understanding the organization of the olfactory system of the trout, and hence of teleosts. Our results also suggest that CR is involved in specific functions in the olfactory system during development.

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.

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.

Anatomy of the Hesse photoreceptor cell axonal system in the central nervous system of amphioxus.

The present study reports the organization of the Hesse cell axonal system in the central nervous system of the amphioxus, with the use of a polyclonal antiserum raised against lamprey gonadotropin-releasing hormone-I (GnRH-I). In the spinal cord, the rhabdomeric photoreceptor cells of the bicellular organs were well labeled with this antibody. These cells sent smooth, straight, lateral processes that bent and became beaded as they passed ventrally and crossed to the contralateral side of the cord. There, the processes of several cells aggregated to give rise to a longitudinal fiber bundle. Beaded collaterals of these processes were directed to ventral neuropil and did not appear to contact giant Rohde cell axons. The crossed projections of the Hesse photoreceptors are compared with those of vertebrate retinal ganglion cells. Other antisera raised against GnRH weakly labeled rhabdomeric photoreceptors located dorsally in the brain, the Joseph cells. The finding that GnRH antibodies label amphioxus photoreceptor cells and axons is not definitive proof that the photoreceptors contain GnRH. Regardless of whether the antibody recognizes amphioxus GnRH, which has not yet been identified by structure, the antibody has revealed the processes of the Hesse photoreceptor cells.

Neuronal Organization of the Brain in the Adult Amphioxus (Branchiostoma lanceolatum): A Study With Acetylated Tubulin Immunohistochemistry.

Amphioxus (Cephalochordata) belongs to the most basal extant chordates, and knowledge of their brain organization appears to be key to deciphering the early stages of evolution of vertebrate brains. Most comprehensive studies of the organization of the central nervous system of adult amphioxus have investigated the spinal cord. Some brain populations have been characterized via neurochemistry and electron microscopy, and the overall cytoarchitecture of the brain was studied by Ekhart et al. (2003; J. Comp. Neurol. 466:319-330) with general staining methods and retrograde transport from the spinal cord. Here, the cytoarchitecture of the brain of adult amphioxus Branchiostoma lanceolatum was reinvestigated by using acetylated tubulin immunohistochemistry, which specifically stains neurons and fibers, in combination with some ancillary methods. This method allowed reproducible staining and mapping of types of neuron, mostly in brain regions caudal to the entrance level of nerve 2, and its comparison with spinal cord populations. The brain populations studied and discussed in detail were the Retzius bipolar cells, lamellate cells, Joseph cells, various types of translumenal cells, somatic motoneurons, Rohde nucleus cells, small ventral multipolar neurons, and Edinger cells. These observations expand our knowledge of the distribution of cell types and provide additional data on the number of cells and the axonal tracts and commissural regions of the adult amphioxus brain. The results of this comprehensive study provide a framework for comparison of complex adult populations with the early brain neuronal populations revealed in developmental studies of the amphioxus.

Somatomotor system of the adult amphioxus (Branchiostoma lanceolatum) revealed by an anticalretinin antiserum: An immunocytochemical study.

In a classic study with silver staining methods, the somatomotor system of the amphioxus spinal cord was described as consisting of three different types of neuron segmentally arranged in two opposite fan-shaped group types (Bone [1960] J Comp Neurol 115:27-64). The present study reports the presence of calretinin-like immunoreactivity in the somatomotor system of the amphioxus, which allows us to reevaluate old descriptions of amphioxus motoneurons. In the spinal cord, two types of calretinin-like immunoreactive (CR-ir) motoneurons, large and small, sent processes toward the ventrolateral region of the cord, where they branched and gave rise to processes coursing longitudinally in the somatomotor bundles. These processes produced a number of long and thin collaterals directed to several neuropil regions. Short collaterals were directed to the region of the neuromuscular contacts at the ventrolateral surface of the cord. The groups of CR-ir motoneurons exhibited a segmental organization and were localized only facing the myomeres, i.e., opposite to the entrance of the dorsal nerve roots, which is at variance with the above-mentioned classical report. CR-ir motoneurons were also observed in the brain between a level just rostral to the nerve III entry and nerve VI. The CR-ir somatomotor bundle ascended to the region of the neuromuscular junction of myomere 1. Additional faintly CR-ir neurons were observed in the region of the lamellate body of the brain. Our results reveal for the first time that calretinin immunoreactivity in the central nervous system of amphioxus was limited to a few types of neuron and that calretinin was not expressed in the peripheral nervous system, unlike vertebrates.

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 Cajal's 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).

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.

Presence of thyrotropin-releasing-hormone-immunoreactive (TRHir) amacrine cells in the retina of anuran and urodele amphibians.

The presence of thyrotropin-releasing-hormone-immunoreactive (TRH-ir) amacrine cells in the retina of amphibians is reported for the first time. The anuran and urodele retinas studied exhibit major differences in the distribution of TRH-ir cells. In the two urodele species investigated, most TRH-ir amacrine cells were located in the ganglion cell layer (GCL). These pear-shaped cells originate a dense TRH-ir dendritic plexus in strata 4-5 of the inner plexiform layer (IPL). A small number of TRH-ir amacrine cells were observed in the inner nuclear layer (INL). Most of these INL TRH-ir cells were multipolar neurons with radiating dendrites that originate a loose plexus in the IPL stratum 1. In the three anuran species investigated, most TRH-ir amacrine cells were located in the INL. Distribution of TRH-ir processes in the IPL of anurans was not so clearly layered as in urodeles, dendrites being observed throughout strata 1-5. In the toad retina THR-ir material was also observed in the outer plexiform layer, which suggests that toads may have some TRH-ir interplexiform neurons. In the frog and toad, TRH-ir fibers were also observed in the optic nerve, although their origin could not be ascertained. The number of TRH-ir amacrine cells per whole retina was higher in anurans than in urodeles, though urodeles have higher cell densities. The marked differences in distribution of TRH-ir amacrine cells observed between anurans and urodeles, and among the three anuran species, suggest different functions of TRH in retinal processing, perhaps related to the different specializations of the visual systems of these species.

Distribution of growth hormone-releasing hormone-like peptide: Immunoreactivity in the central nervous system of the adult zebrafish (Danio rerio).

The distribution of growth hormone-releasing hormone-like peptides (GHRH-LP) in the central nervous system of the zebrafish was investigated by using immunohistochemical techniques with polyclonal antibodies. ELISAs showed that the antiserum raised against salmon (s)GHRH-LP recognized both zebrafish GHRH-LP1 and -2, whereas the antiserum raised against carp (c)GHRH-LP was more sensitive but detected only zebrafish GHRH-LP1. Neither antiserum detected the true GHRH. Large cells in the nucleus lateralis tuberis were immunoreactive with both antisera, which suggests that they contained zebrafish GHRH-LP1, but not excluding GHRH-LP2. Also, immunoreactive fibers, which putatively originated from these hypothalamic neurons, were present in the hypophysis; both antisera detected fibers, although only sGHRH-LP antiserum stained fibers in the neurointermediate lobe. These fibers may have a neuroendocrine role. Candidates for a role in feeding include several areas in which both antisera labeled cells and fibers, implying a strong reaction for GHRH-LP1 and possibly GHRH-LP2. These areas include the isthmus with cells in the secondary gustatory/visceral nucleus, which were also calretinin immunoreactive. Numerous GHRH-LP-immunoreactive fibers (also labeled by both antisera) probably originate from the gustatory/visceral nucleus to innervate the ventral area of the telencephalon, preglomerular nuclei, torus lateralis and hypothalamic diffuse nucleus, habenula, torus semicircularis, and dorsolateral funiculus of the spinal cord. Present results in the zebrafish brain suggest involvement of GHRH-LP in both neuroendocrine and feeding-associated nervous circuits. The present data on the location of the two GHRH-LPs are the first clue to the possible functions of these two hormones.

Development of the inner ear of the brown trout (Salmo trutta fario): II. Cytodifferentiation and innervation of sensory cells.

The study of the sensory organs of the trout labyrinth by means of electron microscopy show that hair cells differentiate gradually in these organs; all of them produce new cells over a long period. The course of cytodifferentiation follows a similar pattern in all organs. Afferent nerve fibers and terminals are found at approximately the same time that sensory cells are being differentiated; the efferent synapses appear latter in development. The maturation of the both types of synapses is described. © 1993 Wiley-Liss, Inc.

Development of the inner ear of the brown trout (Salmo trutta fario): I. Gross morphology and sensory cell proliferation.

The gross development of the trout inner ear between embryonic and juvenile stages was studied by light microscopy. The otocyst has already formed in 3-4 mm embryos. The semicircular canals begin to separate from the utriculo-saccular cavity in 6 mm embryos, the anterior canal first, then the posterior and the horizontal canal later. The formation of the saccular cavity begins in 7 mm embryos, whereas that of the lagena occurs in 18 mm fry. The first macular primordia appear before the separation of cavities. The anterior and horizontal crests arise from the primordium of the utricular macula, and the posterior crest, macula lagena, and macula neglecta arise from that of the saccular macula. The macula lagena and macula neglecta appear later. The sensory areas of the labyrinth and the number of receptor cells grow continuously between the embryonic and juvenile stages. © 1993 Wiley-Liss, Inc.