Detailed organization of nitric oxide synthase, vasopressin and oxytocin immunoreactive cell bodies in the supraoptic nucleus of the female rat.

The anatomical distribution and quantitative relations of cell bodies containing neuronal nitric oxide synthase (nNOS), 8-arginine vasopressin (AVP) and oxytocin (OT) were examined throughout the supraoptic nucleus (SON) of the female rat by means of immunocytochemical and NADPH-diaphorase (NADPH-d) histochemical techniques using a triple labelling methodology. Seven chemically defined populations of neurons containing all combinations of either nNOS, AVP or OT were identified. nNOS-containing (NADPH-d positive) neurons, amounting to about 40% of all neurons counted, were most frequent in central and dorsal regions, and were evenly distributed along the rostro-caudal axis. Two small nNOS-positive neuronal populations were preferentially located dorso-centrally in the nucleus: nNOS-positive neurons containing both AVP- and OT-immunoreactivity, and neurons only containing nNOS. Slightly less than half of all nNOS-positive neurons contained AVP, and a similar share of nNOS-positive neurons contained OT. The occurrence of nNOS-positive/AVP-containing neurons was highest in the caudal half, whereas that of nNOS-positive/OT-neurons was highest in the rostral half of SON. The data demonstrate new findings concerning the anatomical organization and co-localization patterns of nNOS-, AVP- and OT-containing neuronal populations in SON. We conclude that the absolute and relative occurrence of the identified neuronal populations vary markedly in different parts of SON. This is important to take into consideration when performing, and evaluating experimental investigations concerned with neurochemical changes in SON.

Distribution of GABA-immunolabeling in the early zebrafish (Danio rerio) brain.

The spatial and temporal pattern of GABA-expression in the brains of zebrafish (Danio rerio) embryos was studied by means of immunohistochemical techniques. GABA is said to exert neurotrophic actions in the early regulation of the differentiation of the central nervous system. In early stages GABAergic cells form distinct clusters throughout the CNS. As development progresses, more GABAergic clusters appear, and a pattern of GABAergic axonal projections is well defined. Although there is a corresponding pattern of distribution and appearance of GABA-expression in the brain of different teleosts, further studies are needed to establish its role during early morphogenesis of the CNS of vertebrates.

Hypophysiotrophic systems in the brain of the Atlantic salmon. Neuronal innervation of the pituitary and the origin of pituitary dopamine and nonapeptides identified by means of combined carbocyanine tract tracing and immunocytochemistry.

The neuroanatomical organization of neurons projecting to the pituitary and the origin of pituitary dopamine and nonapeptides were investigated in the brain of the Atlantic salmon (Salmo salar). Carbocyanine tract tracing in combination with tyrosine hydroxylase, arginine vasotocin and isotocin immunocytochemistry for double labelling revealed a previously unknown organization of hypophysiotrophic cell groups and their extrahypothalamic projections, and provide the first direct identification in a teleost fish of the origin of the dopaminergic and nonapeptidergic innervation of the pituitary. The present data include identification of (1) hypophysiotrophic neurons in the ventral telencephalon and in the periventricular preoptic nucleus, (2) large (magnocellular) vasotocinergic hypophysiotrophic neurons in the most rostral extension of the preoptic area, (3) a distinct neuronal group located in a supraoptic/suprachiasmatic position in the anterior periventricular nucleus, that seems to be the major source of dopaminergic innervation of the pituitary, (4) the nonapeptidergic hypophysiotrophic neurons in the preoptic nucleus, (5) hypophysiotrophic neurons in the ventral and posterior hypothalamus of which some are of liquor-contacting type, (6) projections from hypophysiotrophic and non-hypophysiotrophic neurons in the preoptic nucleus to extrahypothalamic areas such as thalamic and periventricular pretectal nuclei, and (7) subdivisions within the preoptic nucleus that exhibit different combinations of hypophysiotrophic and extrahypothalamic efferent connections. Together with previous studies of retinohypothalamic projections and neurochemical organization of hypothalamic/preoptic areas, the present data suggest that the preoptic nucleus and the anterior periventricular nucleus in teleosts possess functional subdivisions with features that resemble those of the paraventricular, supraoptic and suprachiasmatic nuclei of other vertebrates. In the Atlantic salmon, specific dopaminergic and nonapeptidergic neuronal subdivisions are proposed to play a role for photoperiod control of endocrine activity.

Histamine-immunoreactive neurons in the brain of the teleost Gasterosteus aculeatus L. Correlation with hypothalamic tyrosine hydroxylase- and serotonin-immunoreactive neurons.

The distribution of putative histaminergic neurons in the brain of a teleost, the three-spined stickleback, was investigated by means of immunocytochemistry using specific antibodies against histamine (HA), and conventional microscopy as well as confocal laser scanning microscopy. Histamine-immunoreactive (HAir) neurons form discrete populations ventral to the nucleus of the posterior recess (NRP) and in the nucleus saccus vasculosus (NSV), which belong to the periventricular hypothalamic nuclei. The neuronal somata are subependymally located, and do not possess apical neurites contacting the cerebrospinal fluid. They give rise to both long-range and local axonal projections. The local projections give rise to a field of dense punctate immunoreaction dorsal to the NRP and lateral to the NSV. Long-range projections are comprised of ascending projections to the thalamus, habenula, preoptic area and dorsal telencephalon; and descending projections via the posterior tuberal nucleus, ventrally to the nucleus interpeduncularis, and dorsally into the central gray. HAir neurons occur together with serotoninergic cerebrospinal fluid-contacting (CSFc) neurons in the NRP, and with tyrosine hydroxylase-immunoreactive (THir) neurons in the NSV. Although HAir elements occur together with THir ones in many brain areas, direct contacts between the two neurotransmitter systems are rare. The putative histaminergic neurons in the brain of the three-spined stickleback constitute a very discrete neuronal system, with a major projection area in the dorsal telencephalon in a region which is considered homologous with the dorsal pallium of land vertebrates.

Nitric oxide synthase in the brain of a teleost.

The presence and distribution of the nitric oxide (NO) converting enzyme, NO synthase (NOS), was investigated in the brain of a teleost, the Atlantic salmon. Both NOS immunoreactive and NADPH diaphorase positive, non-neuronal and neuronal cell bodies, fibers and putative nerve terminals were identified throughout the brain. Even so, the staining was not identical in all regions. NO, synthesized by NOS-like enzymes, may play an important role in a diversity of cellular mechanisms in the brain of the salmon, including in neural systems related to olfactory, visual, hypophysiotrophic, viscero-sensoric and motor functions.

Nitric oxide synthase in the CNS of the Atlantic salmon.

This study describes for the first time the presence and distribution of the nitric oxide (NO) synthesizing enzyme, NO synthase (NOS), in the retina of a teleost. NADPH diaphorase (NADPHd) histochemistry and NOS immunohistochemistry revealed both NOS immunoreactive and NADPHd positive structures in photoreceptor outer segments, amacrine cells, horizontal cells and ganglion cells. Since NO is known to stimulate the synthesis of cGMP, our results implicate an important role for NO in retinal function, especially in cGMP related events in the photoreceptors.

Neural projections of the pineal organ in the larval sea lamprey (Petromyzon marinus L.) revealed by indocarbocyanine dye tracing.

The distribution of the central neural connections of the pineal organ of the larval sea lamprey was investigated by means of anterograde and retrograde tracing with the fluorescent lipophilic dye, DiI (1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate). Pinealofugal projections are well developed in larvae, extending from the posterior commissure into the diencephalon and mesencephalon. Small numbers of neurons were retrogradely labelled in the transition zone between the diencephalon and the mesencephalic tegmentum. These cells may constitute the first pinealopetal system described in anamniotes.

Galanin receptors in the brain of a teleost: autoradiographic distribution of binding sites in the Atlantic salmon.

The distribution of galanin (GAL) binding sites in the brain of the Atlantic salmon (Salmo salar) was investigated by means of radioligand binding in conjunction with autoradiography by using high-performance liquid chromatography (HPLC) characterized radio-iodinated porcine galanin ([125I]GAL). On slide-mounted sections of frozen salmon brain homogenate, [125I]GAL (4 nM) bound rapidly and reversibly to a single population of sites with a Kd of 1.0 +/- 0.08 nM (n = 3) and Bmax of 2.38 +/- 0.19 fmol/mg wet tissue. Specific [125I]GAL binding was found in cellular regions, in fiber tracts, and in neuropil areas throughout the brain, except for in the olfactory bulb, pineal organ, and cerebellum. Autoradiographic microdensitometric measurements revealed high total [125I]GAL binding in the ventral hypothalamus (inferior lobes; around 7-12 fmol/mg tissue), the dorsal spinal cord (between 6 and 12 fmol/mg tissue), sublayers of the optic tectum (around 8 fmol/mg), torus semicircularis (around 7 fmol/mg), and glomerular complex (around 6 fmol/mg). Intermediate densities of [125I]GAL binding (3-5 fmol/mg tissue) were found in the pituitary, telencephalon, dorsolateral thalamic nucleus, and raphe nuclei and in association with the forebrain bundles. Except for in the optic tectum, there is a good concordance of [125I]GAL binding sites and GAL-immunoreactive fiber projections in most brain areas of the salmon. The wide distribution of GAL binding sites provides further evidence that a GAL-like substance might be involved in a diversity of brain functions of teleosts. The topographic distribution of target sites in the hypothalamo-hypophyseal axis indicates that GAL-like substances may have both direct and indirect effect on pituitary functions while in extrahypothalamic areas, functional implications by GAL may include involvement in somatosensory, central gustatory, olfactory, and visual functions. This study provides evidence for the presence of a specific GAL receptor in the brain of the Atlantic salmon. Together the distribution of GAL binding and GAL-like molecules provide a covering delineation of the GAL neuronal system in the brain of the Atlantic salmon. Comparisons with mammals suggest that the GAL receptor molecule has been well preserved during evolution and that GAL-like substances may be present, and even possess similar functional properties, throughout the vertebrate phylogeny.

DiI tracing in combination with immunocytochemistry for analysis of connectivities and chemoarchitectonics of specific neural systems in a teleost, the Atlantic salmon.

An important goal in neuroanatomical research is to identify the neurotransmitters in specific neural pathways. One step towards this goal is to combine experimental neuronal tracing with immunocytochemistry. Unfortunately, optimal procedures for nerve tracing and immunocytochemistry are not always compatible. Carbocyanine compounds have recently been shown to be efficient tracers both in vivo and in paraformaldehyde-prefixed neural tissue. The possibility to apply them to prefixed tissue make them suitable for tracing of neural pathways that are not easily accessible in vivo. We have optimized the procedures for neural tracing with one carbocyanine compound, DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), in the CNS of a teleost fish, and evaluated its compatibility with different immunocytochemical protocols. We have compared several immunocytochemical protocols, taking into account cryostat and vibratome sectioning, glutaraldehyde post-fixation to stabilize DiI, antibodies with different capacity for tissue penetration and the use of detergents, and antibodies with different sensitivity to prolonged paraformaldehyde fixation. We have also evaluated the choice of marker for immunoreactivity and compared indirect immunofluorescence techniques using different fluorophores, and the peroxidase-antiperoxidase (PAP) technique with or without nickel enhancement of the diaminobenzidine reaction product. It appears that DiI tracing of neural connections in the teleost CNS yields very consistent results and that the combination with immunocytochemistry is very reliable. We present four different basic protocols for combined DiI tracing and immunocytochemistry, with notes on their specific applicability. Owing to their reliability, the protocols may prove useful in comparative neuroanatomical studies of other vertebrates, particularly fish and amphibians, as well as in studies of developmental changes and neural plasticity in fish and amphibians.

Galanin-like immunoreactivity in the brain of teleosts: distribution and relation to substance P, vasotocin, and isotocin in the Atlantic salmon (Salmo salar).

The presence of galanin-like substances and their relation to substance P-, vasotocin-, and isotocin-immunoreactive neurons and fibers in the brain of teleosts was investigated with immunohistochemical methods. Two specific antisera against synthetic porcine galanin (GAL) revealed cell bodies and fibers in the brain of four different teleost species (Salmo salar, Carassius carassius, Gasterosteus aculeatus, and Anguilla anguilla). In all four species the main location of galanin immunoreactivity was in the hypothalamo-pituitary region. A detailed study of the distribution of galanin immunoreactivity in S. salar showed that galanin immunoreactive (GALir) perikarya were present in the nucleus preopticus periventricularis, an area that may be compared to the supraoptic nucleus in mammals, and in the nucleus lateralis tuberis, a nucleus involved in pituitary control in fishes that may be compared with the arcuate nucleus in mammals. GALir perikarya were found also in the nucleus recessus lateralis and in the nucleus recessus posterior. Numerous GALir fibers were present in the telencephalon and diencephalon, whereas only small numbers of fibers were found in the brainstem. In contrast to the situation in mammals, no GALir perikarya were observed in the brainstem areas corresponding to the noradrenergic locus coeruleus and serotonergic raphe nuclei in S. salar. We did not find any coexistence of GALir substances with arginine vasotocin or isotocin in neurosecretory neurons, as has been shown for galanin with the mammalian counterparts vasopressin and oxytocin. Also, the galanin-like substance(s) and their structurally closest related peptide family, the tachykinins, belong to separate neuronal systems in teleosts. The presence of GALir neurons in brain areas known to be involved in pituitary control, and a massive GALir innervation of the pituitary, strongly indicate a role for galanin-like substances in pituitary control also in teleosts. Furthermore, the presence of extrahypothalamic GALir fibers suggests involvement of galanin-like substances in other brain functions in teleosts. In conclusion, there are general similarities between teleosts and mammals concerning the distribution of galanin-like substances. However, there seem to be substantial differences in their distribution relative to functionally related peptides within the hypothalamo-pituitary system. Whereas galanin appears to be colocalized and released together with vasopressin and oxytocin in mammals, in teleosts the homologous substances are contained within different sets of neurons that innervate the same target, the pituitary.

Vasopressin- and proctolin-like immunoreactive efferent neurons in blowfly abdominal ganglia: development and ultrastructure.

In the neural sheath of the fused thoracicoabdominal ganglia of the blowfly Calliphora erythrocephala, extensive neurohaemal areas can be seen in the electron microscope. A separate set of neurohaemal areas located in the sheath of the lateral abdominal nerve roots contain neural terminals of at least three morphological types. To determine which bioactive substances are stored and possibly released from the neurons supplying these neurohaemal areas, we applied a large number of antisera raised against different neuropeptides of invertebrate and mammalian type. Antisera to two types of neuropeptides react with neurons innervating the sheath of the abdominal nerve roots: antisera to lysine-vasopressin and proctolin. There are only 14-24 vasopressin-like immunoreactive (VPLI) neurons in the entire nervous system of Calliphora. These are all restricted to a bilateral cluster in the fused abdominal ganglia. From this cluster, the neurohaemal areas in abdominal nerve roots are supplied. Proctolin-like immunoreactivity (PLI) can be seen in a large number of neurons in the nervous system of blowflies. The supply of PLI terminals to the abdominal nerve roots is from 12 to 14 neurons in a bilateral cluster of abdominal PLI neurons. It is clear from light- and electron-microscopic immunocytochemistry that the two antisera label two separate populations of neurons that form overlapping terminals in the neural sheath. The immunoreactive terminals are located just below the permeable acellular basal lamina of the neural sheath. Hence, it is likely that at least two different bioactive peptides can be released neurohormonally into the circulation. An additional set of four efferent PLI neurons send axons into the medial abdominal nerve. These do not form neurohaemal terminals in the nerve root, but may innervate the hindgut. Also in the larval nervous system, VPLI and PLI neurons can be recognized. In the larva, the peptide-containing neurons are segmentally arranged. The 14 larval VPLI neurons supply segmental abdominal nerves with axons that run inside the nerves to their targets. During metamorphosis, the segmental nerves fuse and the VPLI axons invade the neural sheath where they arborize and form varicose terminals. About the same number of PLI neurons could be detected in the abdominal ganglia of larval and adult flies. Only for a set of four caudal PLI neurons could efferent axons be traced in the larva. These axons run inside the medial abdominal nerves. The same four PLI neurons, with the same axonal projections, can be recognized in the adults.