Glycine‐immunoreactive neurons in the brain of a shark (Scyliorhinus canicula L.)

The glycinergic cell populations in the brain of the lesser spotted dogfish were studied by a glycine immunofluorescence method. Numerous glycine‐immunoreactive (Gly‐ir) neurons were observed in different brain nuclei. In the telencephalon, Gly‐ir cells were observed in the olfactory bulb, telencephalic hemispheres, and preoptic region. In the hypothalamus, cerebrospinal fluid‐contacting Gly‐ir neurons were observed in the lateral and posterior recess nuclei. Coronet cells of the saccus vasculosus were Gly‐ir. In the diencephalon, Gly‐ir neurons were observed in the prethalamus and pretectum. In the midbrain, both the optic tectum and lateral mesencephalic nucleus contained numerous Gly‐ir neurons. In the cerebellum, many Golgi cells were Gly‐ir. In the rhombencephalon, Gly‐ir cells were observed in the medial and ventral octavolateral nuclei, vagal lobe, visceromotor nuclei, and reticular formation, including the inferior raphe nucleus. In the spinal cord, some neurons of the marginal nucleus and some cells of the dorsal and ventral horns were Gly‐ir. Comparison of dogfish Gly‐ir cell populations with those reported for the sea lamprey, Siberian sturgeon, and zebrafish revealed some shared features but also notable differences. For example, Gly‐ir cells were observed in the dogfish cerebellum, unlike the case in the Siberian sturgeon and zebrafish, whereas the absence of Gly‐ir neurons in the isthmus is shared by all these species, except for lampreys. Gly‐ir populations in the dogfish hypothalamus and telencephalon are notable in comparison with those of the other jawed vertebrates investigated to date. Together, these results reveal a complex and divergent evolution of glycinergic systems in the major groups of fishes. J. Comp. Neurol. 521: 3057–3082, 2013. © 2013 Wiley Periodicals, Inc.     

Development of galanin-like immunoreactivity in the brain of the brown trout (Salmo trutta fario), with some observations on sexual dimorphism

The development of galanin-like immunoreactive (GAL-ir) cells and fibers was investigated in the brain of brown trout embryos, alevins, juveniles, and adults (some spontaneously releasing their gametes). The earliest GAL-ir neurons appeared in the preoptic region and the primordial hypothalamic lobe of 12-mm embryos. After hatching, new GAL-ir neurons appeared in the lateral, anterior, and posterior tuberal nuclei, and in late alevins, GAL-ir neurons appeared in the area postrema. In juveniles, further GAL-ir populations appeared in the nucleus subglomerulosus and magnocellular preoptic nucleus. The GAL-ir neuronal groups present in juveniles were also observed in sexually mature adults, although the area postrema of males lacked immunoreactive neurons. Moreover, spawning males exhibited GAL-ir somata in the olfactory bulb and habenula, which were never observed in adult females or in developing stages. In adults, numerous GAL-ir fibers were observed in the ventral telencephalon, preoptic area, hypothalamus, neurohypophysis, mesencephalic tegmentum, ventral rhombencephalon, and area postrema. Moderate to low GAL-ir innervation was seen in the olfactory bulbs, dorsomedial telencephalon, epithalamus, medial thalamus, optic tectum, cerebellum, and rhombencephalic alar plate. There were large differences among regions in the GAL-ir innervation establishment time. In embryos, GAL-ir fibers appeared in the preoptic area and hypothalamus, indicating early expression of galanin in hypophysiotrophic centers. The presence of galanin immunoreactivity in the olfactory, reproductive, visual, and sensory-motor centers of the brain suggest that galanin is involved in many other brain functions. Furthermore, the distribution of GAL-ir elements observed throughout trout development indicates that galaninergic system maturation continues until sexual maturity.     

Localization of FMRFamide-like immunoreactivity in the brain of the viviparous skink (Chalcides chalcides)

Neuroanatomical distribution of FMRFamide-like immunoreactivity was investigated in the brain and olfactory system of the viviparous skink, Chalcides chalcides. In the adult brain FMRFamide immunoreactive (ir) perikarya were observed in the diagonal band of Broca, medial septal nucleus, accumbens nucleus, bed nucleus of the anterior commissure, periventricular hypothalamic nucleus, lateral forebrain bundle, and lateral preoptic, subcommissural, suprachiasmatic and lateral hypothalamic areas. This pattern was seen in both male and female brains. Though all major brain areas showed FMRFamide-ir innervation, the densest ir fiber network was observed in the hypothalamus. During development, ir elements were observed for the first time in embryos at mid-pregnancy. FMRFamide perikarya were located along the ventral surface of the vomeronasal nerve, in the olfactory peduncle mediobasally, as well as in the anterior olfactory nucleus and olfactory tubercle. Furthermore, some ir neurons were observed in the rhombencephalic reticular substance; however, the ir fiber network was poorly developed. Later in development FMRFamide-ir neurons appeared also in the bed nucleus of the anterior commissure as well as the rhombencephalic nucleus of solitary tract and the dorsal motor nucleus of vagus nerve. In juveniles, the distribution profile of FMRFamide immunoreactivity was substantially similar to that of the adults, with a less widespread neuronal distribution and a more developed fiber network. Ontogenetic presence of FMRFamide immunoreactivity in the nasal area has been linked to the presence of a nervus terminalis in this reptile.     

Immunocytochemical demonstration of salmon GnRH and chicken GnRH-II in the brain of masu salmon, Oncorhynchus masou.

We have recently developed sensitive and specific radioimmunoassays (RIAs) for salmon gonadotropin-releasing hormone (sGnRH) and chicken GnRH-II (cGnRH-II) and have measured the contents of both GnRHs in the rainbow trout brain. Our results showed that contents of the two GnRHs are variable among different brain regions. Therefore, in order to confirm the differential distribution of the two GnRHs by a different technique, we examined the distribution of immunoreactive sGnRH and cGnRH-II in the brain of masu salmon by using immunocytochemical techniques. sGnRH immunoreactive (ir) cell bodies were scattered in the transitional areas between the olfactory nerve and the olfactory bulb, the ventral olfactory bulb, between the olfactory bulb and the telencephalon, the ventral telencephalon, and the preoptic area. These sGnRH-ir cell bodies were dispersed in a strip-like region running rostrocaudally in the most ventral part of the ventral telencephalon. sGnRH-ir fibers were distributed in the various brain regions from the olfactory bulb to the spinal cord. They were especially abundant in the olfactory bulb, ventral telencephalon, preoptic area, hypothalamus, deep layers of the optic tectum, and thalamus. sGnRH-ir fibers also innervated the pituitary directly. cGnRH-II-ir cell bodies were found in the nucleus of the medial longitudinal fasciculus (nMLF). The distribution of cGnRH-II-ir fibers was similar to that of sGnRH-ir fibers, except that cGnRH-II-ir fibers were absent in the pituitary. The number of cGnRH-II-ir fibers was much fewer than that of sGnRH-ir fibers. The results of the present immunocytochemical study are in basic agreement with those of our previous RIA study. Thus, we suggest that in masu salmon, sGnRH not only regulates gonadotropin (GTH) release from the pituitary but also functions as a neuromodulator in the brain, whereas cGnRH-II functions only as a neuromodulator.     

The organization of CRF neuronal pathways in toads: Evidence that retinal afferents do not contribute significantly to tectal CRF content

Previous work has suggested that the peptide corticotropin-releasing factor (CRF) acts to inhibit visually guided feeding in anurans, but little is known about potential targets for CRF within the subcortical visuomotor circuitry. Here we investigated the relationship between CRF neuronal organization and visual pathways in toads. CRF-immunoreactive (ir) neurons and fibers were widely distributed throughout the ventral subpallial telencephalon and hypothalamus, although few fibers were found in telencephalic areas, such as the striatum, that are known to project to the tectum in anurans. Large populations of CRF-ir cells were observed in the bed nucleus of the stria terminalis and preoptic area as well as in the ventral infundibular hypothalamus. CRF-ir neurons and fibers also were observed in several midbrain and brain stem areas. Colchicine treatment significantly enhanced CRF-ir neurons and fibers throughout the brain, and revealed CRF-ir cell groups in several brain areas (including the dorsal hypothalamus) that were not observed in untreated animals. Intrinsic CRF-immunoreactive neurons were routinely observed in cell layer 8 and sometimes in layer 6 of the optic tectum in both untreated and colchicine-treated animals. CRF was detected in toad optic tectum by radioimmunoassay, although tectal CRF content was less than that of the hypothalamus and forebrain. Unilateral eye ablation did not affect CRF content of the contralateral optic tectum. We conclude that CRF-producing neurons are widely distributed in several areas of the toad brain known to be involved in regulating the behavioral, autonomic and endocrine response to stressors, including the optic tectum and several brain areas known to project to the optic tectum. Furthermore, retinal afferents do not contribute significantly to tectal CRF content.     

Development of NADPH-diaphorase activity in the central nervous system of the cichlid fish, Tilapia mariae

The distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was studied in the cichlid fish Tilapia mariae during ontogenesis by the histochemical reaction of NADPH-diaphorase that indicates, in aldehyde-fixed tissue, the presence of nitric oxide synthase, which is the enzyme responsible for nitric oxide production. The first appearance of NADPH-diaphorase-positive neurons has a striking bilateral symmetry and occurs 20 h after fertilization (stage 8) in the olfactory placodes and in the neural tube where two clusters of positive neurons were seen in the diencephalon and in the rhombomere r4 of the hindbrain. Two days after fertilization (stage 10), the clusters of positive neurons showed labeled axons. The two longitudinal fiber bundles that arose from the diencephalic positive neurons ran caudally in the tract of the postoptic commissure. At stage 12 (3.5 days after fertilization), new populations of NADPH-diaphorase-positive neurons appeared in the telencephalon, in some diencephalic nuclei, and in the hypothalamus. Several trigeminal motor neurons showed strong NADPH-diaphorase activity, whereas the optic tectum and cerebellum were completely free of enzymatic activity. In the hindbrain, clusters of positive neurons were seen in the octavolateral region and in the region defined by the exit of the vagus nerve. In the cervical spinal cord, some ventral putative motor neurons were labeled. At stage 14 (5.5 days after fertilization), several periventricular neurons of the optic tectum and some neurons of the cerebellar lamina were labeled. Dorsal neurons, including a few large superficial neurons were also labeled in the cervical spinal cord. NADPH-diaphorase activity was seen in the neuropil area of the telencephalon, the target of olfactory inputs, and in the sensory dorso-lateral area of the spinal cord.     

Pituitary adenylate cyclase-activating polypeptide in the brain, spinal cord and sensory organs of the zebrafish, Danio rerio, during development

The distribution of Pituitary adenylate cyclase-activating polypeptide (PACAP) was investigated in the brain, pituitary and sensory organs of the zebrafish, Danio rerio, during development, in juvenile and adult specimens, using the immunofluorescence method. In 24 h post fertilization (hpf) embryos, PACAP immunoreactive cells appeared in the rostral telencephalon, dorsal diencephalon, caudal and medial rhombencephalon, spinal cord and retina. At 48 hpf stage, positive cells were present in the dorsal diencephalon, medial rhombencephalon, spinal cord, retina and olfactory placode (Op). At 72 hpf stage, additional immunoreactive elements appeared in the medial telencephalon, hypothalamus, mesencephalic tegmentum, retina and otic sensory epithelium (Ose). At day 5, new immunoreactive cells were found in the anterior rhombencephalon and pituitary pars distalis. At day 13, positive cells were mainly concentrated in the mesencephalic tegmentum and spinal cord. In the telencephalon, diencephalon, rhombencephalon and pituitary, the distribution of positive cells was similar to that previously reported. At 1 month stage, positive cells were detected in the hypothalamus, nucleus of the medial longitudinal fascicle (nMlf), rhombencephalic griseum centrale (Gc) and pituitary pars distalis. At 2-3 month stages, immunoreactive elements were found in several hypothalamic nuclei, in the mesencephalic nucleus isthmi, cerebellum and pituitary. In adults, PACAP immunoreactivity was confined to a few brain regions and the pituitary. PACAP immunoreactivity was transiently expressed in several regions suggesting that the peptide may have a role in the control of cells differentiation and proliferation during zebrafish ontogeny. The finding of positive fibers in the pituitary from day 5 onward indicates that PACAP may function from this stage as a hypophysiotropic peptide.     

Immunohistochemical localization of three different prepro-GnRHs in the brain and pituitary of the European sea bass (Dicentrarchus labrax) using antibodies to the corresponding GnRH-associated peptides

The distribution of the cells expressing three prepro-gonadotrophin-releasing hormones (GnRH), corresponding to salmon GnRH (sGnRH), seabream GnRH (sbGnRH), and chicken GnRH-II (cGnRH-II) forms, was studied in the brain and pituitary of the sea bass (Dicentrarchus labrax) by using immunohistochemistry. To circumvent the cross-reactivity problems of antibodies raised to GnRH decapeptides, we used specific antibodies generated against the different sea bass GnRH-associated peptides (GAP): salmon GAP (sGAP), seabream GAP (sbGAP), and chicken-II GAP (cIIGAP). The salmon GAP immunostaining was mostly detected in terminal nerve neurons but also in ventral telencephalic and preoptic perikarya. Salmon GAP-immunoreactive (ir) fibers were observed mainly in the forebrain, although sGAP-ir projections were also evident in the optic tectum, mesencephalic tegmentum, and ventral rhombencephalon. The pituitary only receives a few sGAP-ir fibers. The seabream GAP-ir cells were mainly detected in the preoptic area. Nevertheless, sbGAP-ir neurons were also found in olfactory bulbs, ventral telencephalon, and ventrolateral hypothalamus. The sbGAP-ir fibers were only observed in the ventral forebrain, innervating strongly the pituitary gland. Finally, chicken-II GAP immunoreactivity was only detected in large synencephalic cells, which are the origin of a profuse innervation reaching the telencephalon, preoptic area, hypothalamus, thalamus, pretectum, posterior tuberculum, mesencephalic tectum and tegmentum, cerebellum, and rhombencephalon. However, no cIIGAP-ir fibers were detected in the hypophysis. These results corroborate the overlapping of sGAP- and sbGAP-expressing cells in the forebrain of the sea bass, and provide, for the first time, unambiguous information on the distribution of projections of the three different GnRH forms expressed in the brain of a single species.     

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.     

An immunocytochemical study of the olfactory projections in the three-spined stickleback, Gasterosteus aculeatus, L

The distribution of olfactory fibers in the brain of the three-spined stickleback was visualized by means of immunohistochemistry. The labeling of the olfactory fibers was produced by serum containing antibodies against somatostatin-14. Olfactory fibers were observed entering the olfactory bulbs, where they terminated in the glomerular layer or collected into fascicles that coursed through the bulbs into the telencephalon without participating in the formation of the glomerules. In the telencephalon the fascicles, which belonged to the medial olfactory tract, formed two fiber systems: ventral descending fibers and dorsal descending fibers. The ventral descending fibers could be followed through the ventral telencephalon to the vicinity of the lateral tuberal nucleus. The dorsal descending fibers coursed via the anterior commissure to the posterior part of the telencephalon. Part of the postcommissural fibers of the dorsal descending system coursed to the posterior zone of the area dorsalis telencephali while others left the telencephalon via the medial forebrain bundle and could be followed to the periventricular hypothalamus. Some axons formed synaptic contacts with unlabeled cell bodies in the nucleus of the terminal nerve which, in this species, is situated immediately behind the bulbs. In addition, an extensive terminal field associated with the dorsal descending fibers was found in the ventromedial aspects of the telencephalon. It is unlikely that the labeling represents immunoreactive somatostatin-14 because: 1) the labeling persisted after the absorption of the antiserum with synthetic somatostatin-14; 2) antiserum against somatostatin-14 from another manufacturer did not have this labeling property; and 3) the production of the absorbable labeling depended on the choice of fixative whereas the production of the unabsorbable labeling did not.     

Immunocytochemical analysis of the dopamine system in the forebrain and midbrain of Raja radiata: evidence for a substantia nigra and ventral tegmental area in cartilaginous fish

The distribution of dopamine-containing cell somata and fibers in the forebrain and midbrain of a cartilaginous fish, Raja radiata, was investigated by means of antibodies directed against dopamine. Many small dopamine immunoreactive neurons are distributed throughout the telencephalon, including the olfactory bulbs. Within the diencephalon and particularly in the hypothalamus, i.e., in the nucleus preopticus, nucleus suprachiasmaticus, the paraventricular organ, lateral hypothalamic area, recessus mamillaris, and nucleus tuberculi posterioris, numerous cell somata stain for dopamine. In the mesencephalon, two distinct cell masses are found, which on the basis of their immunoreactivity for dopamine and their location, may be homologous to the substantia nigra and ventral tegmental area of other vertebrates. Dopamine immunoreactive fibers are found in the glomeruli of the olfactory bulbs, in ventral portions of the telencephalon, where a dense dopaminergic plexus innervates the area superficialis basalis and striatum, and in the diencephalon, where the inferior lobe is the most densely innervated structure. In the mesencephalon, the dopamine immunoreactive fibers are confined predominantly to the periventricular zone and lateral portions of the tectum. We conclude that much of the dopaminergic system in Raja radiata is strikingly similar to that seen in amniotes.     

Development of the histaminergic neurons and expression of histidine decarboxylase mRNA in the zebrafish brain in the absence of all peripheral histaminergic systems

The histamine-storing neural system in adult and developing zebrafish (Danio rerio) was studied with immunocytochemical and chromatographical methods. Furthermore, the gene for histidine decarboxylase was partially cloned and its expression mapped with in situ hybridization. The histamine-storing neurons were only seen in the caudal hypothalamus, around the posterior recess of the diencephalic ventricle. Almost all parts of the brain, except the cerebellum, contained at least some histamine-immunoreactive fibres. The ascending projections had the rostral part of the dorsal telencephalon as a major target. Descending projections terminated in the torus semicircularis, central grey and inferior olive. A prominent innervation of the optic tectum, which has not been reported in other fish, was seen. The in situ hybridization gave a strong signal in cells with the same anatomical position as the histamine-immunoreactive neurons. The first histamine-immunoreactive neurons appeared in the ventral hypothalamus at about 85 h post-fertilization, and at 90 h, immunoreactive fibres terminated in the dorsal telencephalon. The embryonic histamine production described in mammals was lacking in this species. Both immunocytochemical and chromatographical studies indicated that histamine is absent in all other parts of the zebrafish body, and no specific hybridization was seen in any other part of the fish than the hypothalamus. The zebrafish could therefore be a very useful model for pharmacological in vivo studies of the histaminergic system of the brain, since the powerful peripheral actions of histamine should be lacking in this species.     

Organization of vasoactive intestinal peptide-like immunoreactive system in the brain, olfactory organ and retina of the zebrafish, Danio rerio, during development

The localization of vasoactive intestinal peptide (VIP)-like immunoreactivity was investigated in the brain, olfactory system and retina of the zebrafish, Danio rerio, during development and in juvenile specimens, by using the indirect immunofluorescence and the peroxidase-antiperoxidase methods. In 24 h post fertilization (hpf) embryos, VIP-like immunoreactive cells were present in the olfactory pit, the retina, and several regions of the brain, including the dorsal telencephalon, the diencephalon, the tegmentum of the mesencephalon, the caudal rhombencephalon and the anterior pituitary. In 48 hpf embryos, additional VIP-like immunoreactive cell bodies were found in the ventral telencephalon, whereas in the diencephalon VIP-like immunopositive cells were more concentrated within the ventro-caudal hypothalamus. During the 7 day larval period, a dense plexus of VIP-like immunoreactive fibers first appeared in the olfactory bulbs. In 15-day-old larvae, two new groups of positive cells were observed in the periventricular preoptic nucleus and in the dorsal rhombencephalon. In 1 month/2 months old animals, VIP-like immunoreactive elements were confined to the olfactory organ, the olfactory bulbs, the periventricular preoptic nucleus and the pituitary, pars distalis. At 3 months stage, a large number of cells was observed in the periventricular preoptic nucleus. Western immunoblot analysis confirmed that VIP-like peptides, with molecular weight similar to that of synthetic VIP, are present early during the development of zebrafish. These results show that VIP-like immunoreactive structures appear early during ontogeny both in the olfactory pit, retina and brain. Transient expression of positive cells was found in the retina, telencephalon, diencephalon and brainstem. The location of VIP-like immunoreactivity indicates that, during development, VIP could be involved in several neuromodulatory functions, including the processing of visual and olfactory informations, as well as growth or survival promotion activities. The presence of VIP-like immunopositive cells in the pituitary, pars distalis, suggest that, during development, VIP may influence the secretion of pituitary hormones.     

Morphology and axonal projection pattern of neurons in the telencephalon of the fire-bellied toad Bombina orientalis: an anterograde, retrograde, and intracellular biocytin labeling study

The connectivity and cytoarchitecture of telencephalic centers except dorsal and medial pallium were studied in the fire-bellied toad Bombina orientalis by anterograde and retrograde biocytin labeling and intracellular biocytin injection (total of 148 intracellularly labeled neurons or neuron clusters). Our findings suggest the following telencephalic divisions: (1) a central amygdala-bed nucleus of the stria terminalis in the caudal midventral telencephalon, connected to visceral-autonomic centers; (2) a vomeronasal amygdala in the caudolateral ventral telencephalon receiving input from the accessory olfactory bulb and projecting mainly to the preoptic region/hypothalamus; (3) an olfactory amygdala in the caudal pole of the telencephalon lateral to the vomeronasal amygdala receiving input from the main olfactory bulb and projecting to the hypothalamus; (4) a medial amygdala receiving input from the anterior dorsal thalamus and projecting to the medial pallium, septum, and hypothalamus; (5) a ventromedial column formed by a nucleus accumbens and a ventral pallidum projecting to the central amygdala, hypothalamus, and posterior tubercle; (6) a lateral column constituting the dorsal striatum proper rostrally and the dorsal pallidum caudally, and a ventrolateral column constituting the ventral striatum. We conclude that the caudal mediolateral complex consisting of the extended central, vomeronasal, and olfactory amygdala of anurans represents the ancestral condition of the amygdaloid complex. During the evolution of the mammalian telencephalon this complex was shifted medially and involuted. The mammalian basolateral amygdala apparently is an evolutionary new structure, but the medial portion of the amygdalar complex of anurans reveals similarities in input and output with this structure and may serve similar functions.     

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.     

Afferent and efferent connections of the nucleus prethalamicus in the yellowfin goby Acanthogobius flavimanus

The nucleus prethalamicus (PTh) receives fibers from the optic tectum and then projects to the dorsal telencephalon in the yellowfin goby Acanthogobius flavimanus. However, it remained unclear whether the PTh is a visual relay nucleus, because the optic tectum receives not only visual but also other sensory modalities. Furthermore, precise telencephalic regions receiving prethalamic input remained unknown in the goby. We therefore investigated the full set of afferent and efferent connections of the PTh by direct tracer injections into the nucleus. Injections into the PTh labeled cells in the optic tectum, ventromedial thalamic nucleus, central and medial parts of the dorsal telencephalon, and caudal lobe of the cerebellum. We found that the somata of most tecto‐prethalamic neurons are present in the stratum periventriculare. Their dendrites ascend to reach the major retinorecipient layers of the tectum. The PTh is composed of two subnuclei (medial and lateral) and topographic organization was appreciated only for tectal projections to the lateral subnucleus (PTh‐l), which also receives sparse retinal projections. In contrast, the medial subnucleus receives fibers only from the medial tectum. We found that the PTh projects to nine subregions in the dorsal telencephalon and four in the ventral telencephalon. Furthermore, cerebellar injections revealed that cerebello‐prethalamic fibers cross the midline twice to innervate the PTh‐l on both sides. The present study is the first detailed report on the full set of the connections of PTh, which suggests that the PTh relays visual information from the optic tectum to the telencephalon.     

Gonadotropin-releasing hormone (GnRH) immunoreactive system in the brain of the dwarf gourami (Colisa lalia) as revealed by light microscopic immunocytochemistry using a monoclonal antibody to common amino acid sequence of GnRH

The present paper aims to give a morphological basis for the study of the terminal nerve system and its relation to the whole gonadotropin-releasing hormone (GnRH) immunoreactive (ir) neuronal system. We examined the GnRH-ir neuronal system of a tropical fish, the dwarf gourami, by using a recently developed monoclonal antibody against GnRH (LRH13) which recognizes the amino acid sequence common to all known variants of GnRH (Park and Wakabayashi, Endocrinol. Jpn. 33:257-272, '86). The ganglion cells of the terminal nerve (TN-ggl cells) in the transitional area between the olfactory bulb and the telencephalon reacted strongly with the LRH13. A distinct bundle of axons emanating from the TN-ggl cells ran caudally through the ventral telencephalon and the preoptic area. Some of these axons entered the optic nerve and innervated the retina. The remaining axons continued caudally to enter the hypothalamus and the midbrain. A second group of GnRH-ir cell bodies was found in the preoptic area. A distinct bundle of GnRH-ir fibers originating from these cell bodies innervated the pituitary. This pathway is equivalent to the preoptico-infundibular pathway of other vertebrates, and the GnRH in this pathway is presumed to function as hypophysiotrophic hormone to facilitate the release of gonadotropins from the pituitary. The distribution of GnRH-ir fibers in the brain was extensive. Most fibers apparently originated from the TN-ggl cells and covered various brain regions from the olfactory bulb to the spinal cord. They were especially abundant in the olfactory bulb, ventral telencephalon, preoptic area, optic tectum, and some hypothalamic areas. Thus, GnRH might function as a neuromodulator and/or neurotransmitter in these areas. The abundant GnRH-ir fibers in the ventral telencephalon and the preoptic area might affect some aspects of sexual behavior, since these areas have been suggested to be involved in the control of sexual behavior in teleosts.     

Distribution of melanin-concentrating hormone-like immunoreactivity in the central nervous system of Rana esculenta

The distribution of salmon and rat melanin-concentrating hormone (MCH)-like and neuropeptide glutamate-isoleucine (NEI)-like immunoreactivity in the brain and spinal cord of the frog Rana esculenta was studied with immunohistochemistry. In the telencephalon, only fibers showed immunoreactivity in the olfactory bulb, lateral pallium, diagonal band, septum, and the amygdala. Immunoreactive fibers were abundant in all diencephalic structures, except the optic tract, the visual neuropils, and the habenula. Several cells in the central thalamic nucleus and a few in the suprachiasmatic nucleus were stained with the MCH antisera. Cells and their processes were intensely stained in the dorsal hypothalamus with the MCH and NEI antisera. Immunoreactive fibers were found in all tegmental nuclei and the white matter of the mesencephalon. They formed terminal plexuses in the deep layers of the optic tectum and the laminar nucleus of the torus semicircularis. Immunoreactive fibers were sparse in the rhombencephalon and the spinal cord.     

Localization of NADPH-diaphorase in the brain of the chicken

NADPH-diaphorase, an enzyme catalyzed reaction thought to reflect the activity of nitric oxide synthase in the mammalian nervous system, was mapped in the brain of the chicken. Intensely stained neurons and fibers were found in most parts of the telencephalon, in particular in the neostriatum, paleostriatum augmentatum, olfactory tubercle, lobus parolfactorius, hyperstriatum accessorium, and hyperstriatum ventrale. Medial to the nucleus taeniae, an accumulation of stained cells was observed that appeared to merge with a band of stained neurons located dorsal to the occipitomesencephalic tract. These are considered to belong to the nucleus interstitialis of the dorsal olfactory projection. Further caudally, neurons with different staining intensities were found in the lateral hypothalamic area, lateral mammillary nucleus, periventricular organ, ventral tegmental area, medial spiriform nucleus, optic tectum, isthmooptic nucleus, mesencephalic trigeminal nucleus, interpeduncular nucleus, and central gray of the mesencephalon. A particularly dense cluster of NADPH-diaphorase positive neurons was located in the locus coeruleus. It is proposed that these might represent cholinergic cells intermingled with catecholaminergic neurons, thus forming the avian counterpart of the tegmental cholinergic nuclei of mammals. Several NADPH-diaphorase reactive neurons were seen in the parabrachial nucleus and medial and dorsal vestibular nucleus, as well as scattered in the reticular formation. In the caudal medulla, intensely stained cells were grouped around the central canal. Therefore the pattern of expression of NADPH-diaphorase, and thus possibly of nitric oxide synthase, within the avian and mammalian brain might be largely conserved. © 1993 Wiley-Liss, Inc.