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.

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.

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.

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.

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).