Crypt cells of the zebrafish Danio rerio mainly project to the dorsomedial glomerular field of the olfactory bulb.

The olfactory mucosa of the zebrafish consists of 3 morphological types of olfactory receptor neurons (ORNs): ciliated, microvillous, and crypt cells. Previous studies in the zebrafish have revealed differential projections of ciliated and microvillous ORNs, which project to different glomerular fields. However, the bulbar targets of zebrafish crypt cells were not identified. Here, we analyze the relationship between crypt cells of the olfactory epithelium and dorsal glomerular fields of the zebrafish olfactory bulbs, as wells as the connections between these bulbar regions and forebrain regions. For this purpose, a lipophilic carbocyanine tracer (DiI) was used in fixed tissue. Application of DiI to the dorsomedial glomerular field mainly labeled crypt cells in the zebrafish olfactory epithelium. By contrast, application of DiI to the dorsolateral glomerular fields mainly labeled bipolar ORNs and only occasionally crypt cells. Bulbar efferent cells (mitral cells) contacting these dorsal glomerular fields project to different telencephalic areas, with the posterior zone of the dorsal telencephalic area (Dp) as the common target. However, dorsomedial and dorsolateral glomerular fields projected differentially to the ventral telencephalon, the former projecting to the ventrolateral supracommissural region. Retrograde labeling from the ventrolateral supracommissural region revealed mitral cells associated with 2 large glomeruli in the bulbar dorsomedial region, which putatively receives inputs from the crypt cells, indicating the existence of a crypt cell olfactory subsystem with separate projections, in the zebrafish. The comparative significance of the secondary olfactory pathways of zebrafish that convey information from crypt cells is discussed.

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.

Distribution of thyrotropin-releasing hormone immunoreactivity in the brain of the dogfish Scyliorhinus canicula.

To improve knowledge of the peptidergic systems of elasmobranch brains, the distribution of thyrotropin-releasing hormone-immunoreactive (TRHir) neurons and fibers was studied in the brain of the small-spotted dogfish (Scyliorhinus canicula L.). In the olfactory bulbs, small granule neurons richly innervated the olfactory glomeruli. In the telencephalic hemispheres, small TRHir neurons were observed in the superficial dorsal pallium, whereas TRHir fibers were widely distributed in pallial and subpallial regions. In the preoptic region, TRHir neurons formed a caudal ventrolateral group in the preoptic nucleus. In the hypothalamus, the most conspicuous TRHir populations were associated with the lateral hypothalamic recess, but small TRHir populations were found in the posterior tubercle and ventral wall of the posterior recess. The preoptic region and hypothalamus exhibited rich innervation by TRHir fibers. TRHir fibers were observed coursing to the neurohypophysis and the neuroepithelium of the saccus vasculosus, but not to the neurohemal region of the median eminence. Some stellate-like TRHir cells were observed in a few cell cords of the neurointermediate lobe of the hypophysis. The thalamus, pretectum, and midbrain lacked TRHir neurons. Further TRHir neuronal populations were observed in the central gray and superior raphe nucleus of the isthmus, and a few TRHir cells were located in the nucleus of the trigeminal descending tract at the level of the rostral spinal cord. In the brainstem, the central gray, interpeduncular nucleus, secondary visceral region of the isthmus, rhombencephalic raphe, inferior olive, vagal lobe, and Cajal's commissural nucleus were all richly TRHir-innervated. Comparison of the distribution of TRHir neurons observed in the dogfish brain with that observed in teleosts and tetrapods reveals strong resemblance but also interesting differences, indicating the presence of both a conserved basic vertebrate pattern and a number of derived characters.

Distribution of neuropeptide Y immunoreactivity in the central and peripheral nervous systems of amphioxus (Branchiostoma lanceolatum Pallas).

Immunocytochemistry techniques were employed to investigate the distribution of neuropeptide Y-like-immunoreactive (NPY-ir) cells and fibers in the central and peripheral nervous systems of adult amphioxus. NPY-ir neurons of the commissural type were abundant in the brain and present but more scarce in the spinal cord. These neurons gave rise to conspicuous NPY-ir tracts that coursed along the entire length of the nerve cord. Some fibers exhibited conspicuous Herring body-like swellings. In the peripheral nervous system, small NPY-ir neurons and a large number of thin, beaded NPY-ir fibers were observed in the atrial region, indicating the involvement of this substance in visceral regulation. A few NPY-ir fibers, possibly afferent to the spinal cord, coursed in the ventral branches of the spinal nerves of this region, whereas no NPY-ir fibers coursed in the preoral or velar nerves or in the dorsal branches of the other spinal nerves. These results indicate that NPY is widely used as a neuroregulator/neurotransmitter in the central and peripheral nervous systems of this primitive chordate. In addition, this study demonstrates the presence of tall, thin NPY-ir cells in the putative adenohypophyseal homologue, the Hatschek's pit organ, which is located in the roof of the preoral cavity (vestibule).

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.

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 of thyrotropin-releasing hormone immunoreactivity in the brain of larval and adult sea lampreys, Petromyzon marinus L.

This study investigated the distribution of thyrotropin-releasing hormone-immunoreactive (TRHir) neurons and fibers in the brain and retina of lampreys. Our results in the brains of large larvae and upstream-migrating adults of the sea lamprey showed the presence of TRHir neurons mainly in the preoptic region and the hypothalamus. A few TRHir neurons were also found in the striatum. The number and staining intensity of TRHir neurons increased from larval stages to adulthood, and the distribution of TRHir populations was wider in adults. The TRHir fibers were more easily traced in adults. Some TRHir fibers entered the neurohypophysis, although most fibers coursed in the different regions of the brain, mostly in the basal region, from the forebrain to the hindbrain. The presence of TRHir stellate cells was observed in the adenohypophysis. In the retina of adult lampreys, but not in that of larvae, TRHir amacrine cells are present.

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 Gα(olf) 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.

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.