Tyrosine hydroxylase-immunoreactive cell groups in the brain of the teleost fish Gnathonemus petersii.

Different antibodies against tyrosine hydroxylase (TH) were used to obtain detailed information about the distribution, morphology and chemical differentiation of catecholaminergic neurons in the highly differentiated brain of the electric mormyrid fish Gnathonemus petersii. The results show that the distribution of catecholaminergic neurons is much more widespread than was previously thought on the basis of dopamine and noradrenaline immunohistochemistry. Tyrosine hydroxylase-immunoreactive neurons were observed not only in clearly dopaminergic regions (the suprachiasmatic nucleus, the magnocellular hypothalamic nucleus and the area postrema) and noradrenergic cell groups (the locus coeruleus and inferior reticular cell group), but also in regions that do not, or only fragmentarily, display dopamine or noradrenaline immunoreactivity, including the ventral and intermediate telencephalon, the anterior and posterior preoptic cell group, the ventromedial thalamus, the pretectal region and the nucleus of the solitary tract, suggesting that they either represent depleted dopaminergic cell groups or L-dihydroxy phenylalanine-producing nuclei. Most TH-immunoreactive neurons are rather small (< 10 microns) and have only a few slender processes, but neurons in the magnocellular hypothalamic nucleus and the inferior reticular formation are multipolar and larger (10-20 microns), while those of the locus coeruleus are even more than 20 microns in diameter. The hypothalamic paraventricular organ, which is strongly dopamine and noradrenaline immunoreactive, displays minimal TH immunoreactivity, suggesting that its cerebrospinal fluid-contacting neurons do not synthesize catecholamines, but acquire them from external sources. Comparison with other teleosts shows that the catecholaminergic system in the brain of Gnathonemus is similarly organized as in Carassius, Gasterosteus, Anguilla and Aperonotus, with some variations that may partly be due to technical reasons, and partly reflect true species differences. However, TH-immunoreactive neurons in the midbrain tegmentum were not observed, confirming previous conclusions that a major difference between teleosts and mammals concerns the absence of dopaminergic midbrain groups and correlated mesencephalo-telencephalic projections in teleosts.

Distribution of noradrenaline-immunoreactivity in the brain of the mormyrid teleost Gnathonemus petersii.

The distribution of noradrenaline-immunoreactivity in the brain of the mormyrid fish Gnathonemus petersii was studied in order to evaluate the noradrenergic innervation of a number of specialized mormyrid brain regions, including electrosensory centers and a gigantocerebellum. Noradrenaline-immunoreactive (NAi) neurons occur in the hypothalamic paraventricular organ (PVO), the locus coeruleus, and the caudal rhombencephalon. In the PVO, NAi cerebrospinal fluid (CSF)-contacting neurons are located in the same regions where dopamine- and serotonin-containing CSF contacting neurons occur. The locus coeruleus consists, on each side, of at least 30 rather large NAi neurons with ventrolaterally directed dendrites and dorsolaterally coursing axons. In the caudal rhombencephalon, NAi neurons are located in the transition region between the ventromedial motor zone and the dorsolateral sensory zone. The density of NAi fibers is very high in the efferent tract of the locus coeruleus, the medial forebrain bundle, and two telencephalic, one preoptic, and one rhombencephalic subependymal axonal plexus. A marked NAi innervation is present in the dorsomedial and ventral telencephalon, the preoptic region, periventricular hypothalamic and thalamic regions, the midbrain tectum, cerebellar granular layers, the electrosensory lateral line lobe, the rhombencephalic transition region between the sensory and motor zones, and the area postrema. Other regions are more sparsely innervated by NAi fibers, but regions completely devoid of NAi fibers were not observed. Interestingly, NAi fibers form large club endings in some subdivisions of the precerebellar nucleus lateralis valvulae, and parallel fibers in the cerebellar granular layer. Comparison with the distribution of NAi or dopamine-beta-hydroxylase-immunoreactivity in other species shows that all teleosts studied to date have noradrenergic cells in the locus coeruleus and the caudal rhombencephalon. However, NAi CSF-contacting PVO cells have been described only in the teleost Gnathonemus petersii and the lizard Gekko gecko (Smeets and Steinbusch: J. Comp. Neurol. 285:453-466, '89). It is possible that they might pick up catecholamines as well as serotonin from the CSF, into which monoamines might be released by telencephalic and preoptic subependymal axonal plexuses.

An intriguing pitfall in chemical neuroanatomy: specific populations of unspecifically immunoreactive neurons in the brain of the mormyrid fish Gnathonemus petersii.

The present paper describes the location, morphology, ultrastructure and immunocytochemical properties of neurons in the brain of the mormyrid fish Gnathonemus petersii, that appear to be unspecifically immunoreactive to a number of secondary or tertiary antibodies used in immunohistochemical procedures, including rabbit-anti-mouse immunoglobulins (IGGs), rabbit peroxidase-anti-peroxidase IGGs, and rabbit-anti-sheep or sheep peroxidase-anti-peroxidase IGGs. Unspecifically immunoreactive (UI) cells have typically neuronal morphological and ultrastructural characteristics, and occur at four specific locations in the mormyrid brain. A small rhombencephalic group is located rostrolateral to the efferent octaval nucleus, between the fasciculus longitudinalis medialis and the decussation of the lateral lemniscus. A mesencephalic cluster of cells is located in the dorsal midbrain tegmentum against the tractus telencephalo-mesencephalicus. In addition, dispersed UI neurons were observed in the nucleus lateralis of the torus semicircularis and in the preoptic region above the optic chiasm. Remarkably, UI cells are clearly present in a substantial number of brains investigated, but not detectable in others. The present findings point to a curious pitfall in chemical neuroanatomy, the functional significance of which is unknown at present. In several previous studies using the brain of G. petersii, UI cells were abusively included in the description of monoaminergic cell groups. Similar cells have until now not been reported in other vertebrate brains.

Distribution of dopamine immunoreactivity in the brain of the mormyrid teleost Gnathonemus petersii.

The distribution of dopamine-containing cell bodies and fibers was studied with aid of specific antibodies against dopamine in the highly developed brain of the weakly electric fish Gnathonemus petersii. In the telencephalon, dopamine-containing cell bodies were observed in a small area, i.e., area ventralis pars dorsalis and supracommissuralis. In the diencephalon, moderate numbers of dispersed dopamine-immunoreactive cells were present in the preoptic region, while large numbers of dopamine-containing neurons occurred in the hypothalamic paraventricular organ and neighbouring regions. The paraventricular organ, located around small (anterior, intermediate, and posterior) recesses contained many dopamine-immunoreactive cerebrospinal fluid-(CSF)-contacting neurons. Dopamine-containing cells were also observed in a magnocellular hypothalamic cell group, in the nucleus of the lateral recess, and in the nucleus posterior tuberis. In the mesencephalon only a few dopamine-containing cells were observed in a dorsal tegmental (possibly pretectal) area, whereas in ventral mesencephalic regions dopamine-containing cells were lacking. More caudally, dopamine-containing cells were observed in the presumed locus coeruleus, in the caudal region of the reticular formation, and in the presumed area postrema. Dopamine-immunoreactive fiber density was very high in the medioventral hypothalamus and in the preoptic region, where a dense subependymal plexus was observed along the preoptic recess. Such a plexus was also present in the caudal rhombencephalon, where it probably arises from the area postrema. Moderate numbers of dopamine-immunoreactive fibers were present in medioventral parts of the brain along its total rostrocaudal extent as well as in several subnuclei of the torus semicircularis, in the tectum mesencephali, and in the medial part of the dorsal telencephalic area. Other parts of the dorsal telencephalic area, as well as the large cerebellum and the electrosensory lateral line lobe of Gnathonemus, did not contain detectable amounts of dopamine. In spite of the high differentiation of the brain of Gnathonemus, the distribution of catecholamines as visualized with dopamine immunohistochemistry appears to be basically similar to that described in other teleostean and actinopterygian fishes on the basis of formaldehyde-induced fluorescence or tyrosine hydroxylase immunohistochemistry.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of serotonin in the brain of the mormyrid teleost Gnathonemus petersii.

The distribution of serotonin-immunoreactive neurons and fibers was studied in the highly developed brain of the weakly electric fish Gnathonemus petersii with the aid of specific antibodies against serotonin. Serotoninergic cell bodies occur in three regions: the raphe region of the brainstem, the hypothalamus, and the transition zone between the dorsal thalamus and the pretectum. Serotoninergic raphe neurons are clustered in three groups: nucleus raphes superior, intermedius, and inferior. The latter has not been described in other teleosts and thus might be the source of the serotoninergic innervation of specific mormyrid electrosensory brain regions. Most hypothalamic serotoninergic neurons have cerebrospinal-fluid (CSF)-contacting processes and thus belong to the paraventricular organ (PVO), which in Gnathonemus is located around a number of small infundibular recesses. The distribution of serotonin in the PVO precisely matches the distribution of dopamine, as described previously. Serotoninergic cells in the thalamopretectal transition zone also have been described in other teleosts, but not in other vertebrate groups, and thus seem to represent a teleostean specialization. Serotoninergic fiber density is especially high in the medial forebrain bundle and surrounding preoptic and hypothalamic regions as well as in several telencephalic and preoptic subependymal plexus. Serotoninergic fibers appear to be almost completely absent in the large and differentiated corpus and valvula cerebelli. Comparison with the literature on teleostean serotoninergic innervation patterns reveals several mormyrid specializations, including the absence of serotonin in large parts of the mormyrid telencephalic lobes, a differentiated innervation pattern of distinct electrosensory and mechanosensory subnuclei of the torus semicircularis, a refined serotoninergic lamination pattern in the midbrain tectum, and a prominent innervation of the electrosensory lateral line lobe, the associated caudal cerebellar lobe, and the electromotor medullary relay nucleus. A distinct innervation of several types of (pre)motor neurons, such as the Mauthner cells and facial motor neurons, has not been reported previously for other teleosts. Consequently, the distribution of serotoninergic fibers as well as neurons in the mormyrid brain is substantially adapted to the high degree of differentiation of its electrosensory and telencephalic brain regions, but serotoninergic innervation is not involved in the circuitry of the most impressive part of the mormyrid brain; i.e., its large corpus and valvula cerebelli.

Serotonergic neurons in the intestine of two teleosts, Carassius auratus and Oreochromis mossambicus, and the effect of serotonin on transepithelial ion-selectivity and muscle tension.

The application of an antiserum directed against rat serotonin demonstrated serotonin-immunoreactive cell bodies and varicose nerve fibres in the myenteric plexus of both goldfish and tilapia. In the circular muscle layer immunoreactive varicose nerve fibres and a few cell bodies could be detected. A fine network of varicose fibres was observed underlying the epithelial cells. Serotonin immunoreactivity was not observed along incoming mesenterial blood vessels, suggesting that the serotonergic neurons may be intrinsic to the intestine. Immunoreactive endocrine cells were shown in the intestinal epithelium of tilapia but not in goldfish. Serotonin caused a weak contraction of the intestinal wall of the goldfish which could be blocked by tetrodotoxin and by atropine, suggesting that serotonin has an indirect action on muscular contraction. In contrast, serotonin induced a relaxation of the intestinal wall of tilapia which could not be blocked by propranolol or by tetrodotoxin. This indicates that in this species serotonin may act directly on the muscle fibres. The ion-selectivity of the intestinal epithelium of both species was modulated by serotonin. Tetrodotoxin did not inhibit this effect, suggesting that serotonin acts directly on the epithelial cells. The presence of serotonergic fibres in the muscle layer and directly underneath the epithelium, along with the effect of serotonin on muscular tension and on the ion-selectivity of the epithelium, suggests that serotonin may play a role in the regulation of motility and the epithelial function of goldfish and tilapia intestine.

The development of serotonergic raphespinal projections in Xenopus laevis.

The development of serotonin-immunoreactive neurons in the central nervous system of Xenopus laevis larvae has been studied with special emphasis on the development of the raphe nuclei and raphespinal projections. The first serotonergic neurons were observed in the rostral part of the brain stem at stage 25, only 28 hr after fertilization. By stage 28 some 20 serotonin-immunoreactive neurons were found in the rostral part of the brain stem, bearing small protrusions on the ventromedial side of the soma. These initial axonal outgrowths reach the rostral part of the spinal cord at stage 32. By stage 35/36 the growth cones of the descending serotonergic axons in the spinal cord have reached the level of the anus (10th to 15th myotome). Up to stage 45 the majority of the descending serotonergic axons was found in the dorsolateral part of the marginal zone of the spinal cord. After stage 45 some serotonergic axons were also found scattered over other parts of the spinal marginal zone. Collateral branches were first observed in the caudal part of the brain stem at stage 35/36. Later they occurred also in the rostral (stage 43) and caudal (stage 50) spinal cord, usually on fibers in the ventral half of the spinal cord. The number of serotonergic neurons in the central nervous system (brain stem and hypothalamus) increased steadily throughout development until stage 45. After that the total number of serotonergic neurons in the central nervous system increased about two times faster than the number of serotonergic neurons in the raphe nuclei, due to a massive increase of serotonergic neurons in the hypothalamus. The present study shows that young, just differentiated raphe neurons already contain serotonin. The generation of these neurons appears to take place in the ventricular zone (matrix) of the brain stem between the caudal border of the mesencephalon and the entrance of the nervus octavus. From here these neurons seem to migrate to their final destination. The distribution of serotonin-immunoreactive neurons in the brain stem suggests that a superior (not described so far in Anura) and an inferior raphe nucleus can be distinguished in Xenopus. A rostrocaudal gradient seems to be present in the production of serotonergic neurons which project to the spinal cord. Spinal projections from the raphe nuclei are particularly extensive from the nucleus raphes inferior and gradually decrease rostralwards. In the rostral part of the nucleus raphes superior almost no neurons projecting to the spinal cord are found.

Serotonin-immunoreactive cells in the superior cervical ganglion of the rat. Evidence for the existence of separate serotonin- and catecholamine-containing small ganglionic cells.

Superior cervical ganglia of 8 adult male rats were examined by indirect immunofluorescence microscopy, using an antibody to a conjugate of bovine serum albumin and serotonin, and an antibody to a conjugate of bovine serum albumin and noradrenaline. The fixative used was 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3. Consecutive cryostat sections of whole ganglia were alternately stained with these antibodies. Serotonin- as well as catecholamine-immunoreactive, small ganglionic cells were demonstrated, both arranged in clusters. Serotonin-immunoreactive cells were mostly located at the cranial or caudal side of the ganglia. Serotonin-immunoreactive cells provided with processes were easily observed. Only a few mast cells were seen. Catecholamine-immunoreactive cells were rounded without processes. This cell type did not seem to have a preferential position within the ganglia. Intermingling of both immunoreactive, small ganglionic cells was not observed. A considerable variety in the number of both immunoreactive cell types was established. Catecholamine-immunoreactive cells were absent in 3 our of 8 animals. Evidence is provided that the catecholamine-immunoreactive cells are storing noradrenaline. The presented data seem to indicate the presence of 3 different types of small, intensely fluorescent (SIF) cells in the superior cervical ganglion of the rat, viz. a dopamine-storing, a noradrenaline-storing, and a serotonin-storing SIF cell type.

Use of antibodies to norepinephrine and epinephrine in immunohistochemistry.

Antibodies were raised in rabbits and sheep with antigens prepared by coupling NE or E to bovine serum albumin. Tissues were fixed with either 4% paraformaldehyde or 4% paraformaldehyde plus 0.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.3). Cryostat sections were stained by the indirect immunofluorescence technique. Two parenchymal cell types, representing the NE and E-storing cells, were observed in the adrenal medulla. In the central nervous system, fibers and perikarya could be visualized by the antiserum to the NE antigen. In the peripheral nervous system, NE immunoreactive fibers were demonstrated. There was no detectable cross reactivity of NE antibodies with regard to E and 5-HT. Cross reactivity of E antibodies to NE and 5-HT equally could not be demonstrated.

Appearance of tyrosine hydroxylase, aromatic amino-acid decarboxylase, dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase during the ontogenesis of the adrenal medulla: an immunohistochemical study in the rat.

The cellular localization of the enzymes tyrosine hydroxylase (TH), aromatic amino-acid decarboxylase (or dopa decarboxylase, DDC), dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla of adult rats and rat fetuses (14th, 17th, 18th, 19th and 21st day) was examined. In the prenatal stages the medullary blastema and an adjacent part of the primitive sympathetic trunk were also investigated. Tissues were fixed in ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). Cryostat sections (10 micron in thickness) were stained by the indirect immunofluorescence technique. Rabbit antibodies to TH (isolated from human pheochromocytoma), DDC, DBH and PNMT (the latter three isolated from bovine adrenal medulla) were used. Sections incubated with serum of non-immunized rabbits were used as controls. In the adult adrenal medulla, two cell types can be distinguished. One cell type contains only TH, DDC and DBH. The other cell type contains PNMT in addition. It is concluded that these cells correspond to the noradrenaline-(NA-) and adrenaline- (A-)storing cells respectively. In all prenatal stages TH, DDC and DBH are found in the primitive sympathetic trunk, in the medullary blastema, and in the medullary cells which have migrated into the cortical "anlage". PNMT is observed for the first time on the 18th day. Moreover, PNMT could only be demonstrated inside the adrenal gland. From these observations it is concluded that the capacity to synthesize NA is developed even before the "medullary" cells have reached the cortical "anlage". On the contrary, the capacity to synthesize A seems to be acquired only after this contact is established. The hypothesis is put forward that this phenomenon might indicate the induction of PNMT by glucocorticoids secreted by the fetal cortex.